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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.

Basal as well as GTP-dependent adenylate cyclase activity was partially resistant to porcine pancreatic phospholipase A2, although more activity was degraded at 16 than at 2 degrees C. In contrast, isoproterenol-dependent activity was completely destroyed regardless of the temperature. Snake venom phospholipase A2 destroyed approximately 90% of basal and GTP-dependent adenylate cyclase activity at all temperatures. The difference between the lipases is consistent with earlier evidence that elevated temperature facilitates the entry of some forms of phospholipase into the membrane bilayer. The temperature dependence of adenylate cyclase activation by the GTP analog Gpp[NH]p and its pancreatic phospholipase sensitivity were compared. The Arrhenius plots were markedly similar and biphasic with discontinuities at approximately 8 degrees C. The same temperature-dependent phospholipid phase transition might account, therefore, for both adenylate cyclase properties. Only small amounts of membrane phosphatidylethanolamine and phosphatidic acid were hydrolyzed by pancreatic phospholipase in a temperature-dependent manner analogous to adenylate cyclase degradation. These results suggest that specific phospholipids support catalysis and adenylate cyclase activation, but that different phospholipids are required for receptor coupling which may occur in a less viscous part of the membrane.