Isozymes of cAMP-dependent protein kinase present in the rat corpus luteum

Regulatory (R) subunits and their association with catalytic subunits to form cAMP-dependent protein kinase holoenzymes were investigated in corpora lutea of pregnant rats. Following separation by DEAE-cellulose chromatography, R subunits were identified by labeling with 8-N₃[³²P]cAMP and autophosphorylation on one and two-dimensional gel electrophoresis and by reactivity with antisera. DEAE-cellulose elution of R subunits with catalytic subunits as holoenzymes or without catalytic subunits was determined by sedimentation characteristics on sucrose density gradient centrifugation and by cAMP-stimulated kinase activation characteristics on Eadie-Scatchard analysis. We identified the presence of a type I holoenzyme containing RIα (M_r 47,000) subunits, a prominent type II holoenzyme containing RIIβ (M_r 52,000) subunits, and a second more acidic type II holoenzyme peak containing both RIIβ and RIIα (M_r 54,000) subunits. However, the majority of total R subunit activity was associated with a catalytic subunit-free peak of RIα protein which on elution from DEAE-cellulose was associated with cAMP. This report establishes the more basic elution position from DEAE-cellulose of the prominent rat luteal RIIβ holoenzyme in very close proximity to free RIα and presents one of the few reports of a normal tissue containing a large percentage of catalytic subunit-free RIα.