Interaction of uridine diphosphate glucose with calf liver uridine diphosphate glucose dehydrogenase. Significance of hydroxyl groups at C-3, C-4 and C-6 of hexosyl residue.

Analogs of uridine diphosphate glucose (UDPGlc) with a modified hexosyl residue which contained a deoxy-unit at C-3 or C-4 were tested as substrates of calf liver UDPGlc dehydrogenase (EC 1.1.1.22). The 3-deoxyglucose derivative was found not to serve as a substrate for the enzyme whereas the 4-deoxyglucose analog was able to participate in the reaction. The apparent Km of the latter was 5.3 times that of UDPGlc and the relative V was 0.04. The reaction product was identified as uridine diphosphate deoxyhexuronic acid. UDP-deoxyhexoses were non-competitive inhibitors of UDPGlc enzymic oxidation, inhibition increased in the sequence: 2-deoxy-less than 3-and 6-deoxy-less than 4-deoxyglucose derivative. The significance of different HO-groups in hexosyl residue for interaction of UDPGlc with the enzyme is discussed.     

Half-sites oxidation of bovine liver uridine diphosphate glucose dehydrogenase.

6,6-Dithiodinicotinate shows half-of-the-sites reactivity towards the six catalytic-site thiol groups of bovine liver UDP-glucose dehydrogenase. The reagent introduces three intrasubunit disulphide linkages between catalytic-site thiol groups and non-catalytic-site thiol groups and abrogates 60% of the catalytic activity of the hexameric enzyme; excess 2-mercaptoethanol rapidly restores full catalytic activity. These results show the half-of-the-sites behaviour of the enzyme with the reagent and the presence of a non-catalytic-site thiol group capable of forming a disulphide linkage with a catalytic-site thiol group on the same subunit without irreversible denaturation.     

Mechanism of the enzymatic reaction catalyzed by uridine diphosphate glucose dehydrogenase. The chemical synthesis of uridine diphosphate glucose-6-H3 and its oxidation by uridine diphosphate glucose dehydrogenase]

The synthesis of UDP-glucose-6-s-H was performed through condensation of alpha-D-glucopyranosyl phosphate-6-3-H and uridine 5'-phosphomorpholidate. Enzymic oxidation of UDP-glucose-6-3-H with calf liver UDP-glucose dehydrogenase was found to proceed with direct transfer of the hydrogen from C-6 of UDP-glucose onto NAD.     

Rat liver uridine diphosphate glucose dehydrogenase: dual mechanism of regulation in vivo

Rat liver UDPglucose (UDPG) dehydrogenase activity was observed to be decreased after fasting and could be restored to normal levels after refeeding glucose. This could be prevented by prior injection of puromycin, suggesting de novo protein synthesis. Administration of insulin to normal rats on stock diet did not influence the enzyme activity. However, the enzyme activity was decreased in the diabetic condition. Intraperitoneal injection of insulin caused an enhancement of the enzyme activity in diabetic animals. Hepatic UDPG dehydrogenase activity was observed to be decreased on ascorbic acid feeding or intraperitoneal injection of the same. The intraperitoneal injection of either insulin or cAMP to ascorbic acid-treated rats resulted in an increase in enzyme activity reaching normal levels. The insulin-mediated increase could not be prevented by prior injection of puromycin, suggesting a post-translational effect. These results indicate two distinct mechanisms for in vivo regulation of hepatic UDPG dehydrogenase.     

Cyclic uridine diphosphate glucose: a new pyrimidine analog of cyclic ADP ribose

Novel compound 1, as the first example of cyclic ADP-ribose analogs containing a pyrimidine residue, was synthesized by a chemical strategy employing a Mitsunobu reaction for the condensation of the glucosyl moiety on protected uridine, and a Matsuda procedure for the cyclization step.     

Studies on ligand binding to bovine liver uridine diphosphate glucose pyrophosphorylase.

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.     

A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ;mapping' in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5'-dithiobis-(2-nitrobenzoate). 5. NAD(+), UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5'-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4-8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.     

Properties of uridine diphosphate glucose pyrophosphorylase from Golgi apparatus of liver

Golgi apparatus isolated from cat liver contained UDPglucose pyrophosphorylase (UTP:alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.9) activity. The results of washing suggested that pyrophosphorylase was bound firmly to Golgi membranes. Moreover, the enzyme was activated by Triton X-100 in the same extent as galactosyltransferase, a typical Golgi apparatus enzyme. Two-substrate kinetic studies were performed with the enzymes from cytosol and Golgi fractions. The soluble enzyme showed an apparent 2.5-fold greater activity for the glucose 1-phosphate than for UTP, while pyrophosphorylase of Golgi apparatus had the same affinity for the two substrates. A random mechanism was observed with a direct dependence of apparent Michaelis constant values on the concentration of second substrate for soluble enzyme. In contrast, with Golgi enzyme one ligand had no effect on the binding of the other.     

Amino acid sequence of the tryptic peptide containing the catalytic-site thiol group of bovine liver uridine diphosphate glucose dehydrogenase.

The catalytic-site thiol groups of UDP-glucose dehydrogenase from bovine liver were carboxymethylated with iodo[2-14C]acetate or with iodoacetamidofluorescein. After the residual thiol groups were carboxymethylated with iodoacetate, the proteins were digested with trypsin. The 14C-labelled peptide from the carboxymethylated enzyme was purified to homogeneity by successive thick-layer chromatography on silica gel, paper electrophoresis and chromatography, and column chromatography on Bio-Gel P-6. Homogeneous fluoresceincarboxamidomethylated peptide was prepared from a tryptic digest of fluoresceincarboxamidomethylated enzyme by specific adsorption--desorption from Sephadex G-25. The sequences of either peptide determined by the manual Edman dansyl procedure is: Ala-Ser-Val-Gly-Phe-Gly-Gly-Ser-Cys-Phe-Glx-Glx-Gly-Lys.     

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

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.     

Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.     

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

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.     

Uridine diphosphate glucose synthase from calf liver: determinants of enzyme activity in vitro.

The reaction catalyzed by calf liver uridine diphosphate glucose synthase (pyrophosphorylase) (EC 2.7.7.9; UTP + glucose 1-phosphate = UDP-glucose + PPi) is an example of an enzymic reaction in which a nucleoside triphosphate other than ATP is the immediate source of metabolic energy. Kinetic properties of the enzyme, acting in the direction of UCP-glucose formation were investigated in vitro. The reaction was inhibited by UDP-glucose (0.072), Pi (11), UDP (1.6), UDP-xylose (0.87), UDP-glucuronate (1.3), and UDP-galacturonate (0.95). The numbers in parentheses indicate the concentration (mM) required for half-maximal inhibition under the conditions used. Other compounds tested, including ATP, ADP, and AMP, had no effect. Over a range of concentrations of UTP (0.04-0.8 MM) and UDP-glucose (0.05-0.03 mM), the reaction rate was more dependent on the concentration ratio [UDP-glucose]/[UTP] than on the absolute concentration of either compound. Comparison of the kinetic properties in vitro with estimates of metabolite levels in vivo suggests that (1) the enzyme operates in a range far from its maximal rate, and (2) the concentrations of glucose 1-phosphate and Pi and the ratio [UDP-glucose]/[UTP] may be the most important determinants of UDP-glucose synthase activity.