title: Recombinant human glutathione S-transferases catalyse enzymic isomerization of 13-cis-retinoic acid to all-trans-retinoic acid in vitro.
abstract: 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.
authors.affiliation: Department of Pharmacology, School of Medicine, Box 357280, University of Washington, Seattle, WA 98195, USA.
authors.forename: H
authors.initials: H
authors.lastname: Chen
authors.forename: M R
authors.initials: MR
authors.lastname: Juchau
mesh_terms: D000818:Animals; D002384:Catalysis; D004976:Ethacrynic Acid / Q000378:metabolism; D005982:Glutathione Transferase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism*; D015474:Isotretinoin / Q000378:metabolism*; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013552:Swine; D014212:Tretinoin / Q000378:metabolism*
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D014212:Tretinoin; D005982:Glutathione Transferase; D015474:Isotretinoin; D004976:Ethacrynic Acid