New assay for uronosyl 5-epimerases.
Simple assays have been developed for the two uronosyl 5-epimerases which participate in the biosynthesis of heparin and dermatan sulfate (heparosan N-sulfate D-glucuronosyl 5-epimerase and chondroitin D-glucuronosyl 5-epimerase, respectively). Following previously published procedures, substrates labeled with tritium in the C-5 positions of the D-glucuronosyl and L-iduronosyl residues were prepared enzymatically by incubation of O-desulfated heparin and dermatan with 3H2O and crude epimerase preparations from bovine liver and human skin fibroblasts, respectively. In the new assays, 3H2O generated from these substrates during the epimerase reactions was quantitated by the method of Pollard et al. (Anal. Biochem. (1981) 110, 424-430). In this procedure, 3H2O in the aqueous reaction mixture is extracted into a toluene-based organic phase containing 25% isoamyl alcohol, while the polysaccharide substrate remains in the aqueous phase and does not generate scintillations. This procedure is much simpler than that used previously which involves distillation of each reaction mixture and quantitation of the radioactivity in the distillate. The new assays have been validated by the demonstration that conditions of linearity with time and enzyme concentration can be established for both epimerase reactions. Assays of this type should be useful in the study of any enzymatic reaction where 3H2O is formed from a 3H-labeled substrate and the unreacted substrate is not appreciably soluble in the organic phase.
D000818:Animals; D002238:Carbohydrate Epimerases / Q000032:analysis*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003871:Dermatan Sulfate / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D012588:Scintillation Counting; D014316:Tritium; D014867:Water / Q000302:isolation & purification
D014867:Water; D014316:Tritium; D003871:Dermatan Sulfate; D006493:Heparin; D002238:Carbohydrate Epimerases; C038851:chondroitin D-glucuronosyl 5-epimerase; C019591:heparosan N-sulfate D-glucuronosyl 5-epimerase