Castilho R F, Carvalho-Alves P C, Vercesi A E, Ferreira S T
Departamento de Bioquimica, Universidade Estadual de Campinas, Rio de Janeiro, Brazil.
Mol Cell Biochem. 1996 Jun 21;159(2):105-14. doi: 10.1007/BF00420912.
The major protein in the sarcoplasmic reticulum (SR) membrane is the Ca2+ transporting ATPase which carries out active Ca2+ pumping at the expense of ATP hydrolysis. The aim of this work was to elucidate the mechanisms by which oxidative stress induced by Fenton's reaction (Fe(2+)+H2O2-->HO.+OH-+Fe3+) alters the function of SR. ATP hydrolysis by both SR vesicles (SRV) and purified ATPase was inhibited in a dose-dependent manner in the presence of 0-1.5 mM H2O2 plus 50 microM Fe2+ and 6 mM ascorbate. Ca2+ uptake carried out by the Ca(2+)-ATPase in SRV was also inhibited in parallel. The inhibition of hydrolysis and Ca2+ uptake was not prevented by butylhydroxytoluene (BHT) at concentrations which significantly blocked formation of thiobarbituric acid-reactive substances (TBARS), suggesting that inhibition of the ATPase was not due to lipid peroxidation of the SR membrane. In addition, dithiothreitol (DTT) did not prevent inhibition of either ATPase activity or Ca2+ uptake, suggesting that inhibition was not related to oxidation of ATPase thiols. The passive efflux of 45Ca2+ from pre-loaded SR vesicles was greatly increased by oxidative stress and this effect could be only partially prevented (ca 20%) by addition of BHT or DTT. Trifluoperazine (which specifically binds to the Ca(2+)-ATPase, causing conformational changes in the enzyme) fully protected the ATPase activity against oxidative damage. These results suggest that the alterations in function observed upon oxidation of SRV are mainly due to direct effects on the Ca(2+)-ATPase. Electrophoretic analysis of oxidized Ca(2+)-ATPase revealed a decrease in intensity of the silver-stained 110 kDa Ca(2+)-ATPase band and the appearance of low molecular weight peptides (MW < 100 kDa) and high molecular weight protein aggregates. Presence of DTT during oxidation prevented the appearance of protein aggregates and caused a simultaneous increase in the amount of low molecular weight peptides. We propose that impairment of function of the Ca(2+)-pump may be related to aminoacid oxidation and fragmentation of the protein.
肌浆网（SR）膜中的主要蛋白质是Ca2+转运ATP酶，它以ATP水解为代价进行主动Ca2+泵浦。这项工作的目的是阐明芬顿反应（Fe(2+)+H2O2→HO·+OH-+Fe3+）诱导的氧化应激改变SR功能的机制。在存在0 - 1.5 mM H2O2加50 μM Fe2+和6 mM抗坏血酸的情况下，SR囊泡（SRV）和纯化的ATP酶的ATP水解均以剂量依赖的方式受到抑制。SRV中Ca(2+)-ATP酶进行的Ca2+摄取也同时受到抑制。丁基羟基甲苯（BHT）在显著阻断硫代巴比妥酸反应性物质（TBARS）形成的浓度下并不能阻止水解和Ca2+摄取的抑制，这表明ATP酶的抑制不是由于SR膜的脂质过氧化。此外，二硫苏糖醇（DTT）不能阻止ATP酶活性或Ca2+摄取的抑制，这表明抑制与ATP酶硫醇的氧化无关。氧化应激极大地增加了预加载的SR囊泡中45Ca²⁺的被动外流，并且添加BHT或DTT只能部分阻止这种效应（约20%）。三氟拉嗪（它特异性结合Ca(2+)-ATP酶，导致酶的构象变化）完全保护ATP酶活性免受氧化损伤。这些结果表明，SRV氧化时观察到的功能改变主要是由于对Ca(2+)-ATP酶的直接作用。氧化的Ca(2+)-ATP酶的电泳分析显示，银染的110 kDa Ca(2+)-ATP酶条带强度降低，出现了低分子量肽（MW < 100 kDa）和高分子量蛋白质聚集体。氧化过程中存在DTT可防止蛋白质聚集体的出现，并同时导致低分子量肽的量增加。我们提出Ca(2+)-泵功能的损害可能与蛋白质的氨基酸氧化和片段化有关。
