RESUMEN
A UTP-glucose-1-phosphate uridylyltransferase (SpGalU) and a galactokinase (SpGalK) were cloned from Streptococcus pneumoniae TIGR4 and were successfully used to synthesize UDP-galactose (UDP-Gal), UDP-glucose (UDP-Glc), and their derivatives in an efficient one-pot reaction system. The reaction conditions for the one-pot multi-enzyme synthesis were optimized and nine UDP-Glc/Gal derivatives were synthesized. Using this system, six unnatural UDP-Gal derivatives, including UDP-2-deoxy-Galactose and UDP-GalN3 which were not accepted by other approach, can be synthesized efficiently in a one pot fashion. More interestingly, this is the first time it has been reported that UDP-Glc can be synthesized in a simpler one-pot three-enzyme synthesis reaction system.
Asunto(s)
Galactoquinasa/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/metabolismo , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Galactosa/química , Uridina Difosfato Glucosa/química , Técnicas de Química Sintética , Clonación Molecular , Galactoquinasa/genética , Streptococcus pneumoniae/enzimología , Streptococcus pneumoniae/genética , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/metabolismoRESUMEN
We have investigated the applicability of different chemical methods for pyrophosphate bond formation to the synthesis of 5-substituted UDP-galactose and UDP-N-acetylglucosamine derivatives. The use of phosphoromorpholidate chemistry, in conjunction with N-methyl imidazolium chloride as the promoter, was identified as the most reliable synthetic protocol for the preparation of these non-natural sugar-nucleotides. Under these conditions, the primary synthetic targets 5-iodo UDP-galactose and 5-iodo UDP-N-acetylglucosamine were consistently obtained in isolated yields of 40-43%. Both 5-iodo UDP-sugars were used successfully as substrates in the Suzuki-Miyaura cross-coupling with 5-formylthien-2-ylboronic acid under aqueous conditions. Importantly, 5-iodo UDP-GlcNAc and 5-(5-formylthien-2-yl) UDP-GlcNAc showed moderate inhibitory activity against the GlcNAc transferase GnT-V, providing the first examples for the inhibition of a GlcNAc transferase by a base-modified donor analogue.
Asunto(s)
N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Uridina Difosfato/síntesis química , Animales , Células CHO , Cricetinae , Difosfatos/química , Activación Enzimática , Pruebas de Enzimas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Galactosafosfatos/química , Espectroscopía de Resonancia Magnética , N-Acetilglucosaminiltransferasas/química , Proteínas Recombinantes/química , Solventes/química , Tetrazoles/química , Factores de Tiempo , Uridina Difosfato/análogos & derivados , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
A one-step synthesis of two 5-CF(3) UDP-sugars is reported. These non-natural sugar-nucleotides are micromolar inhibitors of two different galactosyltransferases.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glicosiltransferasas/antagonistas & inhibidores , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/farmacología , Dominio Catalítico/efectos de los fármacos , Inhibidores Enzimáticos/química , Glicosiltransferasas/metabolismo , Estructura Molecular , Relación Estructura-Actividad , Uridina Difosfato Galactosa/químicaRESUMEN
Two novel nonisosteric UDP-Gal analogues, (2-deoxy-2-fluoro- and 4-deoxy-4-fluoro-α-D-galactopyranosyl) phosphonoyl phosphates, were synthesized by optimized multistep procedures starting from 3,4,6-tri-O-benzyl-D-galactal and allyl 2,3,6-tri-O-benzyl-α-D-glucopyranoside, respectively. The key steps were a Michaelis-Arbuzov reaction of respective deoxy-fluoro-D-galactopyranosyl acetate with triethyl phosphite followed by a Moffatt-Khorana coupling reaction with UMP-morpholidate. The structure of all new compounds was confirmed by NMR and mass spectroscopies..
Asunto(s)
Desoxiglucosa/análogos & derivados , Organofosfonatos/síntesis química , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Galactosa/síntesis química , Conformación de Carbohidratos , Desoxiglucosa/síntesis química , Desoxiglucosa/química , Organofosfonatos/química , Uridina Difosfato Galactosa/químicaRESUMEN
Galactosyltransferases (GalT) are important molecular targets in a range of therapeutic areas, including infection, inflammation, and cancer. GalT inhibitors are therefore sought after as potential lead compounds for drug discovery. We have recently discovered a new class of GalT inhibitors with a novel mode of action. In this publication, we describe a series of analogues which provide insights, for the first time, into SAR for this new mode of GalT inhibition. We also report that a new C-glycoside, designed as a chemically stable analogue of the most potent inhibitor in this series, retains inhibitory activity against a panel of GalTs. Initial results from cellular studies suggest that despite their polarity, these sugar-nucleotides are taken up by HL-60 cells. Results from molecular modeling studies with a representative bacterial GalT provide a rationale for the differences in bioactivity observed in this series. These findings may provide a blueprint for the rational development of new GalT inhibitors with improved potency.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Galactosiltransferasas/antagonistas & inhibidores , Glicósidos/síntesis química , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Galactosa/síntesis química , Animales , Proteínas Bacterianas/química , Bovinos , Galactosiltransferasas/química , Glicósidos/química , Células HL-60 , Humanos , Modelos Moleculares , Neisseria meningitidis/enzimología , Relación Estructura-Actividad , Uridina Difosfato Galactosa/químicaRESUMEN
Glycosyltransferases (GTs) are a large class of carbohydrate-active enzymes that are involved, in both pro- and eukaryotic organisms, in numerous important biological processes, from cellular adhesion to carcinogenesis. GTs have enormous potential as molecular targets for chemical biology and drug discovery. For the full realisation of this potential, operationally simple and generally applicable GT bioassays, especially for inhibitor screening, are indispensable tools. In order to facilitate the development of GT high-throughput screening assays for the identification of GT inhibitors, we have developed novel, fluorescent derivatives of UDP-galactose (UDP-Gal) that are recognised as donor analogues by several different retaining galactosyltransferases (GalTs). We demonstrate for one of these derivatives that fluorescence emission is quenched upon specific binding to individual GalTs, and that this effect can be used as the read-out in ligand-displacement experiments. The novel fluorophore acts as an excellent sensor for several different enzymes and is suitable for the development of a new type of GalT bioassay, whose modular nature and operational simplicity will significantly facilitate inhibitor screening. Importantly, the structural differences between the natural donor UDP-Gal and the new fluorescent derivatives are minimal, and the general assay principle described herein may therefore also be applicable to other GalTs and/or proteins that use nucleotides or nucleotide conjugates as their cofactor.
Asunto(s)
Colorantes Fluorescentes/química , Galactosiltransferasas/metabolismo , Tiofenos/química , Uridina Difosfato Galactosa/análogos & derivados , Galactosiltransferasas/antagonistas & inhibidores , Cinética , Relación Estructura-Actividad , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/químicaRESUMEN
UDP-galactopyranose mutase (UGM or Glf), which catalyzes the interconversion of UDP-galactopyranose and UDP-galactofuranose, is implicated in the viability and virulence of multiple pathogenic microorganisms. Here we report the synthesis of high-affinity ligands for UGM homologues from Klebsiella pneumoniae and Mycobacterium tuberculosis. The potency of these compounds stems from their ability to access both the substrate binding pocket and an adjacent site.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Galactosa/análogos & derivados , Transferasas Intramoleculares/antagonistas & inhibidores , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato/análogos & derivados , Catálisis , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Galactosa/síntesis química , Galactosa/química , Galactosa/farmacología , Klebsiella pneumoniae/enzimología , Ligandos , Mycobacterium tuberculosis/enzimología , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato/farmacología , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/químicaRESUMEN
Placing an 2-nitrobenzyl group on O-6 of the galactosyl residue in uridine-5'-diphosphogalactose (UDP-Gal) gives 6''-O-2-nitrobenzyl-UDP-Gal that is shown to be inactive as a donor substrate for beta-(1-->4)-galactosyltransferase (GalT). On irradiation at 365 nm, the nitrobenzyl group is completely removed yielding native UDP-Gal that then transfers normally to produce the expected betaGal-(1-->4)-betaGlcNAc disaccharidic linkage. 6''-O-2-Nitrobenzyl-UDP-Gal thus fulfils the minimum requirements of a 'caged' UDP-Gal for application in time-resolved crystallographic studies of beta-(1-->4)-GalT.
Asunto(s)
Nitrobencenos/química , Fotólisis , Uridina Difosfato Galactosa/química , Azúcares de Uridina Difosfato/química , Acetilglucosamina/química , Amino Azúcares/química , Estructura Molecular , N-Acetil-Lactosamina Sintasa/química , Nitrobencenos/síntesis química , Nitrobencenos/efectos de la radiación , Espectrometría de Masa por Ionización de Electrospray , Rayos Ultravioleta , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/efectos de la radiación , Azúcares de Uridina Difosfato/síntesis químicaRESUMEN
The syntheses of a novel uridine diphosphate galactose (UDP-Gal) analog, (UDP-2,4,6-tri-O-acetyl-3-S-acetyl-3-thio-alpha-D-galactopyranose) (11) and the thiolpyridine protected (Uridine 5'-[3-S-(2-S-pyridyl)-3-thio-alpha-D-galactopyranosyl diphosphate) analog (12) are described. The reported synthesis relies on the novel use of thiolpyridine to generate 12 which is a suitably protected intermediate for generating a UDP-thioGal derivative by reduction prior to enzyme transfer via beta-1,4-galactosyltransferase.
Asunto(s)
N-Acetil-Lactosamina Sintasa/metabolismo , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Galactosa/síntesis química , Espectroscopía de Resonancia Magnética , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/metabolismoRESUMEN
The synthesis of a novel analog of uridine diphosphate galactose (UDP-Gal) is described. A sulfur atom was inserted into the 6-position of galactose to give uridine 5'-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-alpha-D-galactopyranosyl diphosphate). This peracetylated thiol analogue of UDP-Gal has been synthesized in nine steps starting from methyl alpha-D-galactopyranoside in an overall yield of 3%.
Asunto(s)
Tiogalactósidos/síntesis química , Uridina Difosfato Galactosa/análogos & derivados , Acetilación , Inhibidores Enzimáticos/síntesis química , Galactosa/química , Galactosiltransferasas/antagonistas & inhibidores , Fosforilación , Uridina Difosfato Galactosa/síntesis químicaRESUMEN
The novel UDP-sugar uridine 5'-(3-deoxy-3-fluoro-D-galactopyranosyl diphosphate) (1) and UDP-(2-deoxy-2-fluoro)-D-galactose (2) have been prepared enzymatically and tested as substrate analogues for the enzyme UDP-galactopyranose mutase (UDP-Galp mutase EC 5.4.99.9). Turnover of both 1 and 2 by UDP-Galp mutase was observed by HPLC and 19F NMR. The HPLC elution profile and 19F chemical shift of the products are consistent with the formation of the predicted furanose forms of 1 and 2. The Km values for compounds 1 and 2 were similar to those of the natural substrate UDP-Galp (0.26 mM for 1, 0.2 mM for 2, and 0.6 mM for UDP-Galp), but the values for kcat were substantially different (1.6/min for 1, 0.02/min for 2, and 1364/min for UDP-Galp). A correlation was also observed between the equilibrium yield of product formed during turnover of UDP-sugar by UDP-Galp mutase (UDP-Galp, compound 1 or compound 2), and the amount of furanose present for the free sugar at thermal equilibrium in aqueous solution, using 1H and 19F NMR spectroscopy. The implications of these results to the mechanism of the unusual enzymatic reaction are discussed.
Asunto(s)
Transferasas Intramoleculares/metabolismo , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Galactosa/síntesis química , Proteínas Bacterianas/metabolismo , Cromatografía Líquida de Alta Presión , Flúor , Cinética , Resonancia Magnética Nuclear Biomolecular , Especificidad por Sustrato , Uridina Difosfato Galactosa/metabolismoRESUMEN
For the enzymatic transfer of galactose, N-acetylglucosamine, and N-acetylgalactosamine, UDP-Gal (1), UDP-GlcNAc (2), and UDP-GalNAc (3) are employed, and UDP serves as a feedback inhibitor. In this paper the synthesis of the novel UDP-sugar analogues 4, 5, and 6 as potential transferase inhibitors is described. Compounds 4-6 feature C-glycosidic hydroxymethylene linkages between the sugar and nucleoside moieties in contrast to the anomeric oxygens in the natural derivatives 1-3.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Transferasas/antagonistas & inhibidores , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato N-Acetilgalactosamina/síntesis química , Uridina Difosfato N-Acetilglucosamina/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Espectroscopía de Resonancia Magnética , Imitación Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Uridina Difosfato Galactosa/química , Uridina Difosfato Galactosa/farmacología , Uridina Difosfato N-Acetilgalactosamina/química , Uridina Difosfato N-Acetilgalactosamina/farmacología , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/farmacologíaRESUMEN
Since the alpha-D-galactose-(1-->3)-D-galactose epitope has been identified to be the major target in the process of hyperacute rejection of xenografts transplanted from nonprimate donors to humans, specific inhibitors of alpha-galactosyltransferases are of broad interest. Using Trypanosoma brucei, a protozoan parasite causing sleeping sickness and Nagana, we have a very useful model system for the investigation of alpha-galactosyltransferase inhibitors, since the variant surface glycoprotein (VSG) accounts for about 10% of the total cell protein an this parasite expresses many different galactosyltransferases including the one catalysing the formation of the Galalpha1-->3Gal epitope. In order to study inhibition of galactosylation on the VSG from Trypanosoma brucei, we designed, synthesized and tested substrate analogues of trypanosomal alpha-galactosyltransferases. Effective inhibitors were a pair of diastereoisomeric UDP-galactose analogs, in which the galactose residue is linked to UDP via a methylene bridge rather than an ester linkage. Hence, galactose cannot be transferred to the respective acceptor substrate VSG or the synthetic acceptor substrate Manalpha1-->6Manalpha1S-(CH2)7-CH3, which was previously proven to replace VSG effectively [Smith et al. (1996) J Biol Chem 271:6476-82]. Inhibitors have been prepared starting from 1-formyl galactal. The final condensation was performed using UMP morpholidate leading to a pair of diastereomeric compounds in 39% or 30% yield, respectively. These compounds were tested using alpha-galactosyltransferases prepared from T. brucei membranes and lactose synthetase from bovine milk. While the K(M)-value for UDP-galactose was determined as 59 microM on bovine lactose synthetase, the K(I)-values for both inhibitors were 0.3 mM and 1.1 mM respectively, showing that these inhibitors are unable to inhibit enzyme activity significantly. However, using the N-glycan specific alpha-galactosyltransferase from trypanosomes, the K(M)-value was determined as 20 microM, while the K(I)-values were 34 microM and 21 microM respectively. Interestingly, other trypanosomal alpha-galactosyltransferases, which modify the GPI membrane anchor, are 2 orders of magnitude less effected by the inhibitor.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Galactosiltransferasas/química , Trypanosoma brucei brucei/química , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato/análogos & derivados , Animales , Bovinos , Inhibidores Enzimáticos/química , Galactosiltransferasas/antagonistas & inhibidores , Lactosa Sintasa/antagonistas & inhibidores , Lactosa Sintasa/química , Ratas , Estereoisomerismo , Uridina Difosfato/síntesis química , Uridina Difosfato/química , Uridina Difosfato Galactosa/químicaRESUMEN
Uridine 5'-diphospho-(2-deoxy-2-fluoro)galactose (UDP-2FGal), prepared and characterized for the first time by a chemoenzymatic method, was found to be a competitive inhibitor of beta-1,4-galactosyltransferase with a Ki value of 149 microM. This study supports that the glycosyltransferase reaction mechanism proceeds through a glycosidic cleavage transition state with sp2 character developed at the anomeric center.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , N-Acetil-Lactosamina Sintasa/antagonistas & inhibidores , Uridina Difosfato Galactosa/análogos & derivados , Inhibidores Enzimáticos/metabolismo , Cinética , Modelos Químicos , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/metabolismoRESUMEN
The chemical synthesis and utilization of two photoaffinity analogs, 125I-labeled 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc and -UDP-GalNAc, is described. Starting with either UDP-GlcNAc or UDP-GalNAc, the synthesis involved the preparation of the 5-mercuri-UDP-HexNAc and then attachment of an allylamine to the 5 position to give 5-(3-amino)allyl-UDP-HexNAc. This was followed by acylation with N-hydroxysuccinimide p-aminosalicylic acid to form the final product, i.e., 5-[3-(p-azidosalicylamido)-1-propenyl]-UDP-GlcNAc or UDP-GalNAc. These products could then be iodinated with chloramine T to give the 125I-derivatives. Both the UDP-GlcNAc and the UDP-GalNAc derivatives reacted in a concentration-dependent manner with a highly purified UDP-HexNAc pyrophosphorylase, and both specifically labeled the subunit(s) of this protein. The labeling of the protein by the UDP-GlcNAc derivative was inhibited in dose-dependent fashion by either unlabeled UDP-GlcNAc or unlabeled UDP-GalNAc. Likewise, labeling with the UDP-GalNAc probe was blocked by either UDP-GlcNAc or UDP-GalNAc. The UDP-GlcNAc probe also specifically labeled a partially purified preparation of GlcNAc transferase I.
Asunto(s)
Marcadores de Afinidad/síntesis química , Glicosiltransferasas/análisis , Nucleotidiltransferasas/análisis , Salicilamidas/síntesis química , Uridina Difosfato Galactosa/análogos & derivados , Uridina Difosfato Glucosa/análogos & derivados , Uridina Difosfato N-Acetilgalactosamina/análogos & derivados , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Animales , Azidas , Fotoquímica , Salicilatos , Porcinos , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Glucosa/síntesis químicaRESUMEN
Bovine beta-(1-->4)-galactosyltransferase (GalT) transfers galactose from UDP-galactose to beta-D-GlcpNAc-terminating oligosaccharides to produce N-acetyllactosamine sequences. We report here the chemical synthesis, structural characterization and enzymatic evaluation of the very labile UDP-2-deoxy-D-lyxo-hexose ("UDP-2-deoxy-galactose," 2) as an alternate donor for GalT. Donor 2 had kinetic parameters, including a Km value of 51 microM, almost identical to those for the natural substrate UDP-galactose when beta-D-GlcpNAc-O(CH2)8COOMe was used as the acceptor. The product of the enzymatic transfer was isolated and confirmed to have the expected 2'-deoxy-N-acetyllactosamine sequence.
Asunto(s)
N-Acetil-Lactosamina Sintasa/metabolismo , Uridina Difosfato Galactosa/análogos & derivados , Animales , Bovinos , Indicadores y Reactivos , Cinética , Espectroscopía de Resonancia Magnética , Especificidad por Sustrato , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/metabolismoRESUMEN
Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Monosacáridos/síntesis química , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato N-Acetilgalactosamina/síntesis química , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Galactosa Oxidasa/metabolismo , Monosacáridos/metabolismo , Oxidación-Reducción , Tritio , Uridina Difosfato Galactosa/metabolismo , Uridina Difosfato N-Acetilgalactosamina/metabolismoRESUMEN
Glycosyltransferases enzymatically transfer monosaccharides from sugar-nucleotides to complex oligosaccharide chains and, as cell surface molecules, exhibit receptor-like activity. We have modified the substate UDP-galactose to produce a compound that has useful absorbance and fluorescence properties upon binding to galactosyltransferase (GalTase). Using strategies similar to those for preparing fluorescent ATP analogs, we were able to synthesize 2,4,6-trinitrophenyl-5'-UDP-galactose (TUG). In solution, the absorbance properties of TUG are pH dependent, with absorbance maxima at 260, 408, and 453 nm and an isobestic point at 353 nm. In the presence of soluble GalTase extracted from bovine milk, TUG exhibited an excitation maximum at 368 nm with emission maxima at 436 and 533 nm; in the absence of GalTase only the 533-nm peak was present. Under enzymatic conditions, TUG acted as a competitive substrate of UDP-galactose with GalTase. Under noncatalytic conditions, the fluorescence emission of TUG at 436 nm increased monotonically with Gal-Tase concentration, with a half-maximal response at approximately 4 microM. This compound may be useful for quantifying GalTase function as both an enzyme and a cell adhesion molecule.
Asunto(s)
Galactosiltransferasas/metabolismo , Trinitrobencenos/aislamiento & purificación , Uridina Difosfato Galactosa , Uridina Difosfato Galactosa/análogos & derivados , Unión Competitiva , Colorantes Fluorescentes , Concentración de Iones de Hidrógeno , Espectrometría de Fluorescencia , Especificidad por Sustrato , Trinitrobencenos/síntesis química , Trinitrobencenos/metabolismo , Uridina Difosfato Galactosa/síntesis química , Uridina Difosfato Galactosa/aislamiento & purificación , Uridina Difosfato Galactosa/metabolismoRESUMEN
UDP[6-3H]galactose of high specificity can be obtained by oxidation of the C-6 hydroxymethyl group of UDP-galactose by galactose oxidase and subsequent reduction by sodium borotritide. One-step purification of the nucleotide sugar involves anion-exchange chromatography on a Pharmacia Mono Q column. Radiolabeled UDP-N-acetylgalactosamine can also be synthesized and purified by this procedure. Both nucleotide sugars can be used for sugar incorporation studies using the appropriate glycosyltransferase.