Determinants of the stimulation of fat cell adenylate cyclase by high concentrations of sodium and magnesium salts. Implications for the role of magnesium in regulation of enzyme activity

Abstract

Stimulation of adenylate cyclase by high concentrations of Mg 2+ has been attributed to either occupation of a divalent cation site on the enzyme or Mg 2+ binding of inhibitory species of uncomplexed ATP. However, the stimulation of adenylate cyclase by other salts led us to reexamine the alleged specificity of Mg 2+. The stimulatory effects of high concentrations of magnesium and sodium salts on adenylate cyclase were compared in rat fat cell membranes (‘ghosts’). Under standard assay conditions of 5 mM MgCl 2 and 1 mM ATP, added MgCl 2 (45 mM), NaCl (90 mM), MgSO 4 (45 mM), and Na 2SO 4 (45 mM) stimulated basal activity in a temperature-dependent manner. At 30°C all four salts produced 2–3-fold stimulation with linear time course to 10 min. At 37°C time courses were nonlinear, and although MgCl 2 and NaCl were stimulatory over 10 min, sulfate salts of both cations had little or no effect. When 10 −4 M GTP was added, however, all four salts became stimulatory at 37°C with linear time courses. In the presence of GTP, MgCl 2 and NaCl were equally effective at equimolar concentrations (100 mM) of Cl −. MgCl 2 and NaCl were stimulatory (in the absence or presence of GTP) over the same range of Cl − concentrations up to 200 mM. In contrast to the results with GTP, magnesium salts enhanced stimulation by the GTP analog 5′-guanylyl-imidodiphosphate (GMP-P(NH)P) much more than did sodium salts. MgCl 2 and MgSO 4 each approximately tripled GMP-P(NH)P stimulation, while NaCl and Na 2SO 4 had only minimal stimulatory effect. None of the salts increased 10 −4 M epinephrine-stimulated adenylate cyclase activity in the absence or presence of GTP. Our results show that the effects of ‘high Mg’ are, under certain conditions, approximated by high concentrations of another cation (Na) and are in part determined by the accompanying anion. Stimulation by high MgCl 2, therefore, appears to be a generalized salt effect rather than a specific effect of Mg 2+. In addition, the degree of stimulation of adenylate cyclase of fat cell ghosts by salts, including those of Mg 2+, is dependent on temperature and the presence or absence of guanine nucleotide and hormone.