Peroxynitrite oxidation of tubulin sulfhydryls inhibits microtubule polymerization

被引:55
作者
Landino, LM [1 ]
Hasan, R [1 ]
McGaw, A [1 ]
Cooley, S [1 ]
Smith, AW [1 ]
Masselam, K [1 ]
Kim, G [1 ]
机构
[1] Coll William & Mary, Dept Chem, Williamsburg, VA 23187 USA
关键词
D O I
10.1006/abbi.2001.2729
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Considerable evidence both in vitro and in vivo implicates protein damage by peroxynitrite as a probable mechanism of cell death. Herein, we report that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dose-dependent inhibition of microtubule polymerization. The extent of cysteine oxidation induced by peroxynitrite correlated well with inhibition of microtubule polymerization. Disulfide bonds between the subunits of the tubulin heterodimer were detected by Western blot as a result of peroxynitrite-induced cysteine oxidation. Addition of disulfide reducing agents including dithiothreitol an beta-mercaptoethanol restored a significant portion the polymerization activity that was lost following peroxynitrite addition. Thus, peroxynitrite-ice disulfide bonds are at least partially responsible for the observed inhibition of polymerization. Sodium bicarbonate protected microtubule protein from the peroxynitrite-induced inhibition of polymerization. Tyrosine nitration of microtubule protein by 1 mM peroxynitrite increased approximately twofold when sodium bicarbonate was present whereas the extent of cysteine oxidation decreased from 7.5 to 6.3 mol cysteine/mol tubulin. These results indicate that cysteine oxidation of tubulin by peroxynitrite, rather than tyrosine nitration, is the primary mechanism of inhibition of microtubule polymerization. (C) 2002 Elsevier Science (USA).
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页码:213 / 220
页数:8
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