Adenylate cyclase activation. Characterization of guanyl nucleotide requirements by direct radioligand-binding methods.
Particulate adenylate cyclase preparations from rat uterine smooth muscle had a single class of [3H]guanyl-5'-yl imidodiphosphate ([3H]GMP.P(NH)P)-binding sites with all of the properties of the guanyl nucleotide-requiring enzyme activation sites (N) which couple hormone receptors and catalytic subunits. These sites bound the radioligand in a reversible manner at low temperature (less than 2 degrees C) but irreversibly at temperatures between 6 and 24 degrees C, properties characteristic of the activation of the enzyme by treatment with GMP.P(NH)P described previously (Krall, J. F., Leshon, S. C., Frolich, M., and Korenman, S. G. (1981) J. Biol. Chem. 256, 5436-5442). Temperature affected the number but not the apparent affinity (Kd approximately 1.0 microM) of binding. The time course of the transition from reversible to irreversible binding was coincident with the irreversible activation of the catalytic subunit. The methyl analog of GTP, guanyl-(beta, gamma-methylene)-diphosphate, a poor irreversible activator compared to GMP.P(NH)P in this enzyme system, was a competitive inhibitor of [3H]GMP.P(NH)P binding but with a 10-fold lower affinity (Kd approximately 10 microM). Using these direct radioligand-binding methods, both an inactive (NL) and active (NL*) form of the guanyl nucleotide-binding activation site were demonstrated, and the transition NL leads to NL* was identified as the temperature-dependent event in catalytic subunit (C) activation. The nondissociability of specific [3H]GMP.P(NH)P binding and the irreversible nature of catalytic subunit activation suggest that a complex of NL*.C characterizes the fully activated state of the smooth muscle enzyme.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D004789:Enzyme Activation; D005260:Female; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D007700:Kinetics; D008024:Ligands; D009130:Muscle, Smooth / Q000201:enzymology; D011485:Protein Binding; D051381:Rats; D013696:Temperature; D014599:Uterus / Q000201:enzymology*
D008024:Ligands; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases