Characterization of Leydig cell protein kinase. Further studies in hormone action

Leydig cell cAMP-dependent protein kinase has been characterized using rapid fractionation and optimal conditions to minimize proteolysis. DEAE-cellulose analysis showed a single Type I peak of cAMP binding and enzyme activity that eluted at 0.1 M KCl. Photoaffinity labelling with 8-azido[32P]cAMP followed by SDS-PAGE showed a doublet with Mr 54000 and 51000 for the peak fraction, while the original extract exhibited only the smaller form. Autophosphorylation revealed a doublet of Mr 54000 +/- 573 and Mr 51000 +/- 710. To titrate the occupancy of regulatory subunits during hCG action, free cAMP receptors were measured by 8-azido[3H]cAMP binding under non-exchange conditions followed by photolysis. hCG treatment caused a dose-related decrease of free receptors and SDS-PAGE analysis of the 8-azido[32P]cAMP regulatory subunit from control and hCG treated cells also showed a hormone dependent decrease in a single band of Mr 50000. These results have shown that the Leydig cell protein kinase behaves as a Type I enzyme on DEAE analysis, but has the physical characteristics of the Type II enzyme. The dose-dependent fall in available receptor sites during hCG stimulation further indicates the central role of cAMP in hormone action in the Leydig cell.