Tong Z, Board P G, Anders M W
Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Avenue, Box 711, Rochester, NY 14642, USA.
Biochem J. 1998 Apr 15;331 ( Pt 2)(Pt 2):371-4. doi: 10.1042/bj3310371.
Dichloroacetic acid (DCA), a common drinking-water contaminant, is hepatocarcinogenic in rats and mice, and is a therapeutic agent used clinically in the management of lactic acidosis. DCA is biotransformed to glyoxylic acid by glutathione-dependent cytosolic enzymes in vitro and is metabolized to glyoxylic acid in vivo. The enzymes that catalyse the oxygenation of DCA to glyoxylic acid have not, however, been identified or characterized. In the present investigation, an enzyme that catalyses the glutathione-dependent oxygenation of DCA was purified to homogeneity (587-fold) from rat liver cytosol. SDS/PAGE and HPLC gel-filtration chromatography showed that the purified enzyme had a molecular mass of 27-28 kDa. Sequence analysis showed that the N-terminus of the purified protein was blocked. An internal sequence of 30 amino acid residues was obtained that matched the recently discovered human glutathione transferase Zeta well [Board, Baker, Chelvanayagam and Jermiin (1997) Biochem. J. 328, 929-935]. Western-blot analysis showed that the purified rat-liver enzyme cross-reacted with rabbit antiserum raised against recombinant human glutathione transferase Zeta. The apparent Km and Vmax values of the purified enzyme with DCA as the variable substrate were 71.4 microM and 1334 nmol/min per mg of protein, respectively; the Km for glutathione was 59 microM. Both the purified rat-liver enzyme and the recombinant human enzyme showed high activity with DCA as the substrate. These results demonstrate that the glutathione-dependent oxygenation of DCA to glyoxylic acid is catalysed by a Zeta-class glutathione transferase.
二氯乙酸（DCA）是一种常见的饮用水污染物，对大鼠和小鼠具有肝脏致癌性，也是临床上用于治疗乳酸性酸中毒的一种治疗剂。在体外，DCA通过谷胱甘肽依赖的胞质酶生物转化为乙醛酸，在体内则代谢为乙醛酸。然而，催化DCA氧化为乙醛酸的酶尚未得到鉴定或表征。在本研究中，从大鼠肝脏胞质溶胶中纯化出一种催化DCA谷胱甘肽依赖氧化反应的酶，使其达到同质状态（纯化了587倍）。SDS/PAGE和HPLC凝胶过滤色谱分析表明，纯化后的酶分子量为27 - 28 kDa。序列分析表明，纯化蛋白的N端被封闭。获得了一段30个氨基酸残基的内部序列，与最近发现的人谷胱甘肽转移酶Zeta匹配良好[博德、贝克、切尔瓦纳亚加姆和杰尔米因（1997年）《生物化学杂志》328卷，929 - 935页]。蛋白质印迹分析表明，纯化的大鼠肝脏酶与针对重组人谷胱甘肽转移酶Zeta产生的兔抗血清发生交叉反应。以DCA为可变底物时，纯化酶的表观Km和Vmax值分别为71.4 microM和每毫克蛋白1334 nmol/min；谷胱甘肽的Km为59 microM。纯化的大鼠肝脏酶和重组人酶以DCA为底物时均表现出高活性。这些结果表明，DCA谷胱甘肽依赖氧化为乙醛酸的反应是由Zeta类谷胱甘肽转移酶催化的。
