Properties of the resolved catalytic unit of skeletal muscle adenylate cyclase.
Attempts were made to resolve the catalytic unit (C) of adenylate cyclase from the guanine nucleotide-binding regulatory protein (G/F) in detergent extracts of pigeon breast muscle. When preparations solubilized in 1% Lubrol 12A9 were fractionated at 33% saturated (NH4)2SO4, catalytic activity precipitated which was insensitive to NaF and guanine nucleotide. G/F was concentrated in a fraction precipitating between 33 and 47% saturated (NH4)2SO4, but was not obtained free of C. C was also resolved from G/F by gel filtration in the presence of 13 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), but recoveries of C were lower than those obtained by salt fractionation. NaF-Activated preparations were also subjected to gel filtration in the presence of Chaps, in which case both the catalytic activity and G/F emerged in the activated state. G/F from such columns was deactivated by removal of NaF by dialysis. NaF and guanine nucleotide sensitivity could be reconstituted in nonactivated preparations of C by G/F in the presence of NaF or guanylylimidodiphosphate. Reconstitution was dependent upon both the amount of C and G/F in the assay. C in both preparations was strongly stimulated by the diterpene, forskolin, whereas the NaF-activated enzyme resolved by gel filtration was only marginally stimulated by this agent. C was only weakly inhibited by the P-site agent 2',5'-dideoxyadenosine. However, when C was stimulated by forskolin, dideoxyadenosine was a potent inhibitor. The NaF-activated catalytic unit was also strongly inhibited by this agent. Preparations of C obtained by (NH4)2SO4 precipitation may be suitable starting material for attempted purification of this component of adenylate cyclase.
D000262:Adenylyl Cyclases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D005591:Chemical Fractionation; D002850:Chromatography, Gel; D005576:Colforsin; D010856:Columbidae; D003839:Deoxyadenosines / Q000031:analogs & derivatives / Q000494:pharmacology; D003902:Detergents; D016048:Dideoxyadenosine / Q000031:analogs & derivatives*; D004224:Diterpenes / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000302:isolation & purification; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology*; D012969:Sodium Fluoride / Q000494:pharmacology; D012995:Solubility
D003839:Deoxyadenosines; D003902:Detergents; D004224:Diterpenes; D005576:Colforsin; D006165:Guanylyl Imidodiphosphate; D016048:Dideoxyadenosine; D012969:Sodium Fluoride; C021008:2',5'-dideoxyadenosine; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases