Organization of Brain Synaptic Vesicle Proteins |
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Authors: | Andrew P. Smith Horace H. Loh |
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Affiliation: | Department of Pharmacology, University of California, San Francisco, California 94122, U.S.A. |
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Abstract: | Abstract: The topographical arrangement of proteins and glycoproteins of mouse brain synaptic vesicles was studied with trypsin and galactose oxidase, reagents known to be impermeable with respect to other membranes. Incubation of vesicles with trypsin at a concentration of 1 μg/ml extensively degraded seven polypeptides of molecular weights (M.W.) (×10-3) 125, 107, 95, 83, 70, 60, and 36; higher concentrations degraded two additional species of 75,000 and 46,000 M.W., while leaving unaffected polypeptides of M.W. 66,000, 55,000, 33,000, 26,000, 22,000, 19,000, and 16,000. All of the trypsin-sensitive species of greater than 70,000 M.W. stained positively with the periodic acid-Schiff reagent; several other glycoproteins, all of M.W. less than 70,000, were identified, and all of these were insensitive to trypsin. Galactose oxidase-NaB3H4 treatment of synaptic vesicles heavily and exclusively labeled material of greater than 70,000 M.W. All of the polypeptides studied were sensitive to each reagent when the synaptic vesicles were first treated with detergents. Extraction of vesicles with 0.05 M-NaOH partially or completely removed a wide variety of polypeptides, including most of those in the M.W. range 46,000–83,000; none of the glycoproteins was solubilized. Essentially the opposite results were obtained when the vesicles were extracted with 0.5% Triton X-100. Most of the vesicle's species were insensitive to several bisimidate cross-linking reagents. These results suggest that: (a) The polypeptides of M.W. 125K, 107K, 95K, 83K, 75K, 70K, 60K, 46K, and 36K are externally oriented in the vesicle, whereas those of 66K, 55K, 33K, 26K, 22K, 19K, and 16K are internally oriented; (b) the vesicles contain two classes of glycoproteins, one consisting of high-molecular-weight, externally oriented species that are rich in galactose, and the other consisting of low-molecular-weight, internally oriented species of relatively low galactose content; (c) the vesicles contain a large class of nonglycosylated species that are relatively loosely attached to the membrane; and (d) most of the vesicles' polypeptides are probably freely mobile in the membrane. The organization of synaptic vesicle proteins is compared with that of the proteins of synaptosomal plasma membrane, with which the vesicle is believed to fuse. |
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Keywords: | Synaptic vesicles Topography Trypsin Galactose oxidase Cross-linking reagents |
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