MacMillan-Crow L A, Crow J P, Thompson J A
Department of Surgery, University of Alabama at Birmingham 35294, USA.
Biochemistry. 1998 Feb 10;37(6):1613-22. doi: 10.1021/bi971894b.
Previous studies from our laboratory have demonstrated that the mitochondrial protein manganese superoxide dismutase is inactivated, tyrosine nitrated, and present as higher molecular mass species during human renal allograft rejection. To elucidate mechanisms whereby tyrosine modifications might result in loss of enzymatic activity and altered structure, the effects of specific biological oxidants on recombinant human manganese superoxide dismutase in vitro have been evaluated. Hydrogen peroxide or nitric oxide had no effect on enzymatic activity, tyrosine modification, or electrophoretic mobility. Exposure to either hypochlorous acid or tetranitromethane (pH 6) inhibited (approximately 50%) enzymatic activity and induced the formation of dityrosine and higher mass species. Treatment with tetranitromethane (pH 8) inhibited enzymatic activity 67% and induced the formation of nitrotyrosine. In contrast, peroxynitrite completely inhibited enzymatic activity and induced formation of both nitrotyrosine and dityrosine along with higher molecular mass species. Combination of real-time spectral analysis and electrospray mass spectroscopy revealed that only three (Y34, Y45, and Y193) of the nine total tyrosine residues in manganese superoxide dismutase were nitrated by peroxynitrite. Inspection of X-ray crystallographic data suggested that neighboring glutamate residues associated with two of these tyrosines may promote targeted nitration by peroxynitrite. Tyr34, which is present in the active site, appeared to be the most susceptible residue to peroxynitrite-mediated nitration. Collectively, these observations are consistent with previous results using chronically rejecting human renal allografts and provide a compelling argument supporting the involvement of peroxynitrite during this pathophysiologic condition.
我们实验室之前的研究表明，在人类肾移植排斥反应期间，线粒体蛋白锰超氧化物歧化酶会失活、酪氨酸硝化，并以更高分子量的形式存在。为了阐明酪氨酸修饰可能导致酶活性丧失和结构改变的机制，已评估了特定生物氧化剂在体外对重组人锰超氧化物歧化酶的影响。过氧化氢或一氧化氮对酶活性、酪氨酸修饰或电泳迁移率没有影响。暴露于次氯酸或四硝基甲烷（pH 6）会抑制（约50%）酶活性，并诱导二酪氨酸和更高分子量物质的形成。用四硝基甲烷（pH 8）处理会抑制67%的酶活性，并诱导硝基酪氨酸的形成。相比之下，过氧亚硝酸盐完全抑制酶活性，并诱导硝基酪氨酸和二酪氨酸以及更高分子量物质的形成。实时光谱分析和电喷雾质谱联用显示，锰超氧化物歧化酶九个酪氨酸残基中只有三个（Y34、Y45和Y193）被过氧亚硝酸盐硝化。对X射线晶体学数据的检查表明，与其中两个酪氨酸相关的相邻谷氨酸残基可能促进过氧亚硝酸盐的靶向硝化。位于活性位点的Tyr34似乎是最易受亚硝酸盐介导硝化作用影响的残基。总的来说，这些观察结果与之前使用慢性排斥的人类肾移植的结果一致，并提供了有力的证据支持过氧亚硝酸盐在这种病理生理状况中的作用。
