Biotransformation of 13-cis- and 9-cis-retinoic acid to all-trans-retinoic acid in rat conceptal homogenates. Evidence for catalysis by a conceptal isomerase.

The purpose of this study was to investigate whether and to what extent the steric isomerization of retinoic acids in conceptal tissues can be attributed to enzymatic catalysis in addition to thiol-dependent, nonenzymatic catalysis. Conversions of 13-cis-retinoic acid and 9-cis-retinoic acid to all-trans-retinoic acid catalyzed by cell-free preparations of conceptal rat tissues (gestational day 12.5) were investigated. Substrates and rat conceptal homogenates (RCH) were incubated in sodium phosphate buffer (0.1 M, pH 7.5) at 37 degrees C in the dark. Incubation mixtures were quantitatively analyzed by HPLC. In RCH-catalyzed reactions, conversions of 13-cis-retinoic acid or 9-cis-retinoic acid to all-trans-retinoic acid were very rapid, in comparison with uncatalyzed isomerization reactions (incubations without RCH). Comparisons of the rates of reactions catalyzed by freshly prepared vs. freshly prepared/dialyzed RCH showed no significant differences, indicating that small, suflhydryl-containing molecules such as reduced glutathione did not significantly contribute to the RCH-catalyzed reactions. Furthermore, at physiological concentrations (2.5-10 mM), reduced glutathione exhibited very low specific catalytic activities, indicating that nonenzymatic, sulfhydryl-dependent catalysis was not a major mechanism in catalyzing interconversions of retinoic acids in vivo. Enzymatic catalysis by RCH of the conversion of 13-cis-retinoic acid to all-trans-retinoic acid was further characterized by showing 1) substrate saturation kinetics, 2) reaction rates that increased proportionally with protein concentrations, and (3) much greater sensitivity of the reactions to heat inactivation and denaturation by urea, compared with nonenzymatic, glutathione-catalyzed reactions. Thus, isomerization of retinoids in conceptal tissues appeared to be under enzymatic control.