Biochemical characterization of the soluble alkaline phosphatase isolated from the venomous snake W. aegyptia.
A soluble form of alkaline phosphatase (ALP) has been identified and purified from Walterinnesia aegyptia venom using an HPLC system Gold 126/1667 equipped with Protein PAK 125 and Protein PAK 60 columns. The enzyme was purified 3.4 fold over crude venom with a yield of 37.3%. On SDS-PAGE under non-reduced conditions the purified enzyme showed three bands of 212 kD, 80 kD, and 55 kD. However, under reducing conditions, the enzyme showed two bands of 80 kD and 55 kD. The specific activity of ALP was 24 U/mg with p-nitrophenylephosphate as the substrate. During isoelectric focusing experiments the ALP exhibited two bands focused at pH 6.2 and 6.8, which suggests that either the enzyme exists as two different isoforms or the two bands in IEF may be two subunits of 80 kD and 55 kD. The kinetic parameters (Km and Vmax) and IC50 of ALP inhibition by L-phenylalanine, L-leucine, imidazole, caffeine, orthophosphate and permanganate were also investigated in the present study. Zinc and cyanide ions at a concentration of 15 mM and 10 mM, respectively, completely inhibited the activity of W. aegyptia ALP.
Al-Saleh
Saad S M
SS
Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, P. O. Box 10219, Riyadh 11433, Saudi Arabia. khansamiullah@hotmail.com
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004546:Elapid Venoms / Q000201:enzymology*; D017815:Elapidae; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007525:Isoelectric Focusing; D012995:Solubility
D004546:Elapid Venoms; D004791:Enzyme Inhibitors; D000469:Alkaline Phosphatase