Effects of pH on allosteric and catalytic properties of the guanosine cyclic 3',5'-phosphate stimulated cyclic nucleotide phosphodiesterase from calf liver.
We have investigated effects of pH on the catalytic and allosteric properties of the cGMP-stimulated cyclic nucleotide phosphodiesterase purified from calf liver. In the "activated" state, i.e., with 0.5 microM [3H]cAMP plus 1 microM cGMP or at saturating substrate concentrations (250 microM [3H]cAMP or [3H]cGMP), hydrolysis was maximal at pH 7.5-8.0 in assays of different pH. Hydrolysis of concentrations of substrate not sufficient to saturate regulatory sites and below the apparent Michaelis constant (Kmapp), i.e., 0.5 microM [3H]cAMP or 0.01 microM [3H]cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 microM [3H]cAMP increased with pH from 7.5 to 9.5, cGMP stimulation of cAMP hydrolysis decreased. As pH increased or decreased from 7.5, Hill coefficients (napp) and Vmax for cAMP decreased. Thus, assay pH affects both catalytic (Vmax) and allosteric (napp) properties. Enzyme was therefore incubated for 5 min at 30 degrees C in the presence of MgCl2 at various pHs before assay at pH 7.5. Prior exposure to different pHs from pH 6.5 to 10.0 did not alter the Vmax or cGMP-stimulated activity (assayed at pH 7.5). Incubation at high (9.0-10.0) pH did, in assays at pH 7.5, markedly increase hydrolysis of 0.5 microM [3H]cAMP and reduce Kmapp and napp. After incubation at pH 10, hydrolysis of 0.5 microM [3H]cAMP was maximally increased and was similar in the presence or absence of cGMP. Thus, after incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulated form. Activation at high pH occurs at 30 degrees C but not 5 degrees C, requires MgCl2, and is prevented but not reversed by ethylenediaminetetraacetic acid.(ABSTRACT TRUNCATED AT 250 WORDS)
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000302:isolation & purification / Q000378:metabolism*; D000494:Allosteric Regulation; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D013379:Substrate Specificity
D000242:Cyclic AMP; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP; D015056:1-Methyl-3-isobutylxanthine