Isolation and Regulation of the Protein Kinase Inhibitor and the Calcium-Dependent Cyclic Nucleotide Phosphodiesterase Regulator in the Sertoli Cell-Enriched Testis1

Abstract

The c[cyclic]AMP-dependent protein kinase inhibitor (PKI) and Ca-dependent cyclic nucleotide phosphodiesterase regulator (CDR) proteins were examined in the Sertoli cell-enriched (SCE) rat testis. PKI activity was stable at 100.degree. C for 10 min whereas the rate of decline of CDR activity at this temperature was first order (t1/2 of 7 min). Greater than 90% of both activities were found in the soluble fraction of the cell with the remaining 10% in the particulate fraction. PKI and CDR could be separated by DEAE-cellulose chromatography and by polyacrylamide gel electrophoresis. Gel filtration chromatography also resulted in their physical separation, CDR eluting slightly before PKI. Molecular weights estimated by gel filtration were 21,400 and 28,800 daltons for PKI and CDR, respectively. Sufficient PKI was present in the SCE testis to inactivate only 7% of the total cAMP-dependent protein kinase, whereas CDR was present in vast excess over the amount required to activate maximally total Ca-dependent phosphodiesterase in the same tissue. The turnover rates for PKI and CDR were estimated following in vivo treatment of SCE rats with cycloheximide (1 mg/kg body wt). PKI activity disappeared with a t1/2 of 10 h whereas CDR activity decreased with a t1/2 of 17 h. The activity of CDR is absolutely dependent on Ca whereas no such modulators were found for PKI. Hypophysectomy (10 days) resulted in a decrease of both testis PKI (4-fold) and CDR (2-fold) activities. Treatment with FSH [follicle stimulating hormone] for 2 or 4 days stimulated the activity of PKI by 2-fold whereas testosterone propionate had no effect. Neither hormone affected the level of CDR in the hypophysectomized animal. The SCE testis contains both PKI and CDR activities. Moreover, despite several physical similarities they are different proteins. FSH may regulate the responsiveness of the Sertoli cell to cAMP via changes in PKI activity.