cAMP-dependent protein kinases in the rat testis: regulatory and catalytic subunit associations

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Abstract

Based upon recent reports that the rat testis exhibits mRNAs for cAMP-dependent protein kinase (A-kinase) regulatory (R) subunits RIα, RIIβ, RIIα, and RIIβ, this study was designed to identify R proteins present in extracts of germ cell-rich testis from adult and Sertoli cell-enriched, germ cell-poor testis from 14–15-day-old rats. Following separation by DEAE-cellulose. R subunits were identified by Mr: (a) upon labeling with 8-N3[32P]cAMP and 32P in an RII phosphorylation reaction and; (b) by Western blot analysis using R-specific antibodies on one- and two-dimensional gel electrophoresis. Elution of R subunits as catalytic (C) subunit-free dimers or in association with C subunits to form holoenzyme was determined by their sedimentation characteristics on sucrose gradient centrifugation in conjunction with their cAMP-stimulated activation characteristics on Eadie-Scatchard analysis. Soluble extracts of testes, from both adult and 14–15 day-old rats, showed the presence of a prominent type I holoenzyme containing RIα subunits (47 kDa, peak 1), a minor type II holoenzyme, containing RIIβ subunits (52 kDa, peak 2), and a second, more abundant, type II holoenzyme peak containing predominately RIIα and, to a lesser extent RIIβ subunits (peak 3). The 53 kDa RIβ protein predicted by mRNA studies was only tentatively identified by Western blot analysis. Testes extracts of 14–15-day-old, but not adult, rats exhibited high levels of C subunit-free RIα, a result not predicted by mRNA studies. This latter results may be attributable to direct RIα regulation or to indirect RIIβ regulation at a time during testis development prior to germ cell maturation.

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    This work was supported by National Institutes of Health grant PO1 HD 21921 (M.H.D.). Preliminary results were presented at the 21st Annual Meeting of the Society for the Study of Reproduction, Seattle, WA, August 1–4, 1988.

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