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PubMed1984

Partial purification and characterization of UDPG:t-cinnamate glucosyltransferase in the root of sweet potato, Ipomoea batatas Lam.

Previously, we isolated t-cinnamoyl-D-glucose as a possible intermediate in chlorogenic acid biosynthesis from sweet potato root. The enzyme which catalyzes the formation of t-cinnamoyl-D-glucose has been purified 539-fold from sweet potato root (Ipomoea batatas Lam.) and characterized. It required UDP-glucose as a glucosyl donor. Its molecular weight was estimated to be 45,000 by gel filtration chromatography through Sephadex G-100. Its Km values were 0.2 mM for t-cinnamic acid and 0.1 mM for UDP-glucose. It also showed activity toward various aromatic carboxylic acids other than t-cinnamic acid with the following relative activities at the concentration of 1.8 mM: t-cinnamic acid, 100; p-coumaric acid, 57; o-coumaric acid, 52; caffeic acid, 15; benzoic acid, 71; ferulic acid, 27; 4-hydroxyl-3-methoxy-benzoic acid, 35. When p-coumaric acid was used as a substrate, the enzyme introduced the glucosyl group exclusively to a carboxyl group, not to a hydroxyl group on a benzene ring. It was inhibited by p-chloromercuribenzoate and HgCl2. Its activity in the extract from sliced root decreased during the first 28 h after slicing, then increased to the original level by 75 h. The apparent decrease seemed to be caused by the appearance of an inhibitory factor of high molecular weight in the tissue extract.

Chemical Phenomena↗
PubMed2003

Anoxia and reoxygenation of human endothelial cells decrease ceramide glucosyltransferase expression and activates caspases.

Endothelial oxidative stress induces cellular activation and sometimes death. Endothelial death can occur via necrosis or apoptosis. Understanding the mechanisms involved in cellular activation and death may lead to therapeutics designed to increase death or preserve cellular function. In the present study, brief periods of anoxia (3 h) followed by varying lengths of reoxygenation (0-5 h) lead to a time-dependent increase in human umbilical vein endothelial cell (HUVEC) caspase activity. Furthermore, ROCK-1 cleavage, which is dependent on caspase-3 activity, was also increased in cells undergoing oxidative stress compared with normoxic cells. Microarray data demonstrated that glucosylceramide synthase (GCS; glucosylceramide transferase), but not acid sphingomyelinase, was modulated by anoxia and reoxygenation. We confirmed that GCS mRNA and protein expression were significantly decreased in a time-dependent fashion following oxidative stress by real-time polymerase chain reaction and Western blot, respectively. Treatment of normoxic cells with the GCS-specific inhibitor, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), increased caspase activity to the same degree as cells undergoing oxidative stress. Fumonisin B1, the N-acyl-sphinganine dehydrogenase (e.g., ceramide synthase) inhibitor significantly attenuated caspase activity in HUVECs undergoing oxidative stress. These data suggest that alterations in GCS expression following brief periods of oxidative stress in human endothelial cells lead to increased caspase activity.

Animals↗
PubMed1994

Identification and characterization of the ecdysteroid UDP-glucosyltransferase gene of the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus.

We have located, cloned, sequenced and characterized the ecdysteroid UDP-glycosyltransferase gene (egt) gene from the baculovirus Lymantria dispar multinucleocapsid nuclear polyhedrosis virus. (LdMNPV), which is specific for the gypsy moth (L. dispar). The egt gene from the related baculovirus Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) disrupts the hormonal balance of the host larva by galactosylating ecdysone, which prevents moulting. The location of the LdMNPV egt gene, determined by hybridization analysis using a cloned coding segment of the AcMNPV egt gene, was mapped to between 79.1 and 80.2 map units on the viral genome. This region contains an open reading frame of 1464 nucleotides capable of encoding a 55K polypeptide. This predicted protein exhibits a 42% amino acid identity with the AcMNPV egt polypeptide. Transcripts of the egt gene were analyzed by Northern blot and primer extension. The egt gene is transcribed from approximately 12 to 48 h, and maximally at about 16 h post-infection. Transcription occurred in the presence of aphidicolin, a viral DNA synthesis inhibitor, but not in the presence of cycloheximide, a protein synthesis inhibitor. Therefore the LdMNPV egt gene is classified as a delayed early gene. The egt gene is transcribed in a clockwise direction with respect to the circular map, and transcription initiates at a single site. Comparisons between the two baculoviral egt proteins and mammalian UDP-glucuronosyltransferases reveal areas which are conserved between the mammalian and baculoviral genes, as well as areas that are only conserved in the viral egt proteins. The LdMNPV protein sequence appears to include a signal peptide, which would allow the protein to be secreted into the haemolymph.

Amino Acid Sequence↗
PubMed2006

The glucosyltransferase UGT72E2 is responsible for monolignol 4-O-glucoside production in Arabidopsis thaliana.

The phenylpropanoid pathway in plants leads to the synthesis of a wide range of soluble secondary metabolites, many of which accumulate as glycosides. In Arabidopsis, a small cluster of three closely related genes, UGT72E1-E3, encode glycosyltransferases shown to glucosylate several phenylpropanoids in vitro, including monolignols, hydroxycinnamic acids and hydroxycinnamic aldehydes. The role of these genes in planta has now been investigated through genetically downregulating the expression of individual genes or silencing the entire cluster. Analysis of these transgenic Arabidopsis plants showed that the levels of coniferyl and sinapyl alcohol 4-O-glucosides that accumulate in light-grown roots were significantly reduced. A 50% reduction in both glucosides was observed in plants in which UGT72E2 was downregulated, whereas silencing the three genes led to a 90% reduction, suggesting some redundancy of function within the cluster. The gene encoding UGT72E2 was constitutively overexpressed in transgenic Arabidopsis to determine whether increased glucosylation of monolignols could influence flux through the soluble phenylpropanoid pathway. Elevated expression of UGT72E2 led to increased accumulation of monolignol glucosides in root tissues and also the appearance of these glucosides in leaves. In particular, coniferyl alcohol 4-O-glucoside accumulated to massive amounts (10 micromol g(-1) FW) in root tissues of these plants. Increased glucosylation of other phenylpropanoids also occurred in plants overexpressing this glycosyltransferase. Significantly changing the pattern of glycosides in the leaves also led to a pronounced change in accumulation of the hydroxycinnamic ester sinapoyl malate. The data demonstrate the plasticity of phenylpropanoid metabolism and the important role that glucosylation of secondary metabolites can play in cellular homeostasis.

Arabidopsis↗
PubMed1980

Glucosyltransferase activities in liver mitochondria. I. Biosynthesis of dolichyl[14C]glucosyl phosphate and [14C]glucosylceramide.

Mitochondria, and specially outer mitochondrial membranes, incorporate D-[14C]glucose from UDP-D-[14C]glucose into products extracted with organic solvents and into a residual precipitate, with a pH optimum of about 6.5 in (2-N-morpholino-ethane)-sulfonic acid (MES) buffer. The chloroform/methanol (2:1, v/v) extract contains two products. The major [14C]glucolipid is stable to mild alkali, but releases [14C]glucose upon mild acid hydrolysis. It is retained on DEAE-cellulose (acetate form) and is eluted with the same ionic strength as an hexosyldolichyl monophosphate diester. This [14C] glucolipid has the same chromatographic behaviour as dolichyl-mannosylphosphate in neutral, acidic and basic solvent systems; and its biosynthesis is greatly increased by exogenous dolichylmonophosphate. The other [14C]glucolipid is stable upon mild acid hydrolysis and is not retained on DEAE-cellulose. On silicic acid it is eluted with acetone. The biosynthesis of this compound is stimulated by exogenous ceramide. This glucolipid has the same chromatographic mobility in different solvent systems as glucosylceramide isolated from the liver of a patient with Gaucher's disease. Biosynthesis of these two glucolipids is inhibited by UDP, but only biosynthesis of dolichylglucosyl monophosphate is reversible with this nucleotide. The biosynthesis of these different glucosylated derivatives is stimulated by the addition of divalent cations (Mn2+, Mg2+). the effect of these two metal ions on dolichylglucosyl monophosphate and glucosylceramide formation is studied in different conditions.

Animals↗
PubMed2000

Production, characterization, and application of monoclonal antibodies which distinguish four glucosyltransferases from Streptococcus sobrinus.

A 1,3-alpha-glucan synthase (GTF-I), a highly branched 1, 6-alpha-glucan synthase (GTF-U) and a 1,6-alpha-glucan synthase (GTF-T) were purified to near homogeneity from the culture fluid of Streptococcus sobrinus strain B13N (serotype d) and characterized. In addition, a crude preparation of a recombinant oligo-isomaltosaccharide synthase (rGTF-S) was prepared from a cell-free extract of Escherichia coli MD124 transformant. Using four homogeneous GTF preparations including previously purified rGTF-S as antigens for immunization, 11 murine hybridomas producing a monoclonal antibody (MAb) were established through the fusion of myeloma cells (P3X63-Ag8-U1) and spleen cells of immunized BALB/c mice. When the immunoreactivities of the resultant MAbs were tested, all five MAbs raised against GTF-I, all three MAbs raised against GTF-T, and two of three MAbs raised against GTF-U reacted specifically with the homologous enzyme alone, while one MAb (B86) raised against GTF-U cross-reacted strongly with all GTFs. Although no MAb monospecific for rGTF-S was obtained, precise recognition of GTF-S was possible using the nonspecific B86 antibody together with the MAbs monospecific for the three glucan synthases. Thus, a set of four typical MAbs (B17, B76, B19 and B86) were successfully used for the identification of gene products expressed in 24 previously constructed E. coli phage clones, and the findings suggested that six phage clones might express a gtfU gene encoding GTF-U which has not been hitherto isolated.

Animals↗
PubMed1988

Immune properties of glucosyltransferases from S. sobrinus.

GTF activity was separated into water-insoluble (GTF-I) and water-soluble (GTF-S) polyglucan-synthesizing enzymes. Each preparation demonstrated a single band on 6% SDS PAGE. Only water-insoluble or water-soluble polyglucan was synthesized by the respective enzyme preparation. Rats were immunized, on Days 1 and 14, with either GTF-I or GTF-S in adjuvant. Animals were bled 13, 35 and 54 days after the initial immunization. Individual antisera were tested against either the GTF-I or the GTF-S for inhibition of radioactive glucose incorporation into glucan, and in gel diffusion, and by Western transfer analyses. The respective antisera reacted with the homologous, but not the heterologous enzyme in gel diffusion and Western transfer. GTF-I activity was not inhibited by antibody to GTF-S, but antibody to GTF-I inhibited GTF-I by 68%. GTF-S was inhibited by more than 60% by each of 3 anti-GTF-S sera. Only one anti-GTF-I serum inhibited GTF-S at as much as a modest 30% level. These data support the antigenic and functional distinctiveness of the GTF enzymes of S. sobrinus 6715.

Animals↗
PubMed1987

Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva.

Two hour in vivo formed enamel pellicle samples and paraffin wax-stimulated saliva samples were collected from 10 volunteers for analyses of glycosyltransferase activity (GTF). GTF activity was recorded by monitoring incorporation of radioactivity from 14C-glucose labeled sucrose into glucan. Pellicle and saliva samples from all 10 subjects demonstrated GTF activity. The GTF activity in the pellicle samples was highest in subjects with high GTF activity producing adhesive glucan in saliva.

Dental Deposits↗
PubMed1978

Binding of glucosyltransferase and glucan synthesis by Streptococcus mutans and other bacteria.

Lyophilized and heat-treated cells from the seven serotypes of Streptococcus mutans were examined for their ability to bind added insoluble-product glucosyl-transferase (GTase) and to synthesize cell-associated glucan from [(14)C]sucrose. Lyophilized cells of serotypes a and g did not synthesize any more additional glucan than did the controls after exposure to GTase. These cells, however, synthesized four- to eightfold-greater quantities of glucan than did the cells of the remaining serotypes. Lyophilized cells of serotypes b, c, d, e, and f synthesized two- to threefold-greater quantities of glucan after exposure to GTase than did the controls without added enzyme. Lyophilized cells of serotypes a and g synthesized 6- to 10-fold-greater quantities of glucan than did heat-treated cells of the same strain after binding of GTase. Lyophilized cells of the remaining serotypes synthesized only 1.6- to 3.3-fold-greater quantities of glucan than did the heat-treated cells. These results demonstrate that heat treatment to inactivate cell-associated GTase does not create additional GTase binding sites in S. mutans and that serotypes a and g are considerably more active in cell-associated glucan synthesis than cells of the other five serotypes. Ten species of gram-positive and gram-negative bacteria from five genera which do not produce in vitro plaque synthesized 10- to 100-fold-less glucan than did the S. mutans strains after exposure to GTase. Of these species, S. sanguis, Actinomyces viscosus, and A. naeslundii synthesized the largest quantities of glucan. Three mutant strains of S. mutans which possess a reduced ability for in vitro adherence but do agglutinate with glucan or dextran synthesized only one-third as much glucan after binding of GTase as the control. These results are discussed in relation to in vitro and in vivo plaque development and the agglutination of S. mutans. The results support earlier findings which indicate that the presence of bacterial species other than S. mutans in smooth-surface dental plaque is due in part to contact of the cells with glucan in the developing plaque and not to the binding of cell-free GTase and the in situ synthesis of glucan. The results obtained with these representative strains of the seven serotypes of S. mutans may not apply to the same extent to other strains within the serotypes.

Escherichia coli↗
PubMed1982

Pyridine analogs inhibit the glucosyltransferase of Streptococcus mutans.

Soluble glucan synthesis catalyzed by dextransucrase preparations from Streptococcus mutans 6715 were inhibited by pyridoxal-5-phosphate and several other pyridine analogs, including pyridoxine, pyridoxamine, pyridoxamine-5-phosphate, pyridoxal, and 4-pyridoxic acid. Pyridine and pyridine-4-carboxaldehyde were not effective inhibitors of the enzyme. Kinetic analyses suggested that pyridoxal-5-phosphate is a noncompetitive inhibitor of dextransucrase. The inactivation was dependent on time, pyridoxal-5-phosphate concentration, and hydrogen ion concentration. Apparent Ki values were 4.9 mM at pH 7.0 and 4.2 mM at pH 5.5. Dextransucrase activity could be restored by dialysis to remove the inhibitors. Maximum inhibition was observed after a 120-min incubation of the enzyme with pyridoxal-5-phosphate. The pH optima for inhibition by pyridoxal-5-phosphate were 4 and 7. The sucrose-dependent adherence of S. mutans cells to saliva-coated hydroxylapatite beads was also inhibited by pyridoxal-5-phosphate but only marginally by the other pyridine anatogs. In addition, pyridoxal-5-phosphate markedly reduced the rate of acid production by intact S. mutans cells from sucrose or glucose substrates. Another pyridoxal-5-phosphate analog, 2-methyl-5-hydroxypyridine, was also effective in preventing the production of acid by S. mutans from sucrose or glucose. When S. mutans cells were preincubated with pyridoxal-5-phosphate or pyridine analogs, significant reductions in the rate of D-glucose uptake were observed. It is suggested that the inhibition of dextransucrase occurs because of a change iun enzyme conformation which results from the binding of the pyridine derivatives. The results suggest that pyridoxal-5-phosphate or structural analogs may ultimately be useful in reducing the incidence of dental caries.

Adhesiveness↗