Chen H, Juchau M R
Department of Pharmacology, School of Medicine, Box 357280, University of Washington, Seattle, WA 98195, USA.
Biochem J. 1998 Nov 15;336 ( Pt 1)(Pt 1):223-6. doi: 10.1042/bj3360223.
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 Km and Vmax values for the reaction were approx. 7 microM 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.
13-顺式视黄酸（13-cRA）向全反式视黄酸（t-RA）的空间异构化被认为是13-cRA所观察到的治疗和致畸活性的一种激活机制。在此，我们研究了重组人谷胱甘肽S-转移酶（GSTs）对13-cRA异构化为t-RA的催化作用。将底物与GST在0.1M磷酸钠缓冲液（pH 7.5）中于37℃在完全黑暗条件下孵育。通过高效液相色谱法定量测定生成的t-RA。在所使用的反应条件下，GSTP1-1在催化异构化反应方面远比人GSTM1-1或人GSTA1-1有效。GSTP1-1催化的反应表现出底物饱和，该反应的Km和Vmax值分别约为7μM和每nmol 650 pmol/min。反应速率随酶浓度增加呈线性增加。热处理和S-癸基谷胱甘肽（一种与GST相关的转移酶活性的有效抑制剂）均抑制该反应。向反应中添加针对人GSTP1-1的多克隆兔抗血清导致t-RA生成显著减少（70 - 80%）。此外，依他尼酸（GST的Pi同工型的选择性底物）也抑制GSTP1-1催化的13-cRA向t-RA的异构化。在相同反应条件下，GSTP1-1在催化9-顺式视黄酸向t-RA的空间异构化方面效果要差得多，表明该酶对将13-cRA转化为t-RA具有立体特异性。这些观察结果表明酶催化是GSTP1-1依赖的13-cRA向t-RA转化的主要机制。纯化的大鼠肝脏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混合物之间的催化活性存在显著的物种差异
