Models of the membrane

Bodunrin O. Ottu
Department of Cell Biology and Genetics,
Room 203, Faculty of Science, University of Lagos.
E: [email protected]

CBG 211
January 2020
Learning objectives
At the end of the lecture, student should be able to understand;
The structure and function of membranes
The different models of membrane structure
 Introduction
Cell Biology is the study of processes carried out by individual
cells such as cell division, organelle inheritance and biogenesis,
signal transduction and motility.
 Introduction
Recall that there are three domains of life.

Archaea and Bacteria (unicellular prokaryotes) lack a

nucleus and other organelles.

Eukaryotes, unicellular or multicellular, have a

nucleus and other membrane-bound organelles.
 Eukaryotes- majorly Fungi, Plants and Animals

Bacterial cell
 Introduction
An essential feature of every cell is
the presence of plasma membranes
that define the boundaries of the cell
and its various internal compartments
ensuring that its contents are retained.

What are the constituents of the

plasma membrane? How are they
organised? We will discuss later on.
Introduction cont’d
The internal volume of the cell exclusive of the nucleus
is called the cytoplasm.
Cytoplasm = organelles + the semifluid cytosol in which
organelles are suspended.
Double membrane e.g. Nucleus, Mitochondrion,
Chloroplast.
Single membrane e.g. Lysosome, Peroxisomes.
No membrane e.g. Ribosome (organelle or not?)
Membrane Functions

Hardin et al., 2012.

Membrane Functions
Biological membranes play five related yet distinct roles
1. They define the boundaries of the cell and its organelles
and act as permeability barriers.
2. They serve as sites for specific biochemical functions,
such as electron transport.
3. Membranes also possess transport proteins that regulate
the movement of substances into and out of the cell and its
organelles.
4. Membranes contain the protein molecules that act as
receptors to detect external signals.
5. They provide mechanisms for cell-to-cell contact,
adhesion and communication.
Models of the membrane
❖ 1880’s- Lord Rayleigh, Agnes Pockels and
many others investigated the spreading of oil
on water.
❖ 1899 - Overton described a lipid barrier
between the eukaryotic cell cytoplasm and the
outside world. The work also focused attention
on the cell surface membrane as the membrane
that is most accessible to experimental study.
❖ 1917 - Based on the experiments of Agnes
Pockels, but with an improved apparatus,
Hardin et al., 2012. Langmuir published a model of how oil
molecules are orientated at the water/air
interface.

Edidin, 2003
 Models of the membrane
❖ 1880’s - Lord Rayleigh, Agnes Pockels and many others
investigated the spreading of oil on water.

❖ 1899 - Overton described a lipid barrier between the eukaryotic

cell cytoplasm and the outside world. The work also focused
attention on the cell surface membrane as the membrane that is
most accessible to experimental study.

❖ 1917 - Based on the experiments of Agnes Pockels, but with an

improved apparatus, Langmuir published a model of how oil
molecules are orientated at the water/air interface.

❖ 1925 - Gorter and Grendel used Langmuir’s

methods to infer that erythrocyte membranes
are bilayers.
❖ 1935 – Jim Danielli and Hugh Davson
described an influential membrane model that
integrates lipids and proteins.

Hardin et al., 2012. Edidin, 2003

Heimburg, 2007
 Models of the membrane
❖ 1959 – J. David Robertson argued that all cell
membranes have a common structure.

❖ 1969 - The fluidity of membrane lipids was

detected by several methods. Lateral and
rotational diffusion of membrane proteins was
demonstrated in several laboratories.

Edidin, 2003
Hardin et al., 2012.
Heimburg, 2007
❖ 1972 - Data on membrane protein composition and
mobility were fused in the fluid mosaic model of cell
membranes.

❖ 1973 - After considerable debate, a consensus was

reached that cell membrane lipids are organized as
bilayers as proposed by Singer and Nicolson.

❖ 1977 - A domain model was proposed, which indicates

that membranes can be mosaic rather than fluid.

❖ 1980s - The importance of membrane trafficking to the

steady-state plasma-membrane structure began to be
appreciated.

❖ 1990s - The fact that membrane lipid and protein

domains have various cell functions began to be
appreciated.

❖ 2003 - We are awaiting a new model that integrates the

numerous features of eukaryotic cell membranes, which
have emerged since they were first characterized.
Edidin, 2003
Breakthrough paper….. Hurray!!!

Singer and Nicholson, 1972

 Models of the membrane

Hardin et al., 2012.

 Review paper

• For a better understanding of the different models

of membrane structure see this article…
• Pdf will be sent to you through your class reps…
Models of the membrane

After the fluid mosaic model…..??? Reece et al., 2010

Models of the membrane

After the fluid mosaic model…..???

Membrane Structure
Membranes comprises of
Lipids
Proteins and
Carbohydrates
Membrane Structure
Membranes comprises of
Lipids
Lipids molecules constitutes about
40% of the mass of most membranes
All lipid molecules are amphiphilic
- i.e., they have a hydrophilic or
polar end and a hydrophobic end.

Nelson and Cox, 2005

 The main classes of membrane lipids are
phospholipids, glycolipids, and sterols.

Hardin et al., 2012.

The main classes of membrane lipids are phospholipids, glycolipids, and sterols.

The main phospholipids are phosphoglycerides or glycerophospholipids

Hardin et al., 2012.

The main classes of membrane lipids are phospholipids, glycolipids, and sterols.

Hardin et al., 2012.

The main classes of membrane lipids are phospholipids, glycolipids, and sterols.

Hardin et al., 2012.

 The main classes of membrane lipids are
phospholipids, glycolipids, and sterols.

Hardin et al., 2012.

Membrane Structure

Nelson and Cox, 2005

 The main classes of
membrane lipids are
phospholipids, glycolipids,
and sterols.

Phospholipids have a
polar head group and
two hydrophobic tails.

Tails can be saturated

Alberts et al., 2008 or unsaturated (one or
more cis-double bonds
which creates a kink).
 The main classes of membrane
lipids are phospholipids,
glycolipids, and sterols.

Phospholipids have a polar

head group and two
hydrophobic tails.

Tails can be saturated or

unsaturated (one or more cis-
double bonds which creates a
kink).

The tails are usually

fatty acids and the
Hardin et al., 2012.
length varies from 14 to
24 carbon atoms.
 Lipid states
o The main phases (mesophases) adopted by pure membrane
lipids when dispersed in water are:

o Lamellar (L), consisting of two lipid layers

whose non-polar moieties are in contact and
away from water. This is the disposition
spontaneously adopted by most phospholipids
and glycolipids.
o Micellar (M), in which the lipids form
spherical droplets whose surface is formed by
the lipid polar headgroups, the hydrophobic
tails providing an oily core. e.g. Gangliosides.
o Inverted hexagonal e.g.
Phosphatidylethanolamine
o Inverted cubic lattices.
Goni, 2014
Membrane Structure
Membranes comprises of
Proteins
The amounts and types of membrane proteins is highly
variable (~50%).
For example, 25% of the membrane mass is protein in
myelin membrane (electrical insulation for nerve cell
axons)
75% of membranes involved in ATP production (such as
the IMM of mitochondria and chloroplasts) is protein.
Three types of membrane proteins – integral proteins,
peripheral proteins and lipid-anchored proteins.
 Integral proteins firmly associates with, and
penetrates the hydrophobic interior of, the lipid bilayer.

They are mostly transmembrane proteins, thus

spanning the membrane e.g. Bacteriorhodopsin

They are removable only by agents that interfere

with hydrophobic interactions, such as detergents,
organic solvents, or denaturants.
Peripheral proteins are not embedded in the lipid
bilayer; they are appendages loosely bound to the
surface of the membrane. e.g. Cytochrome C, Spectrin

They associate with the membrane through

electrostatic interactions and hydrogen bonding
with the hydrophilic domains of integral proteins
and with the polar head groups of membrane
lipids.

They can be released by relatively mild

treatments that interfere with electrostatic
interactions or break hydrogen bonds; a
commonly used agent is carbonate at high pH.
 Lipid-anchored proteins that are located outside
the lipid bilayer, on either the extracellular or
cytoplasmic surface, but are covalently linked to a lipid
molecule that is situated within the bilayer.
 β-barrel

The main classes of membrane proteins, Adapted from Hardin et al., 2012.
Membrane Structure
Membranes comprises of
Carbohydrates
Carbohydrates molecules constitutes between 2-10% of
the mass of the membranes depending on the species
and cell type
Covalently linked to form- glycoproteins, glycolipids.
The carbohydrate of glycoproteins is present as short,
branched hydrophilic oligosaccharides, typically
having fewer than 15 sugars per chain.
Models of the membrane

Reece et al., 2010

 Hardin et al., 2012.

Karp, 2010.
 References
Edidin, M. (2003). Lipids on the frontier: a century of cell-membrane bilayers. Our present picture of
cell membranes. Nature reviews, Molecular cell biology, 4(5): 414-418.
Goñi, F.M., 2014. The basic structure and dynamics of cell membranes: An update of the Singer–
Nicolson model. Biochimica et Biophysica Acta (BBA) - Biomembranes 1838, 1467–1476.
Hardin J., Bertoni, G., Kleinsmith, L. J. and Becker, W. M. (2012). Becker's world of the cell (8th Ed.).
Pearson Benjamin Cummings, Boston. 914pp.
Karp, G. (2010). Cell Biology. 6th Ed. John Wiley and Sons Inc., Singapore. 765pp.
Nelson, D. and Cox, M. (2005). Lehninger principles of Biochemistry (4th Ed.). W.H. Freeman and
Company, New York, 1216pp.
Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V. and Jackson, R. B. (2010).
Campbell Biology. 9th ed. Benjamin Cummings, Boston. E-book.
Function of Nucleus
•Store genes on chromosomes
•Organize genes into chromosomes to allow cell division.
• Transport regulatory factors & gene products via nuclear pores
• Produce messages ( messenger Ribonucleic acid or mRNA)
that code for proteins
• Produce ribosomes in the nucleolus
• Organize the uncoiling of DNA to replicate key genes
NUCLEAR ENVELOPE
• Has two membranes ( unit membrane structure)
• Enclose a flattened sac, connected at the nuclear pore sites.
• Outermost membrane is continuous with the rough endoplasmic
reticulum (RER) and has ribosomes attached
• The space between the outer and inner membranes is also
continuous with rough endoplasmic reticulum space.
• The nuclear envelope is enmeshed in a network of filaments
(nuclear lamina) for stability.
NUCLEAR LAMINA
• Consists of "intermediate filaments", 30-100 nm thick.
• These intermediate filaments are polymers of lamin, ranging from 60-75 Kd
• A-type lamins are inside, next to nucleoplasm
• B-type lamins are near the nuclear membrane (inner).
• They may bind to integral proteins inside that membrane.
• The lamins may be involved in the functional organization of the nucleus.
• They may play a role in assembly and disassembly before and after mitosis.
• Phosphorylation triggers the disassembly of the lamina and causes the nuclear envelope
to break up into vesicles.
• Dephosphorylation reverses this and allows the nucleus to reform
ER; Ribosome;Golgi
How do proteins move to the Golgi
Complex?

SER- Liver cell
POLYRIBOSOME
 rRNA Nomenclature

• Named based on sedimentation rates,

measured in Svedberg units (S)

• Larger sedimentation coefficients indicate

faster sedimentation rates

• Larger rRNAs sediment faster

 rRNA Discovery

• First discovered in the

1930s as part of the
microsome by Albert
Claude

• Characterized as a
ribosomal component
in the 1950s by
George Palade
 RIBOSOME
Small Ribosomal Subunit Large Ribosomal Subunit
• Atomic structure • rRNA largely responsible
published in 2000 for 3D structure

• Decoding center • Peptidyl transferase

composed entirely of center (PTC) composed
rRNA of rRNA

• A, P and E sites made up

mainly of rRNA
 Translation
• Slow rate of synthesis (18 amino acids per
second)
• In bacteria translation and transcription are
coupled. As soon as 5’ end of mRNA is
synthesized translation begins.
• Situation in eukaryotes differs since
transcription and translation occur in different
cellular compartments.
 Ribosomes
• Protein biosynthetic
machinery
• Made of 2 subunits
(bacterial 30S and 50S,
Eukaryotes 40S and 60S)
• Intact ribosome referred to
as 70S ribosome in
Prokaryotes and 80S
ribosome in Eukaryotes
• In bacteria, 20,000
ribosomes per cell, 20% of
cell's mass.
• Mass of ribosomes is
roughly 2/3 RNA
 Prokaryotic Ribosome Structure
• E. coli ribosome is 25 nm
diameter, 2520 kD in mass,
and consists of two unequal
subunits that dissociate at <
1mM Mg2+
• 30S subunit is 930 kD with 21
proteins and a 16S rRNA
• 50S subunit is 1590 kD with 31
proteins and two rRNAs: 23S
rRNA and 5S rRNA
 Eukaryotic Ribosome Structure
• Mitochondrial and chloroplast
ribosomes are quite similar to
prokaryotic ribosomes, reflecting
their supposed prokaryotic origin
• Cytoplasmic ribosomes are larger
and more complex, but many of the
structural and functional properties
are similar
• 40S subunit contains 30 proteins
and 18S RNA.
• 60S subunit contains 40 proteins
and 3 rRNAs.
 Ribosome Assembly

• Assembly is coupled w/ transcription and

pre-rRNA processing
 Ribosome Structure
• Crystal structure of ribosome
is known
• mRNA is associated with the
30S subunit
• Two tRNA binding sites (P and
A sites) are located in the
cavity formed by the
association of the 2 subunits.
• The growing peptide chain
threads through a “tunnel” that
passes through the 40S (30S
in bacteria) subunit.
 Topic:

GOLGI APPARATUS

Lecturer:

Dr. Onyekachi Ogbonnaya Iroanya

 OCCURRENCE OF GOLGI APPARATUS
The Golgi apparatus is a central intracellular membrane-bound organelle
often located adjacent to the nucleus in mammalian cells.

It modifies and packages proteins and lipids into transport carriers and sends
them to the proper locations in the cell.

The Golgi apparatus is also known as the Golgi complex, or simply as

‘the Golgi’.
Its existence was first reported by Camillo Golgi in 1898, when he
described in nerve cells an ‘internal reticular apparatus’ impregnated by a
variant of his chromoargentic staining.

The Golgi apparatus functions as a molecular assembly line in which

membrane proteins undergo extensive post-translational modification.
Golgi apparatus looks like a set of flattened sacs
 STRUCTURE OF GOLGI APPARATUS
There are differences in the organization of the Golgi
apparatus across eukaryotes.

In plants, invertebrates and many protists, Golgi stacks exist as

independent units and are scattered as individual stacks throughout
the cytoplasm.

 These Golgi mini-stacks are functional in glycosylation and in

membrane transport.
STRUCTURE OF GOLGI APPARATUS CONTD
In vertebrates, the organisation of the Golgi is dramatically
different.

 the predominant architecture of the Golgi arises from the

lateral fusion of individual mini-stacks into a twisted
continuous ribbon structure.

This was probably the organisation of the Golgi that Camillo

Golgi was able to detect with his silver stain in 1898.
 STRUCTURE OF GOLGI APPARATUS
The Golgi is made of 5-8 folds called cisternae.

The cisternae contain specific enzymes creating five functional

regions which modify proteins passing through them in a
stereotypical way,
FUNCTIONAL REGIONS OF THE CISTERNAE
They are as follow:

1. Cis-Golgi network: faces the nucleus, forms a connection with the
endoplasmic reticulum and is the entry point into the Golgi apparatus.

2. Cis-Golgi: major processing area allowing biochemical modifications.

3. Medial-Golgi: major processing area allowing biochemical modifications.

4. Trans-Golgi: major processing area allowing biochemical modifications.

5. Trans-Golgi network: exit point for vesicles budding off the Golgi surface,
packages and sorts biochemicals into the vesicles according to their destination.
CHARACTERIZATION OF GOLGI STRUCTURE
The architecture of the Golgi is dramatically influenced by a wide range of cell
processes.

The machinery responsible for promoting these cellular processes either regulate or are
influenced by the morphological status of the Golgi.

The importance of the relationship between signaling pathways and Golgi morphology
has been demonstrated by genome-wide analyses.

20 % of the total kinase and phosphates in the genome are influenced Golgi structure,

 either via fragmentation of the Golgi ribbon or by compaction of the Golgi within the
juxtanuclear region
 CHARACTERIZATION OF GOLGI STRUCTURE
The Golgi ribbon can undergo rapid remodeling events, such as in mitosis or the

repositioning of the Golgi for directed migration.

Golgi dynamics are regulated by membrane flux from the endoplasmic reticulum,

and by interactions between the cytoskeleton and molecular scaffolds on Golgi

membranes.

Both microtubules and actin are essential to maintain the structure of the Golgi and

they respond to signaling events.

Microtubules play a pivotal role in generating and positioning the Golgi ribbon.
CHARACTERIZATION OF GOLGI STRUCTURE
Actin fine-tunes the Golgi architecture, e.g. in regulating the balance of Golgi
stacks and the ribbon.

The centrosome, and also the Golgi organelle itself, function as microtubule
organizing centers (MTOC).

The Golgi MTOC promotes clustering of Golgi stacks following mitosis followed
by the transport of these Golgi elements to the centrosome to promote ribbon
formation.

Loss of microtubules disperses the Golgi whereas loss of actin collapses the Golgi
into a more compact structure at the centrosome.
 GOLGI PROTEINS
A number of Golgi proteins interact with the cytoskeleton.

They are classified as:

1. the golgins,

2. Golgi stacking proteins and

3. additional membrane protein complexes.

 GOLGINS
Golgins are a family of membrane proteins defined by their selective
location at the Golgi apparatus and a high percentage of coiled-coil regions.

They were initially identified as autoantigens recognized by autoantibodies

from serum of patients with systemic autoimmune diseases.

The majority of golgins are recruited from the cytoplasm to the cytoplasmic
face of Golgi membrane proteins by small G proteins of the Arf family,

whereas the remaining golgins are embedded as integral membrane proteins

 GOLGINS

Golgins interact with a wide range of binding partners

including small G proteins, Rabs and Arfs, SNAREs and
microtubule and actin linker proteins and molecular motors.
Figure 1: Golgi proteins which interact with
microtubule and actin cytoskeletal networks.

Key: Shown are the golgins

(diamond), GRASPs (hexagon)
and other scaffold proteins
(quadrilateral) of the Golgi,
their location on the Golgi stack
and whether they interact with
actin filaments (green), myosin
motors (light green),
microtubules (red) or
microtubule motor dynein
(orange).

Source: Kulkarni-Gosavi, 2019

Table 1a. Scaffold molecules of the Golgi and their interactors with
actin and microtubule cytoskeletal network
Golgi components Interactors Function

(A) Both actin and microtubule interactors

Golgin-245 (p230 MACF1, microtubule motor Maintenance of Golgi structure, regulation of

autophagy and minus end directed transport of
Golgi membranes along microtubules

GM130 AKAP450, importin a, Cdc42, Tuba Regulates Golgi as a microtubule organising

center and centrosome
organization; a role in organization of mitotic
spindle

Lava lamp Dynein, spectrin Required for dynein-dependent movement of

Golgi along
microtubules during embryogenesis
 Table 1b. Scaffold molecules of the Golgi and their interactors with
actin and microtubule cytoskeletal network
Golgi Interactors Function
components
(B) Microtubule interactors

GCC185 CLASP1, CLASP2, Arl4a Required for the organization of Golgi MTOC by stabilizing
microtubules on the trans-Golgi

GMAP210 Microtubules, c-tubulin Links microtubules to the cis-Golgi

Formin Microtubules Stabilizes new microtubules during transfer of microtubules from

the cis-Golgi to the GCC185/CLASP complex on trans-Golgi
 Table 1b. Scaffold molecules of the Golgi and their interactors with
actin and microtubule cytoskeletal network
Golgi Interactors Function
components

(C) Actin interactors

GCC88 ITSN-1 Promotes actin assembly at the TGN to regulate the

balance of the Golgi ribbon and mini-stacks
Optineurin Myosin VI Regulates Golgi structure and exocytosis

GOLPH3 Actin filaments, Binds actin filaments to promote Golgi ribbon

MYO18A extension
GRASP65 MENA Role in linking the stacks into the Golgi ribbon by
enhancing actin polymerisation, via MENA
FIGURE 2: THE GOLGI APPARATUS

Source:
https://teachmephysiology.com/
histology/cell-structures/golgi-
apparatus/
 ISOLATION OF THE GOLGI APPARATUS
The study of Golgi structure and function can be facilitated by the isolation
of this organelle from homogenates of tissues or cells.

Liver cells have abundant Golgi membranes because they actively secrete
proteins and lipids; therefore, liver tissue is often the preferred source.
 THE PROTOCOL FOR GOLGI
APPARATUS ISOLATION
The protocol described here is derived from several earlier methods
(Fleischer et al., 1969; Leelavathi et al., 1970; Hino et al., 1978; Hui
et al., 1998; Wang et al., 2006) for obtaining highly purified Golgi
stacks from rat liver and for determining their yield.
 REAGENTS AND MATERIALS FOR GOLGI
APPARATUS ISOLATION
Ethyl alcohol
β-1,4-Galactosyltransferase (GalT) assay mixture
Gradient buffers A–E for Golgi isolation
Protein Assay Dye Reagent Concentrate
Phosphotunastic acid (PTA)/HCl (1% phosphotungstic acid/0.5 M HCl)
Rats
Scintillation fluid
Sodium dodecyl sulfate (SDS) (5% w/v)
Tris (2 M)
Water from a Milli-Q filtration system (Millipore)
 EQUIPMENT FOR GOLGI APPARATUS ISOLATION
Pasteur pipette (plastic) AR200 Automatic Digital Refractometer

Scintillation counter Eppendorf tubes (screw-capped)

Scissors Falcon tubes (50-mL)

Spectrophotometer Heat block at 37˚C

Liquid nitrogen
Stainless-steel receiver
Stainless-steel laboratory test sieve (150-µm
Ultracentrifuge
mesh)
SW-41 rotor
Ultraclear tubes for SW-41 rotor (size 14 mm
Centrifuge × 89 mm)
Conical flask (250-mL)
 Gradient Buffers A–E for Golgi Isolation
Materials Buffer (sucrose concentration)
Potassium Buffer A Buffer B Buffer C Buffer D Buffer E
phosphate [0 M] [0.25 M] [0.5 M] [0.86 M] [1.3 M]
buffer
(0.5 M, pH 6.7)

Sucrose (2 M) 0 mL 6.25 mL 25 mL 43 mL 32.5 mL

MgCl2 (2 M) 0.125 mL 0.125 mL 0.250 mL 0.250 mL 0.125 mL

H2O (ice-cold) 39.9 mL 33.6 mL 54.8 mL 36.8 mL 7.4 mL

Total volume 50 mL 50 mL 100 mL 100 mL 50 mL

Sucrose
0% (w/w) 8.6% (w/w) 16.0% (w/w) 26.4% (w/w) 38.6% (w/w)
concentration

(Source: Tang and Wang, 2016)

 PREPARATION OF GOLGI
1. Fast six female Sprague–Dawley rats for 24 hr, but provide water during this time period.

2. Quickly kill the rats by cervical dislocation. Rapidly transfer the livers into a large
volume of ice-cold buffer C (without protease inhibitors) to cool the liver and wash off the
blood.

3. Weigh the livers.

(A maximum of 50 g of wet liver weight can be used for 12 gradients. Do not overload the
gradients).

4. Transfer the liver into fresh ice-cold buffer C containing protease inhibitors (complete
EDTA free Protease Inhibitor Cocktail Tablets and pepstatin A). Mince the livers into small
pieces (4–5 mm in diameter) with a pair of scissors.
5. Using the base of a 250 mL conical flask, homogenize the tissue by gently
pressing the minced liver through a 150 µm mesh stainless-steel sieve with a
circular motion. If needed, add a small amount of buffer C (containing protease
inhibitors) to the liver to make it easier to press the tissue through the mesh. Collect
the homogenate in a 50 mL Falcon tube.

To maintain the morphology of the Golgi stacks, take caution and be gentle when
homogenizing the tissues.

The final volume should be 50 mL. Keep 200 µL of the homogenate on ice for the
enzyme assay (to be used in Steps 14 and 16).
6. In each of 12 SW-41 Ultraclear tubes, prepare the following
gradient.
i. Add 6 mL of buffer D to the bottom of the tube.
ii. Overlay buffer D with 4.5 mL of liver homogenate.
iii. Overlay the homogenate with 1.8 mL of buffer B, and balance the
tubes to within 0.01 g.
7. Centrifuge at 29,000 rpm (103,800g) in a SW-41 rotor for 60 min at
4˚C.
8. Aspirate and discard the lipids and the cytosol at the top. Use a plastic Pasteur
pipette to collect Golgi that accumulate at the 0.5 M sucrose (homogenate/buffer
C) / 0.86 M sucrose (buffer D) interface.

(The Golgi membranes appear as a cloudy band. In contrast, the lipids at the very
top are colored white, and the cytosol is colored red from the hemoglobin. Avoid
lipid contamination when taking the Golgi fractions, because the presence of lipids
can disrupt the morphology of the Golgi stacks).
9. Pool the Golgi membranes into a 50-mL tube (the total volume should be ~15
mL and the refractive index should be ~ 1.3706 [0.77 M sucrose]). Keep 200 µL
on ice for the enzyme assay (to be used in Steps 14 and 16). Adjust the Golgi
sample to 0.25 M sucrose (refractive index 1.3456) using buffer A. Increase the
volume to 48 mL with buffer B if necessary and confirm that the refractive index
is still 1.3456. (It is critical that the sucrose concentration is accurate).

10. Prepare six additional gradients. For each gradient, overlay 1 mL of buffer E
with 2 mL of buffer C and then 8 mL of the diluted Golgi from Step 9. Balance the
tubes with buffer B.
11. Centrifuge at 8000 rpm (7900g) in a SW-41 rotor for 30 min at 4˚C.

12. Aspirate and discard the top layer. Collect the membranes (thin
cloudy band) at the 0.5 M sucrose (buffer C)/1.3 M sucrose (buffer E)
interface. Gently mix the Golgi membranes (about 1.5 mL–2 mL) with
about 1 volume of buffer A to adjust the sucrose concentration to 0.5 M
(refractive index 1.3574).

(The final volume of the Golgi membranes is normally 3–4 mL, and the
typical protein concentration (as measured in Step 16) is normally 1–2
mg/mL).
13. Aliquot and freeze samples in liquid nitrogen, then store at -80˚C.

(Samples can be thawed and frozen twice without significant loss of enzymatic
activity or loss of morphology. Purified rat liver Golgi can be further used in several
cell-free assays, such as the vesicle transport-assay and the Golgi disassembly and
reassembly assay).
 DETERMINATION OF YIELD OF GA
14. Prepare 1:20 dilutions of the homogenate (Step 5 above) and intermediate (Step 9
above) and Golgi (Step 12 above) fractions using water.

15. Add 80 µL of GalT assay mixture (in duplicate) to screw-capped tubes containing 20
µL of the diluted samples or water (blanks). Vortex and incubate at 37˚C for 30 min.

16. While the GalT reactions are incubating, measure the protein concentration in each
of the three samples using the Protein Assay Dye Reagent Concentrate from Bio-Rad,
and then determine the total amount (mg) of protein that is present in each sample.
17. Stop the reactions by adding 1 mL of ice-cold PTA/HCl to each tube.
Centrifuge at 13,000 rpm for 10 sec.

18. Aspirate and discard the supernatants. Add 1 mL of PTA/HCl. Resuspend the
pellets by vortexing and centrifuge as in Step 17.

19. Aspirate and discard the supernatants. Add 1 mL ice-cold 95 % ethanol, and
resuspend the pellets as in Step 18.

20. Centrifuge as in Step 17 and discard the supernatant. Resuspend the pellets in
50 µL of 2 M Tris followed by 200 µL of 5 % SDS. Shake or vortex until
dissolved.
21. Combine 10 µL of assay mixture (containing 2.5 nmol of UDP-galactose), 40
µL of water, and 200 µL of 5% SDS in a fresh tube.

(This standard will be used to determine the specific activity (SA) of the [3H]UDP-
galactose in the mixture (see Step 23)).

22. Add 1 mL of scintillation fluid to each sample, and vortex. Use the tritium
channel in a scintillation counter to measure the disintegrations per minute (dpm)
of each sample and blank.
23. Calculate the SA of the [3H]UDP-galactose in the mixture (in dpm/nmol) by
subtracting the dpm of the blank from the dpm of the standard, and then dividing
by 2.5 nmol (which is the amount of
UDP-galactose in the standard; see Step 21).

24. For each sample, calculate the GalT activity concentration as follows:
25. For each sample, calculate the total GalT activity as follows:

Total GalT activity (nmol/h) = GalT activity concentration (nmol/h/mL) × volume (mL).

26. Determine the yield of Golgi membranes by dividing the total GalT activity in the

Golgi fraction by the total GalT activity in the homogenate and multiplying the resulting

value by 100%.

27. For each sample, calculate the SA of GalT (in nmol/h/mg) by dividing the value

obtained in Step 25 by the value obtained in Step 16.

28. Determine the purification fold by calculating the factor by which the SA of GalT

increases in the Golgi fraction over the homogenate.

 CHEMICAL COMPOSITION OF THE GOLGI APPARATUS
Golgi complex is rich in chemical substances.

1) Phospholipids: These substances have a composition which is in between the

structure of phospholipids of endoplasmic reticulum and plasma membrane.

2) Enzymes: ATP-ase, CPT-ase, transphorases, etc. enzymes are present.

3) Carbohydrates: Glucose, manose, galactose carbohydrates are seen.

 THE FUNCTION OF THE GOLGI
The Golgi is a vital membrane-bound organelle found in all eukaryotic cells,
including plants, animals, and fungi. Functioning of the Golgi apparatus is often
compared to working of a 'post-office‘, probably because proteins in the Golgi body
are modified, sorted and then packaged before they are secreted.

The primary function of the Golgi is to modify the new proteins synthesized from
the ER present in the cytoplasm, then process and sort them for transportation. It
package proteins and lipids into transport carriers and send them to the proper
locations.
1. Directs / transports the carbohydrates and proteins, needed by the body, to their
proper destination

2. Modifies proteins generated in the endoplasmic reticulum.

3. Breaking down proteins into small, active fragments.

4. Sulfation is one of the important processes handled by the Golgi body. Sulfation of
substances passing through the lumen of Golgi body takes place with the help of
sulfotransferases. It performs sulfation onto the proteoglycans in order to aid in
signaling abilities and giving the molecule a negative charge.
5. Incorporation of phosphate molecules into proteins molecules.

6. It also transports lipids to vital parts of the cell

7. production of lysosomes

8. Synthesis of carbohydrates. Carbohydrate synthesis involves the production of

polysaccharides and glycosaminoglycans (GAGs) which go on to form parts of
connective tissues.
Golgi body plays an important role in the synthesis of proteoglycans. roteoglycans
are found in the extracellular matrix of animal cells.
Secretory or transmembrane proteins are delivered from the endoplasmic
reticulum (ER) to the cis-Golgi network.

Subsequently, the cargo molecules travel through the different cisternae of the
Golgi where they are post-translationally modified by resident enzymes.
Modifications include glycosylation, phosphorylation, sulfation, and proteolysis .

Cargo molecules have been described to move through the Golgi stack in several
different ways.
One possible mechanism is through cisternal maturation, in which the cargo
remains in the cisternae and new cisternae form at the cis side.

The newly formed cisternae then mature by receiving Golgi enzymes via
retrograde transport of COPI vesicles.

Alternatively, proteins are shuttled by COPI vesicles between cisternae, whereas

Golgi resident enzymes reside in the relatively stable cisternal compartments.
Additionally, cargo molecules may flow through transient tubular connections
between adjacent cisternae), or by rapid partitioning between different lipid
domains.

At the transGolgi network (TGN), cargo molecules are sorted and transported to
their proper destinations such as the endosomal-lysosomal system the plasma
membrane, or outside of the cell.

In addition to its function in protein trafficking, the Golgi in mammalian cells
plays an important role in cell polarity and cell cycle control.

This suggests a direct link between membrane trafficking, cell growth, and cell
polarity
 MOLECULAR MECHANISMS OF GOLGI BIOGENESIS
IN MAMMALIAN CELLS
In mammalian cells, the continuous Golgi ribbon needs to be segregated into the
two daughter cells when the cell divides. This is achieved through a three-step
process (Figure 3).

 This involves disassembly (consisting of ribbon unlinking, cisternal unstacking,

and vesiculation), partitioning, and reassembly of Golgi membranes
FIGURE 3. SCHEMATIC ILLUSTRATION OF THE MITOTIC DIVISION
OF THE MAMMALIAN GOLGI.

During interphase, the Golgi

stacks (green) are
interconnected into a
ribbon-like structure near
the centrosomes (red) and
next to the nucleus (blue).
In late G2 phase of the cell cycle, the Golgi ribbon is unlinked upon separation of
the lateral connections between the stacks.

In early mitosis, the cisternae then unstack and further disassemble into vesicular
and tubular membranes.

These mitotic Golgi membranes are then divided into the two daughter cells
where they are reassembled into a functional Golgi.
 MECHANISMS OF MITOTIC GOLGI DISASSEMBLY-

I) UNLINKING THE GOLGI RIBBON

In late G2 phase, the Golgi ribbon is unlinked by severing the tubular connections
between the stacks.

The conversion of the ribbon into stacks depends on the mitogen-activated protein
kinase (MAP kinase), kinase MEK1, but not the classical MEK1 substrates
extracellular signal-regulated kinase (ERK1 or ERK2).
Extracellular signal-regulated kinase 1c (ERK1c) and Polo like kinase 3 (Plk3)/
vaccinia-related kinase 1 (VRK1) were shown to contribute to Golgi ribbon unlinking in
late G2 phase, but their substrates on the Golgi membranes have not yet been identified.

Another study showed that MEK1 causes Golgi ribbon unlinking through mitotic
phosphorylation of Golgi reassembly-stacking protein of 55 kDa (GRASP55) also
known as golgi reassembly-stacking protein 2 (GORASP2) by ERK2.

Expression of a non-phosphorylatable GRASP55 mutant interferes with ribbon

unlinking in late G2, although this result was not confirmed by others.

Precisely how GRASP55 is involved in Golgi ribbon unlinking remains unclear.

Similarly, microinjection of antibodies against GRASP65 also indicates a role for
GRASP65 in severing of the ribbon and the progression from G2 into M-phase.

Besides these kinases, the membrane fission protein C-terminal-binding protein

family (CtBP1/BARS) is important for Golgi ribbon unlinking.

Inhibition of BARS activity prevents severing of the ribbon as well as G2/M

transition, but the underlying molecular mechanisms as well as the signaling
events that activate BARS await further investigation.
 MECHANISMS OF MITOTIC GOLGI
DISASSEMBLY-
II) VESICULATION AND UNSTACKING OF THE
CISTERNAE

Following the conversion of the Golgi ribbon into stacks, the cisternae are unstacked
and vesiculated through COPI-dependent vesicle formation
Vesiculation and cisternal unstacking occurs simultaneously in early mitosis, leading to
rapid conversion of the Golgi into vesicular and tubular membranes.
 Vesiculation of the Golgi cisternae is triggered by an imbalance of membrane budding
and fusion during mitosis.
 FIGURE 4. SCHEMATIC ILLUSTRATION OF PUTATIVE
GOLGI DISASSEMBLY AND REASSEMBLY MECHANISMS
DURING THE CELL CYCLE.
 PARTITIONING THE GOLGI INTO THE
DAUGHTER CELLS
Once disassembled, the mitotic Golgi membranes are present in at least two pools.

In addition to vesicles that are evenly dispersed throughout the cytoplasm, a
significant portion of the mitotic Golgi membranes concentrate at the spindle
poles and associate with astral microtubules.

These spindle-associated membranes are organized in clusters of tubulovesicular

structures with a relatively constant number within each cell.
The mitotic clusters are polarized, with cis-Golgi proteins spatially
separated from those of the trans-Golgi, a similar pattern to that of the Golgi stacks
during interphase.
The spindle association of the mitotic Golgi clusters, which is also
prominent in cells with monoasters, has prompted the idea that Golgi partitioning is
regulated by the spindle.
Upon division, both daughter cells assemble functional Golgi stacks, but the
ribbon of interconnected stacks are only observed in the daughter cell that receives
mitotic Golgi clusters partitioned together with the spindle.
This indicates that the spindle plays a vital role in partitioning an
 POSTMITOTIC GOLGI REASSEMBLY
Upon segregation into the nascent daughter cells, the mitotic Golgi membranes
reassemble during telophase and cytokinesis into two distinct ribbons on opposite sides
of the nucleus

A smaller Golgi ribbon reforms adjacent to the midbody, whereas a larger ribbon is
found in the pericentriolar region on the side away from the cleavage furrow.

The function of the smaller ribbon is not clear, but its positioning next to the mid-body
may facilitate an efficient and polarized delivery of membranes into the cleavage furrow
to seal the plasma membrane during abscission.
Golgi-derived vesicles are directly delivered from both daughter cells into the cleavage furrow
where these membranes fuse.

Golgi is reformed at the end of mitosis by two interrelated processes, formation of flattened
cisternae by membrane fusion and stacking of cisternae.

Postmitotic Golgi membrane fusion is mediated by two AAA-ATPases, the N-ethylmaleimide-

sensitive factor (NSF) and the valosin-containing protein (VCP)/p97 (Cdc48 in yeast).

 Stacking of Golgi cisternae requires the tethering factor p115 that initially links adjacent
membranes and the GRASP proteins that hold the cisternae into stacks.
Endomembrane system
 Endomembrane System
Endomembrane system
-a series of membranes throughout the
cytoplasm
-divides cell into compartments where
different cellular functions occur
1. endoplasmic reticulum
2. Golgi apparatus
3. lysosomes
2
 Endomembrane System
Rough endoplasmic reticulum (RER)
-membranes that create a network of
channels throughout the cytoplasm
-attachment of ribosomes to the
membrane gives a rough appearance
-synthesis of proteins to be secreted, sent
to lysosomes or plasma membrane

3
 Endomembrane System
Smooth endoplasmic reticulum (SER)
-relatively few ribosomes attached
-functions:
-synthesis of membrane lipids
-calcium storage
-detoxification of foreign substances

4
Endomembrane System

5
 Endomembrane System
Golgi apparatus
-flattened stacks of interconnected
membranes
-packaging and distribution of materials to
different parts of the cell
-synthesis of cell wall components

6
7
 Endomembrane System
Lysosomes
-membrane bound vesicles containing
digestive enzymes to break down
macromolecules
-destroy cells or foreign matter that the
cell has engulfed by phagocytosis

8
9
 Endomembrane System
Microbodies
-membrane bound vesicles
-contain enzymes
-not part of the endomembrane system
-glyoxysomes in plants contain enzymes
for converting fats to carbohydrates
-peroxisomes contain oxidative enzymes
and catalase
10
 Endomembrane System
Vacuoles
-membrane-bound structures with various
functions depending on the cell type

There are different types of vacuoles:

-central vacuole in plant cells
-contractile vacuole of some protists
-vacuoles for storage

11
NUCLEIC ACIDS
 Brief history
• 1869: isolated DNA from salmon sperm (Friedrich Miescher)
• 1944: proved DNA is genetic materials (Avery et al.)
• 1953: discovered DNA double helix (Watson and Crick)
• 1968: decoded the genetic codes (Nirenberg)
• 1981: invented DNA sequencing method (Gilbert and Sanger)
• 1987: launched the human genome project
• 2001: accomplished the draft map of human genome
 Genes are composed of nucleic acids
(usually DNA)
• Pneumococcus can be transformed from an
avirulent to a virulent strain

• DNA is the transforming principle

• DNA in bacteriophage particles appears in the

progeny, but very little protein does.
The discovery of DNA double helix

• Chargaff's Rule (A=T,

G=C in DNA)

• Franklin, Wilkins:
X-ray Diffraction
Refined Structure
Structures of nucleic acids
Nucleotides
DNA structures
Sedimentation and Electrophoresis
 Deoxyribonucleic
Deoxyribonucleicacid,
acid,DNA
DNA
Nucleic acid
Ribonucleic
Ribonucleicacid,
acid,RNA
RNA
General Properties of Nucleic Acids
• Acidity
• Amphiphilic molecules; normally acidic because of
phosphate.
• Viscosity
• Solid DNA: white fiber; RNA: white powder. Insoluble in
organic solvents, can be precipitate by ethanol.
• Optical absorption
• UV absorption due to aromatic groups.
• Thermal stability
• Disassociation of dsDNA (double-stranded DNA)
into two ssDNAs (single-stranded DNA).
 phosphate
nucleic acid nucleotides pentose
(Polymer) (Monomer) nucleosides
bases

NH2

N
O
HO P O CH2 N O
O
OH

OH OH
 Composition of DNA and RNA

Nucleic
base ribose
acid

DNA A、G、C、T deoxyribose

RNA A、G、C、U ribose

 The components of DNA and RNA

•DNA and RNA are polymers of nucleotide

units.
• DNA (RNA) consists of 4 kinds of
ribonucleotide units linked together through
covalent bonds.
• Each nucleotide unit is composed of
a nitrogenous base
a pentose sugar
a phosphate group
A simple view of DNA

AGCCTCGCAT
TCGGAGCGTA
Nucleotides
• 3 components to nucleotides:
• Purine or pyrimidine base
• Ribose (RNA) or 2-deoxyribose (DNA)
sugar
• Phosphate
• Base + sugar = Nucleoside
• Base + sugar + phosphate = Nucleotide
Nucleosides =ribose/deoxyribose + bases
•The bases are covalently attached to the 1’ position of a pentose
sugar ring, to form a nucleoside

Glycosidic bond

R
Ribose or 2’-deoxyribose
Adenosine, guanosine, cytidine, thymidine, uridine
Nucleotides = nucleoside + phosphate
•A nucleotide is a nucleoside with one or more phosphate groups bound
covalently to the 3’-, 5’, or ( in ribonucleotides only) the 2’-position. In the
case of 5’-position, up to three phosphates may be attached.

Phosphate ester bonds

Deoxynucleotides Ribonucleotides
(containing deoxyribose) (containing ribose)
BASES NUCLEOSIDES NUCLEOTIDES
Adenine (A) Adenosine Adenosine 5’-triphosphate (ATP)
Deoxyadenosine Deoxyadenosine 5’-triphosphate
(dATP)
Guanine (G) Guanosine Guanosine 5’-triphosphate (GTP)
Deoxyguanosine Deoxy-guanosine 5’-triphosphate
(dGTP)
Cytosine (C) Cytidine Cytidine 5’-triphosphate (CTP)
Deoxycytidine Deoxy-cytidine 5’-triphosphate
(dCTP)
Uracil (U) Uridine Uridine 5’-triphosphate (UTP)
Thymine (T) Thymidine/ Thymidine/deoxythymidie
Deoxythymidie 5’-triphosphate (dTTP)
Types of bases in nucleotides
Pyrimidine
O O
CH3
HN HN

O N O N
H H
Thymine Uracil

Amino- Keto-
Nucleotides: purine bases
NH 2 O

N N
N HN

N N H2 N N N
H H
Adenine Guanine

6-aminopurine A keto-purine
Bases are attached to C1’ of the
sugar via an N-glycosidic bond
NH 2

N
N

N N
HO CH2
5' O
1'
3'
OH
2’-deoxy- Adenosine , a nucleoside
 Phosphate is attached to C5’ of the sugar

1st phosphate is a phosphoester, others are

attached as phosphoanhydrides.

O O O

=
NTP is base
- O–P- O P O-P - O O
O - O- O-
OH OH
phosphoanhydride
phosphoester
g b a
 Structure of a dinucleotide
NH2
The 3’ C of one
nucleotide is linked N
to the 5’ C of the
O 5'
N O
next nucleotide in a
O P O CH2
O
phosphodiester NH2
O
linkage. N
3' N
O H
O N N
5'
P O CH2
O
O
5' cytidylyladenylate
or OH H
5'pCpA 3'
 Nucleic acids are linear chains of
nucleotides
• The 3’ C of one nucleotide is linked to the 5’ C of the
next nucleotide.
• The linkage is by a phosphoester.
• The chain has an orientation defined by the sugar-
phosphate backbone.
• One terminal nucleotide has a “free” 5’ end, and the
other has a “free” 3’ end.
• Thus we designate orientation by 5’ to 3’.
N.B.
• The polymerization of nucleotides to form
nucleic acids occur by condensation reaction
by making phospho-diester bond between 5’
phosphate group of one nucleotide and 3’
hydroxyl group of another nucleotide.

• Polynucleotide chains are always synthesized

in the 5’ to 3’ direction, with a free nucleotide
being added to the 3’ OH group of a growing
chain.
 More on orientation of chains of nucleic acids

• 5’ ACTG 3’ is different from 3’ ACTG 5’

• Unless specified otherwise, a chain is written with the 5’ end on the
left and the 3’ end on the right.
• When complementary strands in DNA are written, usually the top
strand is written 5’ to 3’, left to right, and the bottom strand is written
3’ to 5’, left to right.
5’ GATTCGTACCG
3’ CTAAGCATGGC
Basics of DNA structure
• 2 complementary strands of nucleic acids
• Complementarity is based on H-bonding between
• Keto bases with amino bases
• Pyrimidines with purines
• A pairs with T (or U)
• G pairs with C
• The complementary strands are antiparallel.
• The complementary strands are coiled around each other.
DNA double helix
Essential for replicating DNA and
•Two separate strands transcribing RNA
•Antiparellel (5’→3’
direction) 3’
•Base pairing: hydrogen 5’
bonding that holds two
strands together
•Complementary (sequence)

• Sugar-phosphate backbones
(negatively charged): outside
• Base pairs (stack one above the
other): inside

✓ Stable configuration can be

maintained by hydrogen bond
and base stacking force
(hydrophobic interaction). 3’
5’
 4
3 2 1
7 6
8 5 1
9
4
3 2

A:T G:C

Base pairing
Duplex DNA
• Two strands coil around each other.
• Right-handed coils (B form and A form).
• Coils form major and minor grooves.
• Strands have opposite polarity (antiparallel).
• Opposing bases in strands are complementary.
• Different edges of paired bases are exposed in major and minor grooves.
• Sugar-phosphate backbone is on the outside, bases on the inside
• B-form DNA: base pairs are close to center of long axis of the duplex.
• A-form nucleic acids: base pairs stack away from long axis.
Implications of complementarity
• One chain (strand) of DNA can serve as the template for
synthesis of the complementary chain.
• DNA replication: sequence of nucleotides in one chain of the
duplex determines the sequence of nucleotides in the other
chain.
• Transcription: sequence of nucleotides in one chain of the
duplex determines the sequence of nucleotides in mRNA or
its precursor.
Base pairs in DNA
Major groove Major groove
H H
H N H O CH 3
N N N
O
N N N N
N H N N H N
deoxy- N N
O deoxy-
ribose N H deoxy- O deoxy-
ribose
H ribose ribose

Minor groove Minor groove

Guanine : Cytosine Adenine : Thymine

Forms of DNA
1- B-form helix:
• It is the most common form of DNA in cells.
• Right-handed helix
• Turn every 3.4 nm.
• Each turn contain 10 base pairs (the distance
between each 2 successive bases is 0.34 nm)
• Contain 2 grooves;
• Major groove (wide): provide easy access to
bases
• Minor groove (narrow): provide poor access.
2- A-form DNA:
• Less common form of DNA , more common in
RNA
• Right handed helix
• Each turn contain 11 b.p/turn
• Contain 2 different grooves:
• Major groove: very deep and narrow
• Minor groove: very shallow and wide (binding site for RNA)

3- Z-form DNA:
⚫ Radical change of B-form
⚫ Left handed helix, very extended
⚫ It is GC rich DNA regions.
⚫ The sugar base backbone form Zig-Zag shape
⚫ The B to Z transition of DNA molecule may play a role in
gene regulation.
Forms of nucleic acid duplexes

B-form DNA A-form Z DNA

(e.g. duplex RNA)
Summary of helical parameters for B, A and Z nucleic acids

B A Z
helix sense RH RH LH
bp per turn 10 11 12
vertical rise per bp 3.4 2.56 3.7 Angstroms
rotation per bp +36 +33 -30 degrees
helical diameter 19 23 18 Angstroms
Denaturing and Annealing of DNA
• The DNA double strands can denatured if
heated (95ºC) or treated with chemicals.
• AT regions denature first (2 H bonds)
• GC regions denature last (3 H bonds)

• DNA denaturation is a reversible process, as

denatured strands can re-annealed again if
cooled.

• This process can be monitored using the

hyperchromicity (melting profile).
Hyperchromicity (melting profile)
• It is used to monitor the DNA denaturation and
annealing.

• It is based on the fact that single stranded (SS)

DNA gives higher absorbtion reading than
double stranded (DS) at wavelength 260º.

• Using melting profile we can differentiate

between single stranded and double stranded
DNA.
 Hyperchromicity (melting profile)
Using melting profile we can differentiate between SS DNA and DS
DNA

SS

SS
Ab260

DS

Tm
Temperature

Tm (melting temp.): temp. at which 50% of DS DNA denatured to SS

•Heating of SS DNA: little rise of Ab reading
• Heating of DS DNA: high rise of Ab reading
 Melting profile continue…..
• Melting profile can be also used to give an idea
about the type of base pair rich areas using the
fact that:
• A═T rich regions: denatured first (low melting point)
• G≡C rich regions: denatured last (higher melting
point)

SS
AT rich DNA
Tm1: Small melting temp. of AT rich
GC/AT DNA
DNA
GC rich DNA
Tm2: higher melting temp. of AT/GC
equal DNA

Tm3: Highest melting temp. of GC rich DS

DNA

Tm1 Tm2 Tm3

Hyperchromic shift when DNA is denatured
denaturation by heat or
Native duplex DNA increasing pH Denatured, strands are separate

+
renaturation by cooling or
lowering pH

hyperchromic
lower A higher A
260 260
hypochromic
1.4

A
260
1.2

1.0

T = melting temperature, midpoint of the transition

m
Temperature
 Factors that affect melting temperature
• The melting temperature m = 
(Tm) increases as m = 
• G+C se % G+C
• Ionic strength (or m) se
• Tm decreases as
• Denaturants se
Tm
• Number of mismatches se

Tm = 0.41 (% G+C) + 16.6 log M + 81.5 - 0.7 (% formamide) -1o (% mismatch)

Eukaryotic DNA

• DNA in eukaryotic cells is highly

packed.
• DNA appears in a highly ordered form
called chromosomes during metaphase,
whereas shows a relatively loose form
of chromatin in other phases.
• The basic unit of chromatin is
nucleosome.
• Nucleosomes are composed of DNA
and histone proteins.
The importance of packing of DNA into
chromosomes

➢ Chromosome is a compact form of the DNA

that readily fits inside the cell
➢ To protect DNA from damage
➢ DNA in a chromosome can be transmitted
efficiently to both daughter cells during
cell division
➢ Chromosome confers an overall organization
to each molecule of DNA, which facilitates
gene expression as well as recombination.
• The DNA in a prokaryotic cell is a
supercoil.

• Supercoiling makes the DNA molecule more

compact thus important for its packaging
in cells.
Electrophoresis to measure SIZE
Size markers
DNA samples
-

+
For molecules of the same shape, logM is
inversely proportional to d.

For molecules of the same size, more compact

forms, such as supercoiled DNA, moves faster
than more extended forms, such as linear DNA.
 Markers (DNA Molecular weight Ladder)

Base pairs
(bp)
Alpha-globin
400
gene 300
PCR product 200
217 bp
100

Image of a gel that has undergone electrophoresis

 RNA structure

• It is formed of linear polynucleotide

• It is generally single stranded
• The pentose sugar is Ribose
• Uracile (U) replace Thymine (T) in the pyrimidine bases.

Although RNA is generally single stranded, intra-molecular

H-bond base pairing occur between complementary bases
on the same molecule (secondary structure)
Types of RNA
• Messenger RNA (mRNA):
• Carries genetic information copied from DNA in the form of a
series of 3-base code, each of which specifies a particular
amino acid.
• Transfer RNA (tRNA):
• It is the key that read the code on the mRNA.
• Each amino acid has its own tRNA, which binds to it and
carries it to the growing end of a polypeptide chain.
• Ribosomal RNA (rRNA):
• Associated with a set of proteins to form the ribosomes.
• These complex structures, which physically move along the
mRNA molecule, catalyze the assembly of amino acids into
protein chain.
• They also bind tRNAs that have the specific amino acids
according to the code.
 Other RNAs
• Small nuclear RNA (snRNA)
• Involved in mRNA processing
• Small nucleolar RNA (snoRNA)
• Play a key role in the processing of rRNA molecules
• Small cytoplasmic RNA (scRNA)
• Involved in the selection of proteins for export
• Catalytic RNA or Ribozyme
• Small interfering RNA (siRNA)
• Interfere with the expression of a specific gene
RNA structure
• RNA is a single stranded polynucleotide molecule.

• It can take 3 levels of structure;

• Primary: sequence of nucleotides
• Secondary: hairpin loops (base pairing)
• Tertiary: motifs and 3D foldings
RNA structure
• RNA molecules are largely single-stranded but there
are double-stranded regions.
RNA structure

Transfer RNA (tRNA) structure

Lipids are non-polar (hydrophobic) compounds,
soluble in organic solvents.
Most membrane lipids are amphipathic, having a
non-polar end and a polar end.
Fatty acids consist of a hydrocarbon chain with a
carboxylic acid at one end.
A 16-C fatty acid: CH3(CH2)14-COO-
Non-polar polar

A 16-C fatty acid with one cis double bond

between C atoms 9-10 may be represented as
16:1 cis 9.
Double bonds in
fatty acids usually
have the cis
configuration.
Most naturally
occurring fatty acids
have an even
number
Some of carbon
fatty acids and their common names:
atoms.
14:0 myristic acid; 16:0 palmitic acid; 18:0 stearic
acid; 18:1 cis9 oleic acid
18:2 cis9,12 linoleic acid
18:3 cis9,12,15 -linonenic acid
20:4 cis5,8,11,14 arachidonic acid
20:5 cis5,8,11,14,17 eicosapentaenoic acid (an
There is free rotation about C-C bonds in the fatty
acid hydrocarbon, except where there is a double
bond.
Each cis double bond causes a kink in the chain.
Rotation about other C-C bonds would permit a
more linear structure than shown, but there would
be a kink.
 Glycerophospholipids
Glycerophospholipids
C H 2O H
(phosphoglycerides), are
common constituents of cellular H C OH
membranes.
C H 2O H
They have a glycerol backbone.
Hydroxyls at C1 & C2 are g l y cerol

esterified to fatty acids.

An ester forms
when a hydroxyl F or m ati on of an ester :
reacts with a O O
carboxylic acid,
R 'O H + H O - C - R " R ' - O - C - R '' + H 2O
with loss of
H2O.
 Phosphatidate

In phosphatidate:
fatty acids are esterified to hydroxyls on C1 &
C2
the C3 hydroxyl is esterified to P .
In most glycerophospholipids (phosphoglycerides),

Pi is in turn esterified to OH of a polar head group (X)
: e.g., serine, choline, ethanolamine, glycerol, or
inositol.
The 2 fatty acids tend to be non-identical. They may
differ in length and/or the presence/absence of
Phosphatidylinositol, with inositol as polar head
group, is one glycerophospholipid.
In addition to being a membrane lipid,
phosphatidylinositol has roles in cell signaling.
Phosphatidylcholine, with choline as polar
head group, is another glycerophospholipid.
It is a common membrane lipid.
Each glycerophospholipid
includes
a polar region:

glycerol, carbonyl O
of fatty acids, Pi, & the
polar head group (X)
non-polar hydrocarbon
tails of fatty acids (R1,
R2).
Sphingolipids are derivatives of
the lipid sphingosine, which has a
long hydrocarbon tail, and a polar
domain that includes an amino
group.

Sphingosine may be reversibly

phosphorylated to produce the
signal molecule sphingosine-1-
phosphate.
Other derivatives of sphingosine
are commonly found as
constituents of biological
The amino group of sphingosine
can form an amide bond with a
fatty acid carboxyl, to yield a
ceramide.

In the more complex

sphingolipids, a polar “head
group" is esterified to the
terminal hydroxyl of the
sphingosine moiety of the
Sphingomyelin has
a phosphocholine or
phosphethanolamin
e head group.
Sphingomyelins are
common
constituent of
plasma membranes

Sphingomyelin, with a phosphocholine head group, is

similar in size and shape to the glycerophospholipid
phosphatidyl choline.
A cerebroside is a
sphingolipid
(ceramide) with a
monosaccharide
such as glucose or
galactose as polar
head group.
A ganglioside is a
ceramide with a
polar
head group that is a complex oligosaccharide,
including the acidic sugar derivative sialic acid.
Cerebrosides and gangliosides, collectively called
glycosphingolipids, are commonly found in the
outer leaflet of the plasma membrane bilayer, with
their sugar chains extending out from the cell
Amphipathic lipids in
association with water
form complexes in which
polar regions are in
contact with water and
hydrophobic regions away
from water.
Depending on the lipid, possible molecular
arrangements:
Various micelle structures. E.g., a spherical
micelle is a stable configuration for amphipathic
lipids with a conical shape, such as fatty acids.
A bilayer. This is the most stable configuration
for amphipathic lipids with a cylindrical shape,
such as phospholipids.
Membrane fluidity:
The interior of a lipid
bilayer is normally highly
fluid.
In the liquid crystal state, hydrocarbon chains of
phospholipids are disordered and in constant
motion.
At lower temperature, a membrane containing a
single phospholipid type undergoes transition to a
crystalline state in which fatty acid tails are fully
extended, packing is highly ordered, & van der Waals
interactions between adjacent chains are maximal.
Kinks in fatty acid chains, due to cis double bonds,
interfere with packing in the crystalline state, and
lower the phase transition temperature.
Cholesterol, an
important
constituent of cell
membranes, has a
rigid ring system HO

and a short C h ol ester ol

branched
hydrocarbon tail.
Cholesterol is largely
hydrophobic.
But it has one polar
group, a hydroxyl,
making it amphipathic.
 HO
C h ol ester ol

Cholesterol inserts into bilayer membranes with

its hydroxyl group oriented toward the aqueous
phase & its hydrophobic ring system
adjacent to fatty acid chains of phospholipids.
The OH group of cholesterol forms hydrogen
bonds with polar phospholipid head groups.
Interaction with the relatively rigid
cholesterol decreases the mobility of
hydrocarbon tails of phospholipids.

But the presence of cholesterol in a phospholipid

membrane interferes with close packing of fatty
acid tails in the crystalline state, and thus inhibits
transition to the crystal state.
Phospholipid membranes with a high
concentration of cholesterol have a fluidity
intermediate between the liquid crystal and crystal
states.
Two strategies by which phase changes of
membrane lipids are avoided:
Cholesterol is abundant in membranes, such as
plasma membranes, that include many lipids
with long-chain saturated fatty acids.
In the absence of cholesterol, such membranes
would crystallize at physiological temperatures.
The inner mitochondrial membrane lacks
cholesterol, but includes many phospholipids
whose fatty acids have one or more double
bonds, which lower the melting point to below
physiological temperature.
Lateral mobility of a lipid,
within the plane of a
membrane, is depicted at
right and in an animation.

High speed tracking of individual lipid molecules has

shown that lateral movements are constrained within
small membrane domains.
Hopping from one domain to another occurs less
frequently than rapid movements within a domain.
The apparent constraints on lateral movements of
lipids (and proteins) has been attributed to integral
membrane proteins, anchored to the cytoskeleton,
functioning as a picket fence. See the website of the
Kusumi laboratory.
Flip-flop of lipids (from one
half of a bilayer to the
other) is normally very
slow.
Flip-flop would require the polar head-group of a lipid
to traverse the hydrophobic core of the membrane.
The two leaflets of a bilayer membrane tend to differ
in their lipid composition.
Flippases catalyze flip-flop in membranes where lipid
synthesis occurs.
Some membranes contain enzymes that actively
transport particular lipids from one monolayer to the
other.
 Classification of Lipids

• Fatty acids: are long chain linear (unbranched) hydrocarbon

carboxylic acids

• Triglycerides: are fatty acid esters of glycerol

• Phospholipids: are lipids that contain one or more phosphate

groups

• Glycolipids: have a carbohydrates component

• Eicosanoids: are a family of derivatives of Arachidonic acid

• Steroids: have a basic structure of a perhydrocyclopentanophenanthrene

ring system

• Lipoproteins: are complexes of lipids and proteins that

circulate in the blood.

 Fatty Acids
• long chain linear hydrocarbons carboxylic acids
• Usually have an even number of C atoms (usually 12 to 20)
• The carbons are numbered starting from the carboxylic C.
• They are amphiphilic; they have a polar end and rest of the
molecule is nonpolar
• Fatty acids may be saturated (no double bonds) or
unsaturated
(one or more double bonds)
• All naturally occuring double bonds have a cis-configuration
• 2 or more double bonds exist as non-conjugated double
bonds
• Longer chain and saturation increases melting point of FA
• FAs are ionized at physiological pH
 ώ-3 Fatty Acids

• The highest numbered C is called the ώ -C

• Sometimes FA are classified according to the
position of
the first double bond from the ώ -end
• Most polyunsaturated fatty acids are ώ -6 fatty
acids
• ώ -3 fatty acids are found mainly in fish and fish
products. Also found in flax seeds
• ώ -3 FAs inhibit formation of thromboxane A2 (an
• eicosanoid) required for platelet aggregation and
clot
formation. Thus, ώ -3 FAs decrease the risk of heart
disease
 Triglycerides

• In Triacylglycerol (TG) all 3 –OH of glycerol are

esterified by FAs. Monoacylglygerol and diacylglycerol
have, respectively, 1 and 2 FAs
• Naturally occurring glycerol is L-glycerol.
• TG are the storage form of FA; most dietary fats are
triglycerides
• Physiologically, TG are digested in the small intestine
by
the enzyme pancreatic lipase
• Monoacylglycerols are absorbed through the intestinal
cells, re-converted to TG and assembled into
lipoproteins
 Sphingolipids

• Sphingosine is a derivative of glycerol but it has –

NH2
• instead of -OH at C2 and has a -OH as well as a
long
• chain hydrocarbon on C3
• The –NH2 forms an amide bond with a long chain
FA to
• form a ceramide.
• sphigomyelin is formed when a phosphodiester
bridge
• links the C1 -OH of ceramide to ethanolamine or
choline
• Sphingomyelins are found abundantly in the myelin
• sheath that surrounds the nerve fibers
 Phospholipids

• These are lipids that contain one or more phosphate groups
• PL are the primary components of biomembranes. Other
lipids
in biomembranes are glycolipids and cholesterol. Surfactants
are phopsholipids, mostly phosphatidylcholine
• PL are subclassified based on their parent lipid;
phopshoglycerides or sphingomyelins
• Phosphatidic acid: basic glycerophopholipid. 1,2-
diacylglycerol joined to phosphoric acid by an ester link. This
phosphate can form another ester linkage with an alcohol.
Serine :phosphatidylserine; Choline: phosphatidylcholine
Ethanolamine: phosphatidylethanolamine; Inositol:
phosphatidylinositol; Glycerol: diphosphatidylglycerol
(cardiolipin)
 Glycolipids

.
• Glycolipids are lipids that contain carbohydrates
• Cerebrosides have a monosaccharide attached to
the
C1 -OH of ceramide
• Gangliosides have an oligosaccharide attached to
the
C1 -OH of ceramide
• Cerebrosides and gangliosides are found in the
brain
and nervous tissue
• In biomembranes, glycolipids are oriented
asymmetrically with the sugar units always on the
 Cholesterol

• Cholesterol is an essential component of

biomembranes
and imparts stability to the fluid structure.
• Cholesterol is a steroid. All steroids have the same
basic
structure consisting of 4 hydrocarbon rings linked
together
• Cholesterol has a –OH group which provides the
polarity
and a hydrocarbon group at the other end which adds
to
its hydrophobic nature
• In biomembranes, the –OH of cholesterol is aligned
 Biomembranes

• Make up boundaries of cells and intracellular

organelles (nucleas, golgi, mitochondria, ER, etc.)
• Membranes are dynamic fluid structures
• Composed of a lipid bilayer with proteins
embedded within the bilayer
• Lipids responsible for semipermeability of
biomembranes; hydrophobic chemicals can
penetrate, but most polar molecules are excluded
• Membrane proteins are transporters, channels and
pumps for the selective entry of specific molecules
 Lipid Bilayers

• Phospholipids are amphipathic: They have both hydrophilic

and hydrophobic regions
• The two hydrocarbon chains are parallel to each other, the
polar group is extended in the opposite direction.
• In aqueous solutions, amphipathic molecules arrange
themselves as micelles, bilayers or liposomes.
• These structures are stabilized by hydrophobic interactions
between hydrocarbon chains and hydrogen bonds between
polar head groups and H2O.
• Phospholipids and glycolipids favor the lipid bilayer over
micelles because the interior of a micelle cannot
accommodate 2 hydrocarbon chains of each molecule. Salts
of fatty acids (soaps) prefer to organize as micelles.
 Properties of Lipid Bilayers

• They form spontaneously. The bilayer structure is

inherent in the structures of the constituent lipids.
• They are cooperative structures stabilized by
hydrophobic interactions and hydrogen bonding.
• They are extensive / continuous without beginning
or
end.
• They are enclosed: do not have edges with exposed
hydrocarbons. Thus, they form compartments
• They are non-leaky and self-sealing. Accounts for
their
 Membrane Proteins
• Membrane proteins include pumps, channels, transporters,
receptors and enzymes. Their function include transport and
cellular communication.
• Membrane proteins are classified as peripheral or integral.
• Peripheral proteins are associated to polar head groups of the
lipid bilayer by electrostatic interactions. They can be easily
dissociated by high salt or altering pH.
• Integral proteins are embedded within the bilayer by
hydrophobic interactions with hydrocarbon chains. Their
embedded domains contain aa with hydrophobic side chains.
• Proteins that form channels and transporters have multiple
transmembrane domains
• Certain proteins may be anchored to the bilayer by covalent
bonds between aa of proteins and head groups on lipids
 p er i p h er al
Membrane lipid
proteins may an ch or

be classified as
:
l i p i d b i l ay er
peripheral
integral
M em b r an e
having a i n tegr al P r otei n s
lipid anchor
Peripheral proteins are on the membrane surface.
They are water-soluble, with mostly hydrophilic
surfaces.
Often peripheral proteins can be dislodged by
conditions that disrupt ionic & H-bond interactions,
e.g., extraction with solutions containing high
concentrations of salts, change of pH, and/or
 h y p oth eti cal p r otei n

r egu l ator y catal y ti c m em b r an e

d om ai n d om ai n bi n d i n g

Many proteins have a modular design, with different

segments of the primary structure folding into
domains with different functions.
Some cytosolic proteins have domains that bind to
polar head groups of lipids that transiently exist in a
membrane.
The enzymes that create or degrade these lipids are
subject to signal-mediated regulation, providing a
mechanism for modulating affinity of a protein for a
Glycosylphosphatidylinositols (GPI) are complex
glycolipids that attach some proteins to the outer
surface of the plasma membrane.
The linkage is similar to the following, although the
oligosaccharide composition may vary:
protein (C-term.) - phosphoethanolamine – mannose -
mannose - mannose - N-acetylglucosamine – inositol (of PI in
membrane)

The protein is tethered some distance out from the

membrane surface by the long oligosaccharide chain.
GPI-linked proteins may be released from the outer
cell surface by phospholipases.
 p er i p h er al
lipid
an ch or

l i p i d b i l ay er

M em b r an e
i n tegr al P r otei n s

Integral proteins have domains that extend into the

hydrocarbon core of the membrane.
Often they span the bilayer.
Intramembrane domains have largely hydrophobic
surfaces, that interact with membrane lipids.
 m em b r an e
Amphipathic
detergents are d eter gen t p ol ar
required for sol u b i l i z ati on n on - p ol ar
solubilization of
integral proteins P r otei n w i th
from b ou n d d eter gen t
membranes.
Hydrophobic domains of detergents substitute for
lipids, coating hydrophobic surfaces of integral
proteins.
Polar domains of detergents interact with water.
If detergents are removed, purified integral proteins
tend to aggregate & come out of solution. Their
hydrophobic surfaces associate to minimize contact
Integral protein structure
Atomic-resolution structures have been
determined for a small (but growing) number of
integral membrane proteins.
Integral proteins are difficult to crystallize for X-ray
analysis.
Because of their hydrophobic
transmembrane domains,
detergents must be present C
during crystallization.
m em b r an e

A membrane-spanning -helix is
the most common structural
motif found in integral proteins. N
In an -helix, amino acid R-groups protrude out from
the helically coiled polypeptide backbone.
The largely hydrophobic R-groups of a membrane-
spanning -helix contact the hydrophobic membrane
core, while the more polar peptide backbone is
buried.
Particular amino acids tend to occur at different
positions relative to the surface or interior of the
bilayer in transmembrane segments of integral
proteins.
Residues with aliphatic side-chains (leucine,
isoleucine, alanine, valine) predominate in the
Tyrosine and
tryptophan are
common near the
membrane
surface.
It has been suggested that the polar character of the
tryptophan amide group and the tyrosine hydroxyl,
along with their hydrophobic ring structures, suit
them for localization at the polar/apolar interface.
Lysine & arginine are often at the lipid/water
interface, with the positively charged groups at the
ends of their aliphatic side chains extending toward
the polar membrane surface.
If a hydropathy plot indicates one 20-amino acid
hydrophobic stretch (1 putative transmembrane -
helix), topology studies are expected to confirm
location of N & C termini on opposite sides of
membrane.
If two transmembrane -helices are predicted, N &
C termini should be on the same side. The segment
between the -helices should be on the other side.
Write the name of the appropriate enzyme in
the boxes

F
 Carbohydrates
OBJECTIVES
▪ Understand how Monosaccharides are named
▪ Understand how Carbohydrates form Cyclic
Structures
▪ To describe reactions of Glucose and Other
Monosaccharides
▪ Understand how Polysaccharides are formed
▪ To describe types of bonds in Glycoproteins

1
 Carbohydrates

Compounds containing C, H and O

General formula : Cx(H2O)y

All have C=O and -OH functional groups.

Classified based on
• Size of base carbon chain
• Number of sugar units
• Location of C=O
• Stereochemistry
2
Carbohydrate and sugar structure
Carbohydrates or saccharides are essential
components of living organisms.
(C•H2O)n Where n=3 or greater.
A single saccharide is called a monosaccharide.
Oligosaccharide is a few linked monosaccharides
and are at time associated with proteins
(glycoproteins) or lipids (glycolipids)
Polysaccharides consist of many monosaccharides
i.e. cellulose or glycogen
3
 Types of carbohydrates

Classifications based on number of sugar

units in total chain.
Monosaccharides - single sugar unit
Disaccharides - two sugar units
Oligosaccharides - 2 to 10 sugar units
Polysaccharides - more than 10 units
Chaining relies on ‘bridging’ of oxygen
atoms
glycoside bonds
4
 Monosaccharides

Based on location of C=O

H CH2OH
| |
C=O C=O
| |
H-C-OH HO-C-H
| |
H-C-OH H-C-OH
| |
H-C-OH H-C-OH
| |
CH2OH CH2OH

Aldose Ketose
- aldehyde C=O - ketone C=O
5
 Monosaccharide classifications

Number of carbon atoms in the chain

H
H |
H | C=O
H | C=O |
| C=O | H-C-OH
C=O | H-C-OH |
| H-C-OH | H-C-OH
H-C-OH | H-C-OH |
| H-C-OH | H-C-OH
CH2OH | H-C-OH |
CH2OH | H-C-OH
CH2OH |
CH2OH
triose tetrose pentose hexose
Can be either aldose or ketose sugar.
6
 Examples
CH2OH
|
C=O
|
H HO-C-H
| |
C=O H-C-OH
| |
H-C-OH H-C-OH
| |
CH2OH CH2OH
D-glyceraldehyde D-fructose
triose hexose
aldose ketose
aldotriose sugar ketohexose sugar
7
 Examples
H
|
H C=O
| |
C=O H-C-OH
| |
H-C-OH H-C-OH
| |
H-C-OH HO-C-H
| |
H-C-OH HO-C-H
| |
CH2OH CH2OH
D-ribose L-mannose
pentose, aldose hexose, aldose
aldopentose sugar aldohexose sugar
8
 Stereoisomers

Stereochemistry
Study of the spatial arrangement of molecules.

Stereoisomers have
• the same order and types of bonds.
• different spatial arrangements.
• different properties.
Many biologically important chemicals, like
sugars, exist as stereoisomers. Your body can
tell the difference.
9
 Enantiomers

Pairs of stereoisomers
Designated by D- or L- at the start of the name.
They are mirror images that can’t be
overlapped.

If you don’t believe it,

give it a try!
10
Enantiomers

11
 L- and D- glyceraldehyde

CHO CHO
HO H H OH

C C

CH2OH CH2OH

CHO CHO

HO H H OH

CH2OH CH2OH
12
 Enantiomers

Chiral center.
Asymmetric carbon - 4 different things
are attached to it.
Cl
|

I- C - F
| Chiral center
Br
You must have at least one asymmetric
carbon to have stereoisomers.
13
 Examples

Is the ‘red’ carbon chiral?

H H I
Cl
C=O H3C- C-OH C=C
Cl CH2CH3 Br F

H
|
H C=O HH H
H3C- C-OH | H2N-C-C-C-SH
H-C-OH
H | Cl H Cl
CH2OH

14
 Physical properties

Optical activity
ability to rotate plane polarized light.

dextrorotatory - rotate to right

- use + symbol
- usually D isomers

levorotatory - rotate to left

- use - symbol
- usually L isomers
15
 Plane polarized light

Light is passed through a polarized filter.

A solution of an optical isomer will rotate the
light one direction.
16
 Stereochemistry

Properly drawing enantiomers in 3-D is hard.

Use Fischer Projections
Specific type of formula that designates
the orientation of groups.
H
|
H C=O
| |
C=O H-C-OH
| |
H-C-OH H-C-OH
| |
CH2OH CH2OH
17
 Fischer projections

With this system, a tetrahedral carbon atom is

represented by two crossed lines.
H H
|
C=O O
|
H-C-OH H OH
|
H-C-OH H OH
|
CH2OH CH2OH

A horizontal bond to an asymmetric carbon

designates bonds in the front plane of the page.
Vertical bonds are behind the plane of the page.
18
Some important monosaccharides

D-glyceraldehyde Simplest sugar

D-glucose Most important in diet
D-fructose Sweetest of all sugars
D-galactose Part of milk sugar
D-ribose Used in RNA

note that each is a D- enantiomer

19
 D-glyceraldehyde

Three carbon sugar

Aldose sugar
Triose sugar
H
|
C=O
aldotriose |
H-C-OH
|
CH2OH

20
 D-glucose

• Glucose is an aldohexose sugar.

• Common names include H O

dextrose, grape sugar, blood C
sugar. H C OH

• Most important sugar in our diet.

HO C H
H C OH
• Most abundant organic H C OH
compound found in nature. CH 2OH

• Level in blood can be as high as

0.1%
21
 D-fructose

CH2OH
Another common sugar. |
C=O
|
HO-C-H
It is a ketohexose. |
H-C-OH
|
H-C-OH
Sweetest of all sugars. |
CH2OH

22
 Carbohydrates in cyclic structures

If optical isomers weren’t enough, sugars also

form rings. For many sugars, its the most
common form.

hemiacetal - formed from alcohol and

aldehyde

hemiketal - formed from alcohol and ketone

R OR’’
\ |
C=O + ROH R - C - OH
/ |
R’ R’
23
 Intramolecular cyclization

Cyclization.
Remember - chains can bend and rotate.

CH2OH CH2OH

H
C OH C O

C C O C C OH

C C C C

24
 Intramolecular cyclization

The -OH group that forms can be above or

below the ring resulting in two forms -
anomers

 and  are used to identify the two forms.

•  - OH group is down compared to CH2OH
(trans).
•  - OH group is up compared to CH2OH
(cis).

25
 Intramolecular cyclization

The  and  forms are in equilibrium so one

form can convert to the other -
mutarotation.
Haworth projections can be used to help
see  and  orientations.

O O

26
 Cyclization of D-glucose
CH2 OH  -D - glucose
H O H
H
O OH H
H
C OH OH
H C OH H OH
HO C H
H C OH CH 2 OH  - D - glucose
H C OH H O OH
H
CH 2OH OH H
OH H
H OH
27
 Fischer vs. Haworth projections

 -D-glucose

H C OH CH2 OH
H C OH H O H
H
HO C H O H
OH
H C OH OH OH
HO-CH2 C H
H OH

28
 Cyclization of D-fructose

This can also happen

to ketose sugars. CH2 OH O CH2OH

CH2 OH
H OH −
H OH
C O
OH H
HO C H
H C OH
H C OH CH2 OH O OH
CH 2OH H OH
−
H CH2OH
OH H
29
 D-galactose

• Not found in many biological systems

• Common part of lactose - disaccharide
CH 2 OH
OH O H
H
O OH H
H
C H OH
H C OH H OH
HO C H CH 2 OH
HO C H O OH
OH
H C OH H
OH H
CH 2 OH
H H
H OH
30
 D-glucose vs. D-galactose
D-glucose D-galactose
H O H O
C C
H C OH H C OH
HO C H HO C H
H C OH HO C H
H C OH H C OH
CH 2OH CH 2 OH

Can you find a difference? Your body can!

You can’t digest galactose - it must be
converted to glucose first.
31
 D-ribose

An important sugar used

in genetic material. H
|
This sugar is not used as C=O
|
an energy source but is a part H-C-OH
of the backbone of RNA. |
H-C-OH
|
When the C-2 OH is removed, H-C-OH
the sugar becomes |
CH2OH
deoxyribose which is used
in the backbone of DNA.

32
 Reactions of glucose
and other monosaccharides
Oxidation-Reduction. Required for their
complete metabolic breakdown.

Esterification. Production of phosphate

esters.

Amino derivatives. Used to produce

structural components and glycoprotein.

Glycoside formation. Linkage of

monosaccharides to form polysaccharides.

33
 Oxidation-Reduction.
Aldehyde sugars (reducing sugars) are readily
oxidized and will react with Benedict’s reagent.
H O-
| |
C=O C=O
| + 2 Cu 2+ + 5 OH- | + 2 Cu2O + 3H2O
H-C-OH H-C-OH
|
|
CH2OH CH2OH

This provides a good test for presence of glucose in

urine - forms a red precipitate.

Other tests - Tollen’s or Fehling’s solutions.

34
 Benedict’s reagent

glucose
0.5% 2%

Benedict's
Reagent

35
 Ketone sugars

Ketones are not easy to oxidize except ketoses.

Enediol reaction.
H O
C H C OH CH2OH
H C OH C OH H C O
HO C H HO C H HO C H
H C OH H C OH H C OH
H C OH H C OH H C OH
CH 2OH CH 2OH CH 2OH
So all monosaccharides are reducing sugars.
36
 Esterification

Esters are formed by reaction of hydroxyl

groups (alcohols) with acids.

O O
R OH + C R' R O C R' + H2O
HO

The hydroxyl groups of carbohydrates react

similarly to alcohols.

37
 Esterification

The most important biological esters of

carbohydrates are phosphate esters.
O O
R OH + HO P OH HO P OH + H 2O
OH O-R

Example. Phosphoryl group from ATP forms an

ester with D-glucose, catalyzed by kinases.
kinase
D-glucose + ATP D-glucose-6-phosphate + ADP

38
 Amino derivatives

The replacement of a hydroxyl group on a

carbohydrate results in an amino sugar.
CH2OH CH 2OH

H O OH H O OH
H H
OH H OH H

OH H OH H

H OH H NH 2

-D-glucose -D-2-aminoglucose
(glucosamine)
39
 Amino derivatives

Uses for amino sugars.

Structural components of bacterial cell
walls.
As a component of chitin, a polymer found
in the exoskeleton of insects and
crustaceans.
A major structural unit of chondroitin
sulfate - a component of cartilage.
Component of glycoprotein and glycolipids.
40
 Glycoside formation

 or  -OH group of cyclic monosaccharide

can form link with another one (or more).
CH2OH CH2OH
H O OH H O H
glycosidic bond H
OH H H
H
OH
OH H OH OH
H OH OH H
sugar -O- sugar
CH2OH
CH2OH H O H
H
oxygen bridge H O
H OH
H o
OH H OH
OH H OH H
H OH
+ H2 O
41
 Glycosidic bonds

Type is based on the position of the C-1 OH

 glycosidic bond
- linkage between a C-1  OH and a C-4 OH
 glycosidic bond
- linkage between a C-1  OH and a C-4 OH
 bonds  bonds
O O
O
O
C-4 end can be either up or down depending
on the orientation of the monosaccharide.
42
 Glycosidic bonds

 bonds  bonds

O O

O O

43
 Glycosidic bonds

General format used to describe bond.

OH type carbon# of carbon# of

( or ) ( first sugar second sugar )
As we work through the next few examples
this will become clear.

For disaccharides - the sugar is either  or 

based on form of the
remaining C-1 OH.
44
 -Maltose

Malt sugar. Not common in nature except in

germinating grains.
CH2 OH CH2 OH

H O H H O OH
H H
OH H OH H
O H
OH
H OH H OH

-D-glucose -D-glucose

-D-glucose and -D-glucose,  (1 4) linkage.

45
 -Maltose

It is referred to as -maltose because the

unreacted C-1 on -D-glucose is in the 
position.
CH2 OH CH2 OH

H O H H O OH
H H
OH H OH H
O H
OH
H OH H OH

46
 -Maltose

Uses for -maltose

Ingredient in infant formulas.
Production of beer.
Flavoring - fresh baked aroma.

It is hydrolyzed the in body by:

maltase
maltose + H2O 2 glucose

47
 Cellobiose

Like maltose, it is composed of two molecules of

D-glucose - but with a  (1 4) linkage.

CH2OH

H O OH
H
CH2OH OH H
H
H O O
H
H OH
OH H
OH H

H OH
48
 Cellobiose
CH 2 OH CH 2 OH

H O H H O OH
The difference in H H
OH H OH H
the linkage results OH
O H

in cellobiose H OH H OH
being unusable maltose,  (1 4)
CH2OH

We lack an enzyme H
H
O OH

that can hydrolyze CH2OH OH H

H
cellobiose. H O O
H
H OH
OH H
OH H cellobiose
H OH  (1 4)
49
 Lactose

Milk sugar - dimer of -D-galactose and

either the  or  - D-glucose.
CH2 OH CH2 OH
O O OH
-Lactose OH
H
H
H
OH H O OH H
H H H

H OH H OH

-D-galactose -D-glucose

 (1 4) linkage,  disaccharide.
50
 Lactose

We can’t directly use galactose. It must be

converted to a form of glucose.

Galactosemia - absence of needed enzymes

needed for conversion.

Build up of galactose or a metabolite like

dulcitol (galactitol) causes toxic effects.

Can lead to retardation, cataracts, death.

51
 Lactose

Lactase
Enzyme required to hydrolyze lactose.
Lactose intolerance
Lack or insufficient amount of the
enzyme.

If lactase enters lower

GI, it can cause gas
and cramps.
52
 Sucrose
CH2OH
Table sugar - most
common sugar in all H O H
plants. H
OH H
Sugar cane and beet,
are up to 20% by OH
mass sucrose. H OH
O
Disaccharide of
-glucose and CH2OH O
-fructose.
H OH
H
 (1 2) linkage OH H
CH2OH
53
Sucrose

glucose

fructose

54
 How sweet it is!

Sweetness relative
Sugar to sucrose
lactose 0.16
galactose 0.32
maltose 0.33
sucrose 1.00
fructose 1.73
aspartame 180
saccharin 450
55
 Polysaccharides

Carbohydrate polymers
Storage Polysaccharides
Energy storage - starch and glycogen
Structural Polysaccharides
Used to provide protective walls or
lubricative coating to cells - cellulose
and mucopolysaccharides.
Structural Peptidoglycans
Bacterial cell walls
56
 Starch

Energy storage used by plants

Long repeating chain of -D-glucose

Chains up to 4000 units

Amylose straight chain

major form of starch
Amylopectin branched structure

57
 Amylose starch

Straight chain that forms coils  (1 4)

linkage. Most common type of starch.
CH2OH CH2OH CH2 OH
CH2OH
O O H H O H
H O H H H H
H H H
H OH H
OH H OH H OH H

O O O
O

H OH H OH H OH
H OH

O O O
O

O O O O
O O O O

O O O O O
O O O

O O O
O

58
 Amylose starch

Example showing coiled structure

- 12 glucose units
- hydrogens and side chains are omitted.

59
 Amylopectin starch

Branched structure due to crosslinks.

CH2OH CH2OH CH2OH
CH2OH
O H O H H O H
H O H H H
H H H
H H
H OH H OH H OH
OH
O O O
O

CH2OH H OH CH2OH H OH CH2

H OH CH2OH H OH

O O H H O H
H O H H H H
H H H
H OH H
OH H OH H OH H

O O O
O

H OH H OH H OH
H OH

 (1 6) linkage
at crosslink
60
 Glycogen

• Energy storage of animals.

• Stored in liver and muscles as granules.
• Similar to amylopectin.
O

O  (1 6) linkage
at crosslink
O

O
c
O

O
c O

O
O

O
O

61
 Cellulose

• Most abundant polysaccharide.

•  (1 4) glycosidic linkages.
• Result in long fibers - for plant structure.

CH2OH
CH2OH O
H
CH2OH O H
H O
CH2OH O H OH H
H O
CH2OH O H OH H H
H O
O H OH H H
H O
OH H H OH
H O H
OH H H OH
H
H OH
H
H OH
H OH

62
 Mucopolysaccharides

These materials provide a thin, viscous, jelly-like

coating to cells. H
CH OH
O
2
O
H
The most abundant form is COO-
OH H

hyaluronic acid.
O H NH
H O
CH OH 2 HO C O
H CH3
H O O
H
H H OH
(1 3) COO - OH H
O H NH
H O
HO C O
CH2OH H CH3
(1 4) H O O H
H H OH

COO-
O
OH
H NH
H
Alternating units of
H O
HO
H
C O
CH3
N-acetylglucosamine and
H
H OH D-glucuronic acid.
63
 Structural peptidoglycans
Bacterial cell walls are composed primarily of an
unbranched polymer of alternating units of N-
acetylglucosamine and N-acetylmuramic acid.
CH 3
CH 2OH CH 2OH

H O H O O
R= CH
H H
OH H O OR H
O H H O L-Ala

H NH H NH D-Isoglu
C O C O
(Gly)5 L-Lys
CH 3 CH 3
D-Ala

Peptide crosslinks between the polymer (Gly)5

strands provide extra strength crosslink for

- varies based on bacterium. Staphylococcus
aureus
64
 Glycoproteins

Proteins that carry covalently bound

carbohydrate units.

They have many biological functions.

• immunological protection
• cell-cell recognition
• blood clotting
• host-pathogen interaction
65
 Glycoprotein structure

Carbohydrates only account for 1-30% of

the total weight of a glycoprotein.

The most common monosaccharides are

glucose mannose
galactose fucose
sialic acid
N-acetylgalactosamine
N-acetylglucosamine
66
 Glycoprotein structure
Linking sugars to proteins. threonine

O-glycosidic bonds using CH 2OH CH 3

hydroxyl groups of O
O O C
H
C
H
serine and threonine

polypeptide chain
OH H

H H

H NHCOCH 3

N-glycosidic bonds using

asparagine
side chain amide
CH 2OH O
nitrogen of asparagine O
H
N C C C
residue
H
H H2
OH H
O H

H NHCOCH 3

67
Endosymbiosis and the
Origin of Eukaryotes:

Are mitochondria really just
bacterial symbionts?
Timothy G. Standish, Ph. D.

©1999 Timothy G. Standish

 Outline
Mitochondria - A very brief overview
Endosymbiosis - Theory and evidence
Archaezoa - Eukaryotes lacking
mitochondria
Gene expression - Mitochondrial proteins
coded in the nucleus
Mitochondrial genetic codes
Gene transport - Mitochondria to nucleus
Conclusions
©1999 Timothy G. Standish
 Mitochondria
Mitochondria are organelles found in most
eukaryotic organisms.
The site of Krebs cycle and electron transport
energy producing processes during aerobic
respiration
Are inherited only from the mother during
sexual reproduction in mammals and probably
all other vertebrates.
Because of their mode of inheritance genetic
material found in mitochondria appears to be
useful in determining the maternal lineage of
organisms.
©1999 Timothy G. Standish
 Mitochondria
Outer membrane
Inner membrane

Matrix mtDNA

Inter membrane space

©1999 Timothy G. Standish

 Extranuclear DNA
Mitochondria and chloroplasts have their own DNA
This extranuclear DNA exhibits non-Mendelian
inheritance
Recombination is known between some mt and
ctDNAs
Extranuclear DNA may also be called cytoplasmic DNA
Generally mtDNA and ctDNA is circular and contains
genes for multimeric proteins, some portion of which
are also coded for in the nucleus
Extranuclear DNA has a rate of mutation that is
independent of nuclear DNA
Generally, but not always, all the RNAs needed for
transcription and translation are found in mtDNA and
ctDNA, but only some of the protein genes ©1999 Timothy G. Standish
 mtDNA
Mitochondrial DNA is generally small in animal cells,
about 1.65 kb
In other organisms sizes can be more than an order
of magnitude larger
Plant mtDNA is highly variable in size and content
with the large Arabidopsis mtDNA being 200 kb.
The largest known number of mtDNA protein genes is
97 in the protozoan Riclinomonas mtDNA of 69 kb.
“Most of the genetic information for mitochondrial
biogenesis and function resides in the nuclear
genome, with import into the organelle of nuclear
DNA-specified proteins and in some cases small
RNAs.” (Gray et al.,1999)
©1999 Timothy G. Standish
Endosymbiosis

©1999 Timothy G. Standish

 Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system

©1999 Timothy G. Standish

 Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
Mitochondria

©1999 Timothy G. Standish

 Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
Endoplasmic
Mitochondria Reticulum

Nucleus

Golgi
Chloroplast Body
©1999 Timothy G. Standish
 Origin of Eukaryotes
Two popular theories presupposing naturalism seek to
explain the origin of membrane-bound organelles:
1 Endosymbiosis to explain the origin of mitochondria and
chloroplasts (popularized by Lynn Margulis in 1981)
2 Invagination of the plasma membrane to form the
endomembrane system
Endoplasmic Reticulum
Mitochondria

Nucleus

Golgi Body
Chloroplast
©1999 Timothy G. Standish
How Mitochondria Resemble Bacteria
Most general biology texts list ways in which
mitochondria resemble bacteria. Campbell et al.
(1999) list the following:
Mitochondria resemble bacteria in size and morphology.
They are bounded by a double membrane: the outer thought to
be derived from the engulfing vesicle and the inner from
bacterial plasma membrane.
Some enzymes and inner membrane transport systems
resemble prokaryotic plasma membrane systems.
Mitochondrial division resembles bacterial binary fission
They contain a small circular loop of genetic material (DNA).
Bacterial DNA is also a circular loop.
They produce a small number of proteins using their own
ribosomes which look like bacterial ribosomes.
Their ribosomeal RNA resembles eubacterial rRNA.

©1999 Timothy G. Standish

 How Mitochondria Don’t

Resemble Bacteria
Mitochondria are not always the size or morphology
of bacteria:
– In some Trypanosomes (i.e., Trypanosoma brucei)
mitochondria undergo spectacular changes in
morphology that do not resemble bacteria during different
life cycle stages (Vickermann, 1971)
– Variation in morphology is common in protistans,
“Considerable variation in shape and size of the organelle
can occur.” (Lloyd, 1974 p 1)
Mitochondrial division and distribution of
mitochondria to daughter cells is tightly controlled
by even the simplest eukaryotic cells
©1999 Timothy G. Standish
 How Mitochondria Don’t

Resemble Bacteria
Circular mtDNA replication via D loops is different from
replication of bacterial DNA (Lewin, 1997 p 441).
mtDNA is much smaller than bacterial chromosomes.
Mitochondrial DNA may be linear; examples include:
Plasmodium, C. reinhardtii, Ochromonas, Tetrahymena,
Jakoba (Gray et al., 1999).
Mitochondrial genes may have introns which eubacterial
genes typically lack (these introns are different from nuclear
introns so they cannot have come from that source) (Lewin,
1997 p 721, 888).
The genetic code in many mitochondria is slightly different
from bacteria (Lewin, 1997).

©1999 Timothy G. Standish

Archaezoa

©1999 Timothy G. Standish

 Giardia - A “Missing Link”?
The eukaryotic parasite Giardia has been suggested
as a “missing link” between eukaryotes and
prokaryotes because it lacks mitochondria (Friend,
1966; Adam, 1991) thus serving as an example of
membrane invagination but not endosymbiosis
Giardia also appears to lack smooth endoplasmic
reticulum, peroxisomes and nucleoli (Adam, 1991)
so these must have either been lost or never evolved

©1999 Timothy G. Standish

 A Poor “Missing Link”
As a “missing link” Giardia is not a strong argument
due to its parasitic life cycle which lacks an
independent replicating stage outside of its
vertebrate host
– Transmission is via cysts excreted in feces followed by
ingestion
– As an obligate parasite, to reproduce, Giardia needs other
more derived (advanced?) eukaryotes
Some other free-living Archaezoan may be a better
candidate

©1999 Timothy G. Standish

 Origin of Giardia
Giardia and other eukaryotes lacking mitochondria and
plastids (Metamonada, Microsporidia, and Parabasalia ) have
been grouped by some as “Archaezoa” (Cavalier-Smith, 1983;
Campbell et al., 1999 p 524-6)
This name reflects the belief that these protozoa split from the
group which gained mitochondria prior to that event.
The discovery of a mitochondrial heat shock protein (HSP60)
in Giardia lamblia (Soltys and Gupta, 1994) has called this
interpretation into question.
Other proteins thought to be unique to mitochondria, HSP70
(Germot et al., 1996), chaperonin 60 (HSP60) (Roger et al.,
1996; Horner et al., 1996) and HSP10 (Bui et al., 1996) have
shown up in Giardia’s fellow Archaezoans

©1999 Timothy G. Standish

 Origin of Archaezoa
The authors who reported the presence of mitochondrial
genes in amitochondrial eukaryotes all reinterpreted prevailing
theory in saying that mitochondria must have been present
then lost after they had transferred some of their genetic
information to the nucleus.
The hydrogenosome, a structure involved in carbohydrate
metabolism found in some Archaezoans (Muller, 1992), is now
thought to represent a mitochondria that has lost its genetic
information completely and along with that loss, the ability to
do the Krebs cycle (Palmer, 1997).
Alternative explanations include transfer of genetic material
from other eukaryotes and the denovo production of
hydrogenosomes by primitive eukaryotes.

©1999 Timothy G. Standish

Origin of Archaezoa:

Mitochondrial Acquisition

©1999 Timothy G. Standish

Origin of Archaezoa:

Gene Transfer and Loss

mtGenes

Lost
genetic
materia
©1999 Timothy G. Standish
 Origin of Archaezoa:

Option 1 - Mitochondrial Eukaryote Production

©1999 Timothy G. Standish

 Origin of Archaezoa:

Option 2 - Mitochondrial DNA Loss/

Hydrogenosome production
Hydrogenosome

©1999 Timothy G. Standish

 Origin of Archaezoa:

Option 2A - Mitochondria/Hydrogenosome Loss

©1999 Timothy G. Standish

Gene Transport

©1999 Timothy G. Standish

“All in all then, the host
nucleus seems to be a
tremendous magnet, both
for organellar genes and for
endosymbiotic nuclear
genes.”
Palmer, 1997
©1999 Timothy G. Standish
Steps in Mitochondrial Acquisition:

The Serial Endosymbiosis Theory
Fusion of Rickettsia with either a
nucleus containing Archaezoan
or an archaebacterium
Rickettsia
Host Cell

Primitive
eukaryot
e

DNA
reduction/transf
er to nucleus
Ancestral eukaryote
(assuming a nucleus)
©1999 Timothy G. Standish
Steps in Mitochondrial Acquisition:

The Hydrogen Hypothesis

Fusion of proteobacterium with

an archaebacterium
Hydrogen
producing Hydrogen
proteobacteriu requiring
m archaebacterium

DNA
reduction/transf
er nucleus
production
Ancestral eukaryote
With nucleus containing
both archaebacterium and
proteobacterium genes

©1999 Timothy G. Standish

 Phylogeny
Microsporidia,
Bacteria and Parabasalia Metamonada Eukaryota Bacteria
mtDNA Hydrogenosome/
loss mitochondria
loss
mtDNA
loss
Gene transfer
Cell fusion

Origin of Life
©1999 Timothy G. Standish
 Timing of Gene Transfer
Because gene transfer occurred in eukaryotes
lacking mitochondria, and these are the lowest
branching eukaryotes known:
Gene transfer must have happened very early in
the history of eukaryotes.
The length of time for at least some gene transfer
following acquisition of mitochondria is greatly
shortened.
No plausible mechanism for movement of genes
from the mitochondria to the nucleus exists
although intraspecies transfer of genes is
sometimes invoked to explain the origin of other
individual nuclear genes. ©1999 Timothy G. Standish
 Gene
Expression

©1999 Timothy G. Standish

 Cytoplasmic Production of
Mitochondrial Proteins
Mitochondria produce only a small subset of the
proteins used in the Krebs cycle and electron
transport. The balance come from the nucleus
As mitochondrial genomes vary spectacularly
between different groups of organisms, some of
which may be fairly closely related, if all came from
a common ancestor, different genes coding for
mitochondrial proteins must have been passed
between the nucleus and mitochondria multiple
times

©1999 Timothy G. Standish

 The Unlikely Movement of Genes
Between Mitochondria and the Nucleus
Movement of genes between the
mitochondria and nucleus seems unlikely
for at least two reasons:
1 Mitochondria do not always share the
same genetic code with the cell they are in
2 Mechanisms for transportation of proteins
coded in the nucleus into mitochondria
seem to preclude easy movement of
genes from mitochondria to the nucleus
©1999 Timothy G. Standish
 Protein Production

Mitochondria and Chloroplasts
Cytoplasm

Nucleus G AAAAAA

Export

Mitochondrion Chloroplast

©1999 Timothy G. Standish

 Protein Production

Mitochondria and Chloroplasts
Cytoplasm

Nucleus

Mitochondrion Chloroplast

©1999 Timothy G. Standish

 Protein Production

Mitochondria
Outer membrane
Inner membrane

Matrix

Inter membrane space

©1999 Timothy G. Standish

 Protein Production

Mitochondria
Leader
ATP sequence
binding Outer membrane
P +ADP
receptor

MLSLRQSIRFFKPATRTLCSSRYLL
ATP
P +ADP

Inner membrane
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

M
Peptideas
LS
LR
e cleaves
QS

off the
RFI

leader
F
KP
AT

Inner membrane
RT
L

Inter
CS
SR

membrane
Matrix
YL

space
L

©1999 Timothy G. Standish

 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

ML
SLR
QSI
RFF Inner membrane
KPA
TRT Inter
LC SSR
YL L membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Hsp60
Hsp60
Inner membrane
Inter
Chaperones membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane
Inter
membrane
Matrix Mature protein
space
©1999 Timothy G. Standish
M
L Yeast Cytochrome C
S
L Polar
R
Oxidase Subunit IV Leader
Q Neutral Non-polar
S First 12 residues are sufficient Polar
I Non- for transport to the Basic
polar
R
mitochondria
Acidic
F
F MLSLRQSIRFFKPATRTLCSSRYLL
K
P This leader does not resemble other
A eukaryotic leader sequences, or other
Recognized by peptidase?

T
R
mtProtein leader sequences.
T
L Polar Probably forms an  helix
C
S This would localize specific classes of
S
R amino acids in specific parts of the
Y
helix
L
P There are about 3.6 amino acids per
©1999 Timothy G. Standish
 Yeast Cytochrome C1 Leader
Charged leader sequence
signals for transport to First cut
mitochondria
MFSNLSKRWAQRTLSKTLKGSKSAAGTATSYFE-
KLVTAGVAAAGITASTLLYANSLTAGA--------------
Second cut
Uncharged second leader sequence signals for
transport across inner membrane into the Neutral Non-polar
Cytochrome intermembrane
c functions inspace
electron transport Polar
Basic
and is thus associated with the inner membrane Acidic
on the intermembrane space side
Cytochrome c1 holds an iron containing heme
group and is part of the B-C1 (III) complex
C1 accepts electrons from the Reiske protein
and passes them to cytochrome c ©1999 Timothy G. Standish
 Protein Production

Mitochondria
Outer membrane
Inner membrane

Matrix

Inter membrane space

©1999 Timothy G. Standish

 Protein Production

Mitochondria
Leader
ATP sequence
binding Outer membrane
P +ADP
receptor

ATP
P +ADP

Peptideas
e cleaves
off the Inner membrane
leader
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane

Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane

Peptidease
cleaves off
the second
leader

Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane

Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane

Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Protein Production

Mitochondria
Leader
sequence
binding Outer membrane
receptor

Inner membrane

Mature protein
Inter
membrane
Matrix space
©1999 Timothy G. Standish
 Building a Minimally Functional
Nuclear Mitochondrial Gene
Given that a fragment of DNA travels from the
mitochondria to the nucleus and is inserted into the
nuclear DNA Nuclear DNA

Control SequenceSignal Sequence Mitochondrial Gene

Control Sequence
Additional hurdlesSignalmay include:
Sequence
Resolution of problems resulting from differences
Mitochondrial Gene
between mitochondrial and nuclear introns
Resolution of problems resulting from differences
between mitochondiral and nuclear genetic codes
©1999 Timothy G. Standish
 Additional Requirements
In addition to addition of appropriate control
and leader sequences to mitochondrial
genes, the following would be needed:
Recognition and transport mechanisms in
the cytoplasm
Leader sequence binding receptors
Peptidases that recognize leader
sequences and remove them

©1999 Timothy G. Standish

 No Plausible Mechanism Exists
If genes were to move from the mitochondria to the
nucleus they would have to somehow pick up the
leader sequences necessary to signal for transport
before they could be functional
While leader sequences seem to have meaningful
portions on them, according to Lewin (1997, p 251)
sequence homology between different sequences is
not evident, thus there could be no standard sequence
that was tacked on as genes were moved from
mitochondria to nucleus
Alternatively, if genes for mitochondrial proteins
existed in the nucleus prior to loss of genes in the
mitochondria, the problem remains, where did the
signal sequences come from? And where did the
©1999 Timothy G. Standish
Mitochondrial
Genetic Codes

©1999 Timothy G. Standish

 Variation In Codon Meaning
Lack of variation in codon meanings across almost all phyla
is taken as an indicator that initial assignment must have
occurred early during evolution and all organisms must have
descended from just one individual with the current codon
assignments
Exceptions to the universal code are known in a few single-
celled eukaryotes, mitochondria and at least one prokaryote
Most exceptions are modifications of the stop codons UAA,
UAGOrganism
and UGA Codon/s Common MeaningModified Meaning
Tetrahymena thermophila UAA UAG Stop glutamine
A ciliate
Paramecium UAA UAG Stop glutamine
A ciliate
Euplotes octacarinatus UGA Stop cysteine
A ciliate
Mycoplasma capricolum UGA Stop tryptophan
A bacteria
Candida CUG serine leucine
A yeast
Neutral Non-polar, Polar ©1999 Timothy G. Standish
 Variation in Mitochondrial
Codon Assignment

Platyhelmiths

Echinoderms

Vertebrates
Cytoplasm/

Nematodes

Molluscs
Nucleus

Insects
Yeast/
Plants

Molds

AUA=Met AAA=Asn AUA=Ile UGA/G=Stop

CUN=Thr AAA=Asn
NOTE - This would
mean AUA changed
from Ile to Met, then
AUA=Met changed back to Ile in
AGA/G=Ser the Echinoderms
AAA must have changed from Lys
UGA=Trp to Asn twice
Universal UGA must have changed to Trp then back to
Code stopin mtDNA lower the number of tRNAs
Differences
©1999 Timothy G. Standish
 Problems Resulting From
Differences in Genetic Codes
Changing the genetic code, even of the most
simple genome is very difficult.
Because differences exist in the
mitochondrial genomes of groups following
changes in the mitochondrial genetic code,
mitochondrial genes coding differently must
have been transported to the nucleus.
These mitochondrial genes must have been
edited to remove any problems caused by
differences in the respective genetic codes.
©1999 Timothy G. Standish
 No Modern Examples
Unfortunately for Margulis and S.E.T. [the serial endosymbiotic
theory], no modern examples of prokaryotic endocytosis or
endosymbioses exist . . . She discusses any number of
prokaryotes endosymbiotic in eukaryotes and uses Bdellovibrio
as a model for prokaryotic endocytosis. Bdellovibrios are
predatory (or parasitoid) bacteria that feed on E. coli by
penetrating the cell wall of the latter and then removing nutrient
molecules from E. coli while attached to the outer surface of its
plasma membrane. Although it is perfectly obvious that this is
not an example of one prokaryote being engulfed by another
Margulis continually implies that it is.
P.J. Whitfield, review of “Symbiosis in Cell Evolution,” Biological Journal of the
Linnean Society 18 [1982]:77-78; p 78)

©1999 Timothy G. Standish

 Conclusions
Presence of mitochondrial genes in nuclear DNA
reduces the window of time available for
mitochondrial acquisition in eukaryotes.
Understanding the structure of mitochondrial genes
in the nucleus and how they are expressed makes
the transfer of genes from protomitochondria to the
nucleus appear complex.
Differences between mitochondrial genetic codes
and nuclear genetic codes adds to the complexity
of gene transfer between mitochondria and
nucleus.
As molecular data accumulates, the endosymbiotic
origin of mitochondria appears less probable.
©1999 Timothy G. Standish
The
End

©1999 Timothy G. Standish

Glycolysis Generates ATP by
Catabolizing Glucose to Pyruvate
• Glycolysis (or the glycolytic pathway) is a ten-
step reaction sequence that converts one
glucose molecule into two molecules of pyruvate

• Pyruvate is a three-carbon compound

• Both ATP and NADH are produced

© 2012 Pearson Education, Inc.

Figure 9-6

© 2012 Pearson Education, Inc.

Figure 9-7

© 2012 Pearson Education, Inc.

Glycolysis is present in all organisms
• Glycolysis is common to both aerobic and
anaerobic organisms

• In most cells the enzymes for glycolysis are

found in the cytosol

© 2012 Pearson Education, Inc.

Glycolysis in Overview
• In the absence of oxygen glycolysis leads to
fermentation

• In the presence of oxygen glycolysis leads to

aerobic respiration

© 2012 Pearson Education, Inc.

Important features of the glycolytic
pathway are
• The initial input of ATP (Gly-1)

• The sugar splitting reaction in which glucose is

split into two three-carbon molecules

• The oxidative event that generates NADH (Gly-6)

• The two steps at which the reaction sequence is

coupled to ATP generation (Gly-7 and Gly-10)
© 2012 Pearson Education, Inc.
The glycolytic pathway can be divided
into three phases
• Phase I: the preparatory and cleavage steps

• Phase II: the oxidative sequence, which is the

first ATP-generating event

• Phase III: the second ATP-generating event

© 2012 Pearson Education, Inc.

Phase I: Preparation and Cleavage
• The net result of the first three reactions is to
convert glucose into a doubly phosphorylated
molecule (fructose-1,6-bisphosphate)

• The phosphates are transferred to glucose from

ATP

• ATP hydrolysis is also the driving force that

makes the phosphorylation exergonic and thus
irreversible

© 2012 Pearson Education, Inc.

The first reaction adds a phosphate to
the sixth carbon atom
• The bond formed is a phosphodiester bond, a
lower-energy bond than the phosphoanhydride
bonds in ATP

• The enzyme that catalyzes the reaction is

hexokinase, and is specific for phosphorylation of
other six-carbon sugars as well

• Liver cells also have glucokinase, which is specific

for just glucose
© 2012 Pearson Education, Inc.
The second phosphate is added to
carbon one
• The first carbon of glucose is not as easily
phosphorylated as the sixth

• The Gly-2 reaction first converts glucose-6-

phosphate to fructose-6-phosphate (isomerase),
allowing the Gly-3 reaction to add a phosphate to
carbon one

• This reaction is catalyzed by the enzyme

phosphofructokinase-1 (PFK-1)
© 2012 Pearson Education, Inc.
Summary of Gly-1 to Gly-5
• Glucose is split into two 3-C compounds by
aldolase
• The first phase of the glycolytic pathway can
be summarized as

© 2012 Pearson Education, Inc.

Phase 2: Oxidation and ATP Generation
• The net energy yield of phase one is negative

• Two molecules of ATP have been consumed

per molecule of glucose

• In phase 2, ATP production is linked to an

oxidative event, followed by the generation of
ATP in phase 3

© 2012 Pearson Education, Inc.

Gly-6 and Gly-7
• The oxidation of glyceraldehyde-3-phosphate
to 3-phosphoglycerate is highly exergonic,
and drives

– the reduction of NAD+ to NADH (Gly-6)

– the phosphorylation of ADP with inorganic

phosphate, Pi (Gly-7)

© 2012 Pearson Education, Inc.

Important features of Gly-6 and Gly-7
• NAD+ is an electron acceptor

• The oxidation is coupled to the formation of a

high-energy, doubly phosphorylated intermediate,
1,3-bisphosphoglycerate

• ATP generation by transferring a phosphate

group to ADP from a phosphorylated substrate
such as 1,3-bisphosphoglycerate is called
substrate-level phosphorylation

© 2012 Pearson Education, Inc.

Summary of Gly-6 and Gly-7
• Gly-6 and Gly-7 can be summarized as

• Each reaction involving glyceraldehyde-3-

phosphate occurs twice per starting molecule of
glucose

• The two ATPs invested in the first phase are

recovered in the second phase, for no net ATP
© 2012 Pearson Education, Inc.
Phase 3: Pyruvate Formation and
ATP Generation
• The phosphoester bond of 3-phosphoglycerate is
converted to a phosphoenol bond

• The phosphate group is moved to the adjacent

carbon, forming 2-phosphoglycerate (Gly-8)

• Water is removed from the 2-phosphoglycerate by

the enzyme enolase (Gly-9) generating the high-
energy compound phosphoenolpyruvate (PEP)

© 2012 Pearson Education, Inc.

Phosphoenolpyruvate
• Hydrolysis of the phosphoenol bond of PEP is
one of the most exergonic known in biological
systems

• PEP hydrolysis drives ATP synthesis by

transferring a phosphate to ADP, catalyzed by the
enzyme pyruvate kinase (Gly-10)

• The transfer is irreversible in the direction of

pyruvate and ATP formation

© 2012 Pearson Education, Inc.

Summary of Gly-8 to Gly-1
• The third phase of glycolysis can be summarized
as

© 2012 Pearson Education, Inc.

Summary of Glycolysis
• The two molecules of ATP formed in the second
phosphorylation event (Gly-10) represent the net
yield of ATP for the glycolytic pathway

• .

• The pathway is highly exergonic in the direction

of pyruvate formation; G in a cell is typically
–20 kcal/mol

© 2012 Pearson Education, Inc.

Conservation of Glycolysis
• The glycolytic pathway is one of the most
common and highly conserved metabolic
pathways known

• Virtually all cells have the ability to convert

glucose to pyruvate, extracting energy in the
process

• The next steps depend on the availability of

oxygen

© 2012 Pearson Education, Inc.

Alternative Substrates for Glycolysis
• Glucose is a major substrate for both
fermentations and respiration in a variety of
organisms and some tissues

• But for some organisms and tissues, glucose is

not significant at all

• There are a variety of alternatives to glucose,

which are often converted into an intermediate in
the glucose catabolism pathway

© 2012 Pearson Education, Inc.

Other Sugars and Glycerol Are Also
Catabolized by the Glycolytic Pathway
• Many sugars are available to cells, either
monosaccharides or disaccharides that can be
readily hydrolyzed into monosaccharides

• The monosaccharides are then converted into a

glycolytic intermediate

• Glucose and fructose enter most directly after

phosphorylation on carbon atom 6; mannose and
fructose require more steps
© 2012 Pearson Education, Inc.
Pentoses and glycerol can be channeled
into the glycolytic pathway too
• Phosphorylated pentoses can enter the glycolytic
pathway but must first be converted to hexose
phosphates

• Glycerol, a three-carbon molecule resulting from lipid

breakdown, can enter the cycle

© 2012 Pearson Education, Inc.

Figure 9-9

© 2012 Pearson Education, Inc.

Polysaccharides Are Cleaved to Form
Sugar Phosphates That Also Enter the
Glycolytic Pathway
• Glucose occurs primarily in the form of storage
polysaccharides, most often starch in plants and
glycogen in animals

© 2012 Pearson Education, Inc.

Gluconeogenesis
• The process of glucose synthesis is called
gluconeogenesis

• Glucose is synthesized from three- and four-

carbon precursors (noncarbohydrate)

• Pyruvate and lactate are the most common

starting materials
• Occurs in all organisms and in animals mainly
in liver and kidneys

© 2012 Pearson Education, Inc.

Gluconeogenesis and glycolysis
• Gluconeogenesis occurs by simple reversal of
glycolysis using the same enzyme in both
directions

• But not all the steps are simple reversals of

glycolysis: Gly-1, Gly-3, and Gly-10 are
accomplished by other means

• These are the most exergonic reactions of

glycolysis
• Thermodynamically difficult to reverse

© 2012 Pearson Education, Inc.

© 2012 Pearson Education, Inc.
Gluconeogenesis

• Biosynthetic anabolic pathways are seldom

just a reversal of the corresponding catabolic
pathways
• Glucose to pyruvate has G about
-20kcal/mole
• Reverse process would be about
+20kcal/mole and highly endergonic and
thermodynamically impossible

© 2012 Pearson Education, Inc.

Gluconeogenesis

• Gly1, Gly3 and Gly10 do not simply run in

reverse
– Gluconeogenic pathway has bypass
reactions
– Gly1 and Gly3 you have hydrolytic cleaving
that liberated inorganic phosphate instead
of trying to synthesize ATP.
• Exergonic reaction with G= -3.3 kcal/mole

© 2012 Pearson Education, Inc.

Gluconeogenesis

• Gly 10 is bypassed by a two-reaction

sequence
– Both reactions are driven by hydrolysis of a
phosphoanydride bond
– One from ATP and one from GTP
– Carbon dioxide is added to pyruvate in a
carboxylation reaction
– Next, carboxyl group is removed by
decarboxylation to form PEP

© 2012 Pearson Education, Inc.

Glycolytic vs Gluconeogenesis

• Glycolysis is catabolic producing net two

ATPs per glucose
• Gluconeogenesis in anabolic requiring the
equivalent of six ATPs per molecule of
glucose synthesized
• Gluconeogenesis proceed exergonically in the
direction of glucose synthesis

© 2012 Pearson Education, Inc.

What is happening right now to the
sugars you consume?
• Enzymes begin to break down carbohydrates
in the mouth (sucrase, maltase, lactase, etc)
• Glucose, galactose, and fructose are
absorbed by intestinal epithelial cells.- enter
bloodstream
– Galactosemia
Main sugar in blood is glucose. Regulated

© 2012 Pearson Education, Inc.

Cori Cycle

• Skeletal muscle is main source of blood

lactate
• It can function in either the presence or the
absence of oxygen
• Liver is primary site of gluconeogenesis
• Lactate is converted to glucose and released
into the blood-Cori cycle

© 2012 Pearson Education, Inc.

The Regulation of Glycolysis and
Gluconeogenesis
• Cells have enzymes for both glycolysis and
gluconeogenesis, so the processes must be
regulated
• Spatial regulation keeps the two processes
confined to separate places in the body
– Muscle cells – glycolysis Liver- gluconeogenesis

• There is also temporal regulation in which the

two processes take place at different times in
one cell

© 2012 Pearson Education, Inc.

Regulation of Glycolysis and
Gluconeogenesis
• Both are regulated to function at rates that are
responsive to cellular and organismal needs
for their products. ATP or glucose
• Regulated in reverse manner
• Intracellular conditions know to stimulate one
pathway usually have an inhibitory effect on
the other.

© 2012 Pearson Education, Inc.

Key Enzymes in the Glycolytic and
Gluconeogenic Pathways Are Subject to
Alllosteric Regulation
• Allosteric regulation involves the interconversion
of an enzyme between two forms, one catalytically
active and the other inactive

• The enzyme will be active or not depending on

whether an allosteric effector is bound to the
allosteric site

• The effector might be an activator or inhibitor

© 2012 Pearson Education, Inc.
Figure 9-12

© 2012 Pearson Education, Inc.

Key regulatory enzymes of glycolysis
and gluconeogenesis
• For glycolysis the enzymes are hexokinase,
phosphofructokinase-1 (PFK-1), and pyruvate
kinase

• For gluconeogenesis they are fructose-1,6-

bisphosphatase, and pyruvate carboxylase

• Each of the enzymes is unique to its pathway so

the pathways can be regulated independently

© 2012 Pearson Education, Inc.

Glycolysis and gluconeogenesis are
reciprocally regulated
• AMP and acetyl CoA, the two effectors to which
both pathways are sensitive, have opposite
effects
• AMP activates glycolysis and inhibits
gluconeogenesis
– Low ATP and high AMP cell is low on energy.
Activate glycolysis. High ATP inhibit glycolysis.

© 2012 Pearson Education, Inc.

• Acetyl CoA activate gluconeogenesis but
inhibits glycolysis
• High Acetyl CoA has enough energy inhibit
glycolysis

© 2012 Pearson Education, Inc.

Fructose-2,6-Bisphosphate Is an
Important Regulator of Glycolysis and
Gluconeogenesis
• Fructose-2,6-Bisphosphate (F2,6BP) is the most
important regulator of both glycolysis and
gluconeogenesis- ATP phosphorylation of C-2

• Synthesis of F2,6BP is catalyzed by

phosphofructokinase-2 (PFK-2), not PFK-1

• F2,6BP activates the glycolytic enzyme (PFK-1)

that phosphorylates fructose-6-phosphate and it
inhibits FBPase that catalyzes the reverse reaction
© 2012 Pearson Education, Inc.
Figure 9-13

© 2012 Pearson Education, Inc.

Effect of cAMP on F2,6BP
• cAMP affects the F2,6BP concentration in two
ways

• 1. It inactivates the PFK-2 kinase activity

• 2. It stimulates the F2,6BP phosphatase

activity

• These two effects tend to decrease the

concentration of F2,6BP in the cell

© 2012 Pearson Education, Inc.

Effect of cAMP on hormone regulation
• cAMP level in cells is controlled primarily by
the hormones glucagon and epinephrine
(adrenaline)

• These cause an increase in cAMP

concentration, stimulating gluconeogenesis
when more glucose is needed

© 2012 Pearson Education, Inc.

Novel Roles for Glycolytic Enzymes
• Glycolysis is connected to other cell processes
• Hexokinase (Gly-1) is a transcriptional repressor
in yeast cells under high glucose levels
• Mammals have four isoforms of hexokinase
- One is expressed in highly catabolically active
tumor cells
- Another binds to mitochondria and helps
coordinate glycolysis with mitochondrial
functions
• Many other examples exist

© 2012 Pearson Education, Inc.

Additional functions of other glycolytic
enzymes
• Glyceraldehyde-3-phosphate dehydrogenase (Gly
-6) and enolase (Gly-9) have DNA-binding
abilities

• They can act as transcriptional regulators

• They connect the glycolytic pathway with

processes such as cell division and programmed
cell death

© 2012 Pearson Education, Inc.

Cancer connections
• Phosphoglucoisomerase (PGI; Gly-2) is involved
in cell motility and migration during cancer cell
metastasis

• Metastasis: the release of cells from malignant

tumors into the bloodstream; these can form
secondary tumors throughout the body

• PGI stimulates cell proliferation and migration

© 2012 Pearson Education, Inc.

 2. Citric Acid or Krebs Cycle

Hans Krebs
1900-1981

It is not
necessary to
know the
individual steps

- occur ONLY if oxygen is present and the cell has mitochondria.

- In this stage of cellular respiration, the oxidation of glucose to
CO2 is completed. (this is why we exhale carbon dioxide)
Aerobic cells
use a The Citric
metabolic
wheel – the
Acid Cycle
citric acid
cycle – to
generate
energy by
acetyl CoA
oxidation
 Synthesis of
glycogen Glucose Pentose phosphate
pathway

Glycogen Glucose-6- Ribose, NADPH

phosphate

Degradation of
glycogen

Glycolysis Gluconeogenesis

Ethanol Pyruvate Lactate

Fatty Acids Acetyl Co A Amino Acids

The citric acid

cycle is the final
Most fuel
common pathway
for the oxidation molecules
of fuel molecules enter the
— amino acids, cycle as acetyl
fatty acids, and coenzyme A.
carbohydrates.
 Hans Adolf Krebs.
Names: Biochemist; born in
Germany. Worked in Britain.
The Citric Acid His discovery in 1937 of
Cycle the ‘Krebs cycle’ of
chemical reactions was
Tricarboxylic Acid critical to the
understanding of cell
Cycle metabolism and earned
Krebs Cycle him the 1953 Nobel Prize
for Physiology or Medicine.

In eukaryotes
the reactions
of the citric
acid cycle take
place inside
mitochondria
 An Overview of the Citric Acid Cycle
A four-carbon oxaloacetate condenses with a
two-carbon acetyl unit to yield a six-carbon
citrate.
An isomer of citrate is oxidatively
decarboxylated and five-carbon -ketoglutarate
is formed.
-ketoglutarate is oxidatively decarboxylated to
yield a four-carbon succinate.
Oxaloacetate is then regenerated from succinate.
Two carbon atoms (acetyl CoA) enter the cycle
and two carbon atoms leave the cycle in the
form of two molecules of carbon dioxide.
Three hydride ions (six electrons) are transferred
to three molecules of NAD+, one pair of hydrogen
atoms (two electrons) is transferred to one
molecule of FAD.
The function of the citric acid cycle is
the harvesting of high-energy
electrons from acetyl CoA.
 1. Citrate Synthase
• Citrate formed from acetyl CoA and oxaloacetate
• Only cycle reaction with C-C bond formation
• Addition of C2 unit (acetyl) to the keto double bond of C4
acid, oxaloacetate, to produce C6 compound, citrate

citrate synthase
 2. Aconitase
• Elimination of H2O from citrate to form C=C bond of cis
-aconitate
• Stereospecific addition of H2O to cis-aconitate to form
isocitrate

aconitase aconitase
 3. Isocitrate Dehydrogenase
• Oxidative decarboxylation of isocitrate to

a-ketoglutarate (a metabolically irreversible reaction)
• One of four oxidation-reduction reactions of the cycle
• Hydride ion from the C-2 of isocitrate is transferred to NAD+ to
form NADH
• Oxalosuccinate is decarboxylated to a-ketoglutarate

isocitrate dehydrogenase isocitrate dehydrogenase

4. The -Ketoglutarate Dehydrogenase Complex
• Similar to pyruvate dehydrogenase complex
• Same coenzymes, identical mechanisms
E1 - a-ketoglutarate dehydrogenase (with TPP) E2 –
dihydrolipoyl succinyltransferase (with flexible lipoamide
prosthetic group) E3 -
dihydrolipoyl dehydrogenase (with FAD)

-ketoglutarate
dehydrogenase
 5. Succinyl-CoA Synthetase
• Free energy in thioester bond of succinyl CoA is
conserved as GTP or ATP in higher animals (or ATP in
plants, some bacteria)
• Substrate level phosphorylation reaction

+ HS-
Succinyl-CoA
Synthetase

GTP + ADP GDP + ATP

6. The Succinate Dehydrogenase Complex
• Complex of several polypeptides, an FAD prosthetic group and iron-
sulfur clusters
• Embedded in the inner mitochondrial membrane
• Electrons are transferred from succinate to FAD and then to
ubiquinone (Q) in electron transport chain
• Dehydrogenation is stereospecific; only the trans isomer is formed

Succinate
Dehydrogenase
 7. Fumarase
• Stereospecific trans addition of water to the double
bond of fumarate to form L-malate
• Only the L isomer of malate is formed

Fumarase
 8. Malate Dehydrogenase
Malate is oxidized to form oxaloacetate.

Malate
Dehydrogenase
Stoichiometry of the Citric Acid Cycle
 Functions of the Citric Acid Cycle

• Integration of metabolism. The citric acid cycle is

amphibolic (both catabolic and anabolic).
The cycle is involved in Intermediates of the cycle
the aerobic catabolism of are starting points for many
carbohydrates, lipids and anabolic reactions.
amino acids.

• Yields energy in the form of GTP (ATP).

• Yields reducing power in the form of NADH2 and

FADH2.
Regulation of the Citric Acid Cycle
• Pathway controlled by:
(1) Allosteric modulators
(2) Covalent modification of cycle enzymes
(3) Supply of acetyl CoA (pyruvate dehydrogenase complex)

Three enzymes have regulatory properties

- citrate synthase (is allosterically inhibited by NADH, ATP, succinyl
CoA, citrate – feedback inhibition)
- isocitrate dehydrogenase (allosteric
effectors: (+) ADP; (-) NADH, ATP. Bacterial ICDH can be covalently
modified by kinase/phosphatase)
--ketoglutarate dehydrogenase complex (inhibition by ATP, succinyl
CoA and NADH
Regulation of the citric acid cycle

- NADH, ATP, succinyl

CoA, citrate
Krebs Cycle is a Source of Biosynthetic Precursors
Glucose

Phosphoenol- The citric acid cycle

pyruvate
provides intermediates
for biosyntheses
 • Pre Krebs: The pyruvic
acid (C3) loses a C to
CO2 and use a H + to
form NADH and
becomes Acetyl Co-A
(C2).

Simple put: Pyruvate is converted

to Acetly CoA
– releases 2 CO2
– reduces 2 NAD  2
NADH (moves e-)
– produces 2 acetyl CoA
• Acetyl CoA enters
Krebs cycle
 Krebs Cycle Overview

Citric acid

Aerobic
Occurs in the
matrix (inner
compartment)

• Products
(per glucose)
• 2 ATP
• 6 CO2
• 8 NADH
• 2 FADH2. These energy carriers now enter the electron
transport chain (ETC).
 Count the C & electron carriers!
CO2
pyruvate acetyl CoA
3C 2C
NADH
citrate
NADH 4C 6C

4C reduction

6C
This happens of electron

twice for each carriers CO2
glucose
molecule NADH
4C x2 5C

FADH2 CO2
4C 4C
ATP NADH
ELECTRON TRANSPORT CHAIN


1. Electron transport chains (also called
electron transfer pathways- ETP) are
biochemical reactions that produce ATP,
which is the energy currency of life.

2. An ETP is a series of linked membrane-

embedded electron carrier molecules that
transfer electrons, during a regulated
process of redox reactions, from one
electron carrier molecule to another
releasing and capturing energy for cellular
work.
ETC contd.
3. Only two sources of energy are available to
living organisms:
oxidation-reduction (redox) reactions to
produce ATP in chemotrophs
and using sunlight (photosynthesis) for
energy production in phototrophs.

4. Both chemotrophs and phototrophs utilize

electron transport chains to convert energy
into ATP.
 REDOX REACTIONS
• Cells generate energy by using redox reactions

• Redox reactions are chemical reactions in which

electrons are transferred from a oxidised donor molecule
(reducing agent) e.g. CH3 COOH (acetic acid) to an
electron-deficient acceptor molecule (oxidising agent) e.g.
CH3 CH2OH (ethyl alcohol).

• The two processes always occur simultaneously.

• The reducing agent that donates their electrons becomes

oxidised and the oxidising agent that that accept
electrons become reduced.
REDOX contd.
• When electrons are transferred energy is lost and
cells can capture this energy to do cellular work
such as synthesis of ATP – the energy carrier
molecule that directly supplies energy used to
maintain highly organised cellular structures and
function.

• The underlying force driving these reactions is the

Gibbs free energy of the reactants and products.

• The Gibbs free energy is the energy available (“free”)

to do work. Any reaction that decreases the overall
Gibbs free energy of a system will proceed
spontaneously
 SOME BASIC DEFINITIONS
In an electron transfer reaction, an
element undergoing oxidation loses
electrons, whereas an element gaining
Electrons undergoes reduction.

Remember the formula: OIL RIG where

OIL refers to Oxidation Is Loss and RIG
Reduction Is Gain in electrons.
Basic concepts
When you take hydrogen ions or electrons
away from a molecule, you “oxidize” that
molecule.

When you give hydrogen ions or electrons to a

molecule, you “reduce” that molecule.

When you give phosphate molecules to a

molecule, you “phosphorylate” that
molecule.
Oxidation-reactions

• Reactions of metals or any other organic

compounds with oxygen to give oxides are
labelled as oxidation.

• In other words, oxidation is addition of

oxygen to a compound or removal of
hydrogen from a compound

• Oxidation increases C-O bonds.

Reduction reactions
• The removal of oxygen from metal oxides to give the
metals in their elemental forms is labeled as
reduction.

• Whereas, reduction is addition of hydrogen or

removal of oxygen; it increases C-H bonds.

• The more reduced is a molecule, the more H+ atoms

and more energy it contains.

• Fatty acids have more hydrogen atoms than sugars;

hence yield more energy when oxidised.
 COMPONENTS OF ETC/ETP

• MITOCHONDRIA
• ELECTRONS AND PROTONS
• OXYGEN
• NAD+/NADH (redox coenzymes)
• FLAVIN NUCLEOTIDES (redox
coenzymes)
• COENZYME Q (UBIQUINONE)
• IRON-SULFUR PROTEINS
 BIOENERGETICS
The study of bioenergetics involves the processes
which reduce nicitinamides and flavin nucleotides,
generated from oxidation of carbohydrates and
lipids by molecular oxygen via mitochondrial
electron-transport chain (ETC) and the mechanism
(oxidative phosphorylation) where oxidation is
coupled to ATP synthesis.

Oxidative phosphorylation is central to metabolism

because the free energy of hydrolysis of the ATP
generated is used in the synthesis of biomolecules
and biological activities such as muscle contraction
and transmission of nerve impulses.
 ATP synthase & ETC
• ATP is made by an enzyme called ATP synthase.
The structure of this enzyme and its underlying
genetic code is remarkably similar in all known
forms of life.
• ATP synthase is powered by a transmembrane
electrochemical potential gradient, usually in the
form of a proton gradient.
• The function of the electron transport chain is to
produce this gradient.
• In all living organisms, a series of redox reactions
is used to produce a transmembrane
electrochemical potential gradient.
 UNDERSTANDING THE ELECTRON TRANSFER CHAIN

 The transfer of electrons from a high-energy molecule (the donor) to a

lower-energy molecule (the acceptor) can be spatially separated into a
series of intermediate redox reactions. This is an electron transport
chain.

 Electron transport chains produce energy in the form of a

transmembrane electrochemical potential gradient. This energy is
used to do useful work. The gradient can be used to transport
molecules across membranes. It can be used to produce ATP and
NADH, high-energy molecules that are necessary for growth.

 A small amount of ATP is available from substrate-level

phosphorylation (for example, in glycolysis).

 Some organisms can obtain ATP exclusively by fermentation. In most

organisms, however, the majority of ATP is generated by electron
transport chains.
 ETC IN MITOCHONDRIA
• The cells of all eukaryotes (all animals, plants, fungi,
algae – in other words, all living things except bacteria
and archaea) contain intracellular organelles called
mitochondria that produce ATP.
• Energy sources such as glucose are initially metabolized
in the cytoplasm. The products are imported into
mitochondria.
• Mitochondria continue the process of catabolism using
metabolic pathways including the Krebs cycle, fatty acid
oxidation and amino acid oxidation.
• The end result of these pathways is the production of two
energy-rich electron donors, NADH and FADH2.
ETC IN MITOCHONDRIA- contd.
• Electrons from these donors are passed
through an electron transport chain to oxygen,
which is reduced to water. This is a multi-
step redox process that occurs on the
mitochondrial inner membrane.
• The enzymes that catalyze these reactions
have the remarkable ability to simultaneously
create a proton gradient across the
membrane, producing a thermodynamically
unlikely high-energy state with the potential
to do work
CHEMIOSMOTIC THEORY
• Peter Mitchell, a Britiah biochemist, in 1961,
proposed a mechanism by which the free energy
generated during electron transport drives ATP
synthesis

• As electrons pass through the ETC, protons ae

transported from the matrix and released into the
inter membrane space

• As a result, an electrical potential and proton

gradient (pH) arise across the inner membrane and
this elecrochemical proton gradient is often referred
as protonmotive force
CHEMIOSMO THEORY contd.
• Protons, present in the intermembrane in
excess can pass through the inner
membrane and back into the matrix down
their concentration gradient only through
special channels as the inner membrane is
impermeable to ions (protons)

• As the themodynamically favorable flow of

protons occur through a channel, each of
which contains an ATP synthase activity, an
ATP synthesis occurs.
Chemiosmo theory contd.
Uncouplers:
A variety of molecules such as, dinitrophenol (DNP) and
gramicidin can collapse the proton gradient by equalising the
proton concentration on both sides of the membrane and
according to the chemiosmotic theory, a disrupted proton
gradient dissipates the energy (derived from food) as heat
OXIDATIVE STRESS
• Oxygen can accept single electrons to form
unstable derivatives, referred as reactive
oxygen species (ROS) e.g. Hydrogen
peroxide
• Because ROS are so reactive, they can
seriously damage living cells if formed in
significant ammounts
• They can be kept to the minimum by
antioxidants e.g. Alpha-tocopherol (Vitamin E
), beta-Carotene (Vitamin A) that inhibit the
reaction of molecules with oxygen radicals.
 MITOCHONDRIAL REDOX CARRIERS
• Four membrane-bound complexes have been
identified in mitochondria.
• Each is an extremely complex transmembrane
structure that is embedded in the inner
membrane.
• Three of them are proton pumps. The structures
are electrically connected by lipid-soluble
electron carriers and water-soluble electron
carriers
 PATHWAYS (COMPLEXES) OF ETC

The overall electron transport chain is:

NADH → Complex I → Q → Complex III →cytochrome c →
Complex IV → O2 ↑
Complex II
Complex I (NADH dehydrogenase) removes two electrons from
NADH and transfers them to a lipid-soluble carrier, ubiquinone
(Q). The reduced product, ubiquinol (QH2) is free to diffuse
within the membrane. At the same time, Complex I moves four
protons (H+) across the membrane, producing a proton gradient.
Complex I is also called NADH:ubiquinone oxidoreductase.
 COMPLEX I

1. NADH (nicotinamide adenine dinucleotide

reduced form) is oxidized to NAD+, reducing FMN
(flavin mononucleotide) to FMNH2 in one two-
electron site
2. The next electron carrier is a Fe-S cluster, which
can only accept one electron at a time to reduce
the ferric ion into a ferrous ion.
3. Conveniently, FMNH2 can only be oxidized in
two one-electron steps, through a semiquinone
intermediate.
COMPEX I contd.
4. The electron thus travels from the FMNH2 to the
Fe-S cluster, then from the Fe-S cluster to the
oxidized Q to give the free-radical (semiquinone)
form of Q.
5. This happens again to reduce the semiquinone
form to the ubiquinol form, QH2. During this
process, four protons are translocated across the
inner mitochondrial membrane, from the matrix
to the intermembrane space
6. This creates a proton gradient that will be later
used to generate ATP through oxidative
phosphorylation.
 COMPLEX II
• Complex II (succinate dehydrogenase) is not a proton pump. It
serves to funnel additional electrons into the quinone pool (Q)
by removing electrons from succinate and transferring them (via
FAD) to Q.

• Other electron donors (e.g. fatty acids and glycerol 3-phosphate)

also funnel electrons into Q (via FAD), again without producing a
proton gradient.
 COMPLEX III
• Complex III (cytochrome bc1 complex) removes in a
stepwise fashion two electrons from QH2 and
transfers them to two molecules of cytochrome c, a
water-soluble electron carrier located on the outer
surface of the membrane.

• At the same time, it moves four protons across the

membrane, producing a proton gradient.

• When electron transfer is hindered (by a high

membrane potential, point mutations or respiratory
inhibitors such as antimycin A), Complex III may leak
electrons to oxygen resulting in the formation of a
superoxide.
COMPLEX IV
Complex IV (cytochrome c oxidase) removes four electrons
from four molecules of cytochrome c and transfers them to
molecular oxygen (O2), producing two molecules of water
(H2O). At the same time, it moves four protons across the
membrane, producing a proton gradient.
PROTON PUMP MECHNISMS
• There are three proton pumps: I, III and IV.
The resulting transmembrane proton gradient
is used to make ATP via ATP synthase.
• The trans membrane electrochemical
gradient acts as the intermediate in the
transfer of energy to ATP.
• Oxidation of NADH results in the production
of about 3 molecules per O reduced to water.
• Oxidation of FADH2 yields 2 ATP’s.
 WHAT IS OXIDATIVE
PHOSPHORYLATION?
When you give phosphate molecules to a
molecule, you “phosphorylate” that
molecule.

So, oxidative phosphorylation (very simply)

means the process that couples the
removal of hydrogen ions from one
molecule and giving phosphate molecules
to another molecule.
 MITOCHONDRIA IN OXIDATIVE
PHOSPHORYLATION
Why do we need mitochondria?

The whole idea behind this process is to

get as much ATP out of glucose (or other
food products) as possible. If we have no
oxygen, we get only 4 molecules of ATP
energy packets for each glucose molecule
(in glycolysis).

However, if we have oxygen, then we get

to run the Kreb’s cycle to produce many
more hydrogen ions that can run those ATP
pumps.
 WHY MITOCHONDRIA?
From the Kreb’s cycle we get 24-28 ATP
molecules out of one molecule of glucose
converted to pyruvate (plus the 4
molecules we got out of glycolysis).

So, you can see how much more energy we

can get out of a molecule of glucose if our
mitochondria are working and if we have
oxygen.
ATP synthase complex
• This complex (sometimes termed as COMPLEX V of ETC) is
found in all energy transducing membranes including that of the
mitochondria.
• It contains a proton transport channel, the only way for the
protons to reenter the mitochondrial matrix.
• The energy proton potential gradient is used in the synthesis of
ATP from ADP and Pi
ETC ON CRISTAE
 You've seen this before in
photosynthesis.
Products:
Animation of the ETC McGraw Hill
Animation 34 ATP