Download Isolation and Fractionation 2

Isolation and Fractionation of Subcellular Organelles (continued)
The fraction sedimenting at 10,000 x g, 20 min. consists of three different organelles:
Mitochondria- a number of possible marker enzymes e.g. Succcinate Cyt c reductase
Lysosomes - characterised by a number of acid hydrolases e.g acid phosphatase,
galactosidase
Peroxisomes - marker enzyme catalase
Complete separation of these organelles by differential centrifugation is very difficult
because of their similar size and density. The sedimentation rate of lysosomes and
peroxisomes is very similar and slightly less than that of mitochondria and because of the
heterogeneity of size of each organelle and the variations in organelle density there is
always a great deal of overlap between the fractions.
Continuous density gradient centrifugation
So far I have discussed discontinuous gradients but it is possible to set up linear sucrose
density gradients in centrifuge tubes, which are very stable when centrifuged at high
speed in the cold.
Fig 3. Diagram of a Gradient Former: this apparatus can be used to make linear sucrose
gradients.
The denser sucrose in the compartment on the left hand side mixes with the lighter
sucrose in the mixing compartment when both valves are opened and the peristaltic pump
is switched on. The lighter sucrose runs to the bottom of the tube but is displaced by the
contents of the mixing chamber which becomes progressively denser setting up a linear
gradient in the centrifuge tube. Sucrose has the advantage of being very soluble in water
and is a relatively inert substance. Continuous gradients between varying densities may
be set up using this equipment.
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Table 3. Relationship between sucrose concentration and density
Sucrose Density (g.cm3)
= 1.03
= 1.06
= 1.155
= 1.210
= 1.30
Sucrose Concentration
8.5% (0.25M)
14.4%
34.5%
45.0%
60.0%
Iso-pycnic Centrifugation
This is where the organelles are centrifuged on a linear gradient until they come to rest at
a position in the gradient where their buoyant density is equal to that of the medium. This
can take a long time in a high speed centrifuge. If the density gradient is between 1.10
and 1.26 g.cm3 then the lysosomes will come to equilibrium in a band just above the
mitochondria which, in turn, form a band just above the peroxisomes. These bands can be
pumped off the gradient carefully through a fine needle inserted into the bottom of the
tube and the fractions assayed for marker enzymes but however carefully this is done
there is always overlap between the organelles because of the heterogeneity of the
particles and is therefore not a good method for the routine isolation of one type of
organelle, free from contamination by the others. This is however a good method for
determining the buoyant density of the organelles in sucrose.
Rate Zonal Centrifugation
This is a method which can be used to separate lysosomes and mitochondria on the basis
of their relative sizes rather than density. The 10,000 x g 20 min. pellet is resuspended in
0.25 M sucrose and layered on top of a linear sucrose gradient,  = 1.10 to  = 1.26
g.cm3. The organelles are centrifuged for a shorter time at a slower speed than iso-pycnic
centrifugation. Because of their smaller size the peroxisomes move down the gradient at
a slower rate than mitochondria the distribution of organelles can be investigated by
measuring the activity of marker enzymes:
Fig 4. Distribution of Organelles After Rate-zonal Centrifugation
Distribution of
Enzyme Activities



 =1.26
L = -glucosidase activity (lysosomal marker)
P = Catalase activity (Peroxiosomal marker)
M = Succinate Cytochrome c Reductase (Mitochondrial marker).
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Although this technique separates mitochondria from lysosomes, both fractions tend to be
contaminated by peroxisomes as judged by the distribution of catalase activity.
Selective Modification of the Density of Lysosomes
The size and density of these organelles can be selectively modified by 'indigestible'
substances, which can accumulate in lysosomes as part of their normal function. These
substances are administered to experimental animals by injection into the bloodstream.
These are captured by the liver by endocytosis and become engulfed by lysosomes. It is
possible to increase or decrease the density of lysosomes depending on what is injected.
The most widely used compound is Triton WR1339, which is taken up by liver and
accumulates in lysosomes and decreases the buoyant density of the organelles from 1.21
to 1.11. If you then prepare a 10,000 x g pellet, suspend it in a high-density sucrose
(45%, = 1.21), and then layer on top 34.5% sucrose ( = 1.155) and 14.3% sucrose ( =
1.06) and then centrifuge at 25,000 rpm for 2 hours. The peroxisomes and mitochondria
sediment to the bottom of the tube whilst the less dense lysosomes rise to the interface of
the 14.3% and 34.5% sucrose. The lysosomal layer can be removed, harvested by further
centrifugation and then resuspended in iso-osmotic 0.25 M sucrose.
Conversely lysosomes can be made more dense by injecting Dextran 500 which is also
taken up by the liver. These methods are particularly good for separating lysosomes from
the very similar peroxisomes.
Isolation of Peroxisomes
The major problem is that these organelles are almost identical in size, density and
structure to lysosomes although they are quite different functionally. Hence injection of
WR1339 or Dextran 500 prior to the isolation of the organelles is often used to prevent
contamination. Peroxisomes are very sensitive to changes in hydrostatic pressure and are
hence very fragile.
If the liver is homogenised and centrifuged at low speed such that the lysosomes,
peroxisomes and mitochondria remain in the supernatant. It is possible to treat the
peroxisomes with 1mM glutaraldehyde which cross-links proteins and stabilizes the
peroxisomal membrane. The peroxisomes can be separated on a Percoll gradient where
they come to equilibrium at the interface of a 40% Percoll in 0.25M sucrose and 100%
Percoll ( See below)
Alternative Gradient Media
The most commonly used gradient medium is sucrose, which exerts an osmotic pressure
on the organelles. As we have seen 0.25 M sucrose is iso-osmotic with the contents of
most mammalian cells. All the gradients described so far are HYPEROSMOTIC and thus
have a tendency to cause shrinkage of the organelles as the density of the sucrose
increases because water exits from the organelle to equalize the osmotic pressure inside
and outside the organelle. Because of this, values for organelle densities in sucrose media
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shown in Table 1 do not necessarily reflect the density of the organelle in vivo. The
organelles also tend to become unstable particularly when they are returned to isoosmotic sucrose.
Hence for some applications, synthetic gradient media are used which are dense but exert
a low osmotic pressure. Hence, the use of Percoll, which is a colloidal suspension of
silica particles coated with poly-vinylpyrrolidone, and because it is a colloid rather than a
true solute, it exerts a negligible osmotic effect. Hence the need to add 0.25 M sucrose to
maintain the iso-osmotic conditions for the organelles, The buoyant density of the
organelles in Percoll is a lot less than in sucrose based media.
Nycodenz and Metrizamide - these are water-soluble derivatives of tri-iodobenzoic acid
which exert low osmotic activity and low viscosity and are good for separating lysosomes
and peroxisomes because the buoyant density of these organelles in these media is quite
different to those in sucrose:
Mitochondria  = 1.15 g.cm3
Lysosomes  = 1.14 g.cm3
Peroxisomes  = 1.231 g.cm3
If you select your media carefully you can isolate peroxisomes to accumulate at the
interface of these media at different concentrations.
Peroxisome Function
Peroxisomes are characterised by the presence of high activity of the enzyme known as
CATALASE:
2H2O2  2H2O + O2
Hydrogen peroxide is an active oxygen species which can be very damaging to the cell. It
is generated by a variety of enzymes which oxidise a wide variety of unusual substrates
in the peroxisome. These include D-amino acids, polyamines, Acyl CoA esters and uric
acid, which are metabolised by oxidases thus:
RH2
R
O2
H2O2
Although -oxidation of fatty acids occurs mainly in mitochondria. Very long chain fatty
acyl CoAs are oxidised only in peroxisomes generating medium chain fatty acids which
can be further metabolizesd in mitochondria. The first step in the peroxisomal pathway
generates FADH2 which in turn will produce H2O2. See Lodish et al 3rd Ed. 629-30 for
detail). Hence the need for the highly active catalase in these organelles.
You can find out more about how proteins are targeted to lysosomes and peroxisomes in
Molecular Cell Biology, Lodish et al (3rd Ed.) 708-711 and 837-39 respectively or
(5thEd.) 721 –724 and 693-696
D Davies 2005-10-19
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