Covalent modification of both cAMP binding sites in cAMP-dependent protein kinase I by 8-azidoadenosine 3',5'-monophosphate.
Reconstituted porcine cAMP-dependent protein kinase type I was labeled with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) to study cyclic nucleotide binding and to identify amino acid residues that are either in or in close proximity to the cAMP binding sites. The photoaffinity analogue 8-N3cAMP behaved as cAMP itself with respect to cyclic nucleotide binding. For both cAMP and 8-N3cAMP, 2 mol of nucleotide was bound per mole of type I regulatory subunit monomer (RI), the apparent Kd's observed were approximately 10-17 nM on the basis of either Millipore filtration assays, equilibrium dialysis, or ammonium sulfate precipitation, Scatchard plots showed positive cooperativity, and (4) the Hill coefficients were approximately 1.5-1.6. After photolysis and addition of an excess of cAMP, approximately 1 mol of 8-N3cAMP/mol of RI monomer was covalently incorporated. Tryptic digestion of the labeled protein revealed that two unique tryptic peptides were modified. Proline-271 and tyrosine-371 were identified as the two residues that were covalently modified by 8-N3cAMP in RI. These results contrast with the type II regulatory subunit (RII) where 8-N3cAMP modified covalently a single tyrosine residue [Kerlavage, A. R., & Taylor, S. S. (1980) J. Biol. Chem. 255, 8483-8488]. RI contains two adjacent regions of sequence homology in the COOH-terminal fragment that binds two molecules of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)
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D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; D000249:Adenosine Monophosphate; C021291:8-azidoadenosine 5'-monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin