Matsui K, Nishioka M, Ikeyoshi M, Matsumura Y, Mori T, Kajiwara T
Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan.
Biochim Biophys Acta. 1998 Feb 5;1390(1):8-20. doi: 10.1016/s0005-2760(97)00159-8.
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.
分离出一种编码黄瓜根脂氧合酶的cDNA，并在大肠杆菌中进行表达。当以用吐温20分散的α-亚麻酸为底物时，该酶在pH 5.5时表现出最高活性，但在pH 8.0以上时活性较低。相反，当以用阿拉伯胶乳化的三亚麻酸为底物时，它在pH 9.0时表现出最高活性。当用不同浓度吐温20分散的亚麻酸进行测定时，随着亚麻酸/吐温20比例的增加，直到反应溶液变浑浊之前几乎看不到活性，而之后活性迅速出现。该酶也可作用于磷脂酰胆碱，尽管脂质体经冻融和超声处理后其活性受到强烈影响。向磷脂囊泡中添加脱氧胆酸可显著提高活性。添加游离脂肪酸也被证明可有效提高活性。在后一种情况下，肉豆蔻酸的活性最高。油酸比棕榈酸更能提高活性。这些证据表明，脂氧合酶严格识别反应混合物中磷脂底物的特定物理状态。然而，随着反应的进行，该酶会不可逆地失活，失活速率受添加剂的影响很大。此外，未观察到消耗的氧气与形成的共轭二烯之间的化学计量关系。(c) 1998爱思唯尔科学出版社B.V.
