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.