Incorporation of rat brain adenylate cyclase into artificial phospholipid vesicles.
Adenylate cyclase was solubilized from rat brain particulate fraction with the nonionic detergent, Nonidet P-40. Incubation of detergent-solubilized adenylate cyclase with liposomes prepared from egg yolk phosphatidylcholine results in virtually quantitative incorporation of the enzyme activity into phospholipid vesicles. Incorporation of adenylate cyclase into liposomes results in an approximately 10- to 20-fold purification relative to the solubilized preparation giving a final specific activity of about 50 nmol of cAMP min-1 mg-1. The detergent-solubilized adenylate cyclase migrates as a broad band between 14 and 33% sucrose on density gradient centrifugation, separated from the endogenous phospholipid. Following overnight incubation of the solubilized enzyme with exogenous phospholipid, all enzyme activity is found in a narrow band between 7 and 9% sucrose, co-migrating with the phospholipid. The adenylate cyclase could not be released from the liposomes by extraction with high ionic strength, low ionic strength-EDTA, or sonication. Treatment of liposomal adenylates cyclase with soluble proteases or immobilized trypsin destroys enzyme activity. Thus, it is likely that a functionally important part of the enzyme molecule is exposed on the outer surface of the liposome. Optimal conditions for the incorporation of adenylate cyclase into liposomes, and some effects of manipulating the phospholipid composition on enzyme activity are reported.
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D007700:Kinetics; D008081:Liposomes; D008297:Male; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D010713:Phosphatidylcholines; D051381:Rats; D014357:Trypsin / Q000378:metabolism
D008081:Liposomes; D010713:Phosphatidylcholines; D014357:Trypsin; D000262:Adenylyl Cyclases