Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro

摘要

pThe steric conversion of 13-icis/i-retinoic acid (13-cRA) to all-itrans/i-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 °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 iK/isubm/sub and iV/isubmax/sub values for the reaction were approx. 7 iμ/iM 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 iS/i-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-icis/i-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./p