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PubMed1995

The molecular basis for the recognition of misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase.

The UDP-Glc:glycoprotein glucosyltransferase is a soluble enzyme of the endoplasmic reticulum that glucosylates protein-linked Man7-9GlcNAc2 to form the monoglucosylated derivatives. In vivo the reaction products are immediately deglucosylated by glucosidase II. The glucosyltransferase has a unique property: it glucosylates misfolded, but not native, glycoproteins. It has been proposed that the glucosyltransferase participates, together with calnexin, in the control mechanism by which only properly folded glycoproteins can exit from the endoplasmic reticulum. In this paper it is demonstrated that the glucosyltransferase recognizes two elements in the acceptor substrates: the innermost N-acetylglucosamine unit of the oligosaccharide and protein domains exposed in denatured, but not in native, conformations. Both determinants have to be covalently linked. In many cases the first element is not accessible to macromolecular probes in native conformations. Concerning the protein domains, it is demonstrated here that the glucosyltransferase interacts with hydrophobic amino acids exposed in denatured conformations. More disordered conformations, i.e. those exposing more hydrophobic amino acids, were found to be those having higher glucose acceptor capacity. It is suggested that both accessibility of the innermost N-acetylglucosamine unit and binding to hydrophobic patches determine the exclusive glucosylation of misfolded conformations by the glucosyltransferase.

Acetylglucosamine↗

PubMed1994

The subcellular localization of a bile acid glucosyltransferase in rat liver.

Formation of bile acid glucosides occurs in rat liver homogenate with a specific enzyme activity of 0.014 +/- 0.001 nmol per min per mg protein. Subcellular fractionation of rat liver by differential centrifugation revealed an enrichment of bile acid glucosyltransferase activity both in the mitochondrial-lysosomal fraction and in microsomes with a recovery of 38.8 +/- 4.6% and 37.7 +/- 1.7%, respectively, of enzyme activity in the homogenate. Subfractionation of the mitochondrial-lysosomal fraction after treatment of rats with Triton WR 1339 showed an almost exclusive association of bile acid glucosyltransferase activity with purified lysosomes ("tritosomes"). After subfractionation of microsomes by analytical gradients, bile acid glucosyltransferase was bimodally distributed with peaks at modal densities of 1.09 g/cm3 and 1.16 g/cm3, respectively. If microsomes were pretreated with pyrophosphate, a membrane perturbant known to strip ribosomes, only the peak of bile acid glucosyltransferase at higher density (1.16 g/cm3) and UDP-glucuronosyltransferase (marker of endoplasmic reticulum) shifted to a similar lower equilibrium density. Both enzymes were unaffected in their distribution by pretreatment of microsomes with digitonin. In contrast, markers of plasma membranes (5'-nucleotidase) and the Golgi-complex (galactosyltransferase) shifted to higher equilibrium densities after digitonin treatment, but were unaltered in their distribution after pyrophosphate. Bile acid glucosyltransferase activity in the lower density range with a peak at 1.09 g/cm3 did not show any association with the density distributions of known marker enzymes. In purified microsomal fractions obtained by preparative gradients, bile acid glucosyltransferase activity was enriched in enzyme activity by 1.4-fold in rough and by 2.3-fold in smooth endoplasmic reticulum, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1999

The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor. Isolation, cloning, heterologous expression, and substrate specificity.

The final step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor is the transformation of the labile cyanohydrin into a stable storage form by O-glucosylation of (S)-p-hydroxymandelonitrile at the cyanohydrin function. The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase was isolated from etiolated seedlings of S. bicolor employing Reactive Yellow 3 chromatography with UDP-glucose elution as the critical step. Amino acid sequencing allowed the cloning of a full-length cDNA encoding the glucosyltransferase. Among the few characterized glucosyltransferases, the deduced translation product showed highest overall identity to Zea mays flavonoid-glucosyltransferase (Bz-Mc-2 allele). The substrate specificity of the enzyme was established using isolated recombinant protein. Compared with endogenous p-hydroxymandelonitrile, mandelonitrile, benzyl alcohol, and benzoic acid were utilized at maximum rates of 78, 13, and 4%, respectively. Surprisingly, the monoterpenoid geraniol was glucosylated at a maximum rate of 11% compared with p-hydroxymandelonitrile. The picture that is emerging regarding plant glucosyltransferase substrate specificity is one of limited but extended plasticity toward metabolites of related structure. This in turn ensures that a relatively high, but finite, number of glucosyltransferases can give rise to the large number of glucosides found in plants.

Amino Acid Sequence↗

PubMed1996

Evaluation of three individual glucosyltransferases produced by Streptococcus mutans using monoclonal antibodies.

We previously established murine hybridomas producing a monoclonal antibody monospecific against three glucosyltransferases (I, SI and S) of Streptococcus mutans which contribute to dental caries formation. Here, we developed a new immunochemical technique (cross-dot system) with which individual levels of glucosyltransferases expressed by S. mutans can be evaluated. We also examined glucosyltransferase production and in vitro artificial plaque formation by a reference strain and several clinical isolates of S. mutans. The findings indicate that the levels of glucosyltransferases produced greatly vary with the cells and the culture medium, and that the cells producing high levels of both glucosyltransferase-SI and glucosyltransferase-I enzymes may possess high in vitro artificial plaque forming ability. We suggest that the cross-dot system will be useful for estimating the cariogenic potential of S. mutans isolates.

Antibodies, Monoclonal↗

PubMed1977

Effect of Tween 80 on glucosyltransferase production in Streptococcus mutans.

Glucan production from sucrose by Streptococcus mutans OMZ 176 was stimulated approximately threefold in the presence of 0.1% Tween 80. When OMZ 176 was grown in a medium containing glucose, the glucosyltransferase level in the medium was also increased about fivefold in the presence of 0.1% Tween 80. The glucosyltransferase level increased in proportion to the logarithm of the concentration of Tween 80 in the glucose medium. Tween 80 affected neither bacterial growth nor the activity of glucosyltransferase. The appearance of glucosyltransferase in the glucose medium was inhibited immediately by chloramphenicol and actinomycin D and, after a lag, by rifampin as well. It was observed that the fatty acid composition of the cells grown with Tween 80 was altered. These results suggest that Tween 80 stimulates glucosyltransferase synthesis either directly, or indirectly by promoting glucosyltransferase secretion.

Chloramphenicol↗

PubMed2003

Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila.

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.

Amino Acid Sequence↗

PubMedDate not supplied

Occurrence and characterization of a UDP-glucose:hydroxamic acid glucosyltransferase isolated from wheat (Triticum aestivum) seedlings.

Cyclic hydroxamic acid glucosides are present at high concentrations immediately after germination in wheat (Triticum aestivum L.). Changes in the activity of UDP-Glucose:cyclic hydroxamic acid glucosyltransferase (EC 2.4.1.-) in wheat were investigated using the cyclic hydroxamic acids 2.4-dihydroxy-1,4-benzoxazin-3-one (DIBOA) and its 7-methoxy derivative (DIMBOA) as sugar acceptors. Glucosyltransferase activity on both substrates was detected in dry seeds, with activity increasing after imbibition, peaking in shoots and roots 36-48 hours after imbibition and decreasing thereafter. The transience of glucosyltransferase activity was concurrent with the transient occurrence of the hydroxamic acid glucosides [Nakagawa E., Amano T., Hirai N., and Iwamura H. (1995) Phytochemistry 38, 1349-1354], suggesting that glucosyltransferases regulate the accumulation of hydroxamic acid glucosides in wheat seedlings. Two peaks in activity of UDP-Glucose:DIMBOA glucosyltransferase were detected using a Mono Q column, indicating the presence of at least two isozymes of this glucosyltransferase. The enzyme in the major peak was purified about 1500-fold and shown to be in a monomeric form with a molecular mass of 47 or 49 kDa. The enzyme reacted strongly with DIMBOA, less so with DIBOA. The enzyme of the minor peak on the Mono Q chromatogram, which was also a monomeric enzyme with a molecular mass of 47 kDa, showed similar substrate specificity to that of the major peak enzyme.

Chromatography, Affinity↗

PubMed1997

Regulation of Triacylglucose Fatty Acid Composition (Uridine Diphosphate Glucose:Fatty Acid Glucosyltransferases with Overlapping Chain-Length Specificity).

UDP-glucose (UDP-Glc):fatty acid glucosyltransferases catalyze the UDP-Glc-dependent activation of fatty acids as 1-O-acyl-[beta]-glucoses. 1-O-Acyl-[beta]-glucoses act as acyl donors in the biosynthesis of 2,3,4-tri-O-acylglucoses secreted by wild tomato (Lycopersicon pennellii) glandular trichomes. The acyl composition of L. pennellii 2,3,4-tri-O-acylglucoses is dominated by branched short-chain acids (4:0 and 5:0; approximately 65%) and straight and branched medium-chain-length fatty acids (10:0 and 12:0; approximately 35%). Two operationally soluble UDP-Glc:fatty acid glucosyltransferases (I and II) were separated and partially purified from L. pennellii (LA1376) leaves by polyethylene glycol precipitation followed by DEAE-Sepharose and Cibacron Blue 3GA-agarose chromatography. Whereas both transferases possessed similar affinity for UDP-Glc, glucosyltransferase I showed higher specificity toward short-chain fatty acids (4:0) and glucosyltransferase II showed higher specificity toward medium-chain fatty acids (8:0 and 12:0). The overlapping specificity of UDP-Glc:fatty acid glucosyltransferases for 4:0 to 12:0 fatty acid chain lengths suggests that the mechanism of 6:0 to 9:0 exclusion from acyl substituents of 2,3,4-tri-O-acylglucoses is unlikely to be controlled at the level of fatty acid activation. UDP-Glc:fatty acid glucosyltransferases are also present in cultivated tomato (Lycopersicon esculentum), and activities toward 4:0, 8:0, and 12:0 fatty acids do not appear to be primarily epidermal when assayed in interspecific periclinal chimeras.

Journal Article↗

PubMed1981

Inactivation of D-glucosyltransferases from oral Streptococcus mutans and Streptococcus sanguis by photochemical oxidation.

Cell-free D-glucosyltransferase of D-glucose-grown Streptococcus mutans AHT was completely inactivated in the presence of 0.002% of Methylene Blue at 25 degrees and pH 7.0 after illumination with a 150-W incandescent lamp. The rate of inactivation was decreased at pH values less than 7.0. Histidine was the only amino acid residue modified to a significant extent, and the rates of oxidation of histidine residues and loss of enzyme activity closely agreed. Production of both water-insoluble and -soluble D-glucan fractions from sucrose by the oxidized D-glucosyltransferase preparations was significantly inhibited. Photooxidation with 0.002% of Rose Bengal at pH 7.0 or higher also induced complete inactivation of the D-glucosyltransferase. These results strongly suggest that the imidazole portion of histidine may function as part of the active sites of both D-glucosyltransferase isozymes of S. mutans AHT, which are responsible for the synthesis of (1 goes to 3)- and (1 goes to 6)-alpha-D-glucosidic linkages. The D-glucosyltransferases from S. mutans 6715 and AHT-mutant M1, and Streptococcus sanguis ATCC 10558 were also almost completely inactivated by Methylene Blue-sensitized photooxidation.

Amino Acids↗

PubMed1978

Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings.

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

Acid Anhydride Hydrolases↗

PubMed1998

Molecular cloning and expression of mouse ceramide glucosyltransferase.

Ceramide glucosyltransferase (EC 2.4.1.80) catalyzes the first glycosylation step of glycosphingolipid (GSL) synthesis, the transfer of glucose from UDP-Glucose to hydrophobic ceramide and generate glucosylceramide (GlcCer). We have cloned mouse ceramide glucosyltransferase cDNA from a brain cDNA library by PCR based homology cloning. The nucleotide sequence determination revealed that mouse ceramide glucosyltransferase cDNA encodes 394 amino acids with a calculated molecular mass of 45 kDa. The amino acid sequence of mouse ceramide glucosyltransferase showed 98% identity with the human sequence. Homology searches against currently available databases identified three homologous proteins in Caenorhabditis elegans and one homologous protein in Cyanobacteria. Highly conserved sequences of ceramide glucosyltransferases and the homologs among a wide variety of organisms suggest biological significance of the lipid glucosylation system.

Amino Acid Sequence↗

PubMed2003

An amino acid change near the carboxyl terminus of the Streptococcus gordonii regulatory protein Rgg affects its abilities to bind DNA and influence expression of the glucosyltransferase gene gtfG.

The Streptococcus gordonii glucosyltransferase structural gene, gtfG, is located immediately downstream from its positive transcriptional regulatory determinant, rgg. Recent genetic studies have indicated that the 3' end of rgg is involved either directly as a binding site or indirectly, e.g. by playing a role in secondary structure, in the interaction of Rgg with the gtfG promoter. A previously identified spontaneous mutant with a point mutation near the 3' end of rgg had only approximately 25% of the parental level of glucosyltransferase activity. To determine if this decreased activity was due to a change in the DNA binding site of trans-acting Rgg, or due to a change in the Rgg protein itself, complementation analyses and DNA-binding studies were performed. In Rgg-deficient strains, the chromosomal rgg point mutation did not influence the ability of plasmid-borne rgg to increase glucosyltransferase expression. However, plasmids carrying parental rgg were able to increase glucosyltransferase activity and expression of a gtfG promoter fusion to a greater extent than plasmids carrying the mutant allele, indicating that the mutant Rgg protein had decreased activity. The ability of NH(2)-terminal (hexahistidine) tagged proteins to bind to a 107 bp dsDNA fragment corresponding to the region immediately upstream of gtfG was demonstrated by surface plasmon resonance. Despite their differences in activity, both mutant and parental recombinant Rgg proteins bound to this dsDNA, albeit with different strengths. These studies provide insights into functional domains of S. gordonii Rgg which influence glucosyltransferase expression, and may have implications for Rgg-like regulatory proteins in related bacteria.

Amino Acids↗

PubMed2003

Biochemical and molecular characterization of a novel UDP-glucose:anthocyanin 3'-O-glucosyltransferase, a key enzyme for blue anthocyanin biosynthesis, from gentian.

Gentian (Gentiana triflora) blue petals predominantly contain an unusually blue and stable anthocyanin, delphinidin 3-O-glucosyl-5-O-(6-O-caffeoyl-glucosyl)-3'-O-(6-O-caffeoyl-glucoside) (gentiodelphin). Glucosylation and the subsequent acylation of the 3'-hydroxy group of the B-ring of anthocyanins are important to the stabilization of and the imparting of bluer color to these anthocyanins. The enzymes and their genes involved in these modifications of the B-ring, however, have not been characterized, purified, or isolated to date. In this study, we purified a UDP-glucose (Glc):anthocyanin 3'-O-glucosyltransferase (3'GT) enzyme to homogeneity from gentian blue petals and isolated a cDNA encoding a 3'GT based on the internal amino acid sequences of the purified 3'GT. The deduced amino acid sequence indicates that 3'GT belongs to the same subfamily as a flavonoid 7-O-glucosyltransferase from Schutellaria baicalensis in the plant glucosyltransferase superfamily. Characterization of the enzymatic properties using the recombinant 3'GT protein revealed that, in contrast to most of flavonoid glucosyltransferases, it has strict substrate specificity: 3'GT specifically glucosylates the 3'-hydroxy group of delphinidin-type anthocyanins containing Glc groups at 3 and 5 positions. The enzyme specifically uses UDP-Glc as the sugar donor. The specificity was confirmed by expression of the 3'GT cDNA in transgenic petunia (Petunia hybrida). This is the first report of the gene isolation of a B-ring-specific glucosyltransferase of anthocyanins, which paves the way to modification of flower color by production of blue anthocyanins.

Amino Acid Sequence↗

PubMed1981

Serum galactosylhydroxylysyl glucosyltransferase in acute myocardial infarction and during subsequent collagen scar formation.

Changes in serum galactosylhydroxylysyl glucosyltransferase, an enzyme catalysing one of the intracellular post-translational modifications in collagen biosynthesis, were studied in twenty-four patients with acute myocardial infarction. The enzyme activity was monitored for 18 days from the onset of infarction, and at least a two-peaked pattern was observed. The first peak corresponded to the stage of acute myocardial injury, there being a highly significant correlation between the maximal values for serum glucosyltransferase and alpha-hydroxybutyrate dehydrogenase. An average decreasing in serum glucosyltransferase activity of 41%, was noted during the following 24 h. A new gradual rise in serum glucosyltransferase activity, interpreted as indicating myocardial collagen scar formation, was observed 5 days after the onset of infarction, when the serum enzyme activities indicating myocardial injury had already declined. The average daily values for serum glucosyltransferase between 6 and 18 days correlated highly significantly with the maximal value for serum alpha-hydroxybutyrate dehydrogenase, which serves as a relative estimate of the size of the original myocardial infarction area. The data further suggest that certain other factors including heart failure and/or various drug treatments may also affect the magnitude of this second peak.

Collagen↗

PubMed1997

The role of the Streptococcus mutans glucosyltransferases in the sucrose-dependent attachment to smooth surfaces: essential role of the GtfC enzyme.

Previous results have indicated that the glucosyltransferase activities of mutans streptococci are required for sucrose-dependent colonization of tooth surfaces. We have constructed mutants of Streptococcus mutans GS5 that are altered in varying combinations of the three gtf genes present in this organism. A quantitative in vitro sucrose-dependent attachment system was used to demonstrate that the inactivation of the gtfC gene drastically reduced adherence to smooth surfaces. By contrast, inactivation of the gtfB gene resulted in a smaller, but significant, reduction in attachment while the gtfD mutant was only marginally affected. Furthermore, production of only the glucosyltransferase C enzyme allowed for attachment although at reduced levels compared to the wild-type organism. The results from reintroduction of single copies of each of the gtf genes into a mutant of strain GS5 lacking glucosyltransferase activity also demonstrated the crucial role of the glucosyltransferase C enzyme in colonization. These results suggest a unique role for the glucosyltransferase C enzyme in the sucrose-dependent colonization of tooth surfaces by S. mutans strains.

Bacterial Adhesion↗

PubMed2002

Molecular characterization of a novel glucosyltransferase from Lactobacillus reuteri strain 121 synthesizing a unique, highly branched glucan with alpha-(1-->4) and alpha-(1-->6) glucosidic bonds.

Lactobacillus reuteri strain 121 produces a unique, highly branched, soluble glucan in which the majority of the linkages are of the alpha-(1-->4) glucosidic type. The glucan also contains alpha-(1-->6)-linked glucosyl units and 4,6-disubstituted alpha-glucosyl units at the branching points. Using degenerate primers, based on the amino acid sequences of conserved regions from known glucosyltransferase (gtf) genes from lactic acid bacteria, the L. reuteri strain 121 glucosyltransferase gene (gtfA) was isolated. The gtfA open reading frame (ORF) was 5,343 bp, and it encodes a protein of 1,781 amino acids with a deduced M(r) of 198,637. The deduced amino acid sequence of GTFA revealed clear similarities with other glucosyltransferases. GTFA has a relatively large variable N-terminal domain (702 amino acids) with five unique repeats and a relatively short C-terminal domain (267 amino acids). The gtfA gene was expressed in Escherichia coli, yielding an active GTFA enzyme. With respect to binding type and size distribution, the recombinant GTFA enzyme and the L. reuteri strain 121 culture supernatants synthesized identical glucan polymers. Furthermore, the deduced amino acid sequence of the gtfA ORF and the N-terminal amino acid sequence of the glucosyltransferase isolated from culture supernatants of L. reuteri strain 121 were the same. GTFA is thus responsible for the synthesis of the unique glucan polymer in L. reuteri strain 121. This is the first report on the molecular characterization of a glucosyltransferase from a Lactobacillus strain.

Amino Acid Sequence↗

PubMed1978

Cellular adherence, glucosyltransferase adsorption, and glucan synthesis of Streptococcus mutans AHT mutants.

Streptococcus mutans AHT mutants M1, M2, and M13 failed to adhere to a glass surface, whereas mutants M9 and M35 exhibited decreased and increased adherence, respectively, as compared with the parent strain, when grown in sucrose broth. Extracellular glucosyltransferase prepared from glucose-grown cultures of the adherent strains (wild type, M9, and M35) induced adherence of heat-killed cells of the homologous and heterologous streptococcal strains as well as of Escherichia coli K-12 and uncoated resin particles. The glucosyltransferase was adsorbed on all the streptococcal cells and glucan-coated resins, but not on E. coli cells and the uncoated resins. Glucosyltransferase from the nonadhering mutants (M1, M2, M13) neither was significantly adsorbed on nor induced adherence of any of the cells and resins. Cell-free enzymes from the glucose-grown adherent strains produced water-soluble and water-insoluble glucans, whereas those from the nonadhering mutants produced only water-soluble glucans. Small amounts of alkali-soluble, cell-associated glucan were recovered from the sucrose-grown nonadhering mutants. Thus, the relative proportions of glucosyltransferase isozymes elaborated by the S. mutans mutants, insofar as they affect the physico-chemical properties of the glucans produced, seem to determine the adherence abilities of the cells. The adsorption of glucosyltransferase on glucan molecules on the cell surface is not required for the adherence of S. mutans, but de novo glucan synthesis is important in the adherence process.

Adhesiveness↗

PubMed1979

Preparation of glucosyltransferase from Streptococcus mutans by elution from water-insoluble polysaccharide with a dissociating solvent.

Glucosyltransferase (EC 2.4.1.5) was obtained by dissociation from water-insoluble polysaccharide in the presence of 6 M guanidine-hydrochloride. Water-insoluble polysaccharide was synthesized by cell-free culture supernatants from Streptococcus mutans strain 6715. Gel filtration of the glucosyltransferase on a column of 8% agarose in phosphate buffer, followed by filtration on a column of 4% cross-linked agarose in 6 M guanidine-hydrochloride, gave a 23-fold enrichment of the enzyme. The enriched glucosyltransferase preparation contained 22% carbohydrate and eluted at a position corresponding to a molecular weight of 422,000. Polyacrylamide gel (5%) electrophoresis of this preparation revealed two regions which stained for protein, formed water-insoluble polysaccharide in the presence of sucrose, and precipitated with antisera directed to crude glucosyltransferase preparations. The guanidine-eluted enzyme could be primed by 5 X 10(-5) M dextran T10 (molecular weight, 10,000). High-molecular-weight glucan and a possible glucan-binding protein were also obtained after the final gel filtration step (4% cross-linked agarose) in addition to glucosyltransferase.

Chromatography, Gel↗