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Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage.

ABSTRACT: 1. UDP-glucose dehydrogenase (EC 1.1.1.22) was extracted from epiphysial-plate cartilage of newborn pigs and from whole bovine corneas. 2. Formation of UDP-glucuronic acid was demonstrated by radioautography after separation of the sugar nucleotides by paper chromatography or t.l.c.: in these conditions a radioactive glucuronic acid spot also appears. 3. UDP-xylose prevented the formation in the incubation mixture of both UDP-glucuronic acid and free glucuronic acid. 4. In both tissues the dependence of the enzyme activity on pH and the K(m) values for UDP-glucose and NAD(+) were determined. 5. Inhibition by UDP-xylose with respect to UDP-glucose was investigated. The plots of 1/v versus 1/[UDP-glucose], and of percentage inhibition versus UDP-xylose concentration and the Hill coefficient showed that a co-operative effect existed between UDP-xylose-binding sites. 6. The physiological meaning of the different affinities of cartilage and cornea enzymes for UDP-xylose is discussed and related to the different glycosaminoglycan contents of the two connective tissues studied.

SUBMITTER: Balduini C

PROVIDER: S-EPMC1177692 | biostudies-other | 1973 Jun

REPOSITORIES: biostudies-other

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Project description:1. In incubation mixtures containing digitonin-activated or untreated preparations from rat liver, albumin-solubilized bilirubin as the acceptor substrate and (a) UDP-glucuronic acid, (b) UDP-glucose or (c) UDP-xylose as the sugar donor, formation of the following ester glycosides was demonstrated: with (a), bilirubin beta-d-monoglucuronoside, with (b), bilirubin beta-d-monoglucoside and with (c), bilirubin monoxyloside or mixtures of the mono-and di-xyloside. 2. With UDP-glucuronic acid prolonged incubation and variation of the composition of the incubation mixtures yielded equimolar amounts of azodipyrrole (I) and azodipyrrole beta-d-monoglucuronoside (II) after treatment of the incubation mixtures with the diazonium salt of ethyl anthranilate. The azo-derivatives were identified by t.l.c. by reference to known compounds and by the following chemical tests. After ammonolysis the conjugated azo-derivative (II) yielded d-glucuronic acid and the carboxylic acid amide of azodipyrrole, indicating transfer of a glucuronic acid residue to the carboxylic acid groups of bilirubin. The beta-d-configuration of the sugar moiety and binding at C-1 were demonstrated by enzymic hydrolysis tests. 3. Analogous evidence established the structure of the reaction product obtained with UDP-glucose as the sugar donor, as bilirubin beta-d-monoglucoside. 4. With UDP-xylose as the sugar donor xylosyl transfer to the carboxylic acid groups of bilirubin with attachment at C-1 was demonstrated in an analogous way. A beta-d-configuration is considered very likely, but requires confirmation. 5. Monoxyloside formation was predominant at pH7.4, whereas at decreasing pH values increasing fractions of the substrate were converted into the dixyloside. Prolonged incubation, low concentrations of bilirubin and high concentrations of UDP-xylose favoured diconjugate formation. The available evidence supports the synthesis sequence: bilirubin --> bilirubin monoxyloside --> bilirubin dixyloside.

Project description:1. Digitonin-treated and untreated homogenates, cell extracts and washed microsomal preparations from liver of Wistar R rats are capable of transferring sugar from UDP-glucose or UDP-xylose to bilirubin. No formation of bilirubin glycosides occurred with UDP-galactose or d-glucose, d-xylose or d-glucuronic acid as the sources of sugar. 2. Procedures to assay digitonin-activated and unactivated bilirubin UDP-glucosyltransferase and bilirubin UDP-xylosyltransferase were developed. 3. In digitonin-activated microsomal preparations the transferring enzymes had the following properties. Both enzyme activities were increased 2.5-fold by pretreatment with digitonin. They were optimum at pH6.6-7.2. Michaelis-Menten kinetics were followed with respect to UDP-glucose. In contrast, double-reciprocal plots of enzyme activity against the concentration of UDP-xylose showed two intersecting straight-line sections corresponding to concentration ranges where either bilirubin monoxyloside was formed (at low UDP-xylose concentrations) or where mixtures of both the mono- and di-xyloside were synthesized (at high UDP-xylose concentrations). Both enzyme activities were stimulated by Mg(2+); Ca(2+) was slightly less, and Mn(2+) slightly more, stimulatory than Mg(2+). Of the activities found in standard assay systems containing Mg(2+), 58-78% (substrate UDP-glucose) and 0-38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg(2+)-dependent activities against the concentration of added Mg(2+) were linear. 4. In comparative experiments the relative activities of liver homogenates obtained with UDP-glucuronic acid, UDP-glucose and UDP-xylose were 1:1.5:2.7 for untreated preparations and 1:0.29:0.44 after activation with digitonin. 5. Bilirubin UDP-glucuronyltransferase was protected against denaturation by human serum albumin, whereas bilirubin UDP-xylosyltransferase was not. 6. Digitonin-treated and untreated liver homogenates from Gunn rats were inactive in transferring sugar to bilirubin from UDP-glucuronic acid (in agreement with the work of others), UDP-glucose or UDP-xylose.

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Uridine diphosphate glucose dehydrogenase from cornea and epiphysial-plate cartilage.

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