Lankin V Z, Kühn H, Hiebsch C, Schewe T, Rapoport S M, Tikhaze A K, Gordeeva N T
Biomed Biochim Acta. 1985;44(5):655-64.
The oxygenation of concentrated emulsions (about 260 microM) of arachidonic acid or linoleic acid catalyzed by the purified lipoxygenase from rabbit reticulocytes is strongly stimulated in the presence of low concentrations of beef heart submitochondrial particles or other membrane preparations. Maximal stimulation was observed at a proportion of about 5 mumoles polyenoic acid per mg of membrane protein. Whereas at a constant ratio of arachidonic acid and membranes the reaction rate obeyed the Michaelis-Menten kinetics with an apparent Km value of 75 microM for arachidonic acid, sigmoid kinetics was observed under conditions of constant concentration of membranes and, consequently, varying proportions of arachidonate and membranes. Under conditions of maximal stimulation binding of reticulocyte lipoxygenase to the membranes was insignificant. Moreover, one-third of the arachidonic acid sedimented during ultracentrifugation of the membranes as judged from experiments with [14C] arachidonic acid. From other sedimentation experiments and from the comparison with soybean lipoxygenase which is not stimulated by membranes it is concluded that the emulsion droplets interacting with the membranes are the substrate of the stimulated reaction. The stimulation may be brought about by facilitating the susceptibility of the fatty acids to reticulocyte lipoxygenase. The membrane-stimulated reaction of reticulocyte lipoxygenase was not inhibited by the antioxidant 2,6-di-t-butyl-4-hydroxytoluene (BHT) excluding the participation of free radicals. The stimulatory effect of membranes seems not to be related to their susceptibility towards lipoxygenase attack which varied strongly in order mitochondrialmembranes greater than endoplasmic membranes greater than erythrocyte ghosts. The stimulation by membranes resembles that produced by the detergent sodium cholate at concentrations near to the critical micellar concentration. Moreover, the stimulations by membranes and by cholate do not behave synergistically or abolish each other. The stimulatory effect of membranes did not occur at a proportion of 200 nmoles per mg of membrane protein.
在低浓度的牛心亚线粒体颗粒或其他膜制剂存在的情况下，兔网织红细胞纯化的脂氧合酶催化的花生四烯酸或亚油酸浓缩乳液（约260微摩尔）的氧合作用受到强烈刺激。在每毫克膜蛋白约5微摩尔多不饱和脂肪酸的比例下观察到最大刺激。在花生四烯酸和膜的比例恒定的情况下，反应速率遵循米氏动力学，花生四烯酸的表观Km值为75微摩尔，而在膜浓度恒定且因此花生四烯酸盐和膜的比例不同的条件下观察到S形动力学。在最大刺激条件下，网织红细胞脂氧合酶与膜的结合不明显。此外，根据用[14C]花生四烯酸进行的实验判断，在膜超速离心过程中有三分之一的花生四烯酸沉淀。从其他沉降实验以及与不受膜刺激的大豆脂氧合酶的比较得出结论，与膜相互作用的乳液滴是受刺激反应的底物。这种刺激可能是通过促进脂肪酸对网织红细胞脂氧合酶的敏感性来实现的。网织红细胞脂氧合酶的膜刺激反应不受抗氧化剂2,6-二叔丁基-4-羟基甲苯（BHT）的抑制，排除了自由基的参与。膜的刺激作用似乎与其对脂氧合酶攻击的敏感性无关，其敏感性变化很大，顺序为线粒体内膜大于内质网大于红细胞血影。膜的刺激作用类似于在接近临界胶束浓度的浓度下洗涤剂胆酸钠产生的刺激作用。此外，膜和胆酸盐的刺激作用不会协同或相互消除。在每毫克膜蛋白200纳摩尔的比例下未出现膜的刺激作用。
