Association of binding sites for guanine nucleotides with adenylate cyclase activation in rat pancreatic plasma membranes. Interaction of gastrointestinal hormones.
1. The activation of rat pancreatic adenylate cyclase by guanosine 5'-(beta-gamma-imido)triphosphate (p[NH]ppG) and GTP, and by the two gastrointestinal hormones pancreozymin (as C-terminal octapeptide) and secretin was correlated with the binding of [8-3H]guanosine 5'-(beta-gamma-imido)triphosphate to rat pancreatic plasma membranes. 2. The low basal adenylate cyclase activity was stimulated 17-fold by p[NH]ppG (after a 2 min lag period), 3,5-fold only by GTP, 21-fold by C-terminal octapeptide of pancreozymin, and 8-fold by secretin. GTP inhibited competitively the activation of adenylate cyclase by p[NH]ppG with a Ki,app almost identical with the Ka,app (0.3 micron). p[NH]ppG and GTP enhanced the stimulation by secretin more markedly than that by the C-terminal octapeptide of pancreozymin, leading to the same maximal activity. Both hormones suppressed the lag period of activation by p[NH]ppG. 3. The binding of [8-3H]p[NH]ppG was dependent on time, temperature and Mg2+ and it was also a saturable and reversible process. Scatchard plots with a concavity upward were linearized after co-addition of ATP, Mg2+ and an ATP-regenerating system that abolished low-affinity sites for p[NH]ppG without saturating higher affinity sites, GTP, ITP and UTP inhibited [8-3H]p[NH]ppG binding to the high-affinity sites in concentration ranges identical with those found for adenylate cyclase activation. Considerable binding of [8-3H]p[NH]ppG was still evident at 20 degrees C, but enzyme activation was not observed any more, except in the presence of hormones.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002462:Cell Membrane / Q000201:enzymology; D002766:Cholecystokinin / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007700:Kinetics; D010179:Pancreas / Q000201:enzymology*; D051381:Rats; D012633:Secretin / Q000494:pharmacology*
D012633:Secretin; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D002766:Cholecystokinin; D000262:Adenylyl Cyclases