Formation of lipid-linked sugar compounds in Halobacterium salinarium. Presumed intermediates in glycoprotein synthesis.
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The ability of bacitracin to inhibit the growth of Halobacterium salinarium suggested that glycosylation of the major envelope component, a high molecular weight glycoprotein, might occur via a pathway involving lipid intermediates. This report demonstrates that the cells have enzymatic activities for formation of lipid-linked sugar compounds having the expected properties of such intermediates. Whole cell homogenate catalyzed the transfer of sugar from UDP-glucose, GDP-mannose, and UDP-N-acetyglucosamine to endogenous lipid acceptors. Two lipid products were formed from UDP-glucose, two from GDP-mannose, and one from UDP-N-acetylglucosamine. Characterization of the partially purified lipids by ion exchange chromatography, thin layer chromatography, and mild acid and base hydrolysis showed the major product in each case to have the properties expected for polyisoprenyl phosphoglucose, polyisoprenyl phosphomannose, and polyisoprenyl pyrophospho-N-acetylglucosamine. Estimates of chain length by thin layer chromatography indicate that the lipid has 11 to 12 isoprene identity as a C55-60-polyisoprenyl pyrophospho-N-acetylglucosamine. The N-acetylglucosamine transferase, present in cell envelope preparations, was partially characterized. The enzyme was found to be extremely halophilic, specifically requiring a high concentration of KCl. Optimum activity was obtained at 4 m KCl and partial substitution of K+ by Na+ resulted in a decrease in activity. (+info)
Stimulation of collagen galactosyltransferase and glucosyltransferase activities by lysophosphatidylcholine.
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Lysophosphatidylcholine stimulated the activities of collagen galactosyl- and glucosyl-transferases in chick-embryo extract and its particulate fractions in vitro, whereas essentially no stimulation was noted in the high-speed supernatant, where the enzymes are soluble and membrane-free. The stimulatory effect of lysophosphatidylcholine was masked by 0.1% Triton X-100. In kinetic experiments lysophosphatidylcholine raised the maximum velocities with respect to the substrates and co-substrates, whereas no changes were observed in the apparant Km values. Phospholipase A preincubation of the chick-embryo extract resulted in stimulation of both transferase activities, probably gy generating lysophosphatides from endogenous phospholipids. No stimulation by lysophosphatidylcholine was found when tested with 500-fold-purified glycosyltransferase. The results suggest that collagen glycosyltransferases must be associated with the membrane structures of the cell in order to be stimulated by lysophosphatidylcholine. Lysophosphatidylcholine could have some regulatory significance in vivo, since its concentration in the cell is comparable with that which produced marked stimulation in vitro. (+info)
Donor substrate specificity of recombinant human blood group A, B and hybrid A/B glycosyltransferases expressed in Escherichia coli.
(3/167)
The human blood group A and B glycosyltransferases catalyze the transfer of GalNAc and Gal, to the (O)H-precursor structure Fuc alpha (1-2)Gal beta-OR to form the blood group A and B antigens, respectively. Changing four amino acids (176, 235, 266 and 268) alters the specificity from an A to a B glycosyltransferase. A series of hybrid blood group A/B glycosyltransferases were produced by interchanging these four amino acids in synthetic genes coding for soluble forms of the enzymes and expressed in Escherichia coli. The purified hybrid glycosyltransferases were characterized by two-substrate enzyme kinetic analysis using both UDP-GalNAc and UDP-Gal donor substrates. The A and B glycosyltransferases were screened with other donor substrates and found to also utilize the unnatural donors UDP-GlcNAc and UDP-Glc, respectively. The kinetic data demonstrate the importance of a single amino acid (266) in determining the A vs. B donor specificity. (+info)
Studies on the mechanism of collagen glucosyltransferase reaction.
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The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparations purified about 2500-5000-fold from extract of homogenate of whole chick embryos. Data obtained in experiments on initial velocity and inhibition kinetics of the reaction were consistent with an ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, UDP-glucose and collagen substrate, the addition of Mn2+ being at thermodynamic equilibrium and the binding site of the UDP-glucose to the enzyme not being the same as that for Mn2+ and collagen substrate. Only one metal co-factor seems to be involved in the reaction. The collagen substrate can probably also react in some conditions with enzyme-Mn2+ and with enzyme-Mn2+-UDP, and the UDP with the free enzyme, but in all these instances dead-end complexes are formed. Evidence is presented for an ordered release of the products in the following order: glucosylated collagen, UDP and Mn2+, in which Mn2+ need not leave the enzyme during each catalytic cycle. (+info)
Orotate decreases the inhibitory effect of ethanol on galactose elimination in the perfused rat liver.
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1. The galactose-elimination rate in perfused livers from starved rats was decreased in the presence of ethanol (2-28mM) to one-third of the control values. Orotate injections partly reversed the effect of ethanol, so that the galactose-elimination rate was about two-thirds of the control values. Orotate alone had no effect on the galactose-elimination rate. 2. Ethanol increased [galactose 1-phosphate] and [UDP-galactose], and decreased (UDP-glucose] and [UTP], both with and without orotate. Orotate increased [UTP], [UDP-galactose], both with and without ethanol. The increase of [galactose 1-phosphate] in the presence of ethanol was inhibited by orotate. Orotate alone had no appreciable effect on [galactose 1-phosphate]. 3. Both the effect of ethanol and that of orotate on the galactose-elimination rate can be accounted for by assuming inhibition of galactokinase by galactose 1-phosphate with Ki about 0.2mM, the inhibition being either non-competitive or uncompetitive. 4. The primary effect of ethanol seems to be inhibition of UDP-glucose epimerase (EC 5.1.3.2), followed by accumulation of UDP-galactose, trapping of UDP-glucose and increase of [galactose 1-phosphate]. Orotate decreased the effect of ethanol, probably by increasing [UDP-glucose]. (+info)
Ectogalactosyltransferase studies in fibroblasts and concanavalin A-stimulated lymphocytes.
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In this communication, we have demonstrated that hydrolysis of the nucleotide sugar can cause errors in the detection of an ectoglycosyltransferase. Spleen cell suspensions can incorporate radioactivity when incubated with labeled UDP-galactose, but all the activity is due to decomposition of the nucleotide sugar and uptake of the free sugar. The fibroblast cell lines can incroporate carbohydrate directly from UDP-galactose. Several criteria are presented with can be used to demonstrate that a nucleotide sugar is the direct carbohydrate donor. (+info)
Ectogalactosyltransferase. Presence of enzyme and acceptors on the rat lymphocyte cell surface.
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Experiments are described to demonstrate the existence of ectogalactosyltransferase activity on the lymphocyte surface. The procedures described enable us to exclude the possibility of misleading results due to precursor hydrolysis and intracellular utilization of the free galactose. This depicted transferase is able to catalyse the transfer of a galactosyl residue from UDP-galactose to a nonphagocytosable exogenous acceptor and to endogenous membrane acceptors. The cells galactosylated in this way acquired new agglutinating properties with soybean agglutinin, which proves the external position of the galactosyl residues incorporated on the cell surface. (+info)
Fold recognition study of alpha3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain.
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The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases. (+info)