Identification of minor components of coated vesicles by use of
permeation chromatography |
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| Abstract: | Coated vesicles are thought to be vehicles for the intracellular transport of membranes. Clathrin is the major protein component of coated vesicles. Minor components of these organelles can be identified in highly purified preparations if they can be shown to copurify with clathrin. To show copurification we have made use of the relatively uniform diameter of coated vesicles (50-150 nm) to fractionate conventionally purified coated vesicles according to size in glass bead columns of 200-nm pore size. We have found that bovine brain coated vesicles prepared by the standard procedure of Pearse can be contaminated with large membrane fragments that are removed by permeation chromatography on such glass bead columns. Gel electrophoretic analysis of column fractions shows that only three major polypeptide chains, and a family of polypeptides with molecular weights close to 100,000 are always in constant ratio to clathrin, and are unique to fractions containing coated vesicles. Two other major polypeptides that appear to be components of coated vesicles are also present in other membrane fractions. We have also used permeation chromatography to monitor artifactual membrane trapping during vesicle isolation. Pure radiolabeled synaptic vesicle membranes were added to bovine brain tissue before homogenization. Considerable amounts of the added radioactivity could be recovered in the fractions conventionally pooled in the preparation of coated vesicles. After permeation chromatography, the radioactivity in the coated vesicle peak was reduced essentially to background. |
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