Studies on Characterization and Utilization of Wheat Germ Lipoxygenase

1997 Fiscal Year Final Research Report Summary

• Project/Area Number: 07660172
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 食品科学・栄養科学
• Research Institution: Osaka Women's University
• Principal Investigator: NISHIYAMA Junko Osaka Women's University Faculty of Arts and Sciences Professor, 学芸学部, 教授 (00046583)
• Project Period (FY): 1995 – 1997
• Keywords: Lipoxygenase / Wheat / Liposome / Detergent

Research Abstract

Wheat germ lipoxygenase (W-LOX) was purified by chromatogtaphy techniques. SDS-PAGE exhibited an almost single band. The purified enzyme was characterized, optimum pH of 6.6, molecular weight of 95,000 and Km value of 30 muM toward linoleic acid. The W-LOX contained three isozymes with the same mobility on SDS-PAGE.
The effects of nonionic and ionic detergents on W-LOX were investigated. The activity of enzyme decreased rapidly with an increase in the concetration of these detergents. Especially, Tween 20 and Triton x-100 showed strong inhinition but not for soybean lipoxygenase L-1 (S-LOX). The sucrose ester (SE) showed inhibitory effect increased with an increasing in the carbon chain number of the fatty acid up to 12 (C_<12>). Kinetic study showed that the SE with C_<12> inhibited competitively with Ki of 0.36mM,same as for S-LOX.These results indicate that the W-LOX is labile against detergents but the interaction with SE is similar to S-LOX.
The linoleic acids embedded in the liposomes of soybean phosphatidylcoline (soy-PC) and dipalmitoylphosphatidylcoline (DPPC) were served as the substrate for enzymatic peroxidation. The mean diameter of the liposomes was 31nm. The relationship of the initial velosity and the substrate concentration followed Michaelis-Menten's equation. The turnover of W-LOX for the liposomal substrate was smaller than the miceller one. The v vs. [S] curves shifted with varying molar ratio of linoleic acid to soy-PC in liposomes. The same behaviors were observed in the system consisting of S-LOX and liposomal linoleic acid. The Soy-PC competitively inhibited the S-LOX with the Ki value of 0.20mM,whereas DPPC was no inhibitory effect. The difference in the effect of soy-PC and DPPC on the catalitic activity indicates that the reactivity of LOX toward liposomal substrates depends on the morphology of liposome.