Cross-linking of synaptic vesicle proteins. Effect of ATP.
Synaptic vesicles isolated from bovine brain were subjected to cross-linking with the bifunctional amino group reagent dimethyl adipimidate. The resulting proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The reagent (10 mM) caused partial or complete disappearance from the SDS gel of most of the major polypeptides of the vesicles and the formation of new polymeric species with molecular weights greater than 500000. Using lower concentrations of the adipimidate (2-5 mM) a more selective cross-linking occurred, with the disappearance of a group of protein bands having apparent molecular weight values of 60000-68000, 40000-41000 and 25000-30000. The extent of cross-linking was independent of vesicle concentration in the range 0.3-3.0 mg protein per ml. Addition of ATP or AMP to the cross-linking reaction mixture resulted in a marked reduction in cross-linking of all of the major vesicle polypeptides (apparent molecular weight values 160000, 77000, 55000, 42000, 32000, 28000 and 26000). Several proteins were less affected by ATP or AMP; these were mostly the same vesicle proteins as those which had become cross-linked with low concentrations (2 mM) of dimethyl adipimidate. The ATP effect was markedly reduced if the vesicles were pretreated prior to the cross-linking reaction with alkaline buffer (pH 8.5) in either the presence or the absence of ATP. In the presence of 32P-labeled ATP, several of the vesicle protein bands became phosphorylated, but the extent of their cross-linking did not depend upon the state of phosphorylation of the major phoshorylated proteins. The results are consistent with the presence of aggregated protein complexes and of stabilized arrays of the major proteins within the vesicle membrane.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D004119:Dimethyl Adipimidate / Q000494:pharmacology*; D007096:Imidoesters / Q000494:pharmacology*; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D013572:Synaptic Vesicles / Q000378:metabolism*
D007096:Imidoesters; D009419:Nerve Tissue Proteins; D004119:Dimethyl Adipimidate; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate