Bovine adrenal cortex adenylate cyclase: properties of the particulate enzyme and effects of guanyl nucleotides.
The preparation of a partially purified plasma membrane fraction from bovine adrenal cortex is described. Adenylate cyclase in this particulate preparation retained high sensitivity to ACTH and is also stimulated by 5'-guanylyl-imidodiphosphate [Gpp(NH)p]. GTP, in contrast to Gpp(NH)p, had very little intrinsic activity to stimulate activity to stimulate adenylate cyclase. GTP could however, with high affinity, inhibit the Gpp(NH)p effects on adenylate cyclase. When the concentration of creatine phosphate, a component of the ATP-regenerating system in the adenylate cyclase assay mixture, was lowered from 20 to 2 mM (at 0.1 mM ATP, 5 MM MG2+) GTP, dGTP and other nucleotides like ITP and much less UTP or CTP gained considerable intrinsic activity in the presence of ACTH to stimulate adenylate cyclase. The apparent affinities of the nucleotides for ACTH-stimulated adenylate cyclase from bovine adrenal cortex (at 2 mM creatine phosphate) were, GTP = dGTP greater than Gpp(NH)p greater than Gpp(CH2)p (5'-guanylyl-beta, gamma-methylene-diphosphonate) greater than ITP greater than UTP greater than CTP. These findings indicate that regulatory nucleotide binding sites exist for bovine adrenal cortex adenylate cyclase. Their specificity is similar to the nucleotide sites modulating angiotensin binding in bovine adrenal cortex plasma membranes (Glossmann et al., 1974a). The regulatory nucleotide binding sites for the adrenal cortex adenylate cyclase complex can also be identified under conditions where only Gpp(NH)p has high intrinsic activity (e.g. at 20 mM creatine phosphate) but other nucleotides like GTP act as antagonists. Both stimulants, ACTH and Gpp(NH)p, appear to remain firmly bound to the particulate membrane preparation, as suggested by preincubation experiments.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000032:analysis* / Q000378:metabolism; D000302:Adrenal Cortex / Q000166:cytology / Q000201:enzymology*; D000311:Adrenal Glands / Q000201:enzymology*; D000324:Adrenocorticotropic Hormone / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010761:Phosphorus Radioisotopes; D013268:Stimulation, Chemical; D013997:Time Factors
D006150:Guanine Nucleotides; D010761:Phosphorus Radioisotopes; D000255:Adenosine Triphosphate; D000324:Adrenocorticotropic Hormone; D000262:Adenylyl Cyclases; D008274:Magnesium