THE NUCLEUS

The nucleus is separated from the cytoplasm by an envelope consisting of two membranes.
All chromosomal DNA is held in nucleus, packed into chromatin fibers by it's association
with an equal mass of histone proteins. The nuclear contents communicate with the cytosol
by means of openings in the nuclear envelope called nuclear pores.

Nuclear pores
Nucleoplasm

Nuclear envelope

Nucleolus
Chromatin

Chromatin can condense to form chromosomes. More than one chromosome is present
in eukaryotic cells.
 Basic phospholipid structure

PA=phophatidic acid; PE=phosphatidylethanolamine;

PC =phosphatidylcholine; PG=phosphatidylglycerol
R1,R2 =different fatty acids side chains
 PLASMA MEMBRANE
It is a thin, flexible, phospholipid bilayer with proteins and is about 9 nm in thickness.
It is porous, semipermeable and the limiting membrane.
Membrane lipids comprise the matrix to give form and structure. They
contain the 'amphipathic lipids' that include phospholipids and glycolipids.These lipids
are insoluble in aqueous system.
O

R1 C O CH2

R2 C O CH O

O C O P O CH2 N(CH3)3

O
Tail groups Head Group
(hydrophobic) (hydrophilic function)

Various structures of amphipathic lipids in aqueous environment

Air Monolayer Micelle Liposome Lipid bilayer

Water

Water
Water
Aqueous phase

Encapsulated aqueous phase

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 Plasma membrane protein are of two types

Peripheral or extrinsic proteins Integral or intrinsic proteins

They are tightly bound, penetrate deeply
Weakly bound by ionic bonds to lipid polar
and are not easily displaced.They are func-
heads. Displaced easily.
tional proteins.
e.g-- Cyt C, - lactalbumin
e.g-- Transport carriers, drug receptors, Virus
& hormone receptors, antigens, membrane-
bound enzymes. e.g-- cyt.P450

In 1972 SINGER AND NICOLSON proposed the " FLUID-MOSAIC MODEL" for the membrane proteins.

OUTSIDE
Glycosyl
amino Glycoproteins
glycans
Extrinsic protein

Intrinsic protein

INSIDE

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Functions of the oligosaccharides possibly are receptor sites for hormones. They
define cell type specificity e.g human blood types and possibly define adhesion of
like cells to each other in tissues.

TRANSPORT PROCESSES ACROSS CELL MEMBRANE

Active transport
Passive transpoet
Facilitated diffusion
Group translocation.

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 LYSOSOMES

They are membrane bound vesicles that

0.2-0.5m
contain hydrolytic enzymes involved in
intracellular digestion.

They are spherical bodies, about 0.25 to 0.5m in diameter. They were discovered by De Duve
in 1955. It is a large organelle containing about 30-40 hydrolytic enzymes which are charac-
terised by having acid pH optima. Acidphosphatase is a marker enzyme for this organelle.

Enzyme groups in Lysosomes

Ribonucleases Cathepsins ( proteinase) Deoxyribonucleases

Acid glycosidases Acid phosphatases Sulfatases
Lipases Phospholipases

The median value for pH optima of these enzymes is about pH 5. Lysosomes

are referred to as " suicide bags".

Autophagic process ( self digestion)- Cellular organelles like mitochondria or endoplasmic reticulum
are digested within lysosomes. Lysosomes are active during cell death and cause autolysis ( self
breakdown). e.g - during the metamorphosis of tadpoles to frogs, regression of tadpoles tails is by
lysosomal digestion of tail cells ; Bacteria are digested by lysosomes of white blood cells ; digestion
of acrosome at head of sperm during penetration of the ovum by the sperm etc.
Certain hereditary metabolic diseases e.g TAY-SACHS disease where complex lipids and polysac-
charides accumulate in abnormal amounts are due to defective lysosome actions.
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 MITOCHONDRIA

Mitochondria are the power house of all eukaryiotic cells.They are rod shaped and slightly bent and are
~2-3m long. They are missing in prokaryotes and are the sole site of oxidative phosphorylation in the
cell.They are nearly 800-1000 numbers per cell in liver.

Mitochondria is a double membrane bound

inner membrane folded into cristae
system. The inner membrane by invagination
0.5m
(i.e folding) extends into the matrix & forms
Cristae. All the enzymes of the electron trans-
port system viz. Flavoproteins, Succinic
dehydrogenase, cyt b, c,c1, a and a3 are buried
in the cristae. Inner membrane has knob like
projecting particles called Inner membrane particles.
These (85 A dia) have a coupling factor F1 that has
the matrix space contains a concentrated
solution of many different enzymes. ATPase activity, and hydrophobic channel trans-
versing the inner membrane cd.Fo that connects to F1.
ATPase is the final step in oxidative phosphorylation
catalysing-
ADP + Pi ATP + H2O.
In the matrix, apart from the large no. of soluble enzymes, there are circular double stranded, histone
free mitochondrial DNA about 2-6 in number. Further 70S ribosomes, mitochondrial t-RNA, mRNA &
protein synthesising enzymes are also found. Therefore about 10% of mitochondrial proteins are
synthesised here, while 90% comes from nuclear genes. All these suggest a Prokaryotic origin for
mitochondria ( and also for chloroplasts)

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 Mitochondria

Outer membrane
cytoplasm inside called as matrix
or mitochondrioplasm.

Cristae
Enlarged Inner membrane invaginates to
form cristae.

Inner membrane particles consisting of

Fo and F1.
F1
Fo
Intermembrane space
Outer membrane
Cross section of mitochondria

Mitochondria are sites for :

Electron transport chain
Fatty acid oxidation
Amino acid catabolism
Citric acid cycle.

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 Prokaryotic Origin Theory
Mitochondria and chloroplasts probably evolved from endosymbiotic bacteria.
Ancestral anaerobe --- Anaerobic metabolism is inefficient because fuel is not
completely oxidised. It generates only 2 ATPs per mole
of glucose oxidised.
Aerobic metabolism is efficient
because fuel is oxidised to CO2.
It generates 36 ATP molecules per
mole of glucose oxidised.
Eukaryote Aerobic bacterium

The endosymbiont & host cell share

materials to the advantage of both.

Cyanobacterium uses the energy

of light to synthesize cellular struc-
cyanobacterium tures and fuels from CO2 & H2O.
Host cell with aerobic endosymboint

Symbionts allow further specialization

Cell wall of photosynthetic membranes.Cells
oxidizes fuel efficiently and can obtain
nucleus energy from sunlight.
Mitochondria
Modern photosynthetic eukaryotic cell

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 Lines of evidences

1. Mitochondria and chloroplasts derived from pre-existing mitochondria and chloroplasts,

unlike lysosomes, peroxisomes etc which are derived by simple fission just like bacteria.
Therefore semiautonomous existance.

2. Mitochondrial DNA and chloroplast DNA present for protein synthesis and sequence
similarity with bacterial DNA.

3. 70S ribosomes present similar to bacteria.

4. Enzymes catalysing protein synthesis in these organelles resemble bacterial enzymes.

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 GOLGI BODIES

It is a system of stacked, smooth membrane- bound, flattened sacs ( cisternae) which forms the
golgi complex. It is involved in modifying, sorting and packaging of macromolecules (like proteins)
for the secretion across the cell membrane or for the delivery to other organelles. e.g-- Insulin proteins
have specific signal peptides which caryy them to their destinations( i.e transit peptides).

Around the golgi apparatus there are numerous small membrane-bound vesicles
( 50 nm and larger) that carry materials between the golgi apparatus and different compartments
of the cell.

Lumen
Vesicle

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 ENDOPLASMIC RETICULUM

They are flattened sheets, sacs and tubes of membrane that extend throughout
the cytoplasm of eukaryotic cells, enclosing a large intra-cellular space. The ER
membrane is in structural continuity with the outer membrane of the nuclear
envelope. It specializes in the synthesis and transport of lipids and membrane proteins.

The rough endoplasmic reticulum

generally occurs as flattened sheets
and is studded on its outer face with
ribosomes engaged in protein synthesis.
Nucleus
Lumen Ribosomes

The smooth endoplasmic reticulum is

generally more tubular and lacks attach-
ed ribosomes. It has a major function in
lipid metabolism.

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 PLASMA MEMBRANE

It is the outer boundary of the cell. It is a continuous sheet of phospholipid molecules about
4-5 nm thick in which various proteins are embedded.

Extracellular space
Lipid
bilayer

Protein Channel
Cytoplasm protein

Some of these proteins serve as pumps and channels for transporting specific molecules into
and out of the cell.

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 PEROXISOMES

They are membrane bound vesicles containing

0.2-0.5m oxidative enzymes that generate and destroy
hydrogen peroxide.

These microbodies (peroxisomes in animal cells) are single membrane enclosed cell
organelles. They contain H2O2 producing catalase & oxidases. It is about 0.5m in
diameter. Liver peroxisomes are active in -oxidation of long chain fatty acids (C16,
C18). It is the site of amino acid oxidase action & in plants it is also the site of glyoxylate
cycle reactions. It's size is about 0.5m in diameter.

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 RIBOSOMES

They are nucleo-protein complexes containing about 60% nucleic acid and 40% protein.They
are 15-20nm spherical bodies and take part in protein synthesis. They are closely associated
with endoplasmic reticulum.Ribosomes differ in size. They are 70S in prokaryotes and 80S in
eukaryotes. In prokaryotes, a number of ribosomes associate with mRNA to form Polyribosome.

m-RNA

Ribosomes.

The 70S ribosome in prokaryotes consists of 2 subunits--- 30S and 50S.

The 80S ribosomes in eukaryotes consists of 2 subunits--- 40S and 60S.

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 CYTOSKELETON

Fine structures called microtubules, microfilaments and microtrabaculae form complex

network.
Microtubules are long unbranched slender cylindrical structures ~ 25 nm in
diameter. They are made of protein tubulin ( mw 50000). They play a role in spindle structure
formation during mitosis and meiosis and provide the internal structure to the cell.

Microfilaments are more slender cylindrical structures composed of contractile protein actin
and plays a role in the generation of forces for internal cell motion.

Microtrabecculae are fragile tubes forming transient network in cytosol. They probably have
a role in formimng attachment sites for multienzyme complexes.

Cytosol is a viscous gel where the above form a complex network of linear filaments,
cylinders and tubes. The interaction with other organelles need further study.
 Biochemical Compartmentalisation in cells.

Cytoplasm
Glycolysis. Some run Gluconeogenesis(synthesis of glycogen).
Phosphogluconate pathway. Fatty acid synthesis.
Activation of amino acids --- during protein synthesis.

Mitochondria
Citric acid cycle. Fatty acid oxidation.
Amino acid oxidation ( catabolism).

Nucleus
DNA synthesis.
RNA synthesis.
Lysosomes ( enzymes are active at acid pH and not at neutral pH of cytosol)
Hydrolysis ( breakdown) of DNA. Hydrolysis of RNA.
Hydrolysis of proteins. Hydrolysis of polysaccharides.
Hydrolysis of lipids and phosphatases.

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Endoplasmic Reticulum ( SER and RER)

Lipid synthesis Protein synthesis ( in RER)

Steroid synthesis Detoxification and phospholipid synthesis (in SER)

Peroxisomes
H2O2 producing and removing reactions
Enzyme catalase
Peroxidases

Chloroplasts
Chlorophyll photosynthesis and CO2 fixation.

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 Size and density of some typical
organelles*
----------------------------------
Organelle diameter Density
(µm) (g/cm3)
----------------------------------
Nuclei 5-10 1.4
Mitochondria 1-2 1.1
Lysosomes 1-2 1.1
----------------------------------

http://www.eng.umd.edu/~nsw/ench485/lab10.htm
 ORGANELLES CAN BE ISOLATED BY CENTRIFUGATION
(A) Differential velocity centrifugation:
Settle at different velocities due to difference in size.
Tissue homogenisation or sonication to break open cells in 0.2M sucrose at 40C (ice cold).
(isotonic osmotic pressure avoids bursting of sub-cellular organelles)
Strain through fine gauze
Tissue homogenate
Low speed ~ 3000rpm or 600g, for 3 mins.

Supernatant
Pellet
Unbroken whole cells, nuclei Medium speed~ 20,000g, 20 mins
cytoskeleton, large size plasma
fragments.
Pellet Supernatant
Mitochondria,lysosomes, High speed ~ 80,000g, 1 hr
Peroxisomes, chloroplasts

Pellet Supernatant
Microsomes Very high speed
Ribosomes plus small endoplasmic ~ 1,50,000g, 3 hrs
reticulum membrane fraction.

Pellet Supernatant
Contains "true soluble proteins"
Ribosomes, viruses,large
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of cytoplasm free of anymembra-
ne fragments. viz ' cytosol'
 RATE -ZONAL CENTRIFUGATION
Organelles move according to their size, mass, centrifugal force, difference between density
and friction.
Samples are layered on centrifuge tube containing a low concentration of sucrose solution.
Sucrose solution serves to prevent convection mixing and not for density mixing.
On completion different sized organelles are found in different zones of the centrifuge tube.
If centrifuged for too long, all organelles will end up in pellet at the bottom of the tube.

MARKER ENZYMES
Purity of different organelles can be assesed by the following criterias---
1. Morphology -- microscopic.
2. Assay particular enzyme which is characteristic of the organelle and not found
elsewhere in the cell.
e.g-- Catalase is a good marker for peroxisomes.
Succinate dehydrogenase for mitochondria.
Cathepsin C or acid phosphatase for lysosomes.
Alkaline phosphatase for plasma membrane.

The good indication of the purity/ degree of contamination of any

organelle preparation is ascertained by measuring the activity in these isolated fractions.

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 EQUILIBRIUM DENSITY - GRADIENT CENTRIFUGATION
Isopycnic centrifution -- Organelles of different boyant density ( result of different ratios
of lipids and proteins in each type of organelle) are separated on a density gradient due
to density differences.
A centrifuge tube is filled with a solution, the density of which increases from top to bottom.
Sucrose of different concentrations is layered. When mixture of organelles is layered on top
of the density gradient and centrifuged at high speed, individual organelles sediment until
their bouyant density matches that in the gradient. Each layer is collected separately by
puncturing tube.
Biochemists can obtain purified organelles for study of their role e.g -- lysosomes contain
degradative enzymes, mitochondria contain oxidative enzymes and chloroplasts contain
photosynthetic pigments.
More dense Cesium chloride is used to separate DNA, RNA and proteins also in this way.

High speed centrifugation Less dense

160,000g, 3 hrs component
More dense
component

Tube No 7 6 5 4 3 2 1
Lysoso- Mitochon- Peroxi-
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Types of centrifuges
Clinical centrifuge – a low speed centrifuge going upto 3000 to
5000 rpm

Refrigerated laboratory centrifuge – going up to 15000 to

40,000rpm and having a refrigerated compartment for the rotors.

Ultracentrifuge = A high-speed centrifuge (up to 100,000 rpm)

by means of which large molecules, e.g., of protein or nucleic
acids, are caused to sediment at practicable rates; used for
determinations of molecular weights, separation of large
molecules, criteria of homogeneity of large molecules,
conformational studies, etc. The high speeds are obtained by
making the rotors lightweight by using alloys of titanium and
creating vacuum in the chamber to avoid frictional resistance due
to presence of air
 Ultracentrifugation
The homogenised sample can be centrifuged in an ultracentrifuge. An
ultracentrifuge consists of a refrigerated, evacuated chamber containing
a rotor which is driven by an electrical motor capable of high speed
rotation. Samples are placed in tubes within or attached to the rotor.
Rotational speed may reach up to 100,000 rpm for floor model, 150,000
rpm for bench-top model (Beckman Optima Max-XP), creating
centrifugal speed forces of 800,000g to 1,000,000g. This force causes
sedimentation of macromolecules, and can even cause non-uniform
distributions of small molecules.
Sedimentation depends on mass, shape, and partial specific volume of a
macromolecule, as well as solvent density, rotor size and rate of
rotation. The sedimentation velocity can be monitored during the
experiment to calculate molecular weight. Values of sedimentation
coefficient (S) can be calculated. Large values of S (faster sedimentation
rate) correspond to larger molecular weight. Dense particle sediments
more rapidly. Elongated proteins have larger frictional coefficients, and
sediment more slowly to ensure accuracy.