Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species.
Electrophoretically homogeneous preparations of catalytic subunit (C) of cAMP-dependent protein kinase isolated according to two different procedures from holoenzyme type I and type II from rabbit and from holoenzyme type II from rat skeletal muscle and from bovine cardiac muscle can be separated on carboxymethyl cellulose or on a Mono S column (Pharmacia) by salt gradient elution into two enzymatically active peaks called A and B, which do not interconvert on rechromatography. Cochromatography of peak A fractions or of peak B fractions derived from both holoenzymes respectively yields single enzyme peaks in each case, thus indicating that both represent different entities, which were named CA and CB. The separate character of both enzyme forms is supported by the fact that CB under all conditions is degraded faster by the C-specific protease (E. Alhanaty et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3492-3495) than CA, a phenomenon which is enhanced in both enzyme forms by substrate (Kemptide). The separation of both subtypes from each other is probably based on differences in isoelectric values (delta pH less than or equal to 0.5 units). The reason for the charge difference is not presently known. CA and CB do not differ significantly in their phosphate content. No differences between CA and CB have been detectable so far with respect to their migration in SDS gels, kinetic behavior regarding both substrates and cosubstrate, pH dependence, inhibition by regulatory subunits of holoenzyme type I (rabbit skeletal muscle) and of type II (bovine cardiac muscle), and inhibition by specific-heat and acid-stable inhibitor-modulator. The peptide pattern of both forms after limited proteolysis exhibits small differences.
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007525:Isoelectric Focusing; D009132:Muscles / Q000201:enzymology*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000302:isolation & purification*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D013045:Species Specificity
D047428:Protein Kinase Inhibitors; D011494:Protein Kinases