Properties of the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart.
In the absence of activator, the activator-dependent cyclic nucleotide phosphodiesterase (EC 3.1.4.-) from bovine heart hydrolyzed 3.2-fold more cyclic GMP than cyclic AMP when assayed with 10(-6) M substrate in the presence of 5 mM Mg2+ and 10 muM Ca2+. The addition of saturating amounts of phosphodiesterase activator increased the hydrolysis of cyclic GMP 8-fold and cyclic AMP 6-fold. The pH maxima of the enzyme was rather broad from 6.0 to 7.0 for the hydrolysis of both cyclic GMP and cyclic AMP in the absnece or presence of phosphodiesterase activator. Substrate specificity of the enzyme in the absence or presence of phosphodiesterase activator varied depending on the divalent metal used to support activity in the absence of activator. The enzyme preferentially hydrolyzed cyclic GMP in the presence of Mg2+ while little substrate specificity was observed in the presence of Mn2+, Zn2+, Co2+ or Ni2+. The magnitude of the increase in enzyme activity due to activator and Ca2+ varied depending on the divalent metal used to support enzyme activity without activator. The addition of activator increased the V of cyclic AMP hydrolysis 1.7-fold while causing no significant change in the Km for cyclic AMP. Two apparent Km values for cyclic GMP (1 and 15 muM) were noted in the absence of activator and upon the addition of activator a single apparent Km (3 muM) was observed with a V approx. 2-fold above that in the absence of activator. Cyclic AMP competitively inhibitied the hydrolysis of cyclic GMP with a ki of 50 muM while cyclic GMP non-competitively inhibited the hydrolysis of cyclic AMP with a Ki of 1.8 muM. The results suggest there may be two or more binding sites for cyclic GMP on the enzyme and possibly only one binding site for cyclic AMP.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D006152:Cyclic GMP; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009124:Muscle Proteins / Q000502:physiology*; D009206:Myocardium / Q000201:enzymology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*
D009124:Muscle Proteins; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D008274:Magnesium; D002118:Calcium