Cucumber root lipoxygenase can act on acyl groups in phosphatidylcholine.
A cDNA encoding cucumber root lipoxygenase was isolated and expressed in E. coli. The enzyme showed highest activity at pH 5.5 when alpha-linolenic acid dispersed with Tween 20 was used as a substrate but showed little activity at above pH 8.0. On the contrary, it showed the highest activity at pH 9.0 with trilinolenin emulsified with gum arabic. When the assay was performed with linolenic acid dispersed with different concentrations of Tween 20, little activity which could be seen up to the reaction solution became turbid as the linolenic acid/Tween 20 ratio increased, while the activity rapidly emerged afterward. The enzyme could also act on phosphatidylcholine, although the activity was strongly modified by freeze-thaw and sonication treatment on the lipid vesicles. Addition of deoxycholic acid to the phospholipid vesicles drastically enhanced the activity. Addition of free fatty acid was also revealed to be effective to enhance the activity. In the latter case, myristic acid exerted highest activity. Oleic acid enhanced the activity more highly than palmitic acid did. These lines of evidence suggested that the lipoxygenase strictly recognized a specific physical state of the phospholipid substrate in the reaction mixture. The enzyme was irreversibly inactivated as the reaction proceeded, however, the rate of the inactivation was much influenced by the additives. Furthermore, stoichiometry between consumed oxygen and formed conjugated diene could not be observed. (c) 1998 Elsevier Science B.V.
Matsui
K
K
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan. matsui@agr.yamaguchi-u.ac.jp
Nishioka
M
M
Ikeyoshi
M
M
Matsumura
Y
Y
Mori
T
T
Kajiwara
T
T
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018553:Cucumis sativus / Q000201:enzymology*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D018744:DNA, Plant / Q000737:chemistry / Q000302:isolation & purification; D003840:Deoxycholic Acid / Q000494:pharmacology; D004926:Escherichia coli; D005227:Fatty Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008084:Lipoxygenase / Q000378:metabolism*; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism*; D018517:Plant Roots / Q000201:enzymology; D013499:Surface Properties
D018076:DNA, Complementary; D018744:DNA, Plant; D005227:Fatty Acids; D010713:Phosphatidylcholines; D003840:Deoxycholic Acid; D008084:Lipoxygenase