Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes.
Differential effects of organic hydroperoxides and hydrogen peroxide on proteolysis in human erythrocytes.
复制标题
有机氢过氧化物和过氧化氢对人红细胞蛋白水解的不同影响。
DOI：
10.1021/tx00008a002
复制
发表时间：
1989
影响因子：
4.1
通讯作者：
Novak,RF
中科院分区：
文献类型：
--
作者：
Runge-Morris,M;Frank,P;Novak,RF
已经检查了氢过氧化叔丁基、氢过氧化异丙苯和过氧化氢对人红细胞蛋白水解的影响。有机氢过氧化物有效刺激红细胞和溶血液中蛋白质的降解速率；相反，添加H 2 O 2 在任一系统中均没有显着影响。通过酪氨酸释放监测，相对于对照，叔丁基过氧化氢或异丙苯过氧化氢 (8 mM) 使红细胞中的蛋白质降解速率分别增加 2.3 倍和 4 倍。在溶血液中，与对照相比，8 mM 浓度的叔丁基过氧化氢和异丙苯过氧化氢的蛋白质降解率分别增加了 2 倍和 3 倍。红细胞或溶血液中氢过氧化物刺激的蛋白水解具有浓度依赖性，并在 8 mM 氢过氧化物时达到饱和。反应在 2 小时内呈线性，之后达到稳定水平。与有机氢过氧化物观察到的结果相反，单独添加H 2 O 2 (100或200mM)或在过氧化氢酶抑制剂3-氨基-1,2,4-三唑(50-200mM)存在下添加H 2 O 2 未能刺激蛋白水解。 N-乙酰半胱氨酸 (20 mM) 和二甲基硫脲 (50 mM) 分别抑制红细胞中氢过氧化物刺激的蛋白水解速率约 50% 和约 35%，以及在溶血产物中分别抑制 25% 和 40%。羟基自由基清除剂二甲基亚砜（50 mM）或二甲基呋喃（50 mM）、金属离子螯合剂或自旋陷阱未能显着降低有机氢过氧化物刺激的蛋白水解速率。此外，在受到有机氢过氧化物攻击的红细胞或溶血产物培养物中，钙蛋白酶/钙蛋白酶原系统的抑制剂对蛋白水解速率没有显着影响。采用红细胞孵育的 HPLC 分析来定量组氨酸和酪氨酸的释放。尽管氢过氧化异丙苯产生这些氨基酸的浓度和时间依赖性释放，但H 2 O 2 未能刺激氨基酸释放。这些数据证实了荧光酪氨酸测定的结果。这项研究的结果表明，由于氢过氧化物分解过程中产生的烷基或烷氧基自由基，有机氢过氧化物会在红细胞和溶血产物中产生蛋白质损伤，并且 HPLC 提供了监测这些系统中蛋白质水解的定量方法。蛋白水解被认为是一种防御机制，用于去除因外源性物质损伤而受损的蛋白质，也可能是外源性物质介导的细胞损伤的快速且新颖的指标。
The effects of ieri-butyl hydroperoxide, cumene hydroperoxide, and hydrogen peroxide on proteolysis in human red blood cells have been examined. The organic hydroperoxides effectively stimulated the rate of protein degradation in red cells and in hemolysate; in contrast, H202 addition was without significanteffect in either system. ieri-Butyl hydroperoxide or cumene hydroperoxide (8 mM) increased the rate of protein degradation in red cells 2.3-and 4-fold, respectively, relative to control as monitored by tyrosine release. In hemolysate, ieri-butyl hydroperoxide and cumene hydroperoxide, present at 8 mM, produced a 2-and 3-fold increase in the rate of protein degradation, respectively, as compared to controls. Hydroperoxide-stimulated proteolysis in redcells or in hemolysate was concentration-dependent and reached saturation at 8 mM hydroperoxide. The reaction was linear for 2 h after which a plateau was reached. In contrast to the results observed for the organic hydroperoxides, H202 (100 or 200 mM) addition either alone or in the presence of the catalase inhibitor 3-amino-l, 2, 4-triazole (50-200 mM), failed to stimulate proteolysis. N-Acetylcysteine (20 mM) and dimethylthiourea (50 mM) inhibited the rate of hydroperoxide-stimulated proteolysis in red cells by~ 50 and~ 35%, respectively, and in hemolysate by 25 and 40%, respectively. Thehydroxyl radical scavengers methyl sulfoxide (50 mM) or dimethylfuran (50 mM), metal ionchelators, or spin traps failed to decrease significantly the rate of organic hydroperoxidestimulated proteolysis. In addition, inhibitors of the calpain/procalpain system in red cell or hemolysate incubations challenged by organic hydroperoxide were without significanteffect on the rate of proteolysis. HPLC analysis of red cell incubations was employed to quantify the release of histidine and tyrosine. Whereas cumene hydroperoxide produced a concentration-and time-dependent release of these amino acids, H202 failed to stimulate amino acid release. These data confirm the findings of the fluorometric tyrosine assay. The results of this study reveal that organic hydroperoxides produce protein damage in red cells and hemolysate as a result of alkyl or alkoxyl free radicals generated during hydroperoxide decomposition and that HPLC provides a quantitative approach for monitoring proteolysis in these systems. Proteolysis, which has been suggested as being a defense mechanism for the removal of protein damaged by xenobiotic insult, may also be a rapid and novel index of xenobiotic-mediated cellular injury.