INTERCHAIN DISULFIDE BONDING IN THE REGULATORY SUBUNIT OF CAMP-DEPENDENT PROTEIN KINASE-I

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  • 1 January 1982
    • journal article
    • research article
    • Vol. 257  (5) , 2287-2293
Abstract
The 2 protomers of the purified regulatory subunit from porcine skeletal muscle cAMP-dependent protein kinase I were shown to be covalently cross-linked by interchain disulfide bonding. Limited proteolysis which cleaves the polypeptide chain into 2 fragments demonstrated that the disulfide bonding was associated exclusively with the fragment that corresponded to the NH2-terminal region of the polypeptide chain. This NH2-terminal fragment accounted for .apprx. 15-20% of the molecule. The disulfide bonding was further characterized by alkylating the cysteines in the native regulatory subunit. Following oxidation with performic acid, each regulatory subunit contained 7 cysteic acid residues; however, under denaturing conditions, but without prior reduction, only 5 cysteine residues could be alkylated with iodoacetic acid. Following limited proteolysis, all 5 of these cysteines were associated with the larger COOH-terminal, cAMP binding domain. In contrast, if the denatured subunit was first reduced prior to alkylation, all 7 cysteine residues were alkylated. The 2 cysteines that were only accessible to alkylation after prior reduction were both associated with the NH2-terminal end of the polypeptide chain ultimately with a 5400 peptide. Alkylation of the isolated, denatured NH2-terminal domain with iodoacetic acid resulted in no covalent modification unless the fragment was first reduced with dithiothreitol. The NH2-terminal and COOH-terminal domains were shown to be linked by a region of the polypeptide chain that is rich in both proline and arginine. It is the arginine-rich site that is readily prone to proteolytic cleavage.