title: 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.
authors.affiliation: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia. ssaleh@ksu.edu.sa
authors.forename: Saad S M
authors.initials: SS
authors.lastname: Al-Saleh
authors.forename: Samiullah
authors.initials: S
authors.lastname: Khan
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D000925:Anticoagulants / Q000302:isolation & purification* / Q000494:pharmacology*; D003545:Cysteine / Q000378:metabolism; D004546:Elapid Venoms / Q000737:chemistry* / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008970:Molecular Weight; D043264:Phosphodiesterase I / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000494:pharmacology* / Q000633:toxicity; D012911:Snakes; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000925:Anticoagulants; D004546:Elapid Venoms; D000244:Adenosine Diphosphate; D043264:Phosphodiesterase I; D003545:Cysteine