Characterization of small cAMP-binding fragments of cAMP-dependent protein kinases.
Monomeric cAMP-binding fragments of molecular mass 16,000 and 14,000 daltons were obtained by Sephadex G-75 chromatography of partially trypsin-hydrolyzed regulatory subunits of cAMP-dependent protein kinase isozymes I and II, respectively. The Stokes radii were 19.1 and 16.4 A, the frictional ratios were 1.15 and 1.03, and the sedimentation coefficients were 1.94 and 1.91 S for the 16,000- and 14,000-dalton fragments, respectively. The 16,000-dalton fragment retained specific cyclic nucleotide binding characteristics of the native protein. The specificity of cyclic nucleotide binding to the 14,000-dalton fragment (cAMP greater than cIMP = 8-bromo-cAMP = 8-oxo-cAMP greater than cUMP = cGMP) differed from that of the native subunit (cAMP = 8-oxo-cAMP greater than 8-bromo-cAMP greater than cIMP greater than cUMP = cGMP). The 14,000-dalton fragment bound nearly 1 mol of cAMP/mol of fragment. The binding exchange rate of cAMP was much faster for the 14,000-dalton fragment than for either of the native regulatory subunits or for the 16,000 dalton fragment. Although hemin inhibited cAMP binding to the native regulatory subunits and to the 16,000 dalton fragment, the molecule did not affect cAMP binding to the 14,000-dalton fragment. Both of the native regulatory subunits and the isolated 16,000- and 14,000-dalton fragments could be covalently labeled with the photoaffinity analog, 8-N3-[32P]cAMP. The 14,000-dalton fragment could not be phosphorylated and neither fragment could recombine with the catalytic subunit to inhibit its activity. The results indicate that the functional entities of the regulatory subunit other than cAMP binding are destroyed by trypsin. The properties of the 16,000-dalton fragment suggest that the intact cAMP-binding site is contained in a small trypsin-resistant "core" of the native regulatory subunit. The properties of the 14,000-dalton fragment imply that part of the binding site of the native regulatory subunit was slighlty modified or lost during preparation of this fragment.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D014357:Trypsin
D007527:Isoenzymes; D010446:Peptide Fragments; D000242:Cyclic AMP; D011494:Protein Kinases; D014357:Trypsin