Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration
Oxidative inactivation of gastric peroxidase by site-specific generation of hydroxyl radical and its role in stress-induced gastric ulceration
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DOI：
10.1016/s0891-5849(97)00281-5
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发表时间：
1998-02-01
影响因子：
7.4
通讯作者：
Banerjee, RK
中科院分区：
文献类型：
--
作者：
Das, D;Bandyopadhyay, D;Banerjee, RK
我们先前已经表明，束缚冷应激诱导的大鼠胃溃疡是由金属离子依赖的羟基自由基（OH）的产生引起的。和胃过氧化物酶（GRP）（一种重要的H2 O2清除酶）的氧化失活。为了研究酶的氧化损伤机制，将纯化的酶暴露于OH。含有Cu 2+、抗坏血酸盐和H 2 O 2的发生体系。动力学研究表明，该酶被灭活在一个时间依赖性的过程中，显示饱和相对于Cu 2+浓度。该酶特异性地需要Cu 2+，并且不被相同浓度的Fe 2+、Mn 2+或Zn 2+灭活。对过氧化氢酶的敏感性表明H2 O2在失活中的关键作用。灭活对超氧化物歧化酶不敏感，表明超氧化物没有作用。在D2 O中，除单线态氧参与外，失活速率没有增加。但是，哦。清除剂如苯甲酸盐或甘露醇不能防止失活。结果表明，一个合理的生成OH。作为失活的原因。肽连接或分子内交联的断裂、三级结构的总体变化或在“整体”氧化中发生的固有色氨酸荧光的变化不明显。失活是依赖于pH值和从图的K-obs的失活对pH值，具有7.8的pKa的酶的可电离基团的控制作用可以建议，去质子化有利于失活。氨基酸分析显示失活酶中两个赖氨酸残基的特定损失。竞争动力学研究表明，磷酸吡哆醛，赖氨酸残基的特定改性剂，通过与Cu 2+竞争结合在pH 3上来防止失活。Cu 2+结合基序由至少两个赖氨酸残基组成，其特异性结合Cu 2+并产生OH。自由基氧化赖氨酸残基并扰乱血红素环境以引起失活。我们认为，氧化损伤的视网膜是介导的网站特异性生成的OH。而不是靠“哦”在体相中生成。(C)1998年爱思唯尔科学公司
We have shown earlier that restraint-cold stress-induced gastric ulceration in rats is caused by metal ion-dependent generation of hydroxyl radical (OH.) and oxidative inactivation of the gastric peroxidase (GPO), an important H2O2 scavenging enzyme. To study the mechanism of the oxidative damage of GPO, the purified enzyme was exposed to an OH. generating system containing Cu2+, ascorbate, and H2O2. Kinetic studies indicate that the enzyme is inactivated in a time-dependent process showing saturation with respect to Cu2+ concentration. The enzyme specifically requires Cu2+ and is not inactivated by the same concentration of Fe2+, Mn2+, or Zn2+. Sensitivity to catalase indicates the critical role of H2O2 in the inactivation. Inactivation is insensitive to superoxide dismutase, suggesting no role of superoxide. The rate of inactivation is not increased in D2O excluding the involve ment of singlet oxygen in the process. However, OH. scavengers such as benzoate or mannitol cannot prevent inactivation. The results indicate a plausible generation of OH. within the enzyme molecule as the cause of inactivation. Fragmentation of peptide Linkage or intramolecular crosslinking, gross change of tertiary structure, or change in intrinsic tryptophan fluorescence which occurs in "global" oxidation are not evident. Inactivation is dependent on pH and from a plot of K-obs of inactivation against pH, the controlling role of an ionizable group of the enzyme having a pka of 7.8 could be suggested, deprotonation of which favors inactivation. Amino acid analysis shows a specific loss of two lysine residues in the inactivated enzyme. Competitive kinetic studies indicate that pyridoxal phosphate, a specific modifier of the lysine residue, prevents inactivation by competing with Cu2+ for binding at the GPO. A Cu2+ binding motif consisting at least of two lysine residues exists in GPO, which specifically binds Cu2+ and generates OH.. The radical oxidizes the lysine residues and perturbs the heme environment to cause inactivation. We suggest that oxidative damage of GPO is mediated by site-specific generation of OH. and not by the OH. generated in the bulk phase. (C) 1998 Elsevier Science Inc.