蛇毒の酵素化学的研究(第8報)タイワンハブ毒より毛細血管透過性亢進作用を有するアルギニンエステル水解酵素(ME-1)の精製とその性質

抄録

An arginine ester hydrolase, ME-1, 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, CM-Sephadex C-50 and DEAE-Sephacel. By these procedures, 36.8 mg of purified preparation was obtained from 1g of crude venom. This enzyme hydrolyzed arginine esters, such as tosyl-l-arginine methyl ester (TAME) or benzoyl-l-arginine ethyl ester (BAEE), but did not hydrolyze casein, hemoglobin, tosyl-l-lysine methyl ester (TLME), acetyl-l-tyrosine ethyl ester (ATEE), tosyl-l-argininamide (TAA) or 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-1 was determined to be approximately 27000, and the isoelectric point was found to be pH 5.95 by isoelectric focusing with carrier ampholyte. The esterolytic activity of the final preparation was 481.9 unit/mg. When this enzyme was injected into the skin of a rabbit, capillary premeability increased distinctly. This protein was stable to heat treatment and between pH 4 and 9. Its michaelis constant (K_m) and inhibition constant (K₁) values for TAME or benzamidine were found to be 4.0×10^-3M and 0.292×10^-3M, respectively. This protein had some carbohydrates.