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PubMed1998

Molecular cloning and characterization of the mouse ceramide glucosyltransferase gene.

Ceramide glucosyltransferase (glucosylceramide synthase, GlcT-1, EC 2.4.1.80) catalyzes the first step in glycosphingolipid synthesis, the transfer of glucose from UDP-glucose to ceramide. The product, glucosylceramide, serves as a core structure for over 300 species of glycosphingolipids. The enzyme is a key regulatory factor controlling intracellular levels of ceramide and glycosphingolipids. We have cloned the gene for mouse ceramide glucosyltransferase, Ugcg. The gene spans approximately 32 kb and is composed of 9 exons and 8 introns. The promoter region was found to lack TATA and CAAT boxes but contains Spl binding sites, which are indicators of typical housekeeping genes. In addition to these general transcription factor binding sites, the motifs for AhR, NF-kappaB, AP-2 and GATA-1 binding sites were found.

Animals↗

PubMed1978

A modified assay system for collagen glucosyltransferase.

A simplified assay procedure has been developed for the determination of collagen glucosyltransferase activity in tissue extracts. Using degraded gelatine as acceptor it was possible to isolate the reaction product by precipitation on to a glass fibre disc. Under our conditions degraded gelatine is glucosylated with a reaction rate which is 3--4 times lower compared with the glucosylation of basement membrane derived glycopeptides. Good reproducibility is demonstrated by the coefficient of variation of 4% in the same assay and an interassay variation coefficient below 8%. As the assay allows the testing of large numbers of samples in a few hours, it should prove a useful tool to determine the enzyme level in the tissue of diabetic animals. In humans the activity of the glucosyltransferase could provide a biochemical parameter related to diabetic microangiopathy.

Amino Acids↗

PubMed1977

Gene-dependent flavonoid glucosyltransferase in maize.

A direct relationship between a specific gene and a specific enzyme involved in flavonoid biosynthesis is reported for the gene Bz and uridine diphosphoglucose: quercetin glucosyltransferase in maize pollin, seedlings, and seeds. Ratios are presented for specific activities of the glucosyltransferase from pollen, seed, and seedling tissues homozygous and heterozygous for Bz and homozygous for bz.

Flavonoids↗

PubMed1989

Organization of mitochondrial UDP-glucose:dolichylmonophosphate glucosyltransferase in the outer membrane.

Previous studies have shown the existence of an autonomous mitochondrial UDP-glucose: dolichylmonophosphate glucosyltransferase, located in mitochondrial outer membrane of liver cells. To improve our knowledge about the topographical aspects of glycosylation in mitochondria, we have investigated the organization of this enzyme in intact mitochondria, using controlled proteolysis with trypsin and sensitivity towards amino-acid specific reagents. Our data provides evidence: --for a mitochondrial glucosyltransferase facing the cytoplasmic side of the outer membrane --and for the involvement of histidine and tryptophan residues as well as sulfhydryl groups in the catalytic activity of the enzyme.

Amino Acids↗

PubMed1988

[Stepwise study procedure of sugar substitutes--preliminary study with enzymes. 4. Glucosyltransferases of Streptococcus mutans AHT].

A continuous procedure for the simultaneous enzymatic measurement of the release of free fructose and glucose was adapted to the kinetic conditions of the synthesis of polysaccharides from sucrose by glucosyltransferases from Streptococcus mutans AHT. Initial velocities, Km of sucrose, and the efficiency of the formation of glucans from sucrose can be determined. Longtime incubations with the isolation of soluble and insoluble glucans as an established method were compared with the new procedure. As examples of the effect of sugar substitutes on glucosyltransferases, data on leucrose, nystose, Palatinit, xylitol, leucritol and polyglucose PL-3 are presented. The results provide a preliminary assessment of sugar substitutes such as non-cariogenic sweeteners.

Fructose↗

PubMed1975

Enzymatic synthesis of glucocerebroside by UDP-glucose: ceramide glucosyltransferase during ontogenesis of chicken retina.

The activity of uridine diphosphate-glucose: ceramide glucosyltransferase during chick retina ontogenesis was studied. This enzyme catalyzes the first step in the biosynthetic pathway leading to gangliosides. The glucosyltransferase activity was detected in 8 day old embryos, and was present in adult animals. The highest specific activity was found ca. day 10 of embryonic life. The radioactive product was identified as glucocerebroside by means of radiochromatography. The presence of phosphatidylcholine in the incubation mixtures was found to stimulate the enzyme.

Age Factors↗

PubMed2003

Structural analysis of the functional influence of the surface peptide Gtf-P1 on Streptococcus mutans glucosyltransferase C activity.

Glucosyltransferases (GtfB/C/D) in Streptococcus mutans are responsible for synthesizing water-insoluble and water-soluble glucans from sucrose and play very crucial roles in the formation of dental plaque. A monoclonal antibody against a 19-mer peptide fragment named Gtf-P1 was found in GtfC to reduce the enzyme activity to 50%. However, a similar experiment suggested almost unchanged activity in GtfD, despite of the very high sequence homology between the two enzymes. No further details are yet available to elucidate the biochemical mechanism responsible for such discrimination. For a better understanding of the catalytic behavior of these glucosyltransferases, structural and functional analyses were performed. First, the exact epitope was identified to specify the residue(s) required for monoclonal antibody recognition. The results suggest that the discrimination is determined solely by single residue substitution. Second, based on a combined sequence and secondary structure alignment against known crystal structure of segments from closely related proteins, a three-dimensional homology model for GtfC was built. Structural analysis for the region communicating between Gtf-P1 and the catalytic triad revealed the possibility for an "en bloc" movement of hydrophobic residues, which may transduce the functional influence on enzyme activity from the surface of molecule into the proximity of the active site. Figure Side chain interactions between Gtf-P1 and catalytic Asp-477 in GtfC. Calpha-tracing of GtfC with the two crucial peptides (Gtf-P1, orange; Gtf-P2, blue) and the catalytic triad residues ( red) highlighted to show their relative spatial organization. Side chains for the residues are also depicted according to their atom types. The structure is viewed with the barrel opening facing down

Amino Acid Sequence↗

PubMed2005

A novel glucosyltransferase involved in steroid saponin biosynthesis in Solanum aculeatissimum.

Steroidal saponins are widely distributed in many plant species. Their diverse structures have resulted in a wide range of applications, including drug and medicine production. It has been suggested that the nature of the non-saccharide and oligosaccharide portions of the saponin molecule both contribute to the properties of individual saponins. Despite numerous studies on the occurrence, chemical structure, and varying pharmaceutical activities of steroidal saponins, their biosynthesis pathway is poorly understood. Glycosylation is thought to be the final step in steroidal saponin biosynthesis and it is thought to be involved in regulating the biological activities of saponins. Isolation of the glycosyltransferases that catalyze the transfer of sugar molecules to steroidal compounds will help to clarify the mechanisms that produce diverse saponins and control their activities in plants. In this study, we obtained three cDNAs encoding putative glycosyltransferases from Solanum aculeatissimum. One of the three, SaGT4A showed UDP-glucosyltransferase activity. This is the first cloned glucosyltransferase involved in steroidal saponin biosynthesis. SaGT4A catalyzes the 3-O-glucosylation of steroidal sapogenins, such as diosgenin, nuatigenin, and tigogenin. This enzyme also glucosylates steroidal alkaloids, such as solanidine, solasodine, and tomatidine. Gene expression analysis revealed that the accumulation of SaGT4A transcripts showed a unique response to wounding stress indicating the involvement of SaGT4A in plant defense system.

Amino Acid Sequence↗

PubMed1991

Partial purification, properties, and kinetic studies of UDP-glucose:p-hydroxybenzoate glucosyltransferase from cell cultures of Lithospermum erythrorhizon.

A glucosyltransferase, which catalyzed the transfer of glucose from UDP-glucose (UDPG) to p-hydroxybenzoate (PHB) in cell cultures of Lithospermum erythrorhizon Sieb. et Zucc., Boraginaceae, was purified 219-fold by ammonium sulfate fractionation and chromatography on DEAE-Sephacel, Sephadex G-150, and phenyl-Sepharose Cl-4B. p-Hydroxybenzoic acid O-beta-D-glucoside (PHB-glc) was identified as a product of the enzymatic reaction. This glucosyltransferase has a molecular weight of 47,500 Da, an isoelectric point at pH 5.0, and a pH optimum of 7.8. The enzyme does not sediment at 100,000g. Enzyme activity did not require metal cofactors. The enzyme was highly specific for p-hydroxybenzoate (Km 0.264 mM) and UDP-glucose (Km 0.268 mM). Initial velocity studies suggest that the enzyme reaction mechanism is a sequential rather than a ping-pong mechanism. Product inhibition patterns are consistent with an ordered sequential bi-bi mechanism, where UDPG is the first substrate to bind to the enzyme and UDP the final product released. The data indicate the formation of a dead-end complex between PHB-glc and the enzyme. Uncompetitive inhibition by the substrate PHB can be put down to the formation of an abortive complex between E-UDP and PHB.

Glucosyltransferases↗

PubMed1982

A comparative study of extracellular glucanhydrolase and glucosyltransferase enzyme activities of five different serotypes of oral Streptococcus mutans.

The activities of glucanhydrolase (EC 3.2.1.11) and glucosyltransferase (EC 2.4.1.5) in crude enzyme preparations of 44 strains of Streptococcus mutans of five serotypes were investigated. The strains were grown in a laboratory fermentor for 16 h and the enzymes were isolated by adding solid ammonium sulphate to the culture supernatant, resulting in a 12-fold enrichment of the enzymes. For glucanhydrolase, strains of serotype a showed the lowest total activity (0.768 U, approx. 120 ml), whereas strains of serotype d had an activity 39 times higher (29.9 U). The total activities of strains of serotypes b, c and e were 5.56, 6.30 and 7.06 U, respectively. For glucosyltransferase, strains of type e showed the highest total activity (293 U), whereas differences between strains of the other four types were insignificant (type a: 158 U; type b: 175 U; type c: 191 U; type d: 225 U; approx. 120 ml). A strong correlation was found between the glucanhydrolase activity and the percentage of insoluble glucan synthesized in vitro by the respective strains. This correlation was not substantially changed if the enzyme activities were expressed as specific activities, or as total activities against bacterial weight.

Dextranase↗

PubMed1985

In-vitro inhibition of glucosyltransferase from the dental plaque bacterium Streptococcus mutans by common beverages and food extracts.

Some fruit juices and beverages inhibit the glucosyltransferases of Streptococcus mutans. Inhibition by cocoa, coffee and tea was due partly to gelatin-precipitable tannins and partly to components that exhibited properties of monomeric polyphenols. Charcoal treatment removed all inhibitory activity. Catechin, a known constituent of these beverages, was an effective inhibitor of the enzymes. The effects of the fruit juices were attributable mainly to the inhibition of the glucosyltransferases by the endogenous fructose and glucose. The findings show that naturally-occurring constituents of foods can inhibit extracellular polysaccharide formation from sucrose. Such constituents may play a role in regulating dental plaque formation in vivo and, thereby, may have long-term effects on the development of dental caries.

Beverages↗

PubMed1990

Solubilization of UDPglucose-ceramide glucosyltransferase from the Golgi apparatus.

The choice of a suitable detergent for solubilization of UDPglucose-ceramide glucosyltransferase from Golgi membranes has been investigated. Among the various classes of detergent, CHAPS, a zwitterionic detergent, was used as it produced a substantial activation of the enzyme activity. 30% of the enzyme activity and 50% of proteins were solubilized in the first attempts. Further experiments were conducted with addition of a second detergent, Zwittergent 3-14 which increased enzyme recovery to 45%. Lastly, reducing the concentrations of buffer and divalent cations Mn2+, Mg2+ and introducing glycerol (20%, v/v) allowed 80% of proteins to be solubilized together with 68% of the ceramide glucosyltransferase activity.

Animals↗

PubMed1976

Affinity chromatography of collagen glucosyltransferase on a UDP-glucose derivative coupled to agarose.

UDP-glucuronic acid from the carboxyl group was coupled to agarose via a six-carbon atom spacer, and columns prepared from this material were used in an affinity chromatography of collagen glucosyltransferase. The enzyme was found to have a high affinity for such columns in the presence of Mn2+ in the buffer, whereas a considerably lower affinity was noted in the absence of such ions. The enzyme could be eluted from the column with either EDTA, UDP-glucose, or small peptides prepared from collagen, the peptides being the most effective eluting agent. After elution the enzyme was separated from the peptides by gel filtration. With this procedure a collagen glucosyltransferase putification of about 3000-fold was obtained from extract of chick embryos by relatively simple steps. Collagen galactosyltransferase was found to have no affinity for the column, suggesting that the binding was not only due to the UDP moiety, but that the uronic acid derivate of glucose also contributed to its specificity.

Animals↗

PubMed1977

Characterization of human platelet UDPglucose-collagen glucosyltransferase using a new rapid assay.

A rapid and specific assay has been developed for UDPglucose-collagen glucosyltransferase (UDPglucose: 5-hydroxylysine-collagen glucosyltransferase, EC 2.4.1.66) using galactosylhydroxylysine (Gal-Hyl) as acceptor. Studies with intact human platelets and isolated plasma membranes indicated that about 5--10% of the total activity was surface bound and the rest was of cytoplasmic origin. The two forms of the enzyme had similar broad pH optima (6.5--8.0), Km values for UDPglucose (5 muM) and Gal-Hyl (approx. 4 mM) and for optimal manganese concentrations (25 mM). The soluble form of the enzyme was purified 80-fold. The reaction mechanism was determined as being rapid equilibrium random BiBi + dead end complex or ordered BiBi with UDPglucose being the first substrate to bind. Using Gal-Hyl bound in purified alpha 1 chain of chick skin collagen, a Km value three orders of magnitude less (2 muM) was found than for free Gal-Hyl and the manganese requirement decreased to 2 mM. These results suggest that the binding to the enzyme of Gal-Hyl in the collagen molecule is enhanced by the presence of the protein portion so that the enzyme may be capable of recognizing not only the carbohydrate side chains but also the primary structure of collagen.

Animals↗

PubMed1987

Formation of bile acid glucosides and dolichyl phosphoglucose by microsomal glucosyltransferases in liver, kidney and intestine of man.

Formation of glucosides of the bile acids chenodeoxycholic, ursodeoxycholic, deoxycholic and hyodeoxycholic acids has been detected in microsomes from human liver, kidney and intestinal mucosa. Hepatic and extrahepatic bile acid glucosyltransferase activities were characterized with respect to kinetic parameters and other catalytic properties. Whereas no marked organ-specific differences in the affinities of individual bile acids toward hepatic and extrahepatic glucosyltransferases were observed, microsomes from extrahepatic sources showed twice to 5-times the maximal rates of bile acid glucosidation estimated with microsomes from liver. In addition to bile acid glucoside formation, microsomes from human liver, kidney and intestinal mucosa catalyzed the synthesis of dolichyl phosphoglucose acting as natural glucosyl donor in bile acid glucosidation.

Bile Acids and Salts↗

PubMed1991

Rapid kidney changes resulting from glycosphingolipid depletion by treatment with a glucosyltransferase inhibitor.

The ceramide analog, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, inhibits the glucosylation of ceramide and thus, by virtue of the normal catabolism of the higher glucosphingolipids, leads to a general depletion of cellular glucolipids. In a previous study with chronic administration of this inhibitor in mice, it was found that the kidneys and liver, particularly the former, grew more poorly than the organs of control mice. This study shows that the inhibitor produces rapid decreases in glucolipid concentration in kidney which are maintained for at least 5 days without noticeable harm. The changes were enhanced by inclusion of L-cycloserine in the injection scheme. Cycloserine blocks ketosphinganine synthase and thus slows the synthesis of all sphingolipids. However, sphingomyelin levels did not drop significantly in this study. The glucosyltransferase inhibitor also produced a small decrease in kidney beta-D-glucuronidase and distinct increases in the levels of glucocerebrosidase, galactocerebrosidase and sphingomyelinase. It also produced a small but distinct decrease in the level of glucosyltransferase, after a delay of a few hours, possibly because the inhibitor was metabolized to a covalently inactivating product. Comparison with kidney, liver and brain showed that the kidney was more sensitive to the action of the morpholino inhibitor.

Animals↗

PubMed1988

A novel glucosyltransferase from Streptococcus mutans produces oligo-isomaltosaccharides.

Streptococcus mutans secretes a sucrose-independent branalphang enzyme that utilizes isomaltosaccharides as donors for branalphang formation on dextran. Although the branching enzyme is necessary for the formation of extracellular polysaccharide complexes, the source of the donor for the enzyme is unknown. In this study, we purified a novel glucosyltransferase from S. mutans and characterized its properties. The glucosyltransferase was primer independent 1,6-alpha-D-glucan synthase, which produced oligo-isomaltosaccharides. The enzyme was thought to be a source of donor for the branching enzyme in S. mutans.

Disaccharides↗

PubMed1983

Three kinds of extracellular glucosyltransferases from Streptococcus mutans 6715 (serotype g).

In addition to the 1,3-alpha-D-glucan synthetase (pI 4.9) and the highly-branched 1,6-alpha-D-glucan synthetase (pI 3.9-4.1), Streptococcus mutans 6715 (serotype g) was found to secrete the third glucosyltransferase in multiple forms (pI 5.5-7.0), which exhibited 87% 1,6-alpha-bond-, 6% 1,3-alpha-bond- and 7% 1,3,6-branch-forming activities. The production of this enzyme was extremely enhanced when the organism was grown in Tween 80-supplemented medium. The 3 glucosyltransferases from the same organism were enzymatically and immunologically distinct from each other, and they were commonly found among the serotype g strains.

Chemical Phenomena↗