CAMP-dependent protein kinase isozymes in porcine follicles and corpora lutea

Abstract

Our purpose was to identify regulatory (R) subunits and their associations with catalytic (C) subunits to form cAMP-dependent protein kinase (A-kinase) holoenzymes in select porcine ovarian tissues during follicular differentiation. Soluble extracts of small and preovulatory follicles and corpora lutea (CL) were separated on DEAE-cellulose chromatography. R subunits were labeled with 8-N 3[ 32P]cAMP or with [α- 32P]ATP under RII autophosphorylation conditions and were identified by molecular weight ( M r) determination on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as well as cross-reactivity of unlabeled subunits with anti-R antibodies. A-kinase holoenzymes and C subunit-free R (free R) subunits were distinguished on the basis of DEAE elution position and sedimentation position on sucrose density gradient centrifugation of phosphotransferase and [ 3H]cAMP binding activities. In small and preovulatory follicles and CL we identified a minor peak of type I A-kinase containing RIα ( M r = 47,000) and a major peak of type II A-kinase holoenzyme containing two RII isoforms ( M r = 52,000 and 56,000). Notable amounts of free RIα eluted between the type I and II holoenzymes in all three tissues. Neither of the holoenzymes nor free RIα was regulated as a function of follicular differentiation or CL formation. In contrast, free RII subunits were moderately increased in preovulatory follicles relative to levels in small follicles and CL. We conclude that only the RII subunits are hormonally regulated in developing follicles, and in tissues which express both RI and RII subunits, the RII subunits preferentially associate with C subunits to form the dominant holoenzyme despite the presence of significant amounts of RI.