Skeletal adenylate cyclase: effect of Mg2+, Ca2+, and PTH

Plasma membranes were prepared from mineralized guinea pig bone in order to study Mg2+ and Ca2+ modulation of skeletal adenylate cyclase. Plasma membrane preparation was accomplished by crushing the bone in liquid N2 and subsequent multiple washings in buffer containing EGTA to remove all Ca2+ prior to adenylate cyclase assay. Skeletal adenylate cyclase was found to be dependent on GTP and Mg2+ and responsive to bovine 1-34 PTH. Ca2+ caused a competitive inhibition of Mg2+ -activated skeletal adenylate cyclase. The apparent KaMg was 1.9 +/- 0.3 in the presence of 0.2 microM Ca2+ but increased to a mean of 7.2 +/- 1.3 in the presence of 5.0 microM Ca2+. Analysis of the Ca2+ inhibition curves at concentrations from .05 microM-1.0 mM were consistent with the presence of two Ca2+ inhibition sites, one with an apparent Ki of 1-2 microM and the other with an apparent Ki of approximately 500 microM. Lowering the Mg2+ concentration increased the contribution of the high affinity Ca2+ binding site to the overall Ca2+ inhibition, and raising the Mg2+ concentration had the opposite effect. While bPTH 1-34 enhanced adenylate cyclase activity, it did not increase the affinity of Mg2+ for skeletal adenylate cyclase nor did it alter the KiCa or the pattern of Ca2+ inhibition. These data may explain the skeletal resistance to PTH during Mg deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)