The long-range objective of the proposed research is to elucidate the complete amino acid sequence of the two major porcine cAMP-dependent protein kinases. In conjunction with sequencing a variety of approaches will be used to identify functional sites on the enzymes. 1) Affinity labeling using nucleotide analogues will be used to covalently modify the ATP and cAMP binding sites on the catalytic and regulatory subunits as well as on the holoenzyme. 2) Limited proteolysis will continue to be used both as a tool for defining functional domains on the regulatory subunits and as a complimentary tool for sequencing. 3) Differential labeling of surface lysine residues with acetic anhydride will be used to identify residues that are involved in subunit interactions in the holoenzyme. The effect of ATP on the reactivity of lysine groups in the C-subunit will also be assessed. 4) The cysteine residues in both subunits will be characterized with respect to reactivity, localization in the polypeptide chain, and functional roles. 5) The antigenic sites on RI and RII will be characterized using both serum antibodies and monoclonal antibodies. Sequencing strategy is based on isolation of CNBr fragments and tryptic peptides. The products of limited proteolysis will also be characterized. Sequencing will be carried out using both solid-phase and manual procedures.
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