Purification and subunit composition of a cholinergic synaptic vesicle glycoprotein, phosphointermediate-forming ATPase.
A glycoprotein ATPase in cholinergic synaptic vesicles of Torpedo electric organ was solubilized with octa-ethylene glycol dodecyl ether detergent. Study of potential stabilizing factors identified crude brain phosphatidylserine, glycerol, dithiothreitol, and protease inhibitors as of value in maintaining activity. The ATPase was purified from the solubilized, stabilized material by glycerol density gradient band sedimentation velocity ultracentrifugation, and hydroxylapatite, wheat germ lectin affinity, and size exclusion chromatographies. The pure ATPase had a specific activity of about 37 mumol ATP hydrolyzed/min/mg protein. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified material typically exhibited three polypeptides of molecular masses 110, 104, and 98 kilodaltons (kDa) and a fourth diffuse polypeptide of 60 kDa. This composition suggests that the ATPase is a member of the P-type, or phosphointermediate-forming, family, but it was shown to be distinct from the ouabain-sensitive Na+,K+- and CA2+-stimulated Mg2+-ATPases. The purified vesicle enzyme was rapidly phosphorylated by [gamma-32P]ATP on about 14% of the subunits with molecular weights of 98,000-110,000. About 16% of the ATPase was phosphorylated in whole-vesicle ghosts in a manner consistent with formation of a phosphointermediate, thus confirming the P-type nature of this enzyme.
Yamagata
S K
SK
Department of Chemistry, University of California, Santa Barbara 93106.
Noremberg
K
K
Parsons
S M
SM
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D003902:Detergents; D004355:Drug Stability; D006023:Glycoproteins / Q000302:isolation & purification*; D010275:Parasympathetic Nervous System / Q000032:analysis*; D010718:Phosphatidylserines / Q000494:pharmacology; D012995:Solubility; D013572:Synaptic Vesicles / Q000032:analysis*; D014101:Torpedo
D003902:Detergents; D006023:Glycoproteins; D010718:Phosphatidylserines; D000251:Adenosine Triphosphatases