Abstract
A phosphodiesterase I (EC 3.1.4.1; PDE-I) was purified from Walterinnesia aegyptia venom by preparative native polyacrylamide gel electrophoresis (PAGE). A single protein band was observed in analytical native PAGE and sodium dodecyl sulfate (SDS)-PAGE. PDE-I was a single-chain glycoprotein with an estimated molecular mass of 158 kD (SDS-PAGE). The enzyme was free of 5'-nucleotidase and alkaline phosphatase activities. The optimum pH and temperature were 9.0 and 60 degrees C, respectively. The energy of activation (Ea) was 96.4, the Vmax and Km were 1.14 mu M/min/mg and 1.9x10-3M, respectively, and the Kcat and Ksp were 7s-1 and 60M-1min-1 respectively. Cysteine was a noncompetitive inhibitor, with Ki=6.2x10-3M and an IC50 of 2.6mM, whereas adenosine diphosphate was a competitive inhibitor, with Ki=0.8x10-3 M and an IC50 of 8.3mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg2+ slightly potentiated the activity. PDE-I hydrolyzed thymidine-5'-monophosphate p-nitrophenyl ester most readily, whereas cyclic 3'-5'-AMP was least susceptible to hydrolysis. PDE-I was not lethal to mice at a dose of 4.0mg/kg, ip, but had an anticoagulant effect on human plasma. These findings indicate that W. aegyptia PDE-I shares various characteristics with this enzyme from other snake venoms.