A microassay for UDP-glucose dehydrogenase

An assay for UDP-glucuronic acid [J. Singh, L. R. Schwarz, and F. J. Wiebel, Biochem. J. 189, 369-372 (1980)] has been utilized for determining UDP-glucose dehydrogenase activity. The assay for UDP-glucuronic acid, a product of UDP-glucose dehydrogenase, is based on the fluorometric determination of D-glucuronosyl benzo(a)pyrene. This compound is formed from UDP-glucuronic acid and 3-hydroxybenzo(a)pyrene in a reaction catalyzed by the glycuronosyl transferase of guinea pig microsomes. Unreacted 3-hydroxybenzo(a)pyrene is removed by extraction with chloroform-methanol, and the amount of gluconosylbenzo(a)pyrene formed is determined fluorometrically. Because this assay for UDP-glucose dehydrogenase is about 500 times more sensitive than spectrophotometric assays, it can be used to measure the amount of enzyme extractable from milligram quantities of connective tissue. Some kinetic properties of UDP-glucose dehydrogenase extracted from rabbit tissue have been determined. No evidence of different forms of the enzyme in rabbit liver, cartilage, or corneal stroma was found.