蛇毒の酵素化学的研究(第9報)タイワンハブ毒よりアルギニンエステル水解酵素(ME-2)の精製およびその性質

抄録

An arginine ester hydrolase, ME-2 was isolated from the venom of Trimeresurus mucrosquamatus by gel filtration on Sephadex G-100 and ion exchange chromatographies on CM-Sephadex C-50, DEAE-Sephadex A-50, DEAE-Sephacel. By these procedures, 6.7 mg of purified preparation was obtained from lg of crude venom. This enzyme hydrolyzed arginine esters, such as N-tosyl-L-arginine methyl ester (TAME) or N-benzoyl-L-arginine ethyl ester (BAEE), but did not hydrolyze casein, hemoglobin, N-tosyl-L-lysine methyl ester (TLME), N-acetyl-L-tyrosine ethyl ester (ATEE), N-tosyl-L-argininamide (TAA) or N-benzoyl-L-argininamide (BAA). The esterolytic activity was inhibited by benzamidine but not by diisopropyl fluorophosphate (DFP) or trasylol. The purified preparation was homogeneous as judged by disc electrophoresis on polyacrylamide gel and isoelectric focusing. The molecular weight of ME-2 was determined to be approximately 29800, and the isoelectric point was found to be pH 5.62 by isoelectric focusing with carrier ampholyte. The esterolytic activity of the final preparation was 779.8 units/mg. This enzyme had capillary permeability-increasing and kinin-releasing activities. This protein was stable to heat treatment, and between pH 5 and 9. Its Michaelis constant (K_m) for TAME and inhibition constant (K₁) for benzamidine were found to be 4.27×10^-3 M and 0.194×10^-3 M, respectively. This protein contained a small amount of carbohydrates.