Selective Nitration of Tyr99 in Calmodulin as a Marker of Cellular Conditions of Oxidative Stress

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• 作者：Heather S. Smallwood ; Nadezhda A. Galeva ; Ryan K. Bartlett ; Ramona J. Bieber Urbauer ; Todd D. Williams ; Jeffrey L. Urbauer ; and Thomas C. Squier
• 刊名：Chemical Research in Toxicology
• 出版年：2003
• 出版时间：January 2003
• 年：2003
• 卷：16
• 期：1
• 页码：95 - 102
• 全文大小：107K
• 年卷期：v.16,no.1(January 2003)
• ISSN：1520-5010

文摘

We examined the possible role of methionines as oxidant scavengers that prevent theperoxynitrite-induced nitration of tyrosines within calmodulin (CaM). We used mass spectrometry to investigate the reactivity of peroxynitrite with CaM at physiological pH. Thepossible role of methionines in scavenging peroxynitrite (ONOO^-) was assessed in wild-typeCaM and following substitution of all nine methionines in CaM with leucines. We find thatperoxynitrite selectively nitrates Tyr⁹⁹ at physiological pH, resulting in the formation of between0.05 and 0.25 mol of nitrotyrosine/mol of CaM when the added molar ratio of peroxynitrite perCaM was varied between 2.5 and 15. In wild-type CaM there is a corresponding oxidation ofbetween 0.8 and 2.8 mol of methionine to form methionine sulfoxide. However, following site-directed substitution of all nine methionines in wild-type CaM with leucines, the extent ofnitration by peroxynitrite was unchanged. These results indicate that Tyr⁹⁹ is readily nitratedby peroxynitrite and that methionine side chains do not function as an antioxidant in scavengingperoxynitrite. Thus, separate reactive species are involved in the oxidation of methionine andnitration of Tyr⁹⁹ whose relative concentrations are determined by solution conditions. Thesensitivity of Tyr⁹⁹ in CaM to nitration suggests that CaM-dependent signaling pathways aresensitive to peroxynitrite formation and that nitration of CaM represents a cellular marker ofperoxynitrite-induced changes in cellular function.