Selective covalent binding of acrylonitrile to Cys 186 in rat liver carbonic anhydrase III in vivo.
Selective covalent binding of acrylonitrile to Cys 186 in rat liver carbonic anhydrase III in vivo.
复制标题
体内丙烯腈与大鼠肝脏碳酸酐酶 III 中的 Cys 186 选择性共价结合。
DOI：
10.1021/tx0256883
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Benz,FrederickW
中科院分区：
文献类型：
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作者：
Nerland,DonaldE;Cai,Jian;Benz,FrederickW
反应性化学物质与组织蛋白质的共价结合是一种常见的但知之甚少的毒性机制。蛋白质和蛋白质中化学修饰的特定氨基酸残基的鉴定将有助于我们理解共价结合中涉及的纯化/解毒机制。丙烯腈（AN）是一种商业乙烯基单体，具有急性毒性，易于与组织蛋白结合。AN与组织蛋白的总共价结合与急性毒性高度相关。二维聚丙烯酰胺凝胶电泳和放射自显影被用来定位蛋白质在雄性大鼠肝细胞质中的放射性标记后，管理[2，3 - 14 C] AN在体内。四个强烈标记的斑点在放射自显影图中是突出的，并在约30 kDa处形成明显的"电荷串"。通过基质辅助激光解吸/电离飞行时间（MALDI-TOF）MS的胰蛋白酶肽图谱用于将所有斑点鉴定为碳酸酐酶III（CAIII）。使用胰蛋白酶的HPLC结合MALDI-TOF MS将放射性标记定位于含有氨基酸171 − 187的胰蛋白酶片段T22。该胰蛋白酶片段在大鼠CAIII序列中含有两个Cys残基（Cys181和Cys186）。使用电喷雾电离离子阱MS对肽进行测序，并确定仅Cys186被标记。因此，虽然AN被认为是高度反应性的，我们的数据表明，它不与大鼠CAIII反应不加选择，而是选择性的五个半胱氨酸残基之一。大鼠肝脏CAIII先前已被证明可以保护细胞免受氧化应激。我们的数据表明，CAIII也能够清除活性异生物质，并可能有助于防止共价结合到更关键的大分子。
Covalent binding of reactive chemical species to tissue proteins is a common, but poorly understood, mechanism of toxicity. Identification of the proteins and the specific amino acid residues within the proteins that are chemically modified will aid our understanding of the toxification/detoxification mechanisms involved in covalent binding. Acrylonitrile (AN) is a commercial vinyl monomer that is acutely toxic and readily binds to tissue proteins. Total covalent binding of AN to tissue proteins is highly correlated with acute toxicity. Two-dimensional PAGE and autoradiography were used to locate proteins in male rat liver cytosol that are radiolabeled following administration of [2,3-14C]AN in vivo. Four intensely labeled spots were prominent in the autoradiogram and formed an apparent “charge-train” at approximately 30 kDa. Tryptic peptide mapping by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used to identify all of the spots as carbonic anhydrase III (CAIII). HPLC of the tryptic digests combined with MALDI-TOF MS was used to localize the radiolabel to tryptic fragment T22 containing amino acids 171−187. This tryptic fragment contains two Cys residues (Cys181 and Cys186) in the rat CAIII sequence. Electrospray ionization ion-trap MS was used to sequence the peptide and establish that only Cys186 was labeled. Thus, although AN is considered to be highly reactive, our data indicate that it does not react indiscriminately with rat CAIII but rather is selective for one out of five Cys residues. Rat liver CAIII has previously been shown to protect cells against oxidative stress. Our data suggest that CAIII is also capable of scavenging reactive xenobiotics and may help prevent covalent binding to more critical macromolecules.