Properties of a cyclic nucleotide phosphodiesterase of amphibian oocytes that is activated by calmodulin and calcium, by tryptic proteolysis, and by phospholipids.
A calmodulin-Ca2+-stimulated cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which hydrolyzed both cGMP and cAMP has been purified about 2000-fold from ovaries of the amphibian Xenopus laevis. Gel filtration through Sephadex G-200 indicated a molecular weight of 140,000. A single, major protein band of molecular weight 66,000 was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to the stimulation by calmodulin-Ca2+, the enzyme was activated 5- to 10-fold by proteolysis and by certain phospholipids. Trypsin activation of the enzyme caused a reduction in the native molecular weight to 90,000 and a loss of the capacity to be stimulated by calmodulin-Ca2+ or by phospholipids. The phosphodiesterase was stimulated by low concentrations (0.1 microgram/ml) of lysophosphatidylcholine and lysophosphatidylethanolamine. This response did not require calcium ions. Phosphatidylinositol, fatty acids, progesterone, and phospholipase C had little or no effect on activity. Simultaneous addition of 1 mM 2-chloro-10-(3-aminopropyl)phenothiazine and lysophosphatidylcholine to the enzyme did not diminish the stimulatory effect of the phospholipid. The activation of the enzyme by all three agents resulted in an increase in the maximum velocity of the reaction without significant modification of the apparent Km values for cGMP (5 microM) or cAMP (30 microM). It was suggested that trypsin removed an inhibitory domain from the enzyme and that calmodulin and phospholipids interact with this same domain, eliminating its capacity to inhibit the active center of the enzyme.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D006868:Hydrolysis; D009865:Oocytes / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism; D014982:Xenopus laevis
D002147:Calmodulin; D010743:Phospholipids; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D014357:Trypsin; D002118:Calcium