EFFECT OF LIPID-PEROXIDATION ON MEMBRANE-BOUND CA2+-ATPASE ACTIVITY OF THE INTESTINAL BRUSH-BORDER MEMBRANES
EFFECT OF LIPID-PEROXIDATION ON MEMBRANE-BOUND CA2+-ATPASE ACTIVITY OF THE INTESTINAL BRUSH-BORDER MEMBRANES
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DOI：
10.1016/0005-2736(89)90210-1
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发表时间：
1989-09-04
期刊：
影响因子：
--
通讯作者：
OHYASHIKI, T
中科院分区：
文献类型：
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作者：
OHTA, A;MOHRI, T;OHYASHIKI, T
本文用二硫苏糖醇（DTT）/Fe ~（2+）和叔丁基过氧化氢（t-BuOOH）组成的氧自由基发生体系研究了猪肠刷状缘膜的脂质过氧化和Ca ~（2+）-ATP酶活性。脂质过氧化反应的速度测定硫代巴比妥酸反应物质（TBAR）和共轭二烯的形成。DTT/Fe ~（2+）对Ca ~（2+）-ATP酶活性的抑制分别为20%和50%。抑制程度取决于过氧化氢浓度。硫脲的加入有效地保护了Ca ~（2+）-ATP酶的活性，而过氧化氢酶和超氧化物歧化酶对ATP酶活性的保护作用分别很小和没有。ATP酶活性、ATP和Ca ~（2+）浓度的动力学研究结果表明，这些氧化剂处理后酶活性的降低主要是由于Vmax值的降低。巯基试剂如N-乙基马来酰亚胺、单碘乙酸酯和单碘乙酰胺修饰膜蛋白中的SH基团不引起Ca ~（2+）-ATP酶活性的抑制。从这些结果，它是建议，抑制膜的ATP酶活性的DTT/Fe ~（2+）在存在和不存在的t-BuOOH的处理是依赖于脂质过氧化和氧化修饰的SH基团可能不直接参与ATP酶活性的损失。此外，芘标记的膜的荧光各向异性测量的结果表明，在Ca 2 +-ATP酶活性的变化是部分相关的膜脂质流动性的降低。
We have studied lipid peroxidation and Ca2+-ATPase activity of the porcine intestinal brush-border membranes using a oxygen-radical-generating system consisting of dithiothreitol (DTT)/Fe2+ and tert-butyl hydroperoxide (t-BuOOH). The rates of lipid peroxidation were measured by formation of thiobarbituric acid-reactive substances (TBAR) and conjugated diene. Incubation of the membranes with DTT/Fe2+ in the absence and presence of t-BuOOH resulted in a slight (about 20%) and a marked (about 50%) inhibition of Ca2+-ATPase activity, respectively. The degree of inhibition was dependent on the hydroperoxide concentration. Addition of thiourea effectively protected Ca2+-ATPase activity but catalase and superoxide dismutase showed a slight and no effect on protection of the ATPase activity, respectively. Results of kinetic studies on the ATPase activity ATP and Ca2+ concentrations revealed that the decrease in the enzyme activity by treatment with these oxidizing agents is mainly due to decrease of the Vmax value. Modification of SH groups in the membrane proteins by thiol group reagents such as N-ethylmaleimide, monoiodoacetate and monoiodoacetamide did not induce the inhibition of Ca2+-ATPase activity. From these results, it is suggested that inhibition of the ATPase activity of the membranes by treatment with DTT/Fe2+ in the presence and absence of t-BuOOH is dependent on lipid peroxidation and that oxidative modification of SH groups may be not directly involved to the loss of the ATPase activity. In addition, results of the fluorescence anisotropy measurements of pyrene-labeled membranes suggested that change in the Ca2+-ATPase activity is partly related to a decrease in the membrane lipid fluidity.