Renal Cortical Adenylate Cyclase: Characterization of Magnesium Activation*

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Abstract
The kinetic characteristics of Mg2+ and substrate (Mg-ATP) utilization by renal cortical plasma membrane adenylate cyclase were investigated in the presence or absence of PTH and with either guanosine 5′-triphosphate (GTP) or guanyl-5′-yl-imidodiphosphate [Gpp(NH)p] included in the reaction mixture. Basal- and PTH-stimulated adenylate cyclase activity was found to be dependent on Mg2+. Gpp(NH)p, but not GTP, resulted in hysteretic activation of renal cortical adenylate clase which was abolished with PTH activation. Gpp(NH)p augmented basal adenylate cyclase activity approximately 5-fold greater than GTP. PTH caused a 2-fold increase in activity over basal in the presence of 0.01 mm Gpp(NH)p but a 10-fold increase was seen in the presence of 0.01 mm and 0.10 mm GTP. Maximum velocity (Vmax) under basal conditions and with PTH activation was similar regardless of which guanine nucleotide was included in the reaction mixture. Linear regression analysis of cAMP accumulation with varying Mg2+ concentrations (0.01– 5.0 mm) suggested the presence of two Mg2+ binding sites [activation constant (Ka) for Mg of 2.0 mm and 0.5 mm)]. In these studies it appeared that when Mg2+ was less than 0.5 mm the higher affinity Mg binding site was dominant but at higher Mg2+ concentrations the lower Mg2+ binding site became dominant. An unusual finding was that the affinity of either binding site for Mg2+ was not altered with PTH activation. This was in contrast to findings from experiments using plasma membranes from liver and thyroid where agonist activation of adenylate cyclase did result in an increase in Mg2+ affinity. Basal- and PTH-activated adenylate cyclase activity was also dependent on Mg-ATP. Michaelis constant (Km) for Mg-ATP was 0.4 m in the presence of either 0.01 mm or 0.10 mm GTP or 0.01 mm Gpp(NH)p, although Gpp(NH)p enhanced basal activity so that Vmax was similar to that seen with PTH stimulation. With PTH activation, however, Km for Mg-ATP was reduced to 0.2 mm regardless of the guanine nucleotide present in the reaction mixture. This finding differed from liver adenylate cyclase where Km Mg-ATP was not influenced by glucagon activation. These data indicate that renal cortical adenylate cyclase is dependent on Mg2+ and Mg-ATP. Two apparent Mg2+ binding sites were present, the dominant one being dependent on the prevailing Mg2+ concentration. PTH was not found to alter Mg2+ affinity but did reduce Km for Mg-ATP by 50%(Endocrinology113: 1348, 1983)