Site-specific cyclic nucleotide binding and dissociation of the holoenzyme of cAMP-dependent protein kinase.

The photoaffinity reagent 8-azidoadenosine 3':5'-monophosphate (8-N3cAMP) was previously shown to modify a single tyrosine residue on the type II regulatory subunit of cAMP-dependent protein kinase (Kerlavage, A.R., and Taylor, S.S. (1980) J. Biol. Chem, 255, 8483-8488). In the present studies, the binding stoichiometries of type II holoenzyme for cAMP and 8-N3cAMP were determined using Millipore filtration assays in the absence (Assay A) and presence (Assay B) of 2 M NaCl and histone. The binding stoichiometry of holoenzyme for cAMP was 2 mol/mol with Assay A, and 4 mol/mol with assay B. The binding stoichiometry for 8-N3cAMP was 2 mol/mol with Assay B or with Assay A following photolysis of the holoenzyme:8-N3cAMP mixture. In the absence of photolysis, the binding stoichiometry for 8-N3cAMP was 0.4 mol/mol with Assay A. Both 8-N3cAMP and cAMP fully dissociated the holoenzyme. Holoenzyme, labeled with 8-N3[3H]cAMP on a preparative scale, incorporated 1 mol of 8-N3[3H]cAMP/mol of regulatory subunit (RII) monomer. The labeled RII was separated from catalytic subunit, cleaved with cyanogen bromide, and the resultant peptides were separated by high performance liquid chromatography. A single radioactive peptide was observed which had the same NH2 terminal residue and amino acid composition as the peptide obtained when dissociated RII was labeled with 8-N3cAMP.