Page 207 - Color Atlas of Biochemistry

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198 Organelles

Cell fractionation Particles that are smaller and less dense
than the nuclei can be obtained by step-by-
step acceleration of the gravity on the super-
A. Isolation of cell organelles
natant left over from the first centrifugation.
To investigate the individual compartments of However, this requires very powerful centri-
thecell(seep. 196), various procedures have fuges (high-speed centrifuges and ultracentri-
been developed to enrich and isolate cell or- fuges). The sequence in which the fractions
ganelles. These are mainly based on the size are obtained is: mitochondria, membrane
and density of the various organelles. vesicles,and ribosomes. Finally, the superna-
The isolation of cell components starts tant from the last centrifugation contains the
with disruption of thetissuebeing examined cytosol with the cell’s soluble components, in
and subsequent homogenization of it (break- addition to the buffer.
ing down the cells) in a suitable buffer (see The isolation steps are carried out at low
below). Homogenization using the “Potter” temperatures on principle (usually 0–5 °C), to
(the Potter–Elvehjem homogenizer, a rotating slow down degradation reactions—e. g., due
Teflon pestle in a glass cylinder) is particularly to released enzymes and other influencing
suitable for animal tissue. This method is very factors. The addition of thiols and chelating
gentle and is therefore used to isolate fragile agents protects functional SH groups from
structures and molecules. Other cell disrup- oxidation. Isolated cell organelles quickly
tion procedures include enzymatic lysis with lose their biological activity despite these pre-
the help of enzymes that break down the cell cautions. Nevertheless, it is possible by work-
wall, mechanical disruption by grinding fro- ing carefully to isolate mitochondria that will
zen tissue, cutting or smashing with rotating still take up substrates for a few hours in the
knives, large pressure changes, osmotic test tube and produce ATP via oxidative phos-
shock, and repeated freezing and thawing. phorylation.
To isolate intact organelles, it is important
for the homogenization solution to be iso-
tonic—i. e., theosmotic valueofthe buffer B. Marker molecules
has to be the same as that of the interior of During cell fractionation, it is very important
the cell. If hypotonic solutions were used, the to analyze the purity of the fractions obtained.
organelles would take up water and burst, Whetherornot the intendedorganelle is
while in hypertonic solutions they would present in a particular fraction, and whether
shrink. or not the fraction contains other compo-
Homogenization is followed by coarse fil- nents, can be determined by analyzing
tration through gauze to remove intact cells characteristic marker molecules. These are
and connective-tissue fragments. The actual molecules that occur exclusively or predom-
fractionation of cellular components is then inantly in one type of organelle. For example,
carried out by centrifugation steps,in which the activity of organelle-specific enzymes
the gravitational force (given as multiples of (marker enzymes) is often assessed. The dis-
–2
the earth’s gravity, g =9.81m s )is gradu- tribution of marker enzymes in the cell re-
ally increased (differential centrifugation;see flects the compartmentation of the processes
p. 200). Due to the different shapes and den- they catalyze. These reactions are discussed in
sities of the organelles, this leads to succes- greater detail here under the specific organ-
sive sedimentation of each type out of the elles.
suspension.
Nuclei alreadysedimentatlow accelera-
tions that can be achieved with bench-top
centrifuges. Decanting the residue (the
“supernatant”) and carefully suspending the
sediment (or “pellet”) inanisotonic medium
yields a fraction that is enriched with nuclei.
However, this fraction may still contain other
cellular components as contaminants—e. g.,
fragments of the cytoskeleton.

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