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

Journal Article · Tue Oct 06 04:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00394a042· OSTI ID:5640110

Wada, H; Osborne, Jr, J C; Manganiello, V C

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-stimulated 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.

• Research Organization: National Institutes of Health, Bethesda, MD
• OSTI ID: 5640110
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:20; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
AMP
ANIMALS
AROMATICS
AZAARENES
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
BODY
CALVES
CATTLE
CHEMICAL REACTIONS
CHEMISTRY
DECOMPOSITION
DIGESTIVE SYSTEM
DOMESTIC ANIMALS
ENZYMATIC HYDROLYSIS
ENZYME ACTIVITY
ENZYMES
ESTERASES
GLANDS
GUANOSINE
HETEROCYCLIC COMPOUNDS
HYDROLASES
HYDROLYSIS
KINETICS
LABELLED COMPOUNDS
LIVER
LYSIS
MAMMALS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PH VALUE
PHOSPHODIESTERASES
PURINES
REACTION KINETICS
RIBOSIDES
RUMINANTS
SOLVOLYSIS
STEREOCHEMISTRY
SUBSTRATES
TRITIUM COMPOUNDS
VERTEBRATES