Biology Reference
In-Depth Information
8.
Particularly important is palmitoylation for raft signaling.
9.
Polyunsaturated fatty acyl chains are hidden in clefts on a raft protein surface.
10.
It will be essential to determine the actual structure of lipid rafts as they exist in a
biological membrane.
SUMMARY
Since it is so difficult to study a single protein in a complex biological membrane, methods
have been devised to purify and reconstitute it into a liposome of known composition. These
methods rely on detergents. Detergents are amphipathic molecules that resemble membrane
polar lipids, but are orders of magnitudemorewater soluble. Hundreds of detergents are used
to lyse cells, extract membrane proteins and lipids, and reconstitute them into membranes.
After isolation, the purified membrane is dissolved in an appropriate detergent solution,
and the target integral protein purified, usually by some form of affinity chromatography.
After purification, the protein is concentrated and mixed with a desired lipid that is also
solubilized by the same detergent. Upon rapid removal of the detergent (by dialysis, size-
exclusion chromatography, rapid dilution or polystyrene beads), polar lipids and integral
proteins fall out of solution as proteoliposomes of known composition.
Recently the hot topic in membrane structure/function has become lipid rafts, either
planar rafts or caveolae. It is now clear that if planar lipid rafts exist, they must be extremely
small (
5nm) and fleeting.
Chapter 14 will discuss how solutes enter and leave a cell. Included will be discussions of:
osmosis, simple passive diffusion, facilitated passive diffusion, active transport, Gap Junc-
tions, receptor-mediated endocytosis, phagocytosis, pinocytosis, exocytosis, and apoptotic
membrane blebbing.
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References
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[2] Rosen MJ. Surfactants and Interfacial Phenomena. New York: John Wiley; 1989.
[3] Kunjappu JT. (March). Chemistry World. RSC. Advancing the Chemical Sciences; 2003.
[4] Griffin WC. Classification of surface-active agents by 'HLB'. J Soc Cosmet Chem 1949;1:311.
[5] Griffin WC. Calculation of HLB values of non-ionic surfactants. J Soc Cosmet Chem 1954;5:259.
[6] Wilson L. Methods in Cell Biology: The Cytoskeleton. Cytoskeletal Proteins, Isolation and Characterization,
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[7] Hatefi Y, Hanstein G. Solubilization of particulate proteins and nonelectrolytes by chaotropic agents. Proc
Natl Acad Sci USA 1969;62:1129
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[8] Hochuli E, Bannwarth W, D¨beli H, Gentz R, St ¨ ber D. Genetic approach to facilitate purification of
recombinant proteins with a novel metal chelate adsorbent. Bio Technology 1988;6:1321
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[9] Hengen P. Purification of His-Tag fusion proteins from
Escherichia coli
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[11] Bradford MM. Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing
the principle of protein-dye binding. Anal Biochem 1976;72:248
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[12] Zor T, Selinger Z. Linearization of the Bradford protein assay increases its sensitivity: theoretical and
experimental studies. Anal Biochem 1996;236:302
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