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At least 163 records · Page 9Linked to original sources

PubMed1988

Characterization of a glucosyltransferase activity in liver plasma membrane: modulation by cations and lipidic effectors.

1. Glucosyltransferase activity incorporating [14C]glucose from UDP-[14C]glucose onto endogenous lipidic acceptors was localized primarily in the plasma membrane of liver. 2. Incubation of plasma membrane by phosphatidyl-choline liposomes loaded with dolichyl-phosphate stimulated the enzymatic activity. 3. This enzyme required Mg2+ for maximal catalitic activity. Ca2+ could substitute Mg2+. 4. Mn2+ acted as a partial non-competitive inhibitor of the Mg2+-activated glucosyltransferase. 5. This enzyme can be modulated by neutral and acidic phospholipids; the most efficient were phosphatidyl-serine and phosphatidyl-inositol. 6. The enzymatic activity was not significantly changed by cholesterol alone but it is greatly enhanced by liposomes loaded with dolichyl-phosphate and cholesterol.

Animals↗

PubMed1990

Evidence of partial localization in Golgi apparatus of UDP-glucose collagen glucosyltransferase from chick embryo liver.

1. Collagens are the most important components of the connective tissue. 2. Collagen synthesis involves greater than 12 different enzymes whereas three enzymatic systems are involved in the ordered degradation. 3. Some enzymes are found in the rough endoplasmic reticulum (RER). The subcellular localization of disulfur isomerase, alpha D-glucosidase, proteases, galactosyltransferases and glucosyltransferases specific to collagen is unknown. 4. After having determined the best subcellular fractionation conditions for the chick embryo liver, we demonstrate that the galactosylhydroxylysyl glucosyltransferase specific to collagen is located in the RER and in the Golgi apparatus.

Animals↗

PubMed1992

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.

Animals↗

PubMed1982

Purification and properties of Streptococcus mutans extracellular glucosyltransferase.

Extracellular glucosyltransferase (sucrose:1,6-alpha-D-glucan 3-alpha- and 6-alpha-glucosyltransferase) was purified about 10 000-fold from the culture supernatant of Streptococcus mutans 6715. The enzyme preparation was homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing and ultracentrifugation analyses. The specific activity of the enzyme was 34.9 I.U. per mg of protein and the carbohydrate content was less than 1% (w/w). The molecular weight was determined to be 149 000 +/- 5000 by sedimentation equilibrium experiment. The acidic and basic amino acids of the enzyme comprised 29 and 8.4% of total amino acid, respectively, and the isoelectric point was pH 4.1. The enzyme had the optimum pH of 5.5 and the Km value of 2.4 mM for sucrose. The water-soluble glucan, which was de novo-synthesized from sucrose by the purified enzyme, was analyzed by a gas-liquid chromatography-mass spectroscopy and was found to be 1,6-alpha-D-glucan with highly (35%) branched structure of 1,3,6-linked glucose residue.

Amino Acids↗

PubMed1979

Glucosyltransferase activity in kidney fractions of normal and streptozotocin-diabetic rats.

Glucosyltransferase (UDPglucose: galactosylhydroxylysine-basement membrane glucosyltransferase), an enzyme specifically involved in collagen synthesis, was measured in various kidney fractions of normal, diabetic and underfed rats, using as basis the incorporation of radioactivity into protein during incubation with UDP[U-14C]glucose and alkali-soluble fetal calf-skin collagen. Three criteria of enzyme activity were compared: A, total radioactivity of the washed protein precipitate; B, this figure minus activity incorporated in the absence of the collagen acceptor; and C, radioactivity incorporated into the mixed amino acid fraction, collected by elution with dilute NH4OH from a Dowex 50 resin column after alkaline hydrolysis of the protein. Method A was found satisfactory using whole medulla or isolated glomeruli, since the average proportions of total protein radioactivity recovered in the NH3 fraction were 0.81 and 0.87, respectively, and the deviations were small. There was a larger and variable proportion of nonspecific incorporation using whole cortex. Incubation of a control set of sample without added collagen was found to be unnecessary (Method B). Per mg protein, medulla and glomeruli had more enzyme than did whole cortex. In diabetes, activity was enhanced in the 10,000 X g supernatant fraction of cortex, as previously reported. However, the increase associated with diabetes was even more consistent in the medulla, averaging 3-fold in the 10,000 X g pellet fraction. No increase was found in isolated glomeruli in diabetes. Also, no increase was seen in the kidneys of non-diabetic rats with body weight similar to that of the diabetics.

Animals↗

PubMed1982

Purification and characterization of basic glucosyltransferase from Streptococcus mutans serotype c.

Streptococcus mutans Ingbritt (serotype c) was found to secrete basic glucosyltransferase (sucrose: 1,6-alpha-D-glucan 3-alpha and 6-alpha-glucosyltransferase). The enzyme preparation obtained by ethanol fractionation, DEAE Bio-Gel A chromatography, chromatofocusing and preparative isoelectric focusing was composed of three isozymes with slightly different isoelectric points (pI 8.1-8.4). The molecular weight was estimated to be 151000 by SDS-polyacrylamide gel electrophoresis. The specific activity of the enzyme was 9.8 IU per mg of protein and the optimum pH was 6.5. The enzyme was activated 2.4-fold by commercial dextran T10, and had Km values of 7.1 micro M for the dextran and 4.3 mM for sucrose. Glucan was de novo synthesized from sucrose by the enzyme and found to be 1,6-alpha-D-glucan with 17.7% of 1,3,6-branching structure by a gas-liquid chromatography-mass spectroscopy.

Chromatography, Ion Exchange↗

PubMed1992

De novo glucan synthesis by mutants streptococcal glucosyltransferases present in pellicle promotes firm binding of Streptococcus gordonii to tooth surfaces.

Adherence of 3H-labelled cells of Streptococcus gordonii and Streptococcus milleri to artificial pellicles prepared from saliva supplemented with glucosyltransferases from mutants streptococci was examined using a new assay for sucrose-dependent cell-to-pellicle attachment. Results indicate that S. gordonii, but not S. milleri, could attach tightly to hydroxylapatite surfaces through de novo glucan synthesis by mutants streptococcal glucosyltransferases present in the experimental salivary pellicles.

Bacterial Adhesion↗

PubMed2003

Sterol glucosyltransferases have different functional roles in Pichia pastoris and Yarrowia lipolytica.

Mutants of the methanol-utilizing yeast Pichia pastoris and the alkane-utilizing yeast Yarrowia lipolytica defective in the orthologue of UGT51 (encoding sterol glucosyltransferase) were isolated and compared. These mutants do not contain the specific ergosterol derivate, ergosterol glucoside. We observed that the P. pastoris UGT51 gene is required for pexophagy, the process by which peroxisomes containing methanol-metabolizing enzymes are selectively shipped to and degraded in the vacuole upon shifting methanol-grown cells of this yeast to glucose or ethanol. PpUGT51 is also required for other vacuole related processes. In contrast, the Y. lipolytica UGT51 gene is required for utilization of decane, but not for pexophagy. Thus, sterol glucosyltransferases play different functional roles in P. pastoris and Y. lipolytica.

Alkanes↗

PubMed2004

Detection of UDP-glucose:cyclo-DOPA 5-O-glucosyltransferase activity in four o'clocks (Mirabilis jalapa L.).

Although a pathway for betacyanin biosynthesis has been postulated, most of the catalytic steps have not yet been identified or demonstrated with biochemical evidence. In the postulated pathway, the glucose moiety of betanin is conjugated to the aglycone, betanidin, because the glucosyltransferase (GT) activity that produces betanin has been reported and its cDNA isolated. However, another pathway for betacyanin biosynthesis is proposed in which betanin is formed by GT acting at the 5,6-dihydroxyindoline-2-carboxylic acid (cyclo-DOPA) step, followed by condensation of the product with betalamic acid. Here, we show that GT activity acts upon cyclo-DOPA in the betacyanin synthetic pathway. A crude extract from the petals of four o'clocks (Mirabilis jalapa L.) was mixed with cyclo-DOPA and UDP-glucose. After the reaction was stopped with phosphoric acid, the product was chemically reacted with betalamic acid. In the final reaction mixture, betanin formation was confirmed by HPLC analysis, demonstrating cyclo-DOPA 5-O-glucosyltransferase activity. This activity was correlated with the accumulation of betanin during the development of four o'clock flowers and was detected in another five species of Centrospermae. These results indicate that the glucose moiety of betanin is introduced at the cyclo-DOPA step, which is followed by condensation with betalamic acid, and not at the betanidin aglycone step.

Chromatography, High Pressure Liquid↗

PubMed2004

Detoxification of the cruciferous phytoalexin brassinin in Sclerotinia sclerotiorum requires an inducible glucosyltransferase.

The phytoalexins, brassinin, 1-methoxybrassinin and cyclobrassinin, were metabolized by the stem rot fungus Sclerotinia sclerotiorum into their corresponding glucosyl derivatives displaying no detectable antifungal activity. Importantly, co-incubation of S. sclerotiorum with camalexins, various phytoalexin analogs, and brassinin indicated that a synthetic camalexin derivative could slow down substantially the rate of brassinin detoxification. Furthermore, inducible brassinin glucosyltransferase (BGT) activity was detected in crude cell-free extracts of S. sclerotiorum. BGT activity was induced by the phytoalexin camalexin, and the brassinin analogs methyl tryptamine dithiocarbamate and methyl 1-methyltryptamine dithiocarbamate. The overall results suggest that the fungus S. sclerotiorum in its continuous adaptation and co-evolution with brassinin producing plants, has acquired efficient glucosyltransferase(s) that can disarm some of the most active plant chemical defenses.

Antifungal Agents↗

PubMed2005

The primary in vivo steroidal alkaloid glucosyltransferase from potato.

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to alpha-chaconine or alpha-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of alpha-solanine was increased and alpha-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.

Alkaloids↗

PubMed1977

Metabolism of the polysaccharides of human dental plaque. Part II. Purification and properties of Cladosporium resinae (1 leads to 3)-alpha-D-glucanase, and the enzymic hydrolysis of glucans synthesised by extracellular D-glucosyltransferases of oral streptococci.

Cladosporium resinae (1 leads to 3)-alpha-D-glucanase has been characterized as an endoglucanase capable of completely hydrolysing insoluble (1 leads to 3)-alpha-D-glucans isolated from fungal cell-walls. D-Glucose was the major product, but a small amount of nigerose was also produced. The enzyme was specific for the hydrolysis of (1 leads to 3) bonds that occur in sequence, and nigerotetraose was the smallest substrate that was rapidly attacked. Isolated (1 leads to 3)-alpha-D-glucosidic linkages that occur in mycodextran, isolichein, dextrans, and oligosaccharides derived from dextran were not hydrolysed. Insoluble glucan synthesised from sucrose by culture filtrates of Streptococcus spp. were all hydrolysed to various limits; the range was 11-61%. A soluble glucan, synthesised by an extracellular D-glucosyltransferase of S. mutans OMZ176, was not a substrate, whereas insoluble glucans synthesised by a different D-glucosyltransferase, isolated from S. mutans strains OMZ176 and K1-R, were extensively hydrolysed (84 and 92%, respectively). It is suggested that dextranase-CB, a bacterial endo(1 leads to 6)-alpha-D-glucanase that does not release D-glucose from any substrate, could be used together with C. resinae (1 leads to 3)-alpha-D-glucanase to determine the relative proportions of (1 leads to 6)-linked to (1 leads to 3)-linked sequences of D-glucose residues in the insoluble glucans produce by oral streptococci. The simultaneous action of the two D-glucanoses was highly effective in solubilizing the glucans.

Cladosporium↗

PubMed1981

Oxidized saccharides as inhibitors of alpha-glucan synthesis by Streptococcus mutans glucosyltransferase.

Specific inhibition by periodate-oxidized dextrans of the synthesis of alpha-glucan by S. mutans glucosyltransferase prompted a search for structurally related inhibitors that might be effective as anticaries agents. Clinical dextran derivatives in which from 5 to 50% of the D-glucose units were oxidized acted as potent and specific enzyme-inhibitors, as did 10%-oxidized derivatives of dextran fractions ranging in mol. wt. from 10(4) to 2 X 10(6). Within these limits, differences in oxidation or molecular weight did not significantly affect the high inhibitory potency of the derivatives. In contrast, periodate oxidation of (1 leads to 6)-alpha, (1 leads to 3)-alpha-, and (1 leads to 4)-alpha-linked oligosaccharides containing less than approximately 15 D-glucose units, and of sucrose and structurally related trisaccharides, yielded derivatives that were poor inhibitors. Enzymic hydrolysis of oxidized dextrans caused a loss of their inhibitory power and indicated that, to act as specific inhibitors, oxidized molecules must contain at least 16 to 20 D-glucosyl residues. The similar, minimum size required in order that unoxidized oligosaccharides may act as efficient acceptors in the glucosyltransferase reaction suggests that the inhibitory potencies of oxidized derivatives may reflect their relative abilities to bind at the acceptor site of the enzyme.

Carbohydrates↗

PubMed1979

Improved purification procedure for the extracellular D-glucosyltransferase from Streptococcus mutans 6715.

The exocellular D-glucosyltransferase from Streptococcus mutans 6715 has been highly purified with minimal loss of enzymic activity. The organisms were cultured in trypticase soy-broth that had been treated with invertase and filtered through an ultrafilter fitted with a membrane having a cut-off molecular weight at 10,000. To the growth medium was added Tween 80, which prevented the enzyme from aggregating. The final step in the purification employed insoluble, streptococcal dextran as an affinity support. Two D-glucosyltransferase activities were detected, viz., one that did not adsorb to the insoluble dextran and one that did. The enzymic fraction that had adsorbed to the insoluble dextran in the affinity column was strongly inhibited by added insoluble dextran.

Glucosyltransferases↗

PubMed2002

A base-flipping mechanism for the T4 phage beta-glucosyltransferase and identification of a transition-state analog.

T4 phage beta-glucosyltransferase (BGT) modifies T4 DNA. We crystallized BGT with UDP-glucose and a 13mer DNA fragment containing an abasic site. We obtained two crystal structures of a ternary complex BGT-UDP-DNA at 1.8A and 2.5A resolution, one with a Tris molecule and the other with a metal ion at the active site. Both structures reveal a large distortion in the bound DNA. BGT flips the deoxyribose moiety at the abasic site to an extra-helical position and induces a 40 degrees bend in the DNA with a marked widening of the major groove. The Tris molecule mimics the glucose moiety in its transition state. The base-flipping mechanism, which has so far been observed only for glycosylases, methyltransferases and endonucleases, is now reported for a glucosyltransferase. BGT is unique in binding and inserting a loop into the DNA duplex through the major groove only. Furthermore, BGT compresses the backbone DNA one base further than the target base on the 3'-side.

Bacteriophage T4↗

PubMed2000

Indoxyl-UDPG-glucosyltransferase from Baphicacanthus cusia.

The enzyme catalyzing the transfer of glucose from uridine diphosphate glucose to indoxyl yielding the indoxyl glucoside indican was isolated from Baphicacanthus cusia Bremek (Acanthaceae). The indoxyl-uridine diphosphate glucose (UDPG)-glucosyltransferase was purified to homogeneity in six chromatographic steps. The decisive step for the recovery of a homogeneous enzyme was the application of immobilized metal affinity chromatography yielding an 863-fold purified enzyme. From a total of 60 substances tested, in addition to the natural substrate 3-OH-indole (indoxyl), only 4-OH-, 5-OH-, 6-OH-, and 7-OH-indole were accepted as substrates by the glucosyltransferase. However, the latter substrates were metabolized to varying extent. The optimum pH of the enzyme was 8.5, the optimum temperature was 30 degrees C and the isoelectric point was pH 6.5. The M(r) of the enzyme was determined to be 60 +/- 2 x 10(3). Indoxyl as substrate yielded a K(m) of 1.2 mM, while a K(m) of 1.7 mM was found for UDPG.

Chromatography, Ion Exchange↗

PubMed2001

Structure of the UDP-glucosyltransferase GtfB that modifies the heptapeptide aglycone in the biosynthesis of vancomycin group antibiotics.

BACKGROUND: Members of the vancomycin group of glycopeptide antibiotics have an oxidatively crosslinked heptapeptide scaffold decorated at the hydroxyl groups of 4-OH-Phegly4 or beta-OH-Tyr6 with mono- (residue 6) or disaccharides (residue 4). The disaccharide in vancomycin itself is L-vancosamine-1,2-glucose, and in chloroeremomycin it is L-4-epi-vancosamine-1,2-glucose. The sugars and their substituents play an important role in efficacy, particularly against vancomycin-resistant pathogenic enterococci. RESULTS: The glucosyltransferase, GtfB, that transfers the glucose residue from UDP-glucose to the 4-OH-Phegly4 residue of the vancomycin aglycone, initiating the glycosylation pathway in chloroeremomycin maturation, has been crystallized, and its structure has been determined by X-ray analysis at 1.8 A resolution. The enzyme has a two-domain structure, with a deep interdomain cleft identified as the likely site of UDP-glucose binding. A hydrophobic patch on the surface of the N-terminal domain is proposed to be the binding site of the aglycone substrate. Mutagenesis has revealed Asp332 as the best candidate for the general base in the glucosyltransfer reaction. CONCLUSIONS: The structure of GtfB places it in a growing group of glycosyltransferases, including Escherichia coli MurG and a beta-glucosyltransferase from T4 phage, which together form a subclass of the glycosyltransferase superfamily and give insights into the recognition of the NDP-sugar and aglycone cosubstrates. A single major interdomain linker between the N- and C- terminal domains suggests that reprogramming of sugar transfer or aglycone recognition in the antibiotic glycosyltransferases, including the glycopeptide and also the macrolide antibiotics, will be facilitated by this structural information.

Amino Acid Sequence↗

PubMed1989

Sucrose 6-alpha-D-glucosyltransferase from Streptococcus sobrinus: characterization of a glucosyl-enzyme complex.

A covalent glucosyl-enzyme was isolated from a quenched reaction of Streptococcus sobrinus sucrose 6-alpha-D-glucosyltransferase and radiolabeled sucrose. No complex was observed with heat-inactivated enzyme or when sucrose was replaced with radiolabeled maltose or glucose. The complex was stable at pH 2 in 1% sodium dodecyl sulfate, 6.0 M urea, and 4.0 M guanidine hydrochloride, but became increasingly labile with increased pH (32-min half-life at pH 7.0). D-Glucose was the exclusive radiolabeled compound identified when all radioactivity was released under mild alkaline conditions. Glucosyl-enzyme hydrolysis rates were linearly dependent on hydroxide ion concentration, giving a second-order rate constant of 2.15 x 10(5) M-1 min-1. When compared to the base lability of known glycosyl amino acid derivatives, the pH dependency of the glucosyl-enzyme most closely paralleled a glucosyl linkage to a carboxyl group. A novel application of a carbohydrate high-performance liquid chromatography column in aqueous solution was used to identify the anomeric form of D-glucose released on (i) alkaline hydrolysis of denatured glucosyl-enzyme and (ii) native enzyme hydrolysis of sucrose. The beta-anomer was identified in the former case and the alpha-anomer in the latter. The results with the denatured glucosyl-enzyme are consistent with a beta-glucosyl ester linkage to an aspartic or glutamic acid that hydrolyzes at the ester carbon with retention of anomeric configuration; for native glucosyltransferase catalysis, the data are consistent with a beta-glucosyl covalent intermediate as well, where deglucosylation occurs by attack at the acetal carbon with anomeric inversion.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Radioisotopes↗