Assay and properties of dititonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver.
ABSTRACT: 1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.
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.
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. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.
Project description:Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.
Project description:The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.
Project description:Neoplastic mast cells of mice (including long-established and newly derived lines) were grown in large-volume suspension cultures to provide enough cells for preparation of microsomal fractions. Microsomal preparations from P815Y and P815S cells synthesized (14)C-labelled glycosaminoglycan when incubated with UDP-[(14)C]glucuronic acid and UDP-N-acetylgalactosamine. No significant amount of (14)C-labelled glycosaminoglycan was formed when UDP-N-acetylglucosamine was substituted for the UDP-N-acetylgalactosamine. Microsomal preparations from X163 cells synthesized (14)C-labelled glycosaminoglycan when incubated with UDP-[(14)C]glucuronic acid and either UDP-N-acetylgalactosamine or UDP-N-acetylglucosamine. The (14)C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylgalactosamine was degradable by testicular hyaluronidase, indicating that it was chondroitin-like. The (14)C-labelled glycosaminoglycan formed in the presence of UDP-N-acetylglucosamine was not degradable by testicular hyaluronidase. Microsomal preparations from P815S cells were tested for sulphating activity by incubation with adenosine 3'-phosphate 5'-sulphatophosphate, as well as UDP-[(14)C]glucuronic acid, and UDP-N-acetylgalactosamine. The resulting newly synthesized polysaccharide was shown by chondroitinase ABC digestion to be 70% chondroitin 4-sulphate and 30% chondroitin. The molecular size of this newly synthesized glycosaminoglycan was determined by gel filtration to be larger than 40000 mol.wt. In general, the glycosaminoglycan-synthesizing ability of the microsomal preparations appeared to reflect glycosaminoglycan synthesis by the intact cells.
Project description:1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 188.8.131.52). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.
Project description:1. The thio-beta-d-glucosiduronic acids (thio-beta-glucuronides) of o-aminothiophenol, diethyldithiocarbamic acid, p-nitrothiophenol and thiophenol are formed biosynthetically in broken- and intact-cell preparations of mouse liver. 2. For this biosynthesis to occur in homogenates or microsomal fractions, UDP-glucuronic acid was required during incubation; glucose, glucuronic acid or UDP could not replace it. UDP was a product of the reaction. 3. The biosynthetic mechanism linking glucuronic acid to thiol and carbodithioic groups therefore requires UDP-glucuronyltransferase activity and resembles that forming the various types of O-glucuronides. 4. An analogous enzymic mechanism employing UDP-glucose synthesizes the thio-beta-d-glucosides of diethyldithiocarbamic acid and thiophenol in gut preparations of the mollusc Arion ater; this mechanism resembles that forming the O-glucosides. The thio-beta-d-glucosides are formed also in intact cells. 5. As expected from the distribution of O-glycosides, S-glucuronides of these aglycones were not detectable with the invertebrate, nor were the S-glucosides with the vertebrate. 6. Despite their similar biosyntheses, S- and O-beta-glycosides differ in susceptibility to hydrolysis by beta-glycosidases. Rat preputial-gland beta-glucuronidase hydrolysed thioglucuronides of o-aminothiophenol, diethyldithiocarbamic acid and p-nitrothiophenol, hydrolysis being inhibited by glucarolactone; the thioglucuronide of thiophenol was not hydrolysed by preputial-gland or liver beta-glucuronidase. The two S-glucosides resisted hydrolysis by beta-glucosidase from almond emulsin.
Project description:A radioassay for specific determination of the rates of UDP-glucuronic acid-dependent conversion of bilirubin into the two isomeric (C-8, C-12) bilirubin monoglucuronides and bilirubin diglucuronide is described and illustrated by its application to rat liver microsomes. The method is based on measurement of the relative amounts of radiolabel in unesterified bilirubin and its mono- and di-esterified reaction products after incubation with [14C]bilirubin as substrate. This analysis is performed by the alkaline-methanolysis procedure, combined with one of two t.l.c. systems developed in order to enhance the sensitivity, accuracy and precision of the radioassay. Results for rates of total bilirubin glucuronide formation obtained with the new assay and the standard enzyme assay based on the ethyl anthranilate diazo-method were identical. However, the sensitivity of the latter technique is approx. 10-fold lower than that of the radioassay.
Project description:A particulate enzyme preparation from etiolated pea (Pisum sativum) epicotyls was found to incorporate xylose from UDP-D-xylose into beta-(1----4)-xylan. The ability of this xylan to act as an acceptor for incorporation of [14C]glucuronic acid from UDP-D-[14C]glucuronic acid in a subsequent incubation was very limited, even though glucuronic acid incorporation was greatly prolonged when UDP-D-xylose was present in the same incubation as UDP-D-[14C]glucuronic acid. This indicated that glucuronic acid could not be added to preformed xylan. However, the presence of UDP-D-glucuronic acid inhibited incorporation of [14C]xylose from UDP-D-[14C]xylose into beta-(1----4)-xylan, and neither S-adenosylmethionine nor acetyl-CoA stimulated either the xylosyltransferase or the glucuronyltransferase.