Isolation and characterization of plasma membranes containing LH sensitive adenylate cyclase from a Leydig cell...

An LH sensitive adenylate cyclase from a tumour Leydig cell has been investigated. The plasma membranes, prepared by a 2 phase (dextran-polyethylene glycol) centrifugation method were found to have the following properties: In the presence of LH plus p(NH)ppG (guanosine 5'-beta, gamma-imido triphosphate) or fluoride ions, maximum adenylate cyclase activity was obtained in the plasma membranes with 4 to 6 mM Mg2+ plus 0.33 to 2 mM ATP. LH alone stimulated adenylate cyclase activity 2-fold when compared with basal activity and the time course of cyclic AMP production was linear up to 45 min. With GTP (10(-5)M) and GTP plus LH, adenylate cyclase activity was increased 3 and 6-fold, respectively, for up to 20 min and thereafter declined. In contrast p(NH)ppG (10(-5)M) and p(NH)ppG plus LH increased adenylate cyclase activity 7 and 14-fold which was maintained for at least 45 min. Fluoride ions increased the enzyme activity linearly over 45 min approx 18-fold. When GTP or p(NH)ppG were added alone there was a lag time of activation of approximately 10 min which was abolished by the addition of LH. GTP but not p(NH)ppG at concentrations greater than 10(-4) inhibited basal and LH stimulated adenylate cyclase when compared with 10(-5)M GTP. The tumour Leydig cell adenylate cyclase is thus essentially similar to other hormone sensitive somatic cells. The present study makes it feasible to prepare plasma membranes by a simple method from large quantities of pure Leydig cells.