Substrate specificity and properties of uridine diphosphate glucuronyltransferase purified to apparent homogeneity from phenobarbital-treated rat liver.
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1. The purification to homogeneity of stable highly active preparations of UDP-glucuronyltransferase from liver of phenobarbital-treated rats is briefly described. 2. A single polypeptide was visible after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, of mol.wt.57000. 3. Antiserum raised against the pure enzyme produces a single sharp precipitin line after Ouchterlony double-diffusion analysis. 4. The pure UDP-glucuronyltransferase isolated from livers of untreated and phenobarbital-pretreated rats appears to be the same enzyme. 5. The Km (UDP-glucuronic acid) of the pure enzyme is 5.4 mM. 6. The activity of the pure enzyme towards 2-aminophenol can still be activated 2-3-fold by diethylnitrosamine. 7. UDP-glucose and UDP-galacturonic acid are not substrates for the purified enzyme. 8. The final preparation catalysed the glucuronidation of 4-nitrophenol, 1-naphthol, 2-aminophenol, morphine and 2-aminobenzoate. 9. Activities towards 4-nitrophenol, 1-naphthol and 2-aminophenol were all copurified. The proposed heterogeneity of UDP-glucuronyltransferase is discussed. (+info)
Incorporation of [14C]glucose from UDP[14C]glucose into a phosphoglycolipid by cell-free particulate systems of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423.
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A glucosyl group from uridine diphosphate [U-14C]glucose is incorporated into a phosphoglycolipid, probably a glucosylphosphatidylglycerol, by a disrupted membrane enzyme preparation from a gram-negative, moderately halophilic bacterium, Pseudomonas halosaccharolytica ATCC 29423. The conversion of [14C]phosphatidylglycerol into phosphoglycolipid by the particulate preparation was also enhanced in the presence of non-labelled UDP-glucose. A chemical degradation study of labelled phosphoglycolipid showed the bulk of the radioactivity from UDP[U-14C]glucose to be associated with the glucose moiety, which also appeared to be attached to the hydroxyl group of a second glycerol. (+info)
The mur4 mutant of arabidopsis is partially defective in the de novo synthesis of uridine diphospho L-arabinose.
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To obtain information on the synthesis and function of arabinosylated glycans, the mur4 mutant of Arabidopsis was characterized. This mutation leads to a 50% reduction in the monosaccharide L-arabinose in most organs and affects arabinose-containing pectic cell wall polysaccharides and arabinogalactan proteins. Feeding L-arabinose to mur4 plants restores the cell wall composition to wild-type levels, suggesting a partial defect in the de novo synthesis of UDP-L-arabinose, the activated sugar used by arabinosyltransferases. The defect was traced to the conversion of UDP-D-xylose to UDP-L-arabinose in the microsome fraction of leaf material, indicating that mur4 plants are defective in a membrane-bound UDP-D-xylose 4-epimerase. (+info)
Sugar-nucleotide-binding and autoglycosylating polypeptide(s) from nasturtium fruit: biochemical capacities and potential functions.
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Polypeptide assemblies cross-linked by S-S bonds (molecular mass>200 kDa) and single polypeptides folded with internal S-S cross-links (<41 kDa) have been detected by SDS/PAGE in particulate membranes and soluble extracts of developing cotyledons of nasturtium (Tropaeolum majus L.). When first prepared from fruit homogenates, these polypeptides were found to bind reversibly to UDP-Gal (labelled with [(14)C]Gal or [(3)H]uridine), and to co-precipitate specifically with added xyloglucan from solutions made with 67% ethanol. Initially, the bound UDP-[(14)C]Gal could be replaced (bumped) by adding excess UDP, or exchanged (chased) with UDP-Gal, -Glc, -Man or -Xyl. However, this capacity for turnover was lost during incubation in reaction media, or during SDS/PAGE under reducing conditions, even as the glycone moiety was conserved by autoglycosylation to form a stable 41 kDa polypeptide. Polyclonal antibodies raised to a similar product purified from Arabidopsis bound to all the labelled nasturtium polypeptides in immunoblotting tests. The antibodies also inhibited the binding of nasturtium polypeptides to UDP-Gal, the uptake of UDP-[(14)C]Gal into intact nasturtium membrane vesicles and the incorporation of [(14)C]Gal into nascent xyloglucan within these vesicles. This is the first direct evidence that these polypeptides facilitate the channelling of UDP-activated sugars from the cytoplasm through Golgi vesicle membranes to lumenal sites, where they can be used as substrates for glycosyltransferases to synthesize products such as xyloglucan. (+info)
Uridine diphosphate galactose-4-epimerase. Uridine monophosphate-dependent reduction by alpha- and beta-D-glucose.
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Rates of UMP-dependent reduction of the DPN+ associated with Escherichia coli UDP-galactose-4-epimerase at 27 degrees and 0.2 M ionic strength in 0.1 M Tris-HCl buffer, pH 8.5, are reported. The reaction exhibits excellent pseudo-first order behavior when D-glucose is at anomeric equilibrium. The effects of [UMP] and [glucose] on the observed first order rate constants are consistent with the following equation. The symbols phi are empirical parameters. (See article). The data indicate that the pathway involves random equilibrium binding of UMP and glucose followed by rate-limiting decomposition of the ternary complex to epimerase-DNPH. The binding parameters indicate that the principal activating effect of UMP is not simply to increase the affinity of the enzyme for glucose. UMP appears to increase the reactivity or availability of enzyme-bound DPN+. The kinetic isotope effect for the reaction of D-]1-2H]glucose (kH/kD) is 4.2, which confirms that C-1 is oxidized and that hydride transfer is rate limiting. Both of the purified anomers, alpha- and beta-D-glucose, reduce the enzyme-bound DPN+. As indicated by the deviations from pseudo-first order kinetics because of concurrent mutarotation, the beta anomer is the more reactive, reacting about 4 to 5 times faster than the alpha anomer at concentrations well below saturation. Is is suggested that the lack of stereo-specificity in this reaction may be attributed to the two anomers being productively bound with their opposite faces projecting toward C-4 of bound DPN+. Nonstereospecific oxidation of alpha- and beta-D-glucose may be a model for the mechanism of UDP-hexose epimerization, which also involves nonstereospecific hydride transfer. (+info)
Kinetic basis for the donor nucleotide-sugar specificity of beta1, 4-N-acetylglucosaminyltransferase III.
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The kinetic basis of the donor substrate specificity of beta1, 4-N-acetylglucosaminyltransferase III (GnT-III) was investigated using a purified recombinant enzyme. The enzyme also transfers GalNAc and Glc moieties from their respective UDP-sugars to an acceptor at rates of 0.1-0.2% of that for GlcNAc, but Gal is not transferred at a detectable rate. Kinetic analyses revealed that these inefficient transfers, which are associated with the specificity of the enzyme, are due to the much lower V(max) values, whereas the K(m) values for UDP-GalNAc and UDP-Glc differ only slightly from that for UDP-GlcNAc. It was also found that various other nucleotide-Glc derivatives bind to the enzyme with comparable affinities to those of UDP-GlcNAc and UDP-Glc, although the derivatives do not serve as glycosyl donors. Thus, GnT-III does not appear to distinguish UDP-GlcNAc from other structurally similar nucleotide-sugars by specific binding in the ground state. These findings suggest that the specificity of GnT-III toward the nucleotide-sugar is determined during the catalytic process. This type of specificity may be efficient in preventing a possible mistransfer when other nucleotide-sugars are present in excess over the true donor. (+info)
The sialic acids. XVIII. Subcellular distribution of seven glycosyltransferases in embryonic chicken brain.
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The subcellular distribution of seven glycosyltransferases was studied in embryonic chicken brain. Four of the transferases are required for the synthesis of gangliosides, and three for the synthesis of the terminal trisaccharide units of serum type glycoproteins. Six of the transferases were found only in the particulate fraction of homogenates, while one (UDP-galactose:glycoprotein galactosyltransferase) was found both in the paritculate and soluble fractions of young embryonic chicken brain, but only in the particulate fraction obtained from older embryos. The source of the soluble galactosyltransferase was found to be the fluid surrounding the embryonic brain. Fractionation studies by the Whittaker technique showed that the seven activities were located primarily in the nerve ending (synaptosome)-rich fraction. (+info)
Molecular characterization of Streptococcus pneumoniae type 4, 6B, 8, and 18C capsular polysaccharide gene clusters.
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Capsular polysaccharide (CPS) is a major virulence factor in Streptococcus pneumoniae. CPS gene clusters of S. pneumoniae types 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5' end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3' end of the gene cluster. Comparison of the four dTDP-L-rhamnose pathway genes (rml genes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rml genes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rml genes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5' ends and the relatively conserved rml genes at the 3' ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzx and wzy genes for all sequenced CPS gene clusters by use of motifs. (+info)