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.
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D009130:Muscle, Smooth / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D000262:Adenylyl Cyclases; D007545:Isoproterenol