BS2510 Isolation and Fractionation of Subcellular Organelles

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BS2510


Isolation and Fractionation of Subcellular Organelles


An important aspect of the study of the Biochemistry and Cell Biology of eukaryotes is
the study of the function of cell organelles. This often means that organelles have to be
purified so that

the are free of other cell components with the minimum damage to the
structure and function of the organelle. Various methods can be used for the disruption of
the tissue depending on the size of the cells, the collagen content of the tissue (or in the
ca
se of plant cells, the thickness of the cell walls). Liver is perhaps the most easily
fractionated of all tissues with relatively large cells (10
-
20 µm) which are readily broken.

The homogeniser used is a power
-
driven Teflon homogeniser (Potter
-
Elvehjem
) rotating
in a closely
-
fitting glass tube. The size of the gap between the pestle and the glass is such
that the rotating pestle applies a shearing force which breaks open the cell releasing the
contents without breaking the organelles. The degree of homo
genisation of the tissue
depends on (a), the speed of rotation of the pestle, (b), the clearance between the pestle
and the glass container, (c), the number of strokes of the pestle and (d), the thrust force
applied. If the pestle is too tight or too many
strokes are applied the organelles will be
damaged. The development of the best procedure for the homogenisation of any tissue for
the preparation of intact organelles is a matter of the application of scientific principles
plus trial and error based on e
xperimentation.

Th other important factor for the isolation of intact organelles is the composition of the
homogenisation medium, particularly the osmotic strength of the medium. As with intact
cells, a hypo
-
osmolar medium causes organelle swelling and bre
akage. Usually the
organelles are more susceptible to osmotic damage than the cells from which they are
derived.



The homogenisation should therefore be performed in an iso
-
osmotic medium
containing an inert substance, for example 0.25 M sucrose (or manni
tol). Sometimes
iso
-
osmotic 0.1M KCl is used, reflecting the cytoplasmic concentration of the salt in
most cells.



Usually a dilute buffer is also added to prevent large fluctuations in pH which
may damage organelles. The maintenance of a pH gradient is esp
ecially important in
the function of some organelles.



Chelators of divalent metal ions such as EDTA or EGTA are often added
because such metal ions may damage organelles.


Centrifugation of subcellular organelles


Once the cell
-
free homogenate has been pre
pared the next step is the use of differential
centrifugation to separate out the various organelles on the basis of their size and density
(see Table1).







2


Table 1. Size and Density of Some Sub
-
cellular Organelles from Liver








Size

m



Density (
g/cm
3
)










(in sucrose medium)


Nuclei





3
-

12




>1.30


Mitochondria




0.5
-

2.0




1.17
-

1.21


Lysosomes




0.2
-

0.4




1.20
-

1.22


Peroxisomes




0.2
-

0.5




1.23


Endoplasmic reticulum

vesicles (microsomes)



0.05
-

0.30



1.15 (smooth ER
)










1.22 (rough ER)


Golgi stacks




~ 1.0




1.10
-

1.13


Golgi vesicles




~0.05


Plasma membrane sheets



20




1.15
-

1.19


Plasma membrane vesicles



0.05




< 1.17




Differential Centrifugation


Particles may be separated on the basis of thei
r size and density by differential
centrifugation.


Relative Centrifugal Field (RCF)

This is the force applied to a particle which is in a centrifuge rotor, which is rotating about a
central pivot at a given speed.




RCF(
g
) =1.12 x r x (rpm/1000)
2




Where r = distance of particle from the centre of rotation and rpm is the speed of rotation
in revolutions per minute. It follows that the
g

force is greater at the bottom of the tube than
at the top and therefore particles near the bottom of the tube
sediment faster than those at the
top. This is one reason why some centrifuge rotors are designed to hold the centrifuge tube
at a steep angle, to reduce the difference in
g

force at the top and bottom of the tube.







3


The rate of sedimentation of a part
icle (
v
) is given by the following equation:

where
r
p

and
ρ
p

are, respectively, the radius and density of the particle,
ρ
m

the density of the
medium, g is the centrifugal force and η is the viscosity of the liquid
.


The rate at which a particle is sedimented is therefore dependent on :

(a)

(The radius of the particle
)
2

and is therefore related to the cross
-
sectional are
of the organelle.

(b)


The difference between the density of the organelle and the density of the
medium in which the organelle is suspended. When the two densities are
equal then the organelle will not se
diment regardless of the g force applied
and, indeed if the density of the medium is greater than that of the particle,
then it will tend to float towards the top of the tube.

(c)

The g force applied ~ proportional to (rpm/1000)
2
.

(d)

The sedimentation rate is inv
ersely proportional to the viscosity of the
medium. The higher the viscosity, the slower the rate of sedimentation.


Isolated organelles sediment at different rates related to their size and density (See Table 2)



Table 2. Organelles sedimented by centrif
ugation at increasing speeds


[1]

1,000 x g (10 min)


Unbroken cells, nuclei, plasma membrane sheets, heavy







mitochondria plus smaller, trapped particles.



[2]

3,000 x g (10 min)


Heavy mitochondria, plasma membrane fragments plus






smaller, tra
pped particles.


[3]

10,000 x g (20 min)


Mitochondria, lysosomes, peroxisome, some Golgi







membranes and rough endoplasmic reticulum.


[4]

100,000 x g (60 min)


Membrane vesicles derived from smooth and rough







endoplasmic reticulum, Golgi ves
icles and plasma







membrane vesicles.


[5]

100,000 x g


Supernatant



Cytoplasmic components plus any soluble organelle







components released during homogenisation and







fractionation.








9
)g
-
(
r
2

=

v
m
p
2
p



4

Characterisation of Organelles in Fractions


Organel
les may be identified by their characteristic appearance under the electron
microscope (see Molecular Cell Biology, Chapt. 5 Lodish
et al.
2000,2004) or with
fluorescent
-
tagged antibodies raised against specific organelle components or by
measuring specific

marker enzymes.


Marker Enzymes


Each organelle has a specific role to play in cellular function and it therefore follows that
certain components (including proteins and enzymes) are only found associated with one
organelle. This is not the case for all p
roteins as there is an increasing body of evidence
that some proteins are translocated from one cellular compartment to another as part of
the normal cell function. However there are certain enzymes that are easy to measure
which are located primarily in a

single type of organelle or cell compartment. These are
known as marker enzymes. The genes for these marker enzymes also code for a leader or
signal peptide which directs the particular protein into a specific cellular location. The
assay of marker enzyme
s can be used to track the fate of a particular organelle during a
fractionation procedure.


Let us examine how marker enzymes may be used to identify organelles, starting with a
structure which is not an organelle as such but that does have very specific

and important
functions. i.e. the plasma membrane.


Plasma membrane


The plasma membrane has many essential functions, including transporting nutrients into
the cell and removing waste products, preventing unwanted materials from entering the
cell and pre
venting the loss of essential metabolites as well as maintaining the
intracellular ions, pH and osmotic pressure of the cytoplasm.


A good marker for the hepatic plasma membrane is 5’ nucleotidase:



AMP


adenosine + Pi

This activity is located on the e
xtracellular surface of the plasma membrane of the liver


its function here is not entirely clear.


Plasma membrane fractions are often prepared by homogenisation of tissue in dilute
(1mM) NaHCO
3
. depending on the degree of homogenisation of a tissue the
marker
enzyme tends to be located in large sheets of membrane fragments which tend to
sediment at low speed (1,000 x g) along with nuclei, whole cells and large mitochondria.
However this fraction may be sub
-
fractionated on a discontinouous sucrose gradien
t
where the fraction suspended in 0.25M sucrose which is overlaid on 37% sucrose which,
in turn, is overlaid on 57% sucrose and centrifuged at 75,000 x g for 16 h. The nuclei and
whole cells sediment to the bottom of the tube, mitochondria accumulate at t
he interface
of the 37% and 57% sucrose, while the plasma membrane accumulates at the interface of
the 0.25 M sucrose and the 37% sucrose (Fig 1a). Much smaller, plasma membrane
vesicles can also form on homogenisation which appear in the ‘microsomal’ or 1
00,000 x

5

g pellet along with the endoplasmic reticulum. This fraction can also be sub
-
fractionated
using the discontinous gradient described above with the plasma membrane vesicles
accumulating at the 0.25 M sucrose / 37% sucrose interface (Fig 1b).


Fig 1
a. Subfractionation of ‘nuclear’ pellet, containing plasma membrane fragments




Fig 1b Subfractionation of microsomal pellet, containing plasma membrane vesicles




Why might you need to isolate pure plasma membrane preparations?


To s
tudy various aspects of plasma membrane function for example:



Hormone receptors: adrenergic, glucagon insulin receptors etc



Signal transduction components G
-
proteins, adenylate cyclase



Transport proteins Glut
-
2, Glut
-
4, Na
+
K
+
ATPase, Ion channel prote
ins

Can you think of any other functional components of plasma membranes?


To show the plasma membrane localisation of these proteins you would need to show that
they co
-
fractionate with the 5’nucleotidase. Having established that a protein is
associated w
ith a particuar fraction, it is possible to use this as a first step in a purification
procedure. The next step is often the solubilization of the membrane
-
associated protein
with a detergent.


Endoplasmic Reticulum


The ER is the network of channels for
proteins targeted for specific modifications rather
than cytosolic proteins. There are two types of ER visible under the electron microscope;
the rough ER has ribosomes attached and is the site of protein synthesis while the smooth

6

ER does not have ribosom
es but has enzymes involved in lipid synthesis and steroid
metabolism . A marker enzyme for liver ER is glucose
-
6
-
phosphatase which catalyses the
following reaction:



Glu
-
6
-
P



Glucose

+

Pi


This enzyme plays a key role in the regulation of glucose outpu
t by liver and thus in
control of blood glucose. It is the final enzyme of the gluconeogenic pathway and also in
the mobilisation of glycogen in the liver. If the enzyme activity is determined in a
microsomal fraction the activity is rather low unless a de
tergent is added. This is because
the active site of the enzyme is on the inside of the microsomal vesicles. Glucose
-
6
-
phosphate uptake by the vesicles is rate limiting; there is a specific Glu
-
6
-
P transporter
which is essential for the activity because ot
herwise the ER membrane is impermeable to
the phosphorylated derivatives. The activity of Glu
-
6
-
phosphatase appears much greater
when detergent is added to permeabilise the membrane. This phenomenon is known as
LATENCY and this can also been shown for enzy
mes located in other organelles (e.g.
lysosomes) when exposed to hydrophilic substrates.





Separation of Rough and Smooth Endoplasmic Reticulum


This is relatively easily done, a 10,000 x g, post
-
mitochondrial supernatant is prepared i
n
0.44 M sucrose and layered on to 1.3 M sucrose (44%,


= 1.20) then centrifuged at
105,000 x g for 7 h. The rough microsomes sediment to the bottom of the tube while the
smooth ER remains suspended at the top of the 1.3 M sucrose








DR Davies 2005