Microheterogeneity of type II cAMP-dependent protein kinase in various mammalian species and tissues

Excluding autophosphorylated species, at least six forms of the regulatory subunit of type II cAMP-dependent protein kinase (RII) from various mammalian tissues were identified by sodium dodecyl sulfate (SDS) gel electrophoresis of purified samples and of crude preparations photoaffinity labeled with 8-azido[32P] cAMP and by gel filtration. After autophosphorylation some heart RII forms termed type IIA (bovine, porcine, equine, and dog) shifted to a more slowly migrating band on SDS gels while others termed type IIB (rat, guinea pig, rabbit, and monkey) did not detectably shift. Both subclasses of RII exhibited variation in apparent Mr on SDS gels. Bovine and porcine heart nonautophosphorylated RII had Mr 56,000 and the autophosphorylated RII had Mr 58,000, while dog and equine heart RII had Mr 54,000 and 56,000 while rabbit and guinea pig heart RII had Mr 52,000. More than one RII was found in different tissues of the same species. Rabbit skeletal muscle contained a Mr 56,000 IIB form. Bovine lung contained almost equal amounts of a IIA form apparently identical to that of bovine heart and a Mr 52,000 IIB form similar to that which predominated in bovine brain. Rat adipose tissue, brain, and monkey heart contained predominantly a Mr 51,000 IIB form. The rat liver Mr 56,000 IIB form chromatographed differently from all other RII tested by gel filtration. Several lines of evidence indicated that the various forms of RII were not derived from one another through proteolysis or other processes. Each of the type II forms rapidly incorporated 0.3-1.0 mol of 32P per mol of subunit when incubated with [gamma-32P]ATP and C subunit. Four of the forms tested were similar in the cAMP concentration dependence for activation of their corresponding holoenzymes and inhibited C subunit about equally. Each exhibited two components of [3H]cAMP dissociation, indicating two intrachain cAMP-binding sites, and the dissociation rates for the respective sites were similar.