| Abstract: | Vasopressin-sensitive pig kidney adenylate cyclase is sensitive to several effectors, such as Mg2+, other divalent cations, and guanyl nucleotides. The purpose of the present study was to compare the main characteristics of adenylate cyclase activation by vasopressin, Mg2+, and GMPPNP, respectively. Mg2+ ions were shown to exert at least three different effects on adenylate cyclase. The substrate of the adenylate cyclase reaction is the Mg-ATP complex. Mg2+ interacts with an enzyme regulatory site. Finally, Mg2+ can modulate the hormonal response, with Mg2+ ions affecting the coupling function--that is, the quantitative relationship between receptor occupancy and adenylate cyclase activation. At all the magnesium concentrations tested, from 0.25 mM to 16 mM, adenylate cyclase activation was not a direct function of receptor occupancy. At low Mg2+ concentrations, adenylate cyclase activation dose-response curve to the hormone tended to be superimposable to the hormone dose-binding curve. These results suggest a role of magnesium at the coupling step between the hormone-receptor complex and adenylate cyclase response. Cobalt, but not calcium, ions could exert the same effects as Mg2+ ions on this coupling step. GMPPNP induced considerable adenylate cyclase activation (15 to 35 times the basal value). Activation by GMPPNP was highly time and temperature dependent. At 30 degrees C, a 20 to 60 min preincubation period in the presence of GMPPNP was needed to obtain maximal activation. The higher the dose of GMPPNP in the medium, the longer it took to reach equilibrium. At 15 degrees C, activation was still increasing with time after 3 hr preincubation in the presence of the nucleotide. GMPPNP was active in a 10(-8)M to 10(-5)M concentration range. Unlike the results obtained with lysine vasopressin, the kinetic characteristics of dose-dependent adenylate cyclase activation curves by GMPPNP were unaffected by varying Mg2+ concentrations except for the increase in velocity when raising Mg2+ concentration. It was not clear whether or not the activation processes by the hormone and by GMPPNP had common mechanisms. |
