Regdel D, Schewe T, Rapoport S M
Biomed Biochim Acta. 1985;44(10):1411-28.
A lipoxygenase preparation was obtained from dried green pea seeds. Disc electrophoresis with enzyme staining indicated the presence of only one main isoenzyme corresponding to the isoenzyme PL I according to Yoon and Klein (J. Agric. Food Chem. 24, 955 (1979)), whereas PL II was absent. The assay for pea lipoxygenase has been optimized by using a final concentration of 0.53 mM potassium linoleate in the presence or absence of 0.2% sodium cholate. Without detergent the rho H optimum was 5.9, in its presence 6.8. The formation of conjugated dienes absorbing at 234 nm accounted for 75% of the oxygen uptake. The difference is mainly due to the aerobic formation of oxodienoic acids absorbing at 285 nm via a lipohydroperoxidase activity concomitant with the dioxygenase reaction. Other lipohydroperoxidase products were formed only to a minor extent under aerobic conditions, whereas in the anaerobic lipohydroperoxidase reaction of pea lipoxygenase in the system 13L8-hydroperoxylinoleic acid/linoleic acid fatty acid dimers containing conjugated double bonds were formed additionally. The pea enzyme showed self-inactivation at 37 degrees C, but in contrast to the lipoxygenase from rabbit reticulocytes the self-inactivation appeared only syncatalytically during the aerobic reaction. The antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT, 1 mM) did not protect from self-inactivation. In contrast to the lipoxygenase from soybeans, wheat and rabbit reticulocytes the pea lipoxygenase caused a co-oxidation of Cu-chlorophyllin in the presence of linoleate at 5 degrees C. The co-oxidation was completely inhibited by 1 mM BHT which did not inhibit the dioxygenation of linoleate at this temperature. Unlike the reticulocyte enzyme the pea lipoxygenase failed to attack mitochondrial membranes or to produce inhibition of the respiratory chain. The results lead to the conclusion that a simple classification of lipoxygenases in type I and type II enzymes is not justified. A reaction scheme is proposed to explain both the co-oxidative activity and the aerobic formation of oxodienoic acids by pea lipoxygenase, presuming the dissociation of a linoleic acid radical from the ferrous lipoxygenase as a side reaction of the catalytic cycle.
从干燥的青豌豆种子中获得了一种脂氧合酶制剂。用酶染色的圆盘电泳表明，根据Yoon和Klein（《农业与食品化学杂志》24, 955 (1979)）的研究，仅存在一种与同工酶PL I相对应的主要同工酶，而PL II不存在。通过在存在或不存在0.2%胆酸钠的情况下使用终浓度为0.53 mM的亚油酸酸钾，优化了豌豆脂氧合酶的测定方法。在没有去污剂的情况下，最佳pH值为5.9，在有去污剂的情况下为6.8。在234 nm处吸收的共轭二烯的形成占氧气摄取量的75%。这种差异主要是由于在双加氧酶反应的同时，通过脂氢过氧化物酶活性有氧形成了在285 nm处吸收的氧代二烯酸。在有氧条件下，其他脂氢过氧化物酶产物仅少量形成，而在豌豆脂氧合酶在13L8 - 氢过氧亚油酸/亚油酸系统中的厌氧脂氢过氧化物酶反应中，额外形成了含有共轭双键的脂肪酸二聚体。豌豆酶在37℃下表现出自我失活，但与兔网织红细胞中的脂氧合酶不同，自我失活仅在有氧反应期间协同催化地出现。抗氧化剂2,6 - 二叔丁基 - 4 - 羟基甲苯（BHT，1 mM）不能防止自我失活。与大豆、小麦和兔网织红细胞中的脂氧合酶不同，豌豆脂氧合酶在5℃下亚油酸存在时会导致叶绿素铜钠盐的共氧化。1 mM BHT完全抑制了这种共氧化，但在该温度下不抑制亚油酸的双加氧反应。与网织红细胞酶不同，豌豆脂氧合酶不会攻击线粒体膜或抑制呼吸链。结果得出结论，将脂氧合酶简单分类为I型和II型酶是不合理的。提出了一个反应方案来解释豌豆脂氧合酶的共氧化活性和氧代二烯酸的有氧形成，假定从亚铁脂氧合酶上解离亚油酸自由基是催化循环的一个副反应。
