Enzymatic determination of free glucuronic acid with glucuronolactone reductase. I. Isolation and purification of glucuronolactone reductase from rat kidney.
Glucuronolactone reductase [EC 1.1.1.20] from rat kidney was purified over 300-fold by ammonium sulfate fractionation, chromatography on DEAE-cellulose and hydroxylapatite columns, and preparative isoelectric focusing. The substrate specificity of the enzyme in the reduction reaction was broad, and hexuronic acid was one of the best substrates among monosaccharides. Km values for D-glucuronic acid, D-glucuronolactone, D-galacturonic acid, and L-iduronic acid were 6, 9, 4, and 6 mM, respectively. An investigation of the activity for aldose led to the finding that triose and tetrose served as good substrates for this enzyme. However, the activity for aldopentose or aldohexose was less than 1% of that for D-glucuronic acid at the same concentration. The enzyme was inactive towards most hexosamines (galactosamine, mannosamine, N-acetylglucosamine, N-acetylgalactosamine, and N-acetylmannosamine, but not glucosamine), meso-inositol, D-fructose, and tetrasaccharides from hyaluronic acid and chondroitin 4-sulfate. Trisaccharides from hyaluronic acid and chondroitin 6-sulfate which possess glucuronic acid at the reducing end were poor substrates for the enzyme and the activity towards these 4-substituted glucuronic acids was less than 3% of that towards non-substituted glucuronic acid.
D000818:Animals; D002237:Carbohydrate Dehydrogenases / Q000302:isolation & purification*; D055598:Chemical Phenomena; D002621:Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005965:Glucuronates / Q000032:analysis*; D020723:Glucuronic Acid; D007525:Isoelectric Focusing; D007668:Kidney / Q000201:enzymology*; D008297:Male; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D005965:Glucuronates; D020723:Glucuronic Acid; D002237:Carbohydrate Dehydrogenases; C020645:glucuronolactone reductase