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.
D000818:Animals; D001386:Azides / Q000378:metabolism; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007985:Leydig Cells / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D001386:Azides; D006063:Chorionic Gonadotropin; C017042:8-azidoadenosine-3',5'-monophosphate; D000242:Cyclic AMP; D011494:Protein Kinases