Calcium inhibition of parathyroid adenylate cyclase

Parathyroid adenylate cyclase activity has previously been reported to be inhibited by calcium. However, the concentrations of calcium required to inhibit this enzyme were, in most instances, several orders of magnitude higher than cytoplasmic calcium concentrations. We undertook this study to determine the effects of calcium at submicromolar, as well as higher concentrations on parathyroid adenylate cyclase activity. A partially purified membrane fraction was obtained from normal porcine parathyroid glands using buffers free of divalent cation chelators. Relatively high concentrations of EGTA were then used in the incubation mixture to achieve the desired low ionic calcium concentrations. Addition of 0.5 mM EGTA to the reaction mixture resulted in an approximate 30% increase in basal adenylate cyclase activity and a similar percentage increase in the activity measured in the presence of guanosine triphosphate or NaF, known activators of parathyroid adenylate cyclase. EGTA also stimulated the activity measured in the presence of forskolin, which itself markedly stimulated this enzyme in a concentration-dependent manner. The effects of calcium added in amounts calculated to achieve ionic concentrations of 0.5 X 10(-7) M to 1 X 10(-3) M on the forskolin-activated enzyme activity was investigated. The resultant calcium inhibition curve was found to be stepwise in appearance. Analysis of the data using a mathematical model which assumes multiple, independent calcium-binding sites indicated the presence of two inhibitory sites. One had a high affinity for calcium, Kdiss 0.4-1.5 X 10(-6) M. This site accounted for 50-70% of the calcium-inhibitable activity. The second had a considerably lower affinity, Kdiss = 1.0-5.0 X 10(-4) M. Similar affinities were obtained from experiments in which guanosine triphosphate was used to activate the enzyme instead of forskolin. It is suggested that the high affinity calcium-binding site may be involved in the physiological regulation of parathyroid adenylate cyclase activity and that the previously reported estimates of the calcium sensitivity of parathyroid adenylate cyclase represent composites of the high and low affinity calcium-inhibitable processes.