Characterization of Leydig cell protein kinase

FEBS Letters

Dufau, Maria L.¹  Winters, Christine A.¹ 

[1] Molecular Endocrinology Section, Endocrinology and Reproduction Research Branch, National Institute for Child Health and Human Development, NIH, Bethesda, MD 20205, USA

关键词: cAMP-dependent protein kinase;    Leydig cell;    Hormone action;   

DOI  :  10.1016/0014-5793(84)81243-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 M _r 54000 and 51000 for the peak fraction, while the original extract exhibited only the smaller form. Autophosphorylation revealed a doublet of M _t , 54000 + 573 and M _r 51 000 ± 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 M _r , 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.

【 授权许可】

Unknown