Gas-liquid chromatography of the heptafluorobutyrate derivatives of the O-methyl-glycosides on capillary columns: a method for the quantitative determination of the monosaccharide composition of glycoproteins and glycolipids.
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We have developed a method involving the formation of hepta-fluorobutyrate derivatives of O-methyl-glycosides liberated from glycoproteins and glycolipids following methanolysis. The stable derivatives of the most common monosaccharides of these glycoconjugates (Ara, Rha, Xyl, Fuc, Gal, Man, Glc, GlcNAc, GalNAc, Neu5Ac, KDN) can be separated and quantitatively and reproducibly determined with a high degree of sensitivity level (down to 25 pmol) in the presence of lysine as an internal standard. The GlcNAc residue bound to Asn in N-glycans is quantitatively recovered as two peaks. The latter were easily distinguished from the other GlcNAc residues of N-glycans, thus allowing a considerable improvement of the data on structure of N-glycans obtained from a single carbohydrate analysis. The most common contaminants present in buffers commonly used for the isolation of soluble or membrane-bound glycoproteins (SDS, Triton X-100, DOC, TRIS, glycine, and polyacrylamide or salts, as well as monosaccharide constituents of proteoglycans or degradation products of nucleic acids) do not interfere with these determinations. A carbohydrate analysis of glycoproteins isolated from a SDS/PAGE gel or from PDVF membranes can be performed on microgram amounts without significant interferences. Since fatty acid methyl esters and sphingosine derivatives are separated from the monosaccharide peaks, the complete composition of gangliosides can be achieved in a single step starting from less than 1 microg of the initial compound purified by preparative Silicagel TLC. Using electron impact ionization mass spectrometry, reporter ions for the different classes of O-methyl-glycosides (pentoses, deoxy-hexoses, hexoses, hexosamines, uronic acids, sialic acid, and KDN) allow the identification of these compounds in very complex mixtures. The mass of each compound can be determined in the chemical ionization mode and detection of positive or negative ions. This method presents a considerable improvement compared to those using TMS derivatives. Indeed the heptafluorobutyrate derivatives are stable, and acylation of amino groups is complete. Moreover, there is no interference with contaminants and the separation between fatty acid methyl-esters and O-methyl glycosides is achieved. (+info)
The relationship between the uptake of glucose and 3-O-methylglucose and soluble carbohydrate and polysaccharide in the fungus Dendryphiella salina.
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When mycelium of Dendryphiella salina, pre-incubated in D-[I-14C]mannitol such that this is the only major labelled soluble carbohydrate present, absorbs glucose or the non-metabolized sugar 3-O-methylglucose, there is a specific stimulation of incorporation of 14C into alkali-insoluble, trichloroacetic-acid-soluble (1 leads to 4)-alpha-glucan, probably glycogen. There is also a net increase in the amount of glucan caused by stimulation of synthesis and inhibition of breakdown. Addition of 3-O-methylglucose results in the loss of mannitol into the medium. This loss and the reduced rate of replenishment from the glucan is important in the osmotic regulation of the hyphae. If osmotic adjustment does not occur, the hyphae do not show the specific incorporation of 14C into the glucan. (+info)
Magnetic resonance studies of concanavalin A: location of the binding site of alpha-methyl-D-mannopyranoside.
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The longitudinal nuclear magnetic relaxation times of the methyl protons of alpha-methyl-D-mannopyranoside have been measured at 90 MHz and 270 MHz in solutions of concanavalin A complexed with: (i) Mn2+ and Ca2+; and (ii) Zn2+ and Ca2+. Zn2+ and Mn2+ are known to bind in site S1 and Ca2+ in site S2 of concanavalin A. Both sites must be occupied before monosaccharides will bind to the protein. In order to extract T1p, the paramagnetic contribution to the bound methyl relaxation time, from these observations the relaxation time of uncomplexed sugar was determined. Free Mn2+ contributed insignificantly to the latter value; however, outer sphere relaxation was found to be large at bound Mn2+ concentrations greater than 1 mM. Comparison of the results obtained at the two frequencies allowed the determination of the correlation time for the interaction between the methyl protons and the bound Mn2+ and the distance between them (21.5 +/- 1.2 A). In contrast to previous results from nuclear magnetic resonance studies, this distance is consistent with binding at the cavity proposed for the saccharide binding site by Becker et al. [J. Biol. Chem. 250, 1513 (1975)], although it does not preclude possible binding sites on the surface of the molecule. (+info)
Interaction of the lacZ beta-galactosidase of Escherichia coli with some beta-D-galactopyranoside competitive inhibitors.
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1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501. (+info)
Acetylated methylmannose polysaccharide of Streptomyces griseus. Locations of the acetyl groups.
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The positions of esterification of the 4 to 5 acetyl residues in the acetylated methylmannose-containing polysaccharide from Streptomyces griseus have been established by the methyl replacement technique, wherein ester substituents are specifically replaced with methyl ether substituents. The newly incorporated methyl groups were distinguished from 3-O-methyl groups by the use of polysaccharide containing radioactively labeled endogenous methyl groups. The positions of methyl group localization were established by a proton magnetic resonance study of the intact methyl-replaced polysaccharide combined with an analysis of the constituent monosaccharides by gas-liquid chromatography-electron impact mass spectrometry of their alditol acetate derivatives. These studies demonstrate that the acetyl groups are located at position 6 of approximately half of the 10 contiguous alpha(1 leads to 4)-linked 3-O-methyl-D-mannose residues. Purification of the polysaccharide was accomplished by an added step involving affinity chromatography on a column containing immobilized palmitoyl residues. The affinity of the polysaccharide for this long chain lipid suggests that its plays a role similar to the methylmannose-containing polysaccharide of Mycobacterium smegmatis in its regulation of the bacterium's fatty acid synthetase. (+info)
Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose.
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The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process. (+info)
Evidence for specific and non-covalent binding of lipids to natural and recombinant Mycobacterium bovis BCG hsp60 proteins, and to the Escherichia coli homologue GroEL.
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Heat-shock proteins (Hsps) from various origins are known to share a conserved structure and are assumed to be key partners in the biogenesis of proteins. Fractionation of the mycobacterial Hsp60, a 65 kDa protein also called Cpn60, from Mycobacterium bovis BCG zinc-deficient culture filtrate on phenyl-Sepharose followed by Western blotting revealed the existence of four Hsp60-1 and Hsp60-2 forms, based on their hydrophobicity behaviour. Hsp60-2 species were further purified by ion-exchange chromatography and partial amino acid sequences of cyanogen bromide (CNBr) peptides of purified Hsp60-2 species showed identity with the amino acid sequence deduced from the hsp60-2 gene, indicating that the various Hsp60-2 forms are encoded by the same gene. In addition, the mycobacterial Hsp60-2 was overexpressed in E. coli using the pRR3Hsp60-2 plasmid and analysed on phenyl-Sepharose. The elution pattern of the recombinant Hsp60-2, as well as that of Escherichia coli GroEL, was similar to that of the native Hsp60-2 from the culture filtrate of M. bovis BCG and entirely different from that of the mycobacterial antigen 85. Extraction of mycobacterial Hsp60-2 forms, recombinant BCG Hsp60-2 and E. coli GroEL with organic solvents releases various amounts of non-covalently bound lipids. The presence of lipids on Hsp60-2 was confirmed by labelling M. bovis BCG with radioactive palmitate. The radioactivity was specifically associated with Hsp60 in the aqueous phase and the 19 and 38 kDa lipoproteins in the Triton X-114 phase. Analysis of the lipids extracted from purified Hsp60-2, recombinant BCG Hsp60-2 and E. coli GroEL by TLC showed the same pattern for all the samples. Acid methanolysis of the lipids followed by GC analysis led to the identification of C(16:0), C(18:0) and C(18:1) as the major fatty acyl constituents, and of methylglycoside in these proteins. Altogether, these data demonstrate that lipids are non-covalently bound to Hsp60-2 and homologous proteins. (+info)
Effects of colicin Ia on transport and respiration in Escherichia coli.
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Treatment of Escherichia coli with colicin Ia leads to an inhibition in the active transport of exogenously supplied proline, thiomethyl-beta-D-galactoside and potassium ion. Furthermore, the addition of colicin Ia to cells preloaded with these substances leads to their almost immediate efflux. In contrast, colicin tia treatment enhances by as much as 10-fold the level of accumulation of alpha-methyl-D-glucoside. The colicin Ia-induced stimulation of glucoside accumulation is mediated by the phosphotransferase system. Cells treated with colicin Ia exhibit an increased rate of respiration when glucose is the substrate and a decreased rate when glycerol or succinate is the substrate; The decreased rate of succinate-dependent respiration is probably due to the failure of Ia-treated cells to accumulate succinate. (+info)