3. • Cells contain various organized structures, collectively called as cell
organelles.
• When the cell membrane is disrupted, either by mechanical means or
by lysing the membrane by Tween-20 (a lipid solvent) the organized
particles inside the cell are homogenized.
• This is usually carried out in 0.25M sucrose at pH 7.4.
• The organelles could then be separated by applying differential
centrifugal forces.
• Albert Claude got Nobel prize in 1974 for fractionating subcellular
organelles
4. • Some enzymes are present in
certain organelles only, such
specific enzymes are called as
marker enzymes.
• After centrifugation, the
separated organelles are
identified by detection of marker
enzymes in the sample.
5. Discuss about the following cell organelles
1) Nucleus
2) Endoplasmic reticulum
3) Golgi apparatus
4) Lysosomes
5) peroxisome
6) Mitochondria
7) Plasma membrane
6. 1) NUCLEUS
• It is the most prominent organelle of the cell. All cells in the body
contain nucleus, except mature RBCs in circulation. The uppermost
layer of skin also may not possess a readily identifiable nucleus. In
some cells, nucleus occupies most of the available space, e.g. small
lymphocytes and spermatozoa.
• Nucleus is surrounded by two membranes—the inner one is called
perinuclear membrane with numerous pores and the outer
membrane is continuous with membrane of endoplasmic reticulum.
• Nucleus contains the DNA, the chemical basis of genes, which
governs all the functions of the cell. The very long DNA molecules are
complexed with proteins to form chromatin and are further organized
into chromosomes..
7. DNA replication and RNA synthesis (transcription) are taking place
inside the nucleus
In some cells, a portion of the nucleus may be seen as lighter shaded
area nucleolus.This is the area for RNA processing and ribosome
synthesis. The nucleolus is very prominent in cells actively
synthesizing proteins.
Vesicular transport across membrane is by endocytosis and
exocytosis. Importin and exportin proteins are involved, and it is
helped by RanGAP proteins.
8. 2) ENDOPLASMIC RETICULUM
It is a network of interconnecting membranes enclosing channels or
cisternae, that are continuous from outer nuclear envelope to outer
plasma membrane.
Under electron microscope, the reticular arrangements will have railway
track appearance.
This will be very prominent in cells actively synthesizing proteins
e.g. immunoglobulin secreting plasma cells.
The proteins, glycoproteins and lipoproteins are synthesised in the ER.
Detoxification of various drugs is an important function of ER. Microsomal
cytochrome P-450 hydroxylates drugs, such as benzpyrine, aminopyrine,
aniline, morphine, phenobarbitone, etc.
9. • ER is generally classified into rough and smooth. The rough
appearance is due to ribosomes attached to cytoplasmic side of
membrane where the proteins are being synthesized.
• When cells are fractionated, the complex ER is disrupted in many
places. They are automatically reassembled to form microsomes.
• ERGIC (Endoplasmic reticulum - Golgi intermediate compartment):
The synthesized protein pass through this compartment before going
to the cis Golgi.
10.
11. 3) GOLGI APPARATUS
The Golgi organelle is a
network of flattened smooth
membranes and vesicles. It may
be considered as the converging
area of endoplasmic reticulum
While moving through ER,
carbohydrate groups are
successively added to the
nascent proteins. These
glycoproteins reach the Golgi
area.
12. Golgi apparatus is composed of cis, medial and trans cisternae.
Glycoproteins are generally transported from ER to cis Golgi (proximal
cisterna) then to medial Golgi (intermediate cisterna) and finally to trans
Golgi (distal cisterna) for temporary storage. Trans Golgi is particularly
abundant with vesicles containing glycoproteins.
Newly synthesized proteins are sorted first according to the sorting
signals available in the proteins.
Then they are packaged into transport vesicles having different types
of coat proteins. Finally they are transported into various destinations.
Main function of Golgi apparatus is protein sorting, packaging and
secretion
13. 4) LYSOSOMES
• Lysosomes are tiny organelles
• They are bags of enzymes.
• Endocytic vesicles and phagosomes are fused with lysosome (primary) to form the
secondary lysosome or digestive vacuole.
• Foreign particles are progressively digested inside these vacuoles. Completely hydrolyzed
products are utilized by the cell. As long as the lysosomal membrane is intact, the
encapsulated enzymes can act only locally.
• But when the membrane is disrupted, the released enzymes can hydrolyze external
substrates, leading to tissue damage.
• The lysosomal enzymes have an optimum pH around 5.
• Polysaccharide hydrolyzing enzymes (alpha-glucosidase, alpha-fucosidase, beta-
galactosidase, alpha-mannosidase, betaglucuronidase, hyaluronidase, aryl sulfatase,
lysozyme)
• Protein hydrolyzing enzymes (cathepsins, collagenase, elastase, peptidases)
• Nucleic acid hydrolyzing enzymes (ribonuclease, deoxyribonuclease)
• Lipid hydrolyzing enzymes (fatty acyl esterase, phospholipases)..
14. 5) PEROXISOMES
• The peroxisomes have a granular matrix.
• They are of 0.3–1.5 mm in diameter.
• They contain peroxidases and catalase. They are prominent in
leukocytes and platelets.
• Peroxidation of polyunsaturated fatty acids in vivo may lead to
hydroperoxide formation.
• The free radicals damage molecules, cell membranes, tissues and
genes.
• Catalase and peroxidase are the enzymes present in peroxisomes,
which will destroy the unwanted peroxides and other free radicals
15. 6) MITOCHONDRIA
• They are spherical, oval or rod-like bodies, about 0.5–1 mm in diameter and up to
7 mm in length.
• Erythrocytes do not contain mitochondria. The tail of spermatozoa is fully packed
with mitochondria.
• Mitochondria are the powerhouse of the cell, where energy released from
oxidation of food stuffs is trapped as chemical energy in the form of ATP.
• Mitochondria have two membranes. The inner membrane convolutes into folds
or cristae. The inner mitochondrial membrane contains the enzymes of electron
transport chain. The fluid matrix contains the enzymes of citric acid cycle, urea
cycle and heme synthesis.
• Cytochrome P-450 system present in mitochondrial inner membrane is involved
in steroidogenesis.
• Superoxide dismutase is present in cytosol and mitochondria.
16.
17. • Mitochondria also contain specific DNA. The integral inner
membrane proteins, are made by mitochondrial protein synthesizing
machinery. However, the majority of proteins, especially of outer
membrane are synthesized under the control of cellular DNA. The
division of mitochondria is under the command of mitochondrial
DNA.
• Mitochondrial ribosomes are different from cellular ribosomes.
• Antibiotics inhibiting bacterial protein synthesis do not affect cellular
processes, but do inhibit mitochondrial protein biosynthesis.
• Mitochondria play a role in triggering apoptosis
• Mitochondria are continuously undergoing fission and fusion,
resulting in mixing of contents of mitochondrial particles. Specific
fission and fusion proteins have been identified and abnormalities in
some of these proteins are implicated in diseases like CharcotMarie-
Tooth disease
18.
19. 7) PLASMA MEMBRANE
• The plasma membrane separates the cell from the external environment.
• It has highly selective permeability properties so that the entry and exit of
compounds are regulated. The cellular metabolism is in turn influenced
and probably regulated by the membrane. The membrane is metabolically
very active.
• Membranes are mainly made up of lipids, proteins and small amount of
carbohydrates. The contents of these compounds vary according to the
nature of the membrane. The carbohydrates are present as glycoproteins
and glycolipids.
• Phospholipids are the most common lipids present and they are
amphipathic in nature. Cell membranes also contain cholesterol.
20. Fluid mosaic model
• structure of the biomembranes was described as a fluid mosaic model by
Singer and Nicolson, 1972.
• The phospholipids are arranged in bilayers with the polar head groups oriented
towards the extracellular side and the cytoplasmic side with a hydrophobic core.
• The distribution of the phospholipids is such that choline containing
phospholipids are mainly in the external layer and ethanolamine and serine
containing phospholipids in the inner layer.
• Each leaflet is 25 Å thick, with the head portion 10 Å and tail 15 Å thick. The total
thickness is about 50 to 80 Å. C.
• The lipid bilayer shows free lateral movement of its components, hence the
membrane is said to be fluid in nature. Fluidity enables the membrane to perform
endocytosis and exocytosis.
• components do not freely move from inner to outer layer, or outer to inner layer
(flip-flop movement is restricted). During apoptosis (programed cell death), flip-
flop movement occurs.
21.
22. • The cholesterol content of the membrane alters the fluidity of the
membrane.
• When cholesterol concentration increases, the membrane becomes less
fluid on the outer surface, but more fluid in the hydrophobic core. The
effect of cholesterol on membrane fluidity is different at different
temperatures.
• The nature of the fatty acids also affects the fluidity of the membrane,
the more unsaturated cis fatty acids increase the fluidity.
• The fluidity of the membrane is maintained by the length of the
hydrocarbon chain, degree of unsaturation and nature of the polar head
groups.
• Trans fatty acids (TFA) decrease the fluidity of membranes due to close
packing of hydrocarbon chains.
23. MEMBRANE PROTEINS :
The peripheral proteins exist on the surfaces of the bilayer.
They are attached by ionic and polar bonds to polar heads of the lipids.
Anchoring of proteins to lipid bilayers.
Several peripheral membrane proteins are tethered to the membranes by
covalent linkage with the membrane lipids. Since the lipids are inserted
into the hydrophobic core, the proteins are firmly anchored.
The integral membrane proteins are deeply embedded in the bilayer and
are attached by hydrophobic bonds or van der Waals forces.
Some of the integral membrane proteins span the whole bilayer and they
are called transmembrane proteins .
The hydrophobic side chains of the amino acids are embedded in the
hydrophobic central core of the membrane.
The transmembrane proteins can serve as receptors (for hormones,
growth factors, neurotransmitters), tissue specific antigens, ion channels,
membrane-based enzymes.
25. TRANSPORT MECHANISM
• The permeability of substances across cell membrane is dependent
on their solubility in lipids and not on their molecular size..
• Water soluble compounds are generally impermeable and require
carrier mediated transport.
• An important function of membrane is to withhold unwanted
molecules while permitting entry of molecules necessary for cellular
metabolism.
26. Classified into two types :
1) Passive transport
2) Active transport
3) pumps
Facilitated diffusion
Ion channels
Simple diffusion
27. Simple diffusion :
occurs from higher to lower concentration.
does not require any energy.
it is a slow process
facilitated diffusion :
carrier mediated process.
no energy required
rate of transport is more rapid than simple diffusion
ping and pong states( two conformations)
eg: glucose transporters
28. Ion channels :
are specilaised carrier systems.
allow passage of molecules in accordance with the concentration
gradient.
eg : ligand gated channels- acetyl choline receptor
calcium channels.
Active transport :
requires energy.
unidirectional
requires specialised integral protein- transporters
saturated at higher concentration of solutes.
susceptible to inhibition by specifiv organic or inorganic
compounds.