Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde.
Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)
D000447:Aldehydes / Q000494:pharmacology*; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D046911:Macromolecular Substances; D047428:Protein Kinase Inhibitors; D012265:Ribonucleotides / Q000494:pharmacology; D009764:o-Phthalaldehyde / Q000494:pharmacology*
D000447:Aldehydes; D002413:Cations, Divalent; D046911:Macromolecular Substances; D047428:Protein Kinase Inhibitors; D012265:Ribonucleotides; D004533:Egtazic Acid; D009764:o-Phthalaldehyde; D004492:Edetic Acid; D006152:Cyclic GMP