Salts promote activation of fat cell adenylate cyclase by GTP: Special role for sodium ion

The effects of GTP on adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] of human and rat fat cell membranes ('ghosts' and purified membranes) were examined in the absence and presence of added inorganic salts. With human ghosts GTP alone (0.1 mM) inhibited enzyme activity by 40% at 30°C and had no significant effect at 37°C. At both temperatures Na⁺ salts of Cl^-, N₃^-, and SO₄^2- stimulated activity (up to 4-fold basal activity for 200 mM NaN₃), with maximal effects at salt concentrations of 100-200 mM. Over the same concentration range these salts also allowed temperature-dependent stimulation by GTP. GTP increased the maximal activity produced by salt alone by about 2-fold at 30°C and about 4-fold at 37°C. Na⁺ (added as Cl^-) was much more effective than other alkali metal cations in promoting activation by GTP. Na⁺ salts allowed activation of the human enzyme by the GTP analog 5'-guanylyl imidodiphosphate and also promoted stimulation of rat fat cell adenylate cyclase by both nucleotides. In time course studies of human and rat fat cell ghosts, GTP appeared to sustain an initial high rate of salt-stimulated activity, which in the absence of nucleotide subsequently fell to a lower rate, suggesting that salts might activate adenylate cyclase by promoting the stimulatory effect of endogenous membrane-bound GTP. However, with purified human fat cell membranes and a GTP-free system, salts were still stimulatory and promoted activation by added GTP. These results differ from those of previous reports in other systems in which Na⁺ has promoted only inhibitory effects in GTP regulation of adenylate cyclase.