Covalent modification of an adenosine 3':5'-monophosphate binding site of the regulatory subunit of cAMP-dependent protein kinase II with 8-azidoadenosine 3':5'-monophosphate. Identification of a single modified tyrosine residue.
The regulatory subunit of cAMP-dependent protein kinase II (RII) from porcine heart was modified specifically and covalently using the photoaffinity reagent, 8-azidoadenosine 3':5'-monophosphate (8-N3cAMP). In the presence of excess cAMP, the photo-dependent incorporation of 8-N3cAMP was abolished whereas excess AMP and ATP had no effect. A maximum incorporation of 0.5 mol of 8-N3cAMP was achieved/mol of regulatory subunit monomer (Mr = 55,000). This level of incorporation was obtained when the purified regulatory subunit was treated with urea prior to labeling to remove residual bound cAMP. When the regulatory subunit was labeled with radioactive 8-N3cAMP, cleaved with trypsin, and the tryptic peptides mapped in two dimensions, a single major radioactive peptide was observed. Chemical cleavage of the radioactively labeled RII with cyanogen bromide and subsequent chromatography on Sephadex G-50 also yielded a single major peak of radioactivity. The covalently modified cyanogen bromide peptide subsequently was purified to homogeneity using high performance liquid chromatography. Greater than 90% of the radioactivity that was incorporated into the regulatory subunit was recovered in this cyanogen bromide peptide which had the following sequence: Lys-Arg-Asn-Ile-Ser-His-Tyr (cAMP)-Glu-Glu-Cln-Leu-Val-Lys-Hse. When the Edman degradation of this peptide was carried out, the radioactivity derived from the 8-N3cAMP was released with the tyrosine residue at Step 7 identifying this residue as the specific site of attachment of the photoaffinity reagent.
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides; D001665:Binding Sites; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D046911:Macromolecular Substances; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D013552:Swine; D014443:Tyrosine / Q000032:analysis*
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D046911:Macromolecular Substances; D010446:Peptide Fragments; C017042:8-azidoadenosine-3',5'-monophosphate; D014443:Tyrosine; D000242:Cyclic AMP; D011494:Protein Kinases