Diphosphate Galactose Research Articles

Escherichia coli uridine diphosphate galactose 4-epimerase: circular dichroism of the protein and protein bound dihydronicotinamide adenine dinucleotide

The circular dichroism spectra of E. coli UDP-galactose-4-epimerase in its native (epimerase-NAD+) and reduced (epimerase-NADH.UMP) forms between 190 and 400 nm are presented. The reduced form exhibits a large positive circular dichroism band at 340 nm attributed to NADH in the complex. Relative to the small negative band exhibited at this wavelength by free NADH itself, the rotational strength of enzyme-bound NADH is some 50 times larger than that of free NADH, while the oscillator strengths and other special characteristics are similar. This enhancement reflects dissymetric interactions involving the 340-nm transition and is most consistent with the dihydropyridine ring of NADH being highly immobilized in the reduced complex. In the 200- to 230-nm region both enzyme forms exhibit a negative band at 220 nm and a negative shoulder at 208 nm. The ellipticities of the reduced form are minimally 7% greater at both band positions than those of the native form. The spectra are interpreted to indicate that conversion of the native to the reduced form is accompanied by an increase in alpha-helix structure at the expense of unordered structure.

Specificity of polyprenyl phosphates in the in vitro formation of lipid-linked sugars

Various polyprenyl phosphates were prepared by chemical phosphorylation of native and partially hydrogenated polyprenols. They were tested as lipid acceptors of sugars from nucleoside diphosphate sugars using a microsomal preparation from rat liver and membrane preparations from B. stearothermophilus, S. typhimurium, and Sh. flexneri. With the microsomal glycosyl transferase system, a demand for saturation of the α-isoprene residue of polyprenyl phosphate was observed; the chain length and cis/trans configuration of polyprenyl radical were less important. With bacterial glycosyl transferases, a demand for the unsaturated α-isoprene residue was observed. In B. stearothermophilus, the rate of synthesis of polyprenyl monophosphate glucose did not depend on the chain length of fully unsaturated polyprenyl phosphate. In S. typhimurium, C 55-polyprenyl phosphate was the most effective precursor of polyprenyl diphosphate galactose.

Fluorescence and nucleotide binding properties of Escherichia coli uridine diphosphate galactose 4-epimerase: support for a model for nonstereospecific action

The fluorescence emission spectrum for reduced diphosphopyridine nucleotide (DPNH) in Escherichia coli uridine diphosphate galactose 4-epimerase-DPNH complexes has a maximum at 435 nm, which is about twice as intense when the excitation is at 280 nm as at 340 nm. The fluorescence excitation spectrum monitored at 460 nm has two maxima, one at 340-345 nm and another about twice as intense at 280 nm. The polarization of DPNH fluorescence by these complexes is 0.43-0.44 compared with 0.46 for DPNH immobilized in propylene glycol at -20 degrees C. The small degree of fluorescence depolarization is due to rotational relaxation of the protein, relaxation time 205 ns. The excited-state lifetimes in epimerase-DPNH-nucleotide complexes are 3.5-4.2 ns. The fluorescence data show that the dihydropyridine ring in these complexes is highly immobilized and exhibits no detectable independent motion relative to rotational motions of the protein. The inhibition constants for uridine monophosphate (UMP) and 2,2,6,6-tetramethyl-4-piperidinyl-1-oxyl uridyl pyrophosphate acting as competitive reversible inhibitors of epimerase-DPN+ are 1.2 and 0.2 mM, respectively, at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5. A collection of Ki and Km values for uridine nucleotide inhibitors and substrates indicates that the principle substrate binding interactions involve the nucleotide moieties of substrates. Dissociation constants for uridine nucleotides dissociating from epimerase-DPNH-nucleotide complexes, measured by ultraviolet absorption and fluorescence techniques, are 12 muM for UMP, 14 muM for UDP-hexopyranoses, 4 muM for UDP-pentopyranoses, 27 muM for p-bromoacetamidophenyl uridyl pyrophosphate, 0.14 muM for UDP-4-ketohexopyranose intermediate, and 0.36 muM for UDP-4-ketopentopyranose intermediate at 27 degrees C in 0.1 M sodium bicinate buffer at pH 8.5. Analysis of these data shows conclusively that the major part of the binding free energy for UDP-4-ketopyranose intermediates binding to epimerase-DPNH is attributable to the uridylpyrophosphoryl components and that the glycosyl-binding free energies are much smaller. The data show that the action of this enzyme does not require tight binding between the active site and glycosyl groups of either substrates or intermediates, although there is favorable binding of the uridylpyrophosphoryl components, particularly by epimerase-DPNH. It is postulated that nonstereospecific action results from and depends upon relatively weak, nonspecific active site binding of glycosyl groups in substrates and intermediates and that the uridylpyrophosphoryl groups serve as binding anchors in the epimerization process.

A Review of the Effects of Alcohol on Carbohydrate Metabolism

1. The effects of alcohol (ethanol) on carbohydrate metabolism are reviewed. 2. The metabolism of ethanol by alcohol dehydrogenase leading to a decreased [NAD+]/[NADH] ratio plays an important role in the above effects of ethanol in the liver, whereas effects in brain and other tissues could be caused by acetaldehyde transported from the liver. 3. Ethanol increases peripheral acetate utilization and decreases free coenzyme A in brain. 4. Ethanol inhibits hepatic gluconeogenesis by decreasing the steady-state concentration of pyruvate. 5. Ethanol inhibits glycolysis in liver and brain. In the liver, the inhibition may be at the level of 3-glyceraldehyde phosphate dehydrogenase. 6. Ethanol inhibits the tricarboxylic acid cycle by an undefined mechanism(s) involving decreased pyruvate concentration, increased malate/oxaloacetate ratio or inhibition of citrate synthase and isocitrate dehydrogenase. 7.Ethanol inhibits the pentose phosphate pathway in the liver, but enhances that in the brain. The mechanisms of these actions require investigation. 8. Ethanol causes an initial hyperglycaemia, a later hypoglycaemia and various effects on glucose utilization. 9. Ethanol inhibits galactose metabolism by inhibiting the key enzyme uridine diphosphate galactose 4-epimerase. 10. Ethanol inhibits the metabolism of fructose and sorbitol. 11. It is suggested that further work is required to examine the roles of acetaldehyde and of pyridine nucleotides in the actions of ethanol on brain carbohydrate metabolism.

The metal ion catalyzed decomposition of nucleoside diphosphate sugars.

The metal ion catalysed decomposition of the nucleotide diphosphate sugars, uridine diphosphate glucose, uriding diphosphate galactose, uridine diphosphate N-acetylglucosamine, guanosine diphosphate mannose, and guanosine diphosphate fucose (UDPGlc, UDPGal, UDPGlc-NAc, GDPMan, and GDPFuc, respectively), has been studies as a function of pH. UDPDlc and UDPGal decompose readily to the a,2-cycle phosphate derivative of the sugar and uridine 5'-phosphoric acid (UMP) in the presence of Mn2+. Under all conditions tested, UDPGal decomposes two to three times more rapidly than does UDPGlc. GDPFuc is slowly degraded to free fucose under similar conditions; the other nucleotide diphosphate sugars are stable. The rate of reaction increases with increasing hydroxide ion concentration from pH 6.5 to 7.9 and with metal ion concentration from 10 to 200 mm. Several metal ions are effective catalysts; at pH 7.5 WITH 20 mM UDPGal and 20 mM metal ion, the following apparent first-order rate constants (min-1 x 10(4)) were obtained: Eu3+ 700; Mn2+, 70; Co2+ 27; Zn2+, 22; Ca2+, 3.0; Cu2+, 2.4; and Mg2+, 0. It appears that Mn2+ concentrations that have been used in studies with nucleotide diphosphate sugars at neutral pH can catalyze significant decomposition leading to erroneous interpretation of kinetic and incorporation experiments.

A method for galactose-1-phosphate uridyltransferase assay and the separation of its isozymes by DEAE-cellulose column chromatography

A simplified radioactive assay for galactose-1-phosphate uridyltransferase (EC 2.7.7.12) using a small DEAE-cellulose column for the identification of the endproduct (uridine diphosphate galactose) is described. The enzyme activities in red blood cell hemolysates of normal subjects are in the range of 24 to 33 (average 30.3) μmol UDPgalactose produced per g hemoglobin per h and in fibroblasts 0.39 and 1.39 (average 0.71) nmol per mg protein per min. Furthermore, different isozymes of red blood cell galactose-1-phosphate uridyltransferase were separated on DEAE-cellulose columns. In the case of a normal genotype, most of the enzyme activity is eluted at the earlier fractions with the low molar phosphate buffer, whereas the Duarte variant appeared at later fractions with higher molar phosphate buffer.

The concerted inactivation of Escherichia coli uridine diphosphate galactose 4-epimerase by sugar nucleotide together with a free sugar.

1. The combined effect of the sugar nucleotides UDP-D-fucose or UDP-D-glucuronic acid together with the free sugars D-fucose or L-arabinose is the inactivation of the Escherichia coli enzyme UDP-galactose 4-epimerase (EC 5.1.3.2). The sugar nucleotide or the free sugar alone or the sugar nucleotide plus 5'-Ump do not inactivate the enzyme. 2. The inactivation of the enzyme by its substrate UDP-D-glucose was not affected by the presence of free sugar. 3. In all cases the inactivation observed follows pseudo-first-order kinetics. 4. A comparison of various sugar nucleotides indicates that the hydroxymethyl group at position 6 of the sugar moiety of the natural substrates is important for substrate binding.

Evidence for specific transport of uridine diphosphate galactose across the Golgi membrane of rat mammary gland.

The inhibition of lactose synthesis by UDP-glucose, UDP-glucuronate and, less so, by UDP-N-acetylglucosamine was markedly smaller in preparations of "intact" than of lysed vesicles derived from the Golgi apparatus of lactating rat mammary gland. This constitutes evidence for a specific, probably facilitated, transport of UDP-galactose across the Golgi membrane.

Isolation and characterization of Gal E mutant Ty 21a of Salmonella typhi: a candidate strain for a live, oral typhoid vaccine.

A gal E mutant of Salmonella typhi was isolated; results obtained with Salmonella typhimurium and the mouse as a model for human typhoid fever indicated that this mutant has the potential for use as a live, oral typhoid vaccine. The mutant, Ty 21a, took up galactose from exogenous sources and accumulated sufficient quantities of galactose-1-phosphate and uridine diphosphate galactose to cause lysis of the cells, an event that resulted in the avirulence of the strain. Galactose was incorporated sufficiently into the cell wall of Ty 21a to allow the synthesis of smooth-type lipopolysaccharides, which are necessary for the proper immunogenicity. Cells of strain Ty 21a, when given intraperitoneally, protected mice against lethal challenge with strain Ty 2 of S. typhi.

Kinetic studies on the effect of uridine diphosphate galactose and manganous ions on the reaction between lactose synthetase A protein from human milk and p-hydroxymercuribenzoate.

The inhibition of lactose synthetase A protein by p-hydroxymercuribenzoate at pH7.5 and 25 degrees C, which involves the reaction of one molecule of inhibitor with each molecule of enzyme, was decreased in rate by UDP-galactose, especially in the presence of Mn(2+). Pseudo-first-order rate constants for the reaction between 0.1mm-p-hydroxymercuribenzoate and free enzyme, the enzyme-UDP-galactose complex and the enzyme-Mn(2+)-UDP-galactose complex were 4.4x10(-2), 1.9x10(-2) and 0.3x10(-2)min(-1) respectively. The results also indicated that dissociation constants for UDP-galactose in the enzyme-UDP-galactose and enzyme-Mn(2+)-UDP-galactose complexes were 313 and 16mum respectively, the latter value being similar to the K(m) for UDP-galactose in the lactose synthetase reaction. The protective effect of UDP-galactose and the role of Mn(2+) ions in lactose synthetase are discussed.

Inactivation of Soluble Bovine Milk Galactosyltransferase (Lactose Synthetase) by Sulfhydryl Reagents and Trypsin: PROTECTION BY SUBSTRATES AND PRODUCTS

Abstract Galactosyltransferase exhibited a time-dependent loss of activity when incubated with sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide. Though incubations were continued for periods 20-fold greater than the half-time of inactivation, neither reagent eliminated all catalytic activity, and generally 5 to 10% of the initial activity remained. p-Chloromercuribenzoate inactivated the enzme more rapidly then N-ethylmaleimide, but the inhibition by p-chloromercuribenzoate was reversed by mercaptoethanol. Substrates and substrate analogs protected against inhibition by p-chloromercuribenzoate and N-ethylmaleimide. Ligands giving the most protection against inhibition were analogs of the nucleotide substrate and product, uridine diphosphate galactose and UDP, respectively, in the presence of manganous chloride. When incubated with 10 mm N-ethylmaleimide, galactosyltransferase exhibited an apparent rate constant of inactivation of 0.023 min-1; adding analogs of UDP at concentrations of 2 mm reduced this rate by 30% and the same analogs in the presence of manganous chloride reduced the rate constant of inactivation by 98%. Inhibition of galactosyltransferase by N-ethylmaleimide resulted in a dramatic decrease in Vmax whereas the apparent Km for UDP-galactose remained constant. A similar pattern of protection against inactivation of galactosyltransferase by trypsin was observed suggesting that a conformational change in the protein is responsible for the observed protection.

Studies on the thiol group of lactose synthetase A protein from human milk and on the binding of uridine diphosphate galactose to the enzyme.

The lactose synthetase activity of A protein from human milk was much decreased but not abolished by reaction with thiol-group reagents. Protection experiments indicated that a free thiol group on the enzyme is situated near the UDP-galactose binding site and inactivation of the enzyme with p-hydroxymercuribenzoate was probably due to prevention of UDP-galactose binding. Affinity chromatography showed that the mercuribenzoate substituent also decreased the affinity of A protein for N-acetylglucosamine but complex-formation between A protein-N-acetylglucosamine and alpha-lactalbumin was relatively unaffected. UDP-galactose appears to be bound to the enzyme mainly through its pyrophosphate group with Mn(2+) ion and through the cis hydroxyls of ribose, whereas its hexose moiety has little if any affinity for the enzyme. Lactose synthetase activity remaining after the reaction with thiol-group reagents indicates that a free thiol group is not an essential part of the A protein active site.

Plasma membrane ectoglycosyltransferase activity of L1210 murine leukemic cells.

AbstractL1210 murine leukemia cells after treatment with Cl. perfringens neuraminidase at pH 7.0 incorporated six times more N‐acetylneuraminic acid‐[C14] than control cells when incubated for 30 minutes with cytidine 5′‐monophosphate N‐acetylneuraminic‐[C14] acid and three times more galactose‐[C14] when incubated with uridine diphosphate galactose‐[C14]. These sugars were incorporated in a 10% trichloracetic acid insoluble fraction and more than 75% of the incorporated N‐acetylneuraminic acid‐ [C14] could be removed by further treatment of these cells with neuraminidase. The incorporation of N‐acetylneuraminic acid‐ [C14] as a function of time was divided into two rates: a rapid one, active during the first 30 minutes followed by a slower one, similar to the rate observed with untreated cells. The addition of Ba++ and Ca++ ions at 8.3 mM increased the incorporation of N‐acetylneuraminic acid‐ [C14] by 25% while 8.3 mM EDTA decreased activity by 58% . The addition of Zn++ or Hg++ at similar concentrations abolished the incorporation almost completely. The optimal pH for the incorporation of N‐acetylneuraminic acid‐ [C14] by these neuraminidase treated cells was 6.5. These data suggest that ectoglycosyltransferases are present on the outer surface of the plasma membrane of L1210 cells and are able to catalyze the addition of radiolabeled nucleotide sugars onto macromolecular acceptors (cell surface glycoproteins and glycolipids) prepared by prior incubation of the cells with neuraminidase. Use of these procedures for labeling outer cell surfaces may also prove to be valuable for the study of plasma membrane glycoprotein and glycolipid structure, synthesis, and turnover.

Changes in cerebroside synthetic activities of the brain with development of the rhesus monkey (macaca mulatta)

Cerebroside synthetic activities vary according to age in homogenates of the pons and cortical gray matter from brains of rhesus monkeys. The rate of utilization of uridine diphosphate (UDP) galactose and hydroxy fatty acid (HFA) ceramide for cerebroside synthesis by the pons was very low until 100 days after conception and reached a maximum at about 150 days (15 days before birth). This activity was very low in gray matter.There were marked effects of the type of ceramide and UDP sugar added, as well as of the origin of the brain homogenates from gray matter or pons on the quantity and type of cerebroside synthetized.

Studies on Pyrophosphates II. A New Method for the Synthesis of Nucleotide Coenzymes and Their Analogs

Abstract A new method for the synthesis of unsymmetrical pyrophosphates from the reaction of alkyl phosphorothioates and disilver salt of alkyl phosphates are described. Nucleotide coenzymes such as uridine diphosphate glucose, uridine diphosphate galactose, and flavin adenine dinucleotide was synthesized.

Uridine diphosphate galactose 4-epimerase in human and other mammalian hemolysates

1. 1. In contrast to the requirement for exogenous NAD + in the assay of UDP-galactose 4-epimerase (UDPgalactose epimerase, EC 5.1.3.2), in hemolysates from adults, substantial epimerase activity can be demonstrated in hemolysates from newborn infants without addition of NAD +. It has been shown that erythrocytes of newborns are deficient in stromal NAD nucleosidase; and that a considerable portion of intracellular NAD + remains in the hemolysate. In the preparation of hemolysates of adults, NAD + of intracellular origin is destroyed by activity of stromal NAD nucleosidase. 2. 2. Upon starch gel electrophoresis, two distinctly separate activity bands were found for epimerase in hemolysates from newborn infants and one distinct band, of different mobility, for the enzyme in hemolysates of adults. In the presence of NAD + in the gel, electrophoresis of hemolysates of adults resulted in the two-banded epimerase pattern found for newborns. It is presumed that an NAD +-dependent structural change in the enzyme is involved. 3. 3. Two-banded electrophoretic patterns for epimerase were found in hemolysates of a number of mammals other than man, each set having a different electrophoretic mobility from that of any other.

Glycoprotein synthesis in the adult rat pancreas. I. Subcellular distributions of uridine diphosphate galactose: glycoprotein galactosyltransferase and thiamine pyrophospate phosphohydrolase.

Abstract 1. 1. UDPgalactose:glycoprotein galactosyltransferase in homogenates of adult rat pancreas catalyzed the transfer of galactose to desialylated submaxillary mucins. MnCl2 and Triton X-100 stimulated this reaction and the optimal pH was between 7 and 8. No transfer of galactose from UDPgalactose to N-acetylglucosamine was detected. 2. 2. Approx. 80% of the galactosyltransferase was associated with microsomes; less than 5% of the activity was soluble. The specific activity in the smooth microsomal subfraction was 8-fold greater than the homogenate. No activity was found in zymogen granules or zymogen granule membranes. 3. 3. Thiamine pyrophosphohydrolase was detected in all subcellular fractions. Approx. 60% of this activity was soluble; only 19% was associated with the microsomal fraction. The specific activity in the smooth microsomal subfraction was approx. 4-fold greater than the homogenate.

The Intermediate in the Uridine Diphosphate Galactose 4-Epimerase Reaction: RESOLUTION OF AN APPARENT AMBIGUITY

Abstract Incubation of UDP-galactose with substrate amounts of UDP-galactose 4-epimerase (EC 5.1.3.2) from Escherichia coli for different times between 2 and 120 min, followed by reduction with NaB3H4 invariably yields a mixture of labeled UDP-glucose and UDP-galactose. Chemical degradation of tritiated hexoses, isolated after NaB3H4 reduction of 2-min incubation mixtures, shows all of the tritium associated with carbon 4, in agreement with the results presented earlier using longer incubation times (Maitra, U. S., and Ankel, H. (1972) Proc. Nat. Acad. Sci. U. S. A. 68, 2660). The total amount of label introduced after 2 min of incubation, when enzyme activity is practically unchanged, is considerably less than that after 120 min of incubation, when enzyme activity is reduced to about 50%. The excess of labeled UDP-hexoses after 120 min of incubation can be quantitatively related to the amount of inactive enzyme formed, assuming that during inactivation additional UDP-4-ketohexose is produced in amounts equivalent to the amounts of inactive enzyme.

Immunological investigations of penicillium—II. Primary binding of glycopeptides and glycopeptide derivatives to specific antibodies

An exocellular glycopeptide from Penicillium charlesii has been shown previously to contain a high mol. wt polysaccharide and several oligosaccharides attached to the peptide through seryl and threonyl residues. Guinea pigs immunized with the glycopeptide conjugated to bovine gamma globulin, produced nonprecipitating antibodies to the polysaccharide portion of the glycopeptide. However, a technique was developed for measuring the primary binding of the glycopeptide by the guinea pig antibodies. It was found that the antigen-antibody complex could be precipitated by 40% ammonium sulfate, whereas negligible quantities of the glycopeptide alone or glycopeptide incubated with normal guinea pig sera were precipitated under these conditions. Equations predicting the influence of the ratio of a weighted dissociation constant representing the various antigen-antibody complexes/antibody concentrations, β/[ Ab t] , on antigen binding and the influence of this ratio on inhibition of antigen binding by hapten were derived. The data obtained with the fungal glycopeptide as an antigen were interpreted in terms of this model. The experiments showed that antibodies in the guinea pig sera were directed toward the polysaccharide portion of the glycopeptide, and not toward the peptide portion or the oligosaccharides. Furthermore, a glycopeptide deficient in galactofuranosyl and phosphorus moieties from a uridine diphosphate galactose 4-epimerase negative mutant of P. charlesii competed with wild type glycopeptide for antibody. It appeared that mannopyranosyl residues associated with an, as yet unidentified, acid labile material were the major immunodeterminant groups of the glycopeptide which bound to guinea pig sera. In contrast to its behavior in guinea pig antisera, the polysaccharide portion of the glycopeptide did not compete with the glycopeptide for antibodies in rabbit antisera. In addition the peptide portion of the glycopeptide did not compete with the glycopeptide in the rabbit antisera. Further, dilute acid treatment of the glycopeptide did not destroy its ability to bind to rabbit antibodies. These data suggest that the oligosaccharides attached to the peptide serve as the antigenic determinats which bind to rabbit antibodies.

Substrate stereochemical requirements in the reductive inactivation of uridine diphosphate galactose 4-epimerase by sugar and 5'-uridine monophosphate.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSubstrate stereochemical requirements in the reductive inactivation of uridine diphosphate galactose 4-epimerase by sugar and 5'-uridine monophosphateJeanne Nelson Ketley and Karl A. SchellenbergCite this: Biochemistry 1973, 12, 2, 315–320Publication Date (Print):January 1, 1973Publication History Published online1 May 2002Published inissue 1 January 1973https://doi.org/10.1021/bi00726a022RIGHTS & PERMISSIONSArticle Views29Altmetric-Citations11LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (669 KB) Get e-Alerts Get e-Alerts