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Biomedical subjects

R M Mayer

Publications and source records attributed to R M Mayer.

At least 19 recordsLinked to original sources
PubMed1993

Formation of alpha-(1-->6), alpha-(1-->3), and alpha-(1-->2) glycosidic linkages by dextransucrase from Streptococcus sanguis in acceptor-dependent reactions.

Dextransucrase from Streptococcus sanguis 10558 was found to synthesize alpha-(1-->6), alpha-(1-->3), and alpha-(1-->2) linkages during an acceptor-dependent glucosyl transfer reaction. Normally, new glucosyl residues are added at C-6 of monosaccharide acceptors. However, sugars blocked at C-6 also can serve as good acceptors. The disaccharide and trisaccharide products formed when methyl 6-bromo-6-deoxy-alpha-D-glucopyranoside was used as acceptor were isolated and characterized. Both were found to contain only alpha-(1-->3) glycosidic bonds. This supports the hypothesis that when C-6 is blocked the acceptor binds to the enzyme in a flipped orientation, resulting in an approximate exchange in space of the C-3 and C-6, thereby putting C-3 adjacent to the active site. The second alpha-(1-->3) links in the trisaccharide are formed by a single-chain mechanism without release of the intermediate disaccharide. With maltose as acceptor, new glucosyl residues are added at C-6'. However, if that position is blocked with a bromine atom, the resulting compound, 6'-bromo-6'-deoxy-maltose, can still serve as an acceptor. The product in this case was isolated and characterized. The new glycosidic link was found to be alpha-(1-->2).

Carbohydrate Conformation↗
PubMed1992

Structural determination of alginic acid and the effects of calcium binding as determined by high-field n.m.r.

The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.

Alginates↗
PubMed1992

Antileishmanial drug targeting through glycosylated polymers specifically internalized by macrophage membrane lectins.

Antileishmanial chemotherapy is hampered by the location of the parasite within the phagolysosome of the macrophage, which restricts the bioavailability of many potentially useful antileishmanial drugs. In this study, the possibility of using antileishmanial drugs targeted to the infected macrophages by means of a chemical linkage to a neutral mannose-substituted poly-L-lysine carrier molecule was explored. The study was performed in an in vitro model with Leishmania donovani-infected murine macrophages. The antileishmanial activities of various synthetic constructs were compared with those of the free drugs and the pentavalent antimonial Pentostam, which was used as the positive control. The 50% effective dose of allopurinol riboside linked to the mannosylated poly-L-lysine was below 7.5 x 10(-6) M, while it was up to 3 x 10(-4) M for the free drug, indicating that the drug bound to the polymer was 50 times more active than the free drug. Control experiments with other constructs (e.g., allopurinol riboside linked to the mannose-free polymer) confirmed that the enhancement of activity was indeed achieved by means of the mannose homing device.

Animals↗
PubMed1991

Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl alpha-D-glucopyranoside.

Dextransucrase from Streptococcus sanguis ATCC 10558 was photolabeled using p-azidophenyl alpha-D-glucopyranoside with an apparent rate constant of inactivation of 1.40 min-1. The dissociation constant for this compound, which acts as an acceptor molecule in the enzymatic reaction, is 90 microM. Apparently two acceptor binding sites exist on dextransucrase as shown by (i.) photolabeling the enzyme with p-azidophenyl-alpha-D-[5,6-3H]glucopyranoside and (ii.) fluorescence titration experiments.

Affinity Labels↗
PubMed1990

Stereochemistry and mechanism of the GDP-mannose dehydratase reaction.

The reaction catalyzed by bacterial GDP-mannose dehydratase (E.C. 4.2.1.47), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose (GDP-6-deoxy-D-lyxo-hexos-4-ulose), was studied with (6R)- and (6S)-GDP-D-[4-2H1,6-3H]mannose. Conversion of these stereospecifically labeled substrates in the presence of excess unlabeled GDP-mannose into the 4-keto-6-deoxy derivatives followed by Kuhn-Roth oxidation gave acetic acid samples which were subjected to configurational analysis of the isotopically chiral methyl group. The observed F values of 64 for the material from the (6S) substrate and 31 for that from the (6R) isomer, corresponding to 48% e.e. R and 66% e.e. S configuration, respectively, of the methyl group indicate that (a) the oxidoreductase reaction involves transfer of H-4 to C-6, (b) the transfer is predominantly intramolecular, and (c) the transfer is stereospecific, H-4 replacing the C-6 hydroxyl group with inversion of configuration. A mechanism for the reaction is proposed on the basis of these results.

Carbohydrate Conformation↗
PubMed1986

Dynamic reactivities of dextransucrase.

Dextransucrase, from Streptococcus sanguis ATCC 10558, was immobilized on hydroxylapatite and was "charged" in short pulses with labeled sucrose, as previously described [V. K. Parnaik, G. A. Luzio, D. A. Grahame, S. L. Ditson, and R. M. Mayer (1983) Carbohydr. Res. 121, 257-268]. The "charged" enzyme has been shown to contain both bound glucose and gluco-oligosaccharides. The reactivity of this form of the enzyme has been studied, and shown to have unexpected behavior. Earlier pulse-chase experiments [J. F. Robyt, B. K. Kimble, and T. F. Walseth (1979) Arch. Biochem. Biophys. 165, 634-640; S. L. Ditson and R. M. Mayer (1984) Carbohydr. Res. 126, 170-175], carried out with high concentrations of unlabeled sucrose in the chase, resulted in a rapid decrease in isotope at the reducing termini of enzyme-bound oligosaccharides. However, in the present work, in which the pulsed enzyme was chased with low concentrations of unlabeled sucrose, we observed an increase in the radioactive reducing termini. The possibility that this was due to the enzymatic hydrolysis of dextran has been ruled out. Data presented demonstrate that the enzyme catalyzes the depolymerization of the bound oligosaccharides. Individual glucosyl residues of the oligosaccharides are transferred to acceptors, such as added maltose to form a trisaccharide, or water to form glucose. Similarly, the glucosyl residues can be transferred to added fructose to form sucrose. The studies also provide evidence that the oligosaccharides are slowly released from the enzyme. The ability of the enzyme to catalyze the reverse of the glucosyl transfer reaction involving acceptors was also examined. It was observed that glucose residues transferred by dextransucrase to an acceptor can also be removed to produce sucrose when fructose is added.

Enzymes, Immobilized↗
PubMed1985

Interaction of deoxyhalosucrose derivatives with dextransucrase.

Members of a series of deoxyhalosucrose analogs substituted at one, two, or three primary carbon atoms with bromine or chlorine were prepared. Dextransucrase isolated from Streptococcus sanguis was separately treated with 6-bromo-6-deoxysucrose, 6,6'-dibromo-6,6'-dideoxysucrose, 6,1',6'-tribromotrideoxysucrose, and 6,6'-dichlorodideoxysucrose, in order to determine if they were inactivators. Variation in time of exposure, and in the concentration of the sucrose analogs, did not yield significant irreversible inactivation. In supplementary studies, it was found that the compounds serve as weak, reversible inhibitors.

Glucosyltransferases↗
PubMed1985

Purification, and comparison, of two forms of dextransucrase from Streptococcus sanguis.

A procedure has been developed whereby native and proteolyzed forms of dextransucrase have been purified; it involves gel filtration, and hydroxylapatite chromatography in the presence of 0.10% sodium dodecyl sulfate. This procedure is highly reproducible, and permits approximately 30% recovery of high purity (94% homogeneous) enzyme as an inactive, SDS complex that can be reactivated by the addition of Triton X-100. The purified enzymes have been compared with regard to amino acid compositions, and isoelectric and catalytic properties. An analysis of the structure of their product D-glucans was also made. Although the structural characteristics of the enzyme forms differ, proteolysis does not cause alterations in their catalytic properties.

Amino Acids↗
PubMed1985

Intestinal mucins and cholesterol uptake in vitro.

A mucus material, secreted by intestinal segments, with a high affinity for cholesterol, has been isolated and chemically characterized. The mucin contained 11% carbohydrate, largely as glucosamine, galactose and N-acetylneuraminic acid, and 19% lipid, of which 86% was unesterified fatty acid. The isolated material readily bound cholesterol in a stoichiometric manner. Conditions known to enhance cholesterol absorption in vivo also decreased mucin complexing to cholesterol in vitro. This association of cholesterol and intestinal surface mucin also occurred during incubations of intestinal segments with dispersed cholesterol, resulting in a high level of intestinal adsorption, with little or no cellular absorption of the sterol. However, when cholesterol was solubilized in simple or complex micelles containing bile salts, surface adsorption of cholesterol was reduced and net absorption was increased. The results suggest that surface mucin binding of cholesterol may represent at least one major diffusion limitation to cholesterol absorption in the intestine.

Animals↗
PubMed1984

The origin and composition of multiple forms of dextransucrase from Streptococcus sanguis.

Multiple forms of purified dextransucrase have been observed in the presence of low detergent concentrations ( Luzio , G.A., Grahame , D. A. and Mayer, R.M. (1982) Arch. Biochem. Biophys. 216, 751-757). We now show these forms to arise partly as a result of proteolysis, and partly due to incomplete dissociation of the enzyme. Upon 25 degrees C incubation of the crude enzyme, several new bands appeared with little or no change in total activity. The electrophoretic pattern of aged, crude enzyme was similar to that of partially purified enzyme. Specific detection of dextransucrase on SDS gels revealed a single polypeptide of 174 kDa, which is converted to a 156 kDa protein during the aging process. The observation indicates the occurrence of proteolysis. The polypeptide composition of several of the enzyme forms was determined by two-dimensional electrophoresis. Forms Ia and IIa are composed exclusively of 174 kDa polypeptides. Forms III and IVa consist of 156 kDa units, as does the newly observed form Ic. It is likely that form Ib contains both 174 and 156 kDa polypeptides. The results indicate that incomplete dissociation of aggregates of the 174 kDa unit accounts for all of the bands observed on native gels run on fresh culture extracts. Additional enzyme forms result from aggregation of the 156 kDa proteolysis product alone, and from aggregation with unproteolyzed units to form hybrid aggregates.

Glucosyltransferases↗
PubMed1983

A D-glucosylated form of dextransucrase: preparation and characteristics.

Dextransucrase was treated with [14C]sucrose, and the product applied to gel-permeation columns. In the absence of the detergents SDS and Triton X-100, poor recovery of enzyme was observed; however, that enzyme which was recovered was labeled. In the presence of detergents, recovery was increased, but the material appeared to be a large aggregate (mol. wt. greater than 5 X 10(6) ). In addition, the ratio of D-glucose to enzyme suggested that a polymer had been formed. Disc-gel electrophoresis in the presence of a mixture of SDS and Triton X-100 showed similar results, and indicated that the aggregate was disrupted upon treatment with dextranase. Native enzyme that had been immobilized on hydroxylapatite could also be labeled with [14C]sucrose, and the labeling followed saturation kinetics. The labeled protein could be released from the gel with 8M urea, but was aggregated. Radioactive sugars, free from protein, could be released by heating the labeled enzyme. The sugars released consisted of a mixture of D-glucose with oligosaccharides having an average chain-length of 17 D-glucosyl residues. The significance of these observations is discussed.

Chromatography, Gel↗
PubMed1983

A D-glucosylated form of dextransucrase: demonstration of partial reactions.

A D-glucosylated form of dextransucrase, whose preparation and characteristics have just been reported in Carbohydr. Res., was employed in a series of studies designed to explore the question of whether the bound sugars participate in the reactions catalyzed by the enzyme. When exposed to maltose, a good acceptor-substrate, monomeric D-glucosyl groups were rapidly transferred to the disaccharide, affording a trisaccharide. In the absence of an acceptor, monomeric D-glucose was released from the enzyme by hydrolysis. In a reaction with D-fructose, the charged enzyme catalyzed the formation of sucrose. Finally, in the presence of unlabeled sucrose, monomeric D-glucosyl groups were chased into enzyme-associated oligomers. Evidence is also presented which indicates that the various pathways for the bound D-glucosyl groups are competitive. The significance of these observations is discussed.

Glucose↗