Purification and characterization of phosphodiesterase i from Walterinnesia aegyptia venom

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°C, respectively. The energy of activation (Ea) was 96.4, the V(max) and K(m) were 1.14 µM/min/mg and 1.9 × 10(-3) M, respectively, and the K(cat) and K(sp) were 7 s(-1) and 60 M(-1) min(-1) respectively. Cysteine was a noncompetitive inhibitor, with K(i) = 6.2 × 10(-3) M and an IC(50) of 2.6 mM, whereas adenosine diphosphate was a competitive inhibitor, with K(i) = 0.8 × 10(-3) M and an IC(50) of 8.3 mM. Glutathione, o-phenanthroline, zinc, and ethylenediamine tetraacetic acid (EDTA) inhibited PDE-I activity whereas Mg(2+) 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 4.0 mg/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.