Studies of functional domains of the regulatory subunit from cAMP-dependent protein kinase isozyme I.
Homogenous regulatory subunit from rabbit skeletal muscle cAMP-dependent protein kinase (isozyme I) was partially hydrolyzed with low (1 g/1300 g) or high (1 g/6 g) concentrations of trypsin. After treatment with low trypsin two main peptides (Mr = 35,000 and 12,000) were produced. The cAMP-binding activity (2 mol cAMP/mol of subunit monomer) was recovered in the monomeric Mr = 35,000 peptide. The ability of either fragment to inhibit catalytic subunit activity was lost. Treatment of the regulatory subunit with a high concentration of trypsin yielded three main fragments (Mr = 32,000, 16,000, and 6,000) which could be resolved by Sephadex G-75 and purified further on DEAE-cellulose columns. One of the peptides (Mr = 32,000) bound 2 mol cAMP/mol fragment. The Mr = 16,000 fragment was very labile and bound cAMP with an undetermined stoichiometry. Cyclic AMP dissociation curves for the native regulatory subunit and its Mr = 32,000 component were similar and suggested the presence of two nonidentical binding sites in each monomer. Using the same procedure, the Mr = 16,000 fragment or homogenous cGMP-dependent protein kinase appeared to contain a single type of binding site. Purified Mr = 32,000 fragment was readily converted to the Mr = 16,000 fragment using high trypsin as assessed by protein bands on SDS-disc gels or by following transfer of radioactivity from Mr = 32,000 peptide covalently labeled with 8-N3-[32P] cAMP to radiolabeled Mr = 16,000 fragment. The smallest regulatory subunit fragment (Mr = 6,000) did not bind cAMP, but was dimeric and could be part of the dimerization domain in the native protein. A model is presented to explain the possible structural-functional relationships of the regulatory subunit.
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology*; D010446:Peptide Fragments; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013329:Structure-Activity Relationship; D013552:Swine; D014357:Trypsin
D007527:Isoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin