Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro
Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro
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DOI：
10.1042/bj3360223
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发表时间：
1998-11-15
影响因子：
4.1
通讯作者：
Juchau, MR
中科院分区：
文献类型：
--
作者：
Chen, H;Juchau, MR
13-顺式视黄酸 (13-cRA) 向全反式视黄酸 (t-RA) 的空间转化已被提议作为观察到的 13-cRA 治疗和致畸活性的激活机制。在这里，我们研究了重组人谷胱甘肽 S-转移酶 (GST) 对 13-cRA 异构化为 t-RA 的催化作用。将底物与 GST 在 0.1 M 磷酸钠缓冲液（pH 7.5）中于 37°C、完全黑暗中孵育。通过 HPLC 定量测量生成的 t-RA。在所使用的反应条件下，GSTP1-1在催化异构化反应方面比人GSTM1-1或人GSTA1-1更有效。 GSTP1-1 催化的反应显示底物饱和，反应的 K-m 和 V-max 值约为。分别为 7 μM 和 650 pmol/min/nmol。反应速率随着酶浓度的增加而线性增加。该反应受到热处理和 S-癸基谷胱甘肽（与 GST 相关的转移酶活性的有效抑制剂）的抑制。在反应中添加针对人 GSTP1-1 的多克隆兔抗血清导致 t-RA 的产生显着减少 (70-80%)。此外，GST Pi 同工型的选择性底物依他尼酸也抑制 GSTP1-1 催化的 13-cRA 异构化为 t-RA。在相同的反应条件下，GSTP1-1在催化9-顺式视黄酸向t-RA的空间转化方面效果较差，表明该酶对于13-cRA向t-RA的转化具有立体专一性。这些观察结果表明酶催化是 GSTP1-1 依赖性 13-cRA 转化为 t-RA 的主要机制。纯化的大鼠肝 GST Pi 同工酶催化的反应比人 GSTP1-1 催化的反应进行得更慢。比较研究还表明，各种纯化的哺乳动物肝脏 GST 混合物的催化活性存在明显的物种差异。
The steric conversion of 13-cis-retinoic acid (13-cRA) to all-trans-retinoic acid (t-RA) has been proposed as an activation mechanism for the observed therapeutic and teratogenic activities of 13-cRA. Here we have investigated the catalysis of isomerization of 13-cRA to t-RA by recombinant human glutathione S-transferases (GSTs). Substrate was incubated with GST in 0.1 M sodium phosphate buffer, pH 7.5, at 37 degrees C in total darkness. The t-RA generated was measured quantitatively by HPLC. Under the reaction conditions used, GSTP1-1 was far more effective than human GSTM1-1 or human GSTA1-1 in catalysing the isomerization reaction. The reaction catalysed by GSTP1-1 showed substrate saturation and the K-m and V-max values for the reaction were approx. 7 mu M and 650 pmol/min per nmol respectively. The reaction rate increased linearly with increasing enzyme concentration. The reaction was inhibited both by heat treatment and by S-decylglutathione (a potent inhibitor of transferase activity associated with GST). Additions of polyclonal rabbit antiserum for human GSTP1-1 to the reaction resulted in a significant decrease in generation of t-RA (70-80 %). In addition, ethacrynic acid, a selective substrate for Pi isoforms of GST, also inhibited the isomerization of 13-cRA to t-RA catalysed by GSTP1-1. Under the same reaction conditions, GSTP1-1 was much less effective in catalysing the steric conversion of 9-cis-retinoic acid to t-RA, indicating that the enzyme was stereospecific for the conversion of 13-cRA to t-RA. These observations suggest that enzymic catalysis was the primary mechanism for the GSTP1-1-dependent conversion of 13-cRA to t-RA. Reactions catalysed by a purified rat hepatic GST Pi isoenzyme proceeded more slowly than reactions catalysed by human GSTP1-1. Comparative studies also showed that there were marked species differences in catalytic activities between various purified mammalian hepatic GST mixtures.