Effects of pH on allosteric and catalytic properties of the guanosine cyclic 3',5'-phosphate stimulated cyclic nucleotide phosphodiesterase from calf liver.

The authors have investigated effects on 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 ..mu..M (/sup 3/H)cAMP plus 1 ..mu..M cGMP or at saturating substrate concentrations (250 ..mu..M (/sup 3/H)cAMP or (/sup 3/H)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 (K/sub m/sup app/), i.e., 0.5 ..mu..M (/sup 3/H)cAMP or 0.01 ..mu..M (/sup 3/H)cGMP, was maximal at pH 9.5. Although hydrolysis of 0.5 ..mu..M (/sup 3/H)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 (n/sub app/) and V/sub max/ for cAMP decreased. Thus, assay pH affects both catalytic (V/sub max/) and allosteric (n/sub app/) properties. Enzyme was therefore incubated for 5 min at 30/sup 0/C in the presence of MgCl/sub 2/ 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 V/sub max/ or cGMP-stimulated activity. After incubation at high pH, the phosphodiesterase acquires characteristics of the cGMP-stimulatedmore » form. Activation at high pH occurs at 30/sup 0/C but not 5/sup 0/C, requires MgCl/sub 2/, and is prevented but not reversed by ethylenediaminetetraacetic acid. These results indicate that incubation at high pH in the absence of substrates and/or effectors promotes allosteric transitions (n/sub app/) and a decrease in K/sub m/sup app/ in the absence of changes in V/sub max/ and suggest independent regulation of topographical features and domains responsible for these properties.« less