scholarly journals Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 605-618 ◽  
Author(s):  
K. P. M. Heirwegh ◽  
M. Van De Vijver ◽  
J. Fevery
Keyword(s):  
Rat Liver ◽  
Metal Ion ◽  
Glucuronic Acid ◽  
Uridine Diphosphate ◽  
Bivalent Metal ◽  
Bivalent Metal Ions ◽  
Liver Homogenates ◽  
Cell Fractions ◽  
Biliary Excretion Rate ◽  
Substrate Concentrations

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°C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of Pi) 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. Mn2+ was slightly more, and Ca2+ somewhat less, stimulatory than Mg2+. The Mg2+-dependent fraction showed Michaelis–Menten kinetics with respect to the added Mg2+. 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.

scholarly journals Enzymic transfer of glucose and xylose from uridine diphosphate glucose and uridine diphosphate xylose to bilirubin by untreated and digitonin-activated preparations from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 619-633 ◽  
Author(s):  
J. Fevery ◽  
P. Leroy ◽  
K. P. M. Heirwegh
Keyword(s):  
Enzyme Activities ◽  
Metal Ion ◽  
Glucuronic Acid ◽  
Uridine Diphosphate ◽  
No Formation ◽  
Cell Extracts ◽  
Straight Line ◽  
Standard Assay ◽  
Diphosphate Glucose ◽  
Liver Homogenates

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 Mg2+; Ca2+ was slightly less, and Mn2+ slightly more, stimulatory than Mg2+. Of the activities found in standard assay systems containing Mg2+, 58–78% (substrate UDP-glucose) and 0–38% (substrate UDP-xylose) were independent of added bivalent metal ion. Double-reciprocal plots of the Mg2+-dependent activities against the concentration of added Mg2+ 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.

scholarly journals The properties of uridine diphosphate glucuronyltransferase(s) which catalyse the synthesis of steroid glucuronides in microsomal fractions from guinea-pig liver

1976 ◽  
Vol 157 (3) ◽  
pp. 667-673 ◽  
Author(s):  
D Zakim ◽  
D A Vessey
Keyword(s):  
Guinea Pig ◽  
Glucuronic Acid ◽  
Large Family ◽  
Uridine Diphosphate ◽  
Bivalent Metal ◽  
Pig Liver ◽  
Bivalent Metal Ions ◽  
Triton X ◽  
Related Proteins ◽  
Guinea Pig Liver

The properties of the UDP-glucuronyltransferase(s) of guinea-pig liver that catalyse the synthesis of steroid glucuronides were examined. There are many similarities between apparently different substrate-specific forms of these enzymes in that all are activated by bivalent metal ions, and all contain at least 2 thiol groups important for enzyme activity. On the other hand, there are significant differences between the enzymes conjugating steroids and those conjugating non-steroids. Only the latter are activated by UDP-N-acetylglucosamine, which enhances their relatively poor affinity for UDP-glucuronic acid. The steroid-conjugating forms of UDP-glucuronyltransferase are not activated by UDP-N-acetylglucosamine and have relatively high apparent affinities for UDP-glucuronic acid. The rate of glucuronidation of testosterone was inhibited by treatment with phospholipase A. Treatment with cholate or Triton X-100 did not enhance the rates of glucuronidation of any steroid tested. The data indicate several similarities between different forms of UDP-glucuronyltransferase, suggesting that there is a large family of related proteins. At the same time there are important differences in the parameters that modulate the rates of different glucuronidation reactions.

THE ACID-SOLUBLE NUCLEOTIDES OF SALMON LIVER

1958 ◽  
Vol 36 (5) ◽  
pp. 465-473 ◽  
Author(s):  
H. Tsuyuki ◽  
Violet M. Chang ◽  
D. R. Idler
Keyword(s):  
Low Temperature ◽  
Anion Exchange ◽  
Rat Liver ◽  
Glucuronic Acid ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Uridine Diphosphate ◽  
Carbohydrate Storage ◽  
Diphosphate Glucose

The acid-soluble nucleotides of spring salmon liver have been separated by anion-exchange chromatography at low temperature and characterized. Under these conditions the relatively labile uridine-5′-diphosphate nucleotides of acetylglucosamine, galactose, and glucuronic acid were obtained intact, a fact that is further substantiated by the complete absence of uridine-5′-diphosphate. The occurrence of these uridine diphosphate compounds and the absence of uridine diphosphate glucose is discussed in relation to the previously postulated role of inositol as a carbohydrate storage product. A new peptide-containing nucleotide, succinoadenosine-5′-phosphosulphate (peptide), was found in the fraction which immediately follows adenosine-5′-diphosphate. The parent base of this nucleotide, succinoadenine, was also isolated. The nucleotide pattern is simpler than that reported by other investigators for rat liver and wheat.

Membrane translocation and regulation of uridine diphosphate-glucuronic acid uptake in rat liver microsomal vesicles

1995 ◽  
Vol 108 (1) ◽  
pp. 183-192 ◽  
Author(s):  
Carl L. Berg ◽  
Anna Radominska ◽  
Roger Lester ◽  
John L. Gollan
Keyword(s):  
Rat Liver ◽  
Glucuronic Acid ◽  
Acid Uptake ◽  
Uridine Diphosphate ◽  
Membrane Translocation

scholarly journals Stimulation of defective Gunn-rat liver uridine diphosphate glucuronyltransferase activity in vitro by alkyl ketones

1979 ◽  
Vol 177 (3) ◽  
pp. 993-995 ◽  
Author(s):  
E N Lalani ◽  
B Burchell
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Wistar Rat ◽  
Uridine Diphosphate ◽  
Gunn Rat ◽  
Alkyl Ketone ◽  
Genetic Deficiency ◽  
Liver Homogenates ◽  
Stimulation Of

Addition of alkyl ketone (10mM) to Gunn-rat liver homogenates increased UDP-glucuronyltransferase activity towards 2-aminophenol by 10–20 fold, up to enhanced values of enzyme activity observed with similarly treated Wistar-rat liver homogenates. Alkyl ketones also activate the defective enzyme purified from Gunn-rat liver. This genetic deficiency of UDP-glucuronyltransferase activity is no longer apparent when assayed in the presence of alkyl ketones.

Degradation of Perfluoropolyether Fluids With Metal Ions

1999 ◽  
Vol 121 (2) ◽  
pp. 348-351 ◽  
Author(s):  
Takashi Fukuchi
Keyword(s):  
Metal Ions ◽  
Reaction Mechanisms ◽  
Metal Ion ◽  
Chemical Degradation ◽  
Bivalent Metal ◽  
Ether Cleavage ◽  
Cleavage Process ◽  
Metal Atoms ◽  
Bivalent Metal Ions ◽  
Fomblin Z

Chemical degradation of lubricant fluids of perfluoropolyether (PFPE), such as Fomblin Z-DOL, Fomblin AM-2001, Fomblin Z-25, Demnum SP-3, and Demnum SA, with bivalent metal ions was examined. The respective reaction mechanisms involve an ether cleavage process which is caused by the coordination of metal ions with oxygen atoms in the main chains of the PFPE. Metal ions were evaluated for their ability to degrade the PFPE, and the results demonstrate that the ions ranking in terms of this ability is as follows: Cu2+ > Ni2+ > Co2+ > Fe2+ > Mn2+ > Mg2+. This trend can be explained by the ionization potentials of the metal atoms. For an identical metal ion, the Demnum lubricants showed a greater stability than the Fomblin lubricants.

scholarly journals Structures of bilirubin conjugates synthesized in vitro from bilirubin and uridine diphosphate glucuronic acid, uridine diphosphate glucose or uridine diphosphate xylose by preparations from rat liver

1972 ◽  
Vol 129 (3) ◽  
pp. 635-644 ◽  
Author(s):  
J. Fevery ◽  
P. Leroy ◽  
M. Van De Vijver ◽  
K. P. M. Heirwegh
Keyword(s):  
Carboxylic Acid ◽  
Rat Liver ◽  
Glucuronic Acid ◽  
Diazonium Salt ◽  
Acid Amide ◽  
Uridine Diphosphate ◽  
Prolonged Incubation ◽  
Carboxylic Acid Groups ◽  
Decreasing Ph

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 β-d-monoglucuronoside, with (b), bilirubin β-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 β-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 β-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 β-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 β-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.

scholarly journals Activation of membrane-bound high-affinity calcium ion-sensitive adenosine triphosphatase of human erythrocytes by bivalent metal ions

1975 ◽  
Vol 147 (2) ◽  
pp. 359-361 ◽  
Author(s):  
H Pfleger ◽  
H U Wolf
Keyword(s):  
Metal Ions ◽  
Calcium Ion ◽  
Metal Ion ◽  
Adenosine Triphosphatase ◽  
Erythrocyte Membranes ◽  
Bivalent Metal ◽  
High Affinity ◽  
Human Erythrocyte Membranes ◽  
Bivalent Metal Ions ◽  
Membrane Bound

The Ca2+-sensitive ATPase (adenosine triphosphatase) of human erythrocyte membranes is activated, not only by Ca2+ ions, but also by a series of other bivalent metal ions including Sr2+, Ba2+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+ and Pb2+. The degree of activation is dependent on the radius of the ion rather than on its nature, in contrast with the dissociation constant of the enzyme--metal ion complex.

scholarly journals The specificity of yeast low-Km cyclic AMP phosphodiesterase towards free bivalent metal ions and the diastereoisomers of cyclic adenosine phosphorothioate

1985 ◽  
Vol 226 (3) ◽  
pp. 897-900 ◽  
Author(s):  
K Suoranta ◽  
J Londesborough
Keyword(s):  
Cyclic Amp ◽  
Metal Ions ◽  
Metal Ion ◽  
Relative Activity ◽  
Bivalent Metal ◽  
Cyclic Amp Phosphodiesterase ◽  
Bivalent Metal Ions

The relative activity of a zinc-containing cyclic AMP phosphodiesterase towards the (Sp)- compared with the (Rp)-diastereoisomer of cyclic adensine phosphorothioate varied with the identity of the free bivalent metal ion from more than 35 to 0.074 along the series Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Cd2+, showing that this ion, and not the tightly bound zinc, bonds to the phosphorothioate moiety of the substrate.

scholarly journals Characterization of jack-bean α-D-mannosidase as a zinc metalloenzyme

1975 ◽  
Vol 147 (1) ◽  
pp. 83-90 ◽  
Author(s):  
S M Snaith
Keyword(s):  
Gel Electrophoresis ◽  
Metal Ion ◽  
Chelating Agents ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Chromatography ◽  
Enzyme Molecule ◽  
Bivalent Metal ◽  
Jack Bean ◽  
Bivalent Metal Ions

1. Two methods were used to obtain α-mannosidase free from unbound Zn2+, (a) by removal of excess of metal ion from preparations purified in the presence of Zn2+ and (b) by purification under conditions that eliminate the need to add Zn2+. 2. The purified enzyme is homogeneous on ultracentrifugation, polyacrylamide-gel electrophoresis and gel chromatography. 3. The molecular weight is estimated to be 230 000. 4. The enzyme contains between 470 and 565 mug of zinc/g of protein, corresponding to between 1.7 and 2 atoms of zinc/enzyme molecule. The contents of other metals are much lower. 5. The enzyme is inactivated by chelating agents and activity is restored by Zn2+. 6. No other metal ion was found to replace Zn2+ with retention of activity. Some bivalent metal ions, e.g. Cu2+, rapidly inactivate the enzyme. 7. The results indicate that jack-bean α-mannosidase exists naturally as a zinc-protein complex and may be considered as a metalloenzyme.

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