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.

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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1158475PMC
http://dx.doi.org/10.1042/bj2050249DOI Listing

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