HYDROXYL RADICAL-MEDIATED REDUCTION OF CA2+-ATPASE ACTIVITY OF MASSETER MUSCLE SARCOPLASMIC-RETICULUM
HYDROXYL RADICAL-MEDIATED REDUCTION OF CA2+-ATPASE ACTIVITY OF MASSETER MUSCLE SARCOPLASMIC-RETICULUM
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DOI：
10.1254/jjp.67.21
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发表时间：
1995-01-01
期刊：
影响因子：
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通讯作者：
OKABE, E
中科院分区：
文献类型：
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作者：
LEE, CI;OKABE, E
为了了解氧自由基对Ca 2 +-ATP酶的影响，我们以犬咬肌肌浆网（SR）微粒体为模型系统，探讨了氧化对生物膜的影响，并研究了芬顿试剂（H2 O2/FeSO 4）产生的羟基自由基（;OH）对Ca 2 +-ATP酶的影响。H2 O2（10 mM）单独对Ca 2 +-ATP酶活性没有影响;在FeSO 4（0.2 mM）存在下，H2 O2抑制酶活性。在不存在SR囊泡的情况下，通过高灵敏度电子自旋共振光谱和自旋捕获5，5-二甲基-1-吡咯啉-N-氧化物（DMPO）验证了在Ca 2 +-ATP酶测定中使用的条件下由H2 O2/FeSO 4产生的氧自由基物质;观察到DMPO-OH自旋加合物的特征1：2：2：1四重峰（A（N）=A（H）（β）=1.49 mT）。Ca 2 +-ATP酶活性与DMPO-OH的计算信号强度呈负相关，DMPO-OH的计算信号强度指示产生的OH自由基的量。过氧化氢酶、二甲基亚砜和二甲基硫脲能有效抑制芬顿试剂的作用，巯基还原剂半胱氨酸和二硫苏糖醇也能抑制芬顿试剂的作用。巯基修饰剂对氯汞苯甲酸盐和5，5 '-二硫代双（2-硝基苯甲酸）对Ca 2 +-ATP酶活性有抑制作用，而在芬顿试剂存在下，巯基修饰剂对Ca 2 +-ATP酶活性的抑制作用增强。结果表明，OH自由基引起的氧化损伤可能主要是由于咬肌SR囊泡ATP酶分子上的关键巯基发生了修饰。
To understand the effect of oxygen free radicals on Ca2+-ATPase, we used sarcoplasmic reticulum (SR) microsomes of canine masseter muscle as a model system in which to explore the effects of oxidation on a biological membrane, and we investigated the effect of hydroxyl radicals (;OH) generated from Fenton's reagent (H2O2/FeSO4). H2O2 (10 mM) alone had no effect on Ca2+-ATPase activity; in the presence of FeSO4 (0.2 mM), H2O2 inhibited the enzyme activity. Oxygen free radical species generated from H2O2/FeSO4 under the conditions employed in the Ca2+-ATPase assay were verified by highly sensitive electron spin resonance spectroscopy and the spin-trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) in the absence of SR vesicles; the 1:2:2:1 quartet (A(N)=A(H)(beta)=1.49 mT), characteristic of the DMPO-OH spin adduct, was observed. The Ca2+-ATPase activity was inversely correlated with the calculated signal intensity of DMPO-OH, which is indicative of the amount of OH radical generated. The effect of Fenton's reagent was effectively inhibited by catalase, dimethylsulfoxide, and dimethylthiourea; the effect was also inhibited by sulfhydryl (SH) group reducing agents, cysteine and dithiothreitol. The SH group modifying agents, p-chloromercuric benzoate and 5,5'-dithiobis(2-nitrobenzoic acid) depressed Ca2+-ATPase activity; the effects of the SH group modifying agents used were potentiated in the presence of Fenton's reagent. It is suggested that OH radical-induced oxidant injury may be caused primarily by modification of the key SH group(s) on the ATPase molecule of masseter muscle SR vesicles.