Properties of a cGMP-dependent monomeric protein kinase from bovine aorta.
A form of cGMP-dependent protein kinase (cGK) that was different from previously described cGK was purified from bovine aorta smooth muscle. The partial amino-terminal sequencing of this enzyme indicated that it was derived by endogenous proteolysis of the type I beta isozyme of cGK. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this form migrated as a smaller protein (Mr = 70,000) than the parent cGK (Mr = 80,000), and since the calculated nondenatured Mr was approximately 89,000 compared to Mr = 170,000 for the dimeric native enzyme, it represented a monomeric form of cGK. The monomer bound approximately 2 mol of [3H]cGMP per mol of monomer, although it had only one rapid component in [3H]cGMP dissociation assays as compared to one rapid and one slow component for the native cGK. The specific catalytic activity of the kinase was similar to that of the native enzyme, suggesting that the catalytic domain was essentially intact. The monomeric cGK incorporated significant 32P when incubated with Mg2+ and [gamma-32P]ATP in the presence of cGMP, although the phosphorylation proceeded at a slower rate than that obtained with native cGK. In contrast to previous reports of monomeric forms of cGK, this monomer was highly cGMP-dependent, although it had a slightly higher Ka (0.8 microM) for cGMP than that of the native enzyme (0.4 microM) and a low Hill coefficient of 1.0 (1.6 for the native enzyme). The cGMP dependence of the monomer did not decrease with dilution, implying that the cGMP dependence was not due to monomer-monomer interactions in the assay. The results indicated that the catalytic domain, cGMP binding domain(s), and inhibitory domain of cGK interact primarily within the same subunit rather than between subunits of the dimer as previously hypothesized for dimeric cGK.
Wolfe
L
L
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232.
Francis
S H
SH
Corbin
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000201:enzymology*; D002417:Cattle; D006152:Cyclic GMP / Q000502:physiology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000648:ultrastructure; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism; D011487:Protein Conformation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D046911:Macromolecular Substances; D009124:Muscle Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; D006152:Cyclic GMP