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Characterization of Leydig cell protein kinase
Author(s) -
Winters Christine A.,
Dufau Maria L.
Publication year - 1984
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(84)81243-0
Subject(s) - autophosphorylation , protein kinase a , receptor , leydig cell , protein subunit , chemistry , photoaffinity labeling , proteolysis , enzyme , stimulation , biochemistry , hormone , endocrinology , biology , luteinizing hormone , gene
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[ 32 P]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[ 3 H]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[ 32 P]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.