Biology Reference
In-Depth Information
liquids, often polyethylene glycol and dextran [45] . In one example Yoshida and Kawata [46]
reported that 'mung bean tonoplast has a high partition coefficient for the polyethylene
glycol-enriched upper phase and the smooth endoplasmic reticulum has a high partition coef-
ficient for the Dextran-enriched lower phase'. Two phase separation of membrane fractions is
at best poorly understood and is presently only a curiosity that will never likely receive wide
application.
Silica Particles
Silica particles have been used to isolate plasma membrane (PM) fractions [47] . Affinity is
provided by charge attraction as silica microbeads are positively charged while PM fractions
are negatively charged. Silica microbeads are also dense and can be easily centrifuged away
from other, non-bound and hence lighter, membranes. The procedure begins by binding
whole cells to the dense silica particles. Upon cell lysis, large open sheets of PM remain
bound to the silica and are isolated away from the unbound internal membranes by gentle
centrifugation. Using this procedure, Chaney and Jacobson reported that PM from Dictyoste-
lium discoideum was obtained in high yield (70
80%) and purified 10 to 17-fold [47] .
e
Separation by Size
It is relatively easy to separate intact organelles from intact cells and even from one
another on the basis of size and this is normally accomplished by differential centrifugation.
However, separation on the basis of size can also be achieved by common filter techniques
including Millipore Filters, Sephadex (size exclusion chromatography) and PAGE electro-
phoresis (polyacrylamide gel electrophoresis). These techniques have much more important
applications in other aspects of bioscience, but have occasionally been used for membrane
separations. For example, Millipore Filters are extensively used to remove contaminants
from laboratory water and to size liposomes (Chapter 13), Sephadex to separate non-
sequestered solutes from solutes sequestered inside liposomes (Chapter 13), and PAGE elec-
trophoresis for protein purification.
Membrane-Specific 'Tricks'
Membrane purification is as much an art as it is a science. A large number of what could be
best described as 'tricks' have been employed for very specific applications. These applica-
tions have no general use in membrane purification procedures. Three examples follow:
1. It is often difficult to separate microsomes derived from the rough endoplasmic reticulum
(RER) from the smooth endoplasmic reticulum (SER). However, in the presence of 15 mM
CsCl, RER microsomes aggregate, greatly facilitating the separation.
2. Rat liver lysosomes can be hard to separate from mitochondria and peroxisomes due to
their similar sedimentation coefficients. Nobel Prize recipient Christian de Duve (in
Physiology or Medicine , 1974) reported that if the detergent TritonWR 1339 was injected into
the rat prior to organelle extraction, lysosomes accumulated the detergent making them
lighter [48] . With detergent, lysosome density decreased from 1.21 to 1.12, facilitating their
separation from the unmodified mitochondrial and peroxisomal fractions.
3. Finally, an ER separation has been reported based on the presence of an ER enzyme,
glucose 6-phosphatase. Glucose 6-phosphate, the substrate for the enzyme, enters the ER
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