Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets.
A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band Mr = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg2+ and Mn2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S0.5 values of 50 +/- 12 microM for cyclic AMP and 35 +/- 15 microM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.
Grant
P G
PG
Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140.
Mannarino
A F
AF
Colman
R W
RW
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000097:blood* / Q000302:isolation & purification; D001792:Blood Platelets / Q000201:enzymology*; D002147:Calmodulin / Q000502:physiology; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D006152:Cyclic GMP / Q000502:physiology*; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics
D002147:Calmodulin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP