Analysis of UDP-Sugars from Cultured Cells and Small Tissue Samples.

UDP-sugars are important substrates for the synthesis of various cellular glycans and glycoconjugates, many of which play essential roles in the pathobiology of diseases associated with deranged glucose metabolism, such as cancer and type 2 diabetes. Hence, their analysis from cultured cells and especially from tissue samples can give valuable information. This chapter describes a method for UDP-sugar isolation from various sources utilizing ion-pair solid-phase extraction with graphitized carbon cartridges, and their analysis using anion-exchange high-performance liquid chromatography.

Quantitative HPLC-MS analysis of nucleotide sugars in plant cells following off-line SPE sample preparation.

An analytical workflow was developed for the absolute quantification of uridine diphosphate (UDP)-sugars in plant material in order to compare their metabolism both in wild-type Arabidopsis thaliana and mutated plants (ugd2,3) possessing genetic alterations within the UDP-glucose dehydrogenase genes involved in UDP-sugar metabolism. UDP-sugars were extracted from fresh plant material by chloroform-methanol-water extraction and further purified by solid-phase extraction with a porous graphitic carbon adsorbent with extraction efficiencies between 80 ± 5 % and 90 ± 5 %. Quantitative determination of the UDP-sugars was accomplished through HPLC separation with a porous graphitic carbon column (Hypercarb(TM)) which was interfaced to electrospray ionization Orbitrap mass spectrometry. The problem of instable retention times due to redox processes on the stationary phase were circumvented by grounding of the column effluent and incorporation of a column regeneration procedure using acetonitrile-water containing 0.10 % trifluoroacetic acid. The method was calibrated using external calibration and UDP as internal standard. Calibration functions were approximated by first- or second-order regression analysis for concentrations spanning three orders of magnitude. Upon injecting sample volumes of 2.65 µL, the limits of detection for the UDP-sugars were in the 70 nmol L(-1) range. Six different UDP-sugars, including UDP-glucose, UDP-galactose, UDP-arabinose, UDP-xylose, UDP-glucuronic acid, and UDP-galacturonic acid were found in concentrations of 0.4 to 38 µg/g plant material. Data evaluation by analysis of variance (ANOVA) revealed statistically significant differences in UDP-sugar concentrations between wild-type and mutant plants, which were found to conclusively mirror the impaired metabolic pathways in the mutant plants.

Borate-aided anion exchange high-performance liquid chromatography of uridine diphosphate-sugars in brain, heart, adipose and liver tissues.

In this paper we describe a method optimized for the purification of uridine diphosphate (UDP)-sugars from liver, adipose tissue, brain, and heart, with highly reproducible up to 85% recoveries. Rapid tissue homogenization in cold ethanol, lipid removal by butanol extraction, and purification with a graphitized carbon column resulted in isolation of picomolar quantities of the UDP-sugars from 10 to 30mg of tissue. The UDP-sugars were baseline separated from each other, and from all major nucleotides using a CarboPac PA1 anion exchange column eluted with a gradient of acetate and borate buffers. The extraction and purification protocol produced samples with few unidentified peaks. UDP-N-acetylglucosamine was a dominant UDP-sugar in all the rat tissues studied. However, brain and adipose tissue showed high UDP-glucose levels, equal to that of UDP-N-acetylglucosamine. The UDP-N-acetylglucosamine showed 2.3-2.7 times higher levels than UDP-N-acetylgalactosamine in all tissues, and about the same ratio was found between UDP-glucose and UDP-galactose in adipose tissue and brain (2.6 and 2.8, respectively). Interestingly, the UDP-glucose/UDP-galactose ratio was markedly lower in liver (1.1) and heart (1.7). The UDP-N-acetylglucosamine/UDP-glucuronic acid ratio was also constant, between 9.7 and 7.7, except in liver with the ratio as low as 1.8. The distinct UDP-glucose/galactose ratio, and the abundance of UDP-glucuronic acid may reflect the specific role of liver in glycogen synthesis, and metabolism of hormones and xenobiotics, respectively, using these UDP-sugars as substrates.

Enzymatic synthesis and purification of uridine diphospho-beta-l-arabinopyranose, a substrate for the biosynthesis of plant polysaccharides.

Many plant cell wall components such as the polysaccharides xylans and pectins or the glycoproteins arabinogalactan proteins and extensins contain arabinosyl residues. The arabinosyl substituents are thought to be incorporated into these wall polymers by the action of arabinosyltransferases using UDP-l-arabinose as the precursor. UDP-l-arabinose is not commercially available and therefore a procedure for generating UDP-l-arabinose was developed for use in studies on the biosynthesis of the arabinose-containing polymers. In this procedure UDP-d-xylose is incubated with an enzyme preparation from wheat germ and the nucleotide sugars in the reaction mixture are extracted. High-performance anion-exchange chromatography of the extract resolves two major UV-absorbing components: one corresponding to UDP-xylose and a second that elutes earlier. TLC analysis of collected and hydrolyzed fractions demonstrated the presence of l-arabinose in the early eluting fraction. Further analysis by NMR identified the compound as UDP-beta-l-arabinopyranose. The procedure reported here provides an efficient method for preparing either radioactive UDP-l-[(14)C]arabinose or nonradioactive UDP-l-arabinose and can also be used as an assay for UDP-xylose-4-epimerase activity.

Enzymatic synthesis and purification of [(3)H]uridine diphosphate galacturonic acid for use in studying Golgi-localized transporters.

Uridine 5'-diphosphate galacturonic acid (UDP-GalA) is a substrate for the galacturonosyltransferases that synthesize the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Pectin synthesis occurs in the Golgi and it is hypothesized that UDP-GalA is transported into the lumen of the Golgi by membrane-localized transporters. To study the transport and metabolism of UDP-GalA in the Golgi, UDP-GalA labeled in the uridine moiety is required. Here we present a high-yield method for the synthesis of [(3)H]UDP-GalA from [(3)H]UTP and Glc-1-P by sequential reactions catalyzed by UDP-Glc pyrophosphorylase, UDP-Glc dehydrogenase, and UDP-GlcA-4-epimerase and the separation of the reaction products over a Dionex CarboPac PA1 anion-exchange column using high-performance anion-exchange chromatography (HPAEC). Approximately half of the [(3)H]UTP was converted into [(3)H]UDP-GalA and the remaining 50% was recovered as [(3)H]UDP-GlcA. Both products were purified and the identity of the [(3)H]UDP-GalA was confirmed by its conversion into [(3)H]UDP-GlcA by UDP-GlcA-4-epimerase. The enzymatic synthesis of diverse nucleotide sugars radiolabeled in the nucleotide by the use of nucleotide-converting enzymes, combined with the high-resolution separation of the nucleotide sugars and their purification by HPAEC, can provide unique substrates required for the study of diverse nucleotide sugar transporters.

A novel UDP-sugar, UDP-3-ketoglucosamine or UDP-4-ketoglucosamine, from bovine heart muscle reduces metmyoglobin with NAD(P)H.

3-Ketoglucose and similar ketosugars have been identified in microorganisms only and little is known about their functions. UDP-sugars are widely found as an intermediate in sugar metabolism in living organisms. Yet what role UDP-sugars play, or whether they play a direct role in metabolism, is still unknown. UDP-sugars were isolated and purified from bovine heart muscle, and a UDP-sugar fraction capable of NAD(P)H-dependent catalytic reduction of metmyoglobin was detected. Subsequent identification revealed that the active UDP-sugar was UDP-3- or UDP-4-ketoglucosamine. These compounds were purified from bovine cardiac muscle by ultrafiltration, anion-exchange column chromatography and reverse-phase chromatography. They were further characterized by determination of their chemical reducing activity, by comparison with synthetic UDP-3- or UDP-4-ketoglucosamine standards using high-performance liquid chromatography, by estimation of molecular mass using fast atom bombardment mass spectrometry, and by Fourier transform infrared microspectroscopy and electron probe microanalysis. The results suggest that UDP-3- or UDP-4-ketoglucosamine reduces metmyoglobin in bovine cardiac muscle. It is important that the reducing activity displayed by this ketosugar is not the effect of UDP-3- or UDP-4-ketoglucosamine alone but depends on NAD(P)H. In other words, this action of UDP-3- or UDP-4-ketoglucosamine is catalytic.

Enzymatic synthesis and purification of uridine diphosphate [14C]galacturonic acid: a substrate for pectin biosynthesis.

Pectins are complex polysaccharides that contain 1,4-linked alpha-D-galactosyluronic acid residues found in the primary wall of all higher plant cells. The pectic polysaccharides play critical roles in cell wall structure and in plant growth and development. As a first step in studying pectin biosynthesis a method was developed to routinely generate and purify UDP-[U-14C]galacturonic acid (UDP-[14C]GalA), the nucleotide sugar substrate for homogalacturonan biosynthesis. UDP-[14C]GalA was enzymatically synthesized by 4-epimerization of commercially available UDP-[U-14C]glucuronic acid (UDP-[14C]GlcA) using a particulate preparation from radish roots. The resulting mixture of UDP-[14C]GalA and UDP-[14C]GlcA was separated by high-performance anion-exchange chromatography using a Dionex CarboPac PA1 anion-exchange column. The UDP-sugars were detected by their absorbance at 262 nm or by pulsed amperometric detection following postcolumn addition of NaOH. The yield of UDP-[14C]GalA obtained using this procedure was 16% of the starting UDP-[14C]GlcA. Establishment of a reliable method to synthesize and purify UDP-[14C]GalA will facilitate the identification and purification of the galacturonosyltransferase(s) involved in pectin biosynthesis.

Separation and analysis of 4'-epimeric UDP-sugars, nucleotides, and sugar phosphates by anion-exchange high-performance liquid chromatography with conductimetric detection.

An anion-exchange HPLC method coupled with conductimetric detection was developed for the analysis of UDP-sugars, nucleotides, and sugar phosphates, each in a single chromatographic run. The analysis was applicable to concentrations over 50 pmol. The utility of this technique was demonstrated by the measurement of UDP-sugar 4'-epimerase activity of cell-free extracts from wild-type and mutant Neisseria meningitidis serogroup B strains. Additionally, the method has been applied to the analysis of the intermediates of glycolysis in human erythrocytes.

Isolation and characterization of sugar nucleotides from Mycobacterium smegmatis.

The cell walls of Mycobacterium smegmatis contained a number of serologically active polysaccharides such as arabinomannan, mannan, and glucan in addition to arabinogalactan. The biosynthetic pathways of these polysaccharides are not well understood. Characterization of the sugar nucleotide pool of M. smegmatis showed the presence of (uridine diphosphate-) UDP-glucose, UDP-galactose, UDP-mannose, UDP-arabinose and UDP-hexuronic acid(s). It is suggested that these compounds may be intermediates in the biosynthesis of a number of mycobacterial polysaccharides.

Stimulation of the methylcoenzyme M reduction by uridine-5'-diphospho-sugars in cell-free extracts of Methanobacterium thermoautotrophicum (strain delta H).

The hydrogen-dependent reduction of methylcoenzyme M catalyzed by coenzyme-depleted cell-free extracts of Methanobacterium thermoautotrophicum was stimulated by micromolar concentrations of a UDP-disaccharide present in the organism. The compound was isolated and identified as UDP-1-O-alpha-D-2-acetamido-2-deoxyglucopyranose (UDPGlcpNAc) glycosidically linked to 2-acetamido-2-deoxymannopyranosyluronic acid. Maximal stimulation was observed when both the UDP-disaccharide and mercaptoheptanoylthreonine phosphate were present in the reaction mixtures. The UDP derivative isolated was not specific in its action: other UDP-sugars tested in micromolar concentrations stimulated the methylcoenzyme M reduction to the same extent. The activated sugars presumably substitute for ATP, which is usually required in much higher concentrations to activate the methylcoenzyme M reductase system.

Production of a streptomycin-Park nucleotide complex by Streptomyces griseus.

A compound (compound X) with antibacterial activity was isolated from early-exponential-phase cultures of the streptomycin producer Streptomyces griseus and from protoplast cultures of the same strain. The protoplast cultures produced a larger amount of compound X than did the young hyphae. Both the mycelia and the protoplasts incorporated 14C-labeled myo-inositol, a precursor of streptomycin, into compound X, which has an amino acid content related to that of the cell wall peptidoglycan of the producer strain. Compound X may contain a streptomycin molecule covalently bound to a cell wall precursor unit, i.e., to a Park nucleotide (J. T. Park, J. Biol. Chem. 194:897-904, 1952), through the glutamic acid of the pentapeptide component.

Occurrence in Bacillus megaterium of uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine, intermediates in the biosynthesis of galactosamine-6-phosphate polymer.

We isolated two galactosamine derivatives from Bacillus megaterium sporulating cells by lectin affinity chromatography followed by DEAE-Sephadex A-25 chromatography. From chemical analyses and measurements of these compounds, it was determined that one was uridine 5'-diphospho-N-acetylgalactosamine and that the other was uridine 5'-diphosphogalactosamine. They appeared in the middle stage of sporulation and disappeared during the period when galactosamine-6-phosphate is deposited on the forespore surface. These results suggest that uridine 5'-diphospho-N-acetylgalactosamine and uridine 5'-diphosphogalactosamine are intermediates in the biosynthesis of the galactosamine-6-phosphate polymer, a backbone structure of the exosporium.

Isolation and identification of uridine(5')-diphospho(1)-2,3-diacetamido-2,3-dideoxy-alpha-D- glucopyra nuronic acid from Pseudomonas aeruginosa P1-III.

A new uridine nucleotide was isolated from Pseudomonas aeruginosa P1-III (Habs serotype 5). On the basis of 13C-n.m.r. and p.m.r. spectroscopy, mass spectrometry, i.r.-absorption spectroscopy and circular dichrometry, the structure of the new compound was unequivocally identified as uridine(5')-diphospho(1)-2,3-diacetamido-2,3-dideoxy-alpha-D-gl ucopyranuronic acid.

A simplified procedure for synthesizing large quantities of highly purified uridine [beta-32P]diphospho-N-acetylglucosamine.

A simplified procedure for the synthesis of [beta-32P]UDP-N-acetylglucosamine is described. This novel method utilizes commercially available enzymes, chemical acetylation, and preparative thin-layer chromatography and can be used to synthesize millicurie quantities of the sugar nucleotide at an extremely high specific activity and purity.

Separation and analysis of 4'-epimeric UDP-sugars by borate high-performance liquid chromatography.

Separation of UDP-glucose from UDP-galactose, of UDP-N-acetylglucosamine from UDP-N-acetylgalactosamine, of UDP-glucuronate from UDP-galacturonate, or of UDP-glucosamine from UDP-galactosamine was achieved within 10-45 min by isocratic anion-exchange high-performance liquid chromatography (HPLC) using a flow rate of 2 ml/min. The eluants were composed of borate as complex-forming and eluting agent and of glycerol for protection of the alkali-labile silica packing of the column. This borate HPLC was suitable for the analysis of 4'-epimeric UDP-sugars in the range of 2 to 100 nmol. The applicability of this technique was demonstrated by determination of the relative amounts of 4'-epimeric UDP-amino sugars formed in rat liver after administration of D-galactosamine. Since a high salt content of UDP-sugar samples can interfere with borate HPLC, desalting was performed on a 1-ml C18 cartridge using triethylammonium hydrogen carbonate buffer. This procedure enabled the complete separation of various nucleotides from salts within 10 min prior to HPLC.