Phospholipid and guanine nucleotides sensitive properties of the smooth muscle adenylate cyclase catalytic unit.

The adenylate cyclase catalytic unit was partially purified from uterine smooth muscle by chromatography on columns of SM-2 Bio-Beads and Sepharose 6B. Stimulation of catalysis by forskolin was much greater in the presence of Mn2+ than in the presence of Mg2+. Neither NaF nor guanine nucleotide stimulated catalysis in the presence of Mg2+ or Mn2+. These properties indicated the catalytic unit was not sensitive to regulation by the GS regulatory protein. Guanine nucleotide inhibited catalysis, however, and was a competitive inhibitor of the ATP substrate (Ki approximately 50 microM). Since inhibition affected Km but not Vmax, the catalytic unit also seemed insensitive to regulation by the Gi regulatory protein, which does not act like a competitive inhibitor in other enzyme systems. The catalytic unit was also phospholipid sensitive. Only phosphatidic acid (Pho-A) had a direct effect on catalysis and was a potent inhibitor. Its effects were antagonized by the concomitant addition of phosphatidylcholine (Pho-C) but not by phosphatidylethanolamine, phosphatidylserine, or phosphatidylinositol. Acyl chain composition had a marked effect on Pho-C binding when this was determined by antagonism of Pho-A-dependent inhibition. These properties suggest the catalytic unit has both polar head group and acyl chain requirements for phospholipid binding.

Phospholipase A2 sensitivity of uterine smooth muscle membrane phospholipids and adenylate cyclase activity. Effect of temperature on the action of phospholipase present in excess.

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.

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.

Kinetics and thermodynamics of activation of pretreatment with guanosine 5'-[beta, gamma-imido]triphosphate of smooth-muscle adenylate cyclase.

Catalytic subunits (C) of uterine smooth-muscle adenylate cyclase were activated (C*) by incubating the enzyme with the GTP analogue guanosine 5'-[betagamma-imido]triphosphate (p[NH]ppG), followed by treatment with GTP and washing at 2 degrees C. Activation (C-->C*) proceeded in a time- and temperature-dependent manner as disclosed by subsequent assay of the pretreated particles at 37 degrees C. The properties of the activated subunits were a function of the pretreatment temperature and not those of the enzyme assay performed at 37 degrees C. Over the range 6-24 degrees C, activation by pretreatment with p[NH]ppG followed simple Michaelis-Menten kinetics, and increase in temperature increased the concentration of catalytic subunits in the C* state and decreased K(m) for the guanosine nucleotide. Characterization of the temperature-dependent effects of pretreatment with p[NH]ppG suggested that activation of the catalytic subunit at the temperature in situ (37 degrees C) was moderately endergonic (DeltaH(0) approximately 8kJ.mol(-1)) and accompanied by an increase in entropy (DeltaS(0) approximately 146J.mol(-1).K(-1)). The beta-adrenergic catecholamine receptor, reflected by isoproterenol's effect on activation by pretreatment with p[NH]ppG, increased the concentration of catalytic subunits in the C* state but had an insignificant (P>0.05) effect on the K(m) at every temperature. This result suggested that formation of the receptor-hormone complex produced an increase in the first-order rate constant without an appreciable effect on the actual catalytic-subunit activation step. The primary function of the beta-adrenergic catecholamine receptor under these conditions appeared to be regulation of the concentration of activation sites available for binding of p[NH]ppG.