COMPARTMENTATION OF THE TYPE-I REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN-KINASE IN CARDIAC VENTRICULAR MUSCLE

The species-dependent compartmentation of type I cAMP-dependent protei n kinase (PKA I) and its dissociated regulatory subunit (RI) was exami ned in the heart by biochemical and immunohistochemical means, PKA I a nd RI were resolved from type II cAMP-dependent protein kinase and its regulatory subunit by DEAE-Sephacel chromatography of the supernatant and Triton X-100 soluble particulate fractions of heart homogenates, The relative amounts of holoenzymes and subunits were determined by cA MP-binding, protein kinase, 8-N-3-[P-32]cAMP photoaffinity labeling, a nd Western blot assays, Rat, rabbit, and guinea pig hearts all contain ed PKA I to varying degrees, but only in the supernatant fractions, Si gnificant amounts of dissociated RI were found in the supernatant frac tions, and to a lesser extent the particulate fractions, of these spec ies, In contrast, though no PRA I was detected in the supernatant or p articulate fractions of pig and beef heart, half of the cAMP-binding a ctivity in the particulate fraction was attributed to RI, The results suggest that RI may associate with membrane fractions when it is not a ssociated with the PKA catalytic subunit, Immunohistochemical studies of tissue sections from pig, beef, and rat cardiac ventricle using ant ibodies directed against RI also revealed species-dependent localizati on of RI, Cardiac myocyte intercalated discs were stained in pig and b eef sections with additional sarcolemmal staining in beef sections, Ra t ventricle, which contained large amounts of supernatant PKA I, showe d nuclear staining, The localization of RI to cardiac myocyte intercal ated discs and sarcolemma in certain species suggests a role(s) for th is subunit in mediating cAMP-regulated events in these regions. (C) 19 97 Academic Press.