A specific substrate from rabbit cerebellum for guanosine 3':5'-monophosphate-dependent protein kinase. II. Kinetic studies on its phosphorylation by guanosine 3':5'-monophosphate-dependent and adenosine 3':5'-monophosphate-dependent protein kinases.
Kinetic studies on the activity of purified cGMP-dependent protein kinase and catalytic subunit of cAMP-dependent protein kinase have been carried out using a protein termed G-substrate (see preceding paper) as the phosphate acceptor. Each enzyme catalyzed the phosphorylation of 2.0-2.1 mol of 32P/mol of G-substrate, with phosphorylation occurring primarily at threonine residues. When phosphorylation was carried out in the simultaneous presence of the two enzymes, the stoichiometry increased only slightly, to a value of 2.4, suggesting that both enzymes phosphorylated the same two sites. Initial rate studies on the phosphorylation of G-substrate by cGMP-dependent protein kinase yielded a Km of 0.21 microM and a Vmax of 2.2 mumol/min/mg. Similar studies with the cAMP-dependent protein kinase yielded a Km of 5.8 microM and a Vmax of 2.3 mumol/min/mg. cGMP-dependent protein kinase thus exhibited a high degree of specificity towards this substrate which was apparently based on selective substrate binding rather than catalytic efficacy. The activity of cGMP-dependent protein kinase towards G-substrate was maximal at pH 7.5-8.0 and a Mg2+ concentration of 1-3 mM. Activity declined sharply at high ionic strength (greater than 20 mM KCl).
D000818:Animals; D002417:Cattle; D002531:Cerebellum / Q000032:analysis*; D000242:Cyclic AMP / Q000494:pharmacology; D006152:Cyclic GMP / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
C029152:G-substrate; D009419:Nerve Tissue Proteins; D015636:Magnesium Chloride; D011189:Potassium Chloride; D000242:Cyclic AMP; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium