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Cell Fractionation & Marker Enzymes

The document discusses cell fractionation techniques to isolate cell organelles. Key steps include homogenization to disrupt cells, filtration to remove intact cells, and differential centrifugation with increasing gravitational forces to separate organelles by density. Nuclei pellet first, then mitochondria, membranes, and ribosomes. Marker enzymes identify organelles, like DNA for nuclei and ATP synthase for mitochondria. Cell fractionation is important for studying cellular processes and diseases of organelles.

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Vivek P
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213 views

Cell Fractionation & Marker Enzymes

The document discusses cell fractionation techniques to isolate cell organelles. Key steps include homogenization to disrupt cells, filtration to remove intact cells, and differential centrifugation with increasing gravitational forces to separate organelles by density. Nuclei pellet first, then mitochondria, membranes, and ribosomes. Marker enzymes identify organelles, like DNA for nuclei and ATP synthase for mitochondria. Cell fractionation is important for studying cellular processes and diseases of organelles.

Uploaded by

Vivek P
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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CELL FRACTIONATION & MARKER

ENZYMES
CELL ORGENELLES
• Cell contains various organized structures
• Collectively k/s Cell Organelles
• Mitochondria
• Nucleus
• Endoplasmic Reticulum(Rough & Smooth)
• Golgi complex
• Peroxisome
• Lysosome
• Plasma Membrane
STRUCTURE OF CELL
SEPARATION OF CELL ORGANELLES
• Cell organelles can be isolated
• By a technique k/s Cell
fractionation
• Discovered by Albert Claude
• Got Nobel Prize in 1974
CELL FRACTIONATION
1. Isolation of cell organelles
• To investigate the individual compartments of
the cell
• Various procedures have been developed
• To enrich and isolate cell organelles
• Which are mainly based on the size & density
of the various organelles
STEPS OF ISOLATION
1. Disruption & Homogenization
• Homogenization of the tissue done by using
suitable buffer (isotonic to cytoplasm)
• 0.25M sucrose at pH 7.4 is used
• Homogenization is carried out in homogenizer
HOMOGENIZER
• Potter–Elvehjem Homogenizer,
a rotating Teflon pestle in a
glass cylinder
• Particularly suitable for animal
tissue
• This method is very gentle
• Therefore, used to isolate
fragile structures & molecules
OTHER METHODS

• Enzymatic Lysis  enzymes that break down the


cell wall
• Mechanical disruption grinding /cutting/
smashing frozen tissue with rotating knives,

large pressure changes, osmotic shock, and


repeated freezing & thawing
2. Filtration
• Homogenization is followed by coarse
filtration
• through gauze to remove intact cells &
connective-tissue fragments
3. Differential Centrifugation
• Actual Cell fractionation is then
• carried out by Centrifugation steps
• in which the gravitational force is gradually
increased (Differential centrifugation)
• Due to the different shapes and densities
of the organelles,
• Successive sedimentation of each type out of
the suspension occurs
SEPARATION BY CENTRIFUGATION.
• Nuclei sediments at low accelerations
• can be achieved with bench-top centrifuges
• By decanting the “supernatant” & carefully
suspending the “pellet” in an isotonic medium
• yields a fraction that is enriched with nuclei.
• It may also contain other cytoskeleton
fragments
• Particles, smaller & less dense than nuclei
• can be obtained by step-by step acceleration of
the gravity on the supernatant
• this requires very powerful centrifuges
• Like High-speed centrifuges / ultracentrifuges
• Sequence in which the fractions are obtained :
Mitochondria, Membrane Vesicles & Ribosomes.
• Finally, the last supernatant contains
Cytosol with cell’s soluble components
PRECAUTIONS CARRIED OUT
• Isolation steps are carried out at low temp.
(usually 0–5 °C)
• To slow down degradation reactions
• e. g., due to released enzymes and other factors.
• Isolation of mitochondria should be carefully done
• It will take up substrates for a few hours in the
test tube
• Also produces ATP via oxidative phosphorylation.
A. ISOLATION OF CELL ORGANELLES
B. MARKER MOLECULES
• During cell fractionation, it is very important to analyze the
purity of the fractions obtained.
• To know the intended orgennelle present in a particular
fraction marker molecules are identified.
• They occur exclusively or predominantly in one type of
organelle.
• For example,
• Activity of organelle-specific enzymes marker enzymes is often
assessed.
• The distribution of marker enzymes in the cell reflects
• the compartmentation of the processes they catalyze.
B. MARKER MOLECULES
SUMMARY OF CELL FRACTIONATION
CELL ORGANELLES & THEIR MARKER ENZYMES

Subcellular Pellet formed at the Marker


organelle centrifugal force of
Marker enzyme
Nucleus 600–750 x g, 10 min DNA
Nucleus 600–750 x g, 10 min Inner membrane,
Mitochondria 10,000–15,000 x ATP synthasse
g, 10 min
Lysosome 18,000–25,000 x g, 10 min Cathepsin
Golgi complex 35,000–40,000 x g, 30 min Galactosyltransferase
Microsomes 75,000–100,000 x g, 100 Glucose-6-
min phosphatase
Cytoplasm Supernatant Lactate
dehydrogenase
CLINICAL IMPORTANCE
• To determine the molecular details of complex cellular
processes
• To study protein synthesis, DNA replication, RNA splicing, cell
cycle, mitosis and various types of intracellular transports
• To study specific diseases of the cell organelles

(OXPHOS disease- due to mutation in Mt.DNA)


• To study SARS COV-2 virus which particularly deactivate
disease fighting function of lysosomes

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