Characterization of a novel isozyme of cGMP-dependent protein kinase from bovine aorta.
Two isozymic forms of cGMP-dependent protein kinase (designated types I alpha and I beta) were purified to homogeneity from bovine aorta smooth muscle. Type I alpha was apparently the same as the well characterized bovine lung cGMP-dependent protein kinase. Type I beta had a subunit Mr = 80,000 compared with Mr = 78,000 for type I alpha, and both forms were dimeric with similar calculated native Mr (170,000-178,000). Both enzymes contained two cGMP-binding sites per subunit, exhibited similar specificities for the peptide substrates tested, photoaffinity labeled with 8-N3[32P] cAMP, and catalyzed autophosphorylation. Silver-stained peptide maps of types I alpha and I beta were similar but not identical; however, autoradiographs of peptide maps of these enzymes prelabeled by either autophosphorylation or photoaffinity labeling showed clearly different patterns. The amino-terminal sequence of a breakdown product of type I beta could not be aligned confidently with any of the published sequence of bovine lung cGMP-dependent protein kinase. [3H]cGMP dissociation curves for types I alpha and I beta were both biphasic, but the dissociation rate of the slow component of type I beta was faster than the corresponding component of type I alpha. The concentration of cGMP required for half-maximal activation (K alpha) was slightly lower for type I alpha than for type I beta (0.29 and 0.44 microM, respectively), and the two enzymes had similar K alpha values for cAMP (16 and 18 microM, respectively). Types I alpha and I beta exhibited different K alpha values for several cGMP analogs. The abundance of type I beta in specific tissues suggested that it could have an important physiological role.
Wolfe
L
L
Department of Molecular Physiology, Vanderbilt University, Nashville, Tennessee 37232-0295.
Corbin
J D
JD
Francis
S H
SH
D000818:Animals; D001011:Aorta / Q000201:enzymology; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002852:Chromatography, Ion Exchange; D006152:Cyclic GMP / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010449:Peptide Mapping; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D009712:Nucleotides, Cyclic; D011494:Protein Kinases; D006152:Cyclic GMP