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 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 Studies on glycogen synthesis in pigeon liver homogenates. Glycogen synthesis from glucose monophosphates and uridine diphosphate glucose

1967 ◽  
Vol 105 (2) ◽  
pp. 515-519 ◽  
Author(s):  
V. N. Nigam
Keyword(s):  
Time Course ◽  
Initial Rate ◽  
Glycogen Synthesis ◽  
Uridine Diphosphate ◽  
Cytoplasmic Fraction ◽  
Uridine Diphosphate Glucose ◽  
Diphosphate Glucose ◽  
Liver Homogenates ◽  
Pigeon Liver ◽  
Glycogen Formation

Comparative time-course studies of glycogen synthesis from glucose 6-phosphate, glucose 1-phosphate and UDP-glucose show that glucose 1-phosphate forms glycogen at an initial rate faster than that obtained with glucose 6-phosphate and UDP-glucose. After 5min. the rates from glucose monophosphates are considerably slower. 2,4-Dinitrophenol decreases glycogen synthesis from both glucose monophosphates, whereas arsenate and EDTA increase glycogen synthesis from glucose 1-phosphate and inhibit the reaction from glucose 6-phosphate, galactose and galactose 1-phosphate. Mitochondria-free pigeon liver cytoplasmic fraction forms less glycogen from glucose monophosphates than does the whole homogenate. 2-Deoxyglucose 6-phosphate inhibits glycogen synthesis from glucose monophosphates. Glycogen formation from UDP-glucose is relatively unaffected by dinitrophenol, by arsenate, by EDTA, by 2-deoxyglucose 6-phosphate and by the removal of mitochondria from the whole homogenate.

A DFT study on the catalytic mechanism of UDP-glucose dehydrogenase

2010 ◽  
Vol 88 (8) ◽  
pp. 804-814 ◽  
Author(s):  
WenJuan Huang ◽  
Jorge Llano ◽  
James W. Gauld
Keyword(s):  
Pathogenic Bacteria ◽  
Catalytic Mechanism ◽  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Polarizable Continuum Model ◽  
Uridine Diphosphate ◽  
Reaction Field ◽  
Enzyme Active Site ◽  
Site Model ◽  
Diphosphate Glucose

Uridine 5′-diphosphate glucuronic acid (UDPGlcUA) is a key intermediary metabolite in many species, including pathogenic bacteria and humans. It is biosynthesized from UDP-glucose (UDPGlc) by uridine diphosphate glucose dehydrogenase (UDPGlcDH) via a twofold two-electron–one-proton oxidation that successively transforms the 6-hydroxymethyl of glucopyranose into a formyl, and the latter into the final carboxylic function. The catalytic mechanism of UDPGlcDH was investigated using a large enzyme active-site model in combination with the B3LYP method and the polarizable continuum model (IEF-PCM) self-consistent reaction field. The latter was used to correct for the long-range electrostatic effect of the protein environment. The overall mechanism consists of four catalytic steps: (i) NAD+-dependent oxidation of glucose to glucuronaldehyde, (ii) nucleophilic addition of Cys260–SH to glucuronaldehyde to form a 6-thiohemiacetal intermediate, (iii) NAD+-dependent oxidation of the 6-thiohemiacetal to form a 6-thioester intermediate, and finally, (iv) hydrolysis of the 6-thioester to give glucuronic acid. In addition, this study also provides insight into the debated roles of Lys204 and Asp264, and the most likely protonation state of a reactive Michaelis complex of UDPGlcDH.

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.

scholarly journals The binding of oxidized and reduced nicotinamide–adenine dinucleotides to bovine liver uridine diphosphate glucose dehydrogenase

1974 ◽  
Vol 141 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Paul A. Gainey ◽  
Charles F. Phelps
Keyword(s):  
Glucuronic Acid ◽  
Glucose Dehydrogenase ◽  
Gel Filtration ◽  
Bovine Liver ◽  
Uridine Diphosphate ◽  
Protein Fluorescence ◽  
Diphosphate Glucose ◽  
Ph Dependent ◽  
Uridine Diphosphate Glucose Dehydrogenase ◽  
Biphasic Profile

The binding of NAD+and NADH to bovine liver UDP-glucose dehydrogenase was studied by using gel-filtration and fluorescence-titration methods. The enzyme bound 0.5mol of NAD+and 2 mol of NADH/mol of subunit at saturating concentrations of both substrate and product. The dissociation constant for NADH was 4.3μm. The binding of NAD+to the enzyme resulted in a small quench of protein fluorescence whereas the binding of NADH resulted in a much larger (60–70%) quench of protein fluorescence. The binding of NADH to the enzyme was pH-dependent. At pH8.1 a biphasic profile was obtained on titrating the enzyme with NADH, whereas at pH8.8 the titration profile was hyperbolic. UDP-xylose, and to a lesser extent UDP-glucuronic acid, lowered the apparent affinity of the enzyme for NADH.

HISTOCHEMICAL INVESTIGATION OF THE EFFECTS OF OESTROGEN, PROGESTERONE AND RELAXIN ON GLYCOGEN, AMYLOPHOSPHORYLASE, TRANSGLYCOSYLASE AND URIDINE DIPHOSPHATE GLUCOSE-GLYCOGEN TRANSFERASE IN UTERI OF MICE

1965 ◽  
Vol 32 (2) ◽  
pp. 245-257 ◽  
Author(s):  
KATHLEEN HALL
Keyword(s):  
Enzyme Activities ◽  
Glycogen Synthesis ◽  
Glandular Epithelium ◽  
Uridine Diphosphate ◽  
Phosphorylase Activity ◽  
Uridine Diphosphate Glucose ◽  
Ovariectomized Mice ◽  
Diphosphate Glucose ◽  
Arterial Muscle

SUMMARY (1) The effects of combinations of oestrogen, progesterone and relaxin on glycogen content, and on amylophosphorylase, transglycosylase and uridine diphosphate glucose-glycogen glucosyl transferase activities in the corpus uteri of intact and ovariectomized virgin mice were investigated by histochemical techniques. (2) Glycogen and the enzyme activities were localized to myometrial and arterial muscle fibres, mobilized leucocytes when present, and luminal and glandular epithelium. Transglycosylase activity was not found in glandular epithelium and no information was obtained about UDPG-glycogen synthesis in epithelium or in leucocytes; otherwise the distribution of the three activities appeared to be similar. (3) In untreated ovariectomized mice no glycogen was detected in vivo and phosphorylase activity was low. In untreated intact mice little histochemically detectable glycogen was found in myometrial muscle at any stage of the cycle and almost no UDPG-synthesized glycogen; amylophosphorylase activity appeared to be increased during pro-oestrus and oestrus. (4) Oestrogen produced increased amounts of glycogen in vivo and stimulated phosphorylase activity in both muscle layers in intact and ovariectomized mice; UDPG-glycogen synthesis was probably also increased. (5) Relaxin had no detectable effect on myometrial glycogen or on phosphorylase activity in non-primed ovariectomized mice, but both were increased when relaxin was given to oestrogen-primed ovariectomized mice or to intact mice at pro-oestrus or oestrus. Only small increases were detected in UDPG-glycogen synthesis. (6) In both intact and ovariectomized oestrogen-primed mice progesterone had a differential action on the two layers of the myometrium: it increased both glycogenolysis and phosphorylase activity in the longitudinal fibres, but inhibited phosphorylase activity in the circular fibres without resulting in glycogen synthesis in vivo. Results on UDPG-glycogen synthesis were inconclusive. Simultaneous administration of relaxin prevented the inhibitory action of progesterone on glycogen and phosphorylase activity in the circular muscle layer and UDPG-glycogen synthesis was also high in these mice. (7) No consistent effects of the hormones were detected on glycogen or enzyme activities in arterial muscle. (8) The histochemical tests visualized total phosphorylase activity but gave no information about hormonal influence on phosphorylase a and b ratios.

ENZYMES OF LACTOSE BIOSYNTHESIS IN NORMAL AND HORMONALLY STIMULATED RABBIT MAMMARY GLANDS

1968 ◽  
Vol 40 (1) ◽  
pp. 81-84 ◽  
Author(s):  
R. J. HEITZMAN
Keyword(s):  
Enzyme Activities ◽  
Deoxyribonucleic Acid ◽  
Late Pregnancy ◽  
Mammary Glands ◽  
Uridine Diphosphate ◽  
Early Lactation ◽  
Uridine Diphosphate Glucose ◽  
Pregnancy And Lactation ◽  
Diphosphate Glucose ◽  
The Difference

SUMMARY The activities of uridine diphosphate glucose (UDPG) pyrophosphorylase and UDPG-4′-epimerase in mammary glands of rabbits were determined in late pregnancy and lactation. The activities in animals during the last 4 days of pregnancy and during days 0–4, 5–9 and 11–21 of lactation increased but the difference in the activities was significant between the days 5–9 and 11–21 only and for the pyrophosphorylase activity between days for 0–4 and 5–9. Prolactin and cortisol acetate given daily for 3 or 5 days to rabbits pseudopregnant for 15 days caused increases in enzyme activities that were several times greater than those found in controls. The enzyme activities in the stimulated glands were similar to those observed in early lactation. The levels of deoxyribonucleic acid/g. wet tissue were the same in the stimulated and lactating glands.

scholarly journals Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase

1976 ◽  
Vol 159 (1) ◽  
pp. 65-70 ◽  
Author(s):  
R A J Stevens ◽  
C F Phelps
Keyword(s):  
Ligand Binding ◽  
Metal Ion ◽  
Bovine Liver ◽  
Uridine Diphosphate ◽  
Uridine Diphosphate Glucose ◽  
Substrate Analogue ◽  
Diphosphate Glucose ◽  
Enzyme Intermediate

A procedure for the preparation of crystalline UDP-glucose pyrophosphorylase is described. K(s) values for UDP-glucose and UTP were determined as 7 and 20 muM respectively, the latter being confirmed by three methods. By assuming an octameric structure, 1 mol of enzyme subunit bound 1 mol of substrate. The metal-ion activator, Mg2+, did not affect the equilibrium between nucleotide and enzyme. A substrate analogue, alphabeta-methylene-UTP, was synthesized and had the same K(s) value as UTP. In its presence, the K(s) for glucose 1-phosphate decreased by two orders of magnitude, thus confirming a compulsory binding order and excluding an uridylated enzyme intermediate. The results are discussed with respect to their implications in vivo.

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