Biomedical subjects

C F Brewer

Publications and source records attributed to C F Brewer.

At least 55 records · Page 3Linked to original sources

PubMed1990

Binding and precipitating activities of Lotus tetragonolobus isolectins with L-fucosyl oligosaccharides. Formation of unique homogeneous cross-linked lattices observed by electron microscopy.

We have recently observed that certain asparagine-linked oligosaccharides are multivalent and capable of binding and precipitating with the D-mannose-specific lectin concanavalin A [cf. Bhattacharyya, L., & Brewer, C. F. (1989) Eur. J. Biochem. 178, 721-726] and with a variety of D-galactose-specific lectins [Bhattacharyya, L., Haraldsson, M., & Brewer, C. F. (1988) Biochemistry 27, 1034-1041]. In the present study, we have examined the binding and precipitating activities of a variety of mono- and biantennary L-fucosyl oligosaccharides with three L-fucose-specific isolectins from Lotus tetragonolobus, LTL-A, LTL-B, and LTL-C. The results show that certain difucosyl biantennary oligosaccharides are capable of cross-linking and precipitating with tetrameric isolectins, LTL-A and LTL-C, but not with dimeric isolectin, LTL-B. Quantitative precipitation analyses show that biantennary oligosaccharides containing the Lewis(x) antigen (or type 2 chain of Lewis(a)), Gal beta (1-4)[Fuc alpha (1-3)]GlcNAc, at the nonreducing terminus of each arm are bivalent ligands. However, a biantennary oligosaccharide containing a Lewis(x) determinant on one arm and a type 2 chain of blood group H(O) determinant, Fuc alpha (1-2)Gal beta (1-4)GlcNAc, on the other arm and a monoantennary oligosaccharide containing two fucose residues (analogue of the Lewis(y) antigen) bind but do not precipitate with the isolectins, indicating that the positions and linkage of fucose residues are critical for cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding, Competitive↗

PubMed1990

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei.

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl beta-D-cellotrioside (Km = 48 microM, kcat = 0.7 min-1) with release of the beta-cellobiose (retention of configuration). The same enzyme catalyzes the trans-hydration of cellobial (Km = 116 microM, kcat = 1.16 min-1) and lactal (Km = 135 microM, kcat = 1.35 min-1), presumably with glycosyl oxo-carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl beta-cellotrioside, but products formed from these prochiral substrates are again of beta configuration. Cellobiohydrolase II from the same microorganism hydrolyzes methyl beta-D-cellotetraoside (Km = 4 microM, kcat = 112 min-1) with inversion of configuration to produce alpha-cellobiose. The other reaction product, methyl beta-cellobioside, is in turn partly hydrolysed by cellobiohydrolase II to form methyl beta-D-glucoside and D-glucose, presumably the alpha-anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si-direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed1990

Hydrolysis of beta-D-glucopyranosyl fluoride to alpha-D-glucose catalyzed by Aspergillus niger alpha-D-glucosidase.

Aspergillus niger alpha-D-glucosidase, crystallized and free of detectable activity for beta-D-glucosides, catalyzes the slow hydrolysis of beta-D-glucopyranosyl fluoride to form alpha-D-glucose. Maximal initial rates, V, for the hydrolysis of beta-D-glucosyl fluoride, p-nitrophenyl alpha-D-glucopyranoside, and alpha-D-glucopyranosyl fluoride are 0.27, 0.75, and 78.5 mumol.min-1.mg-1, respectively, with corresponding V/K constants of 0.0068, 1.44, and 41.3. Independent lines of evidence make clear that the reaction stems from beta-D-glucosyl fluoride and not from a contaminating trace of alpha-D-glucosyl fluoride, and is catalyzed by the alpha-D-glucosidase and not by an accompanying trace of beta-D-glucosidase or glucoamylase. Maltotriose competitively inhibits the hydrolysis, and beta-D-glucosyl fluoride in turn competitively inhibits the hydrolysis of p-nitrophenyl alpha-D-glucopyranoside, indicating that beta-D-glucosyl fluoride is bound at the same site as known substrates for the alpha-glucosidase. Present findings provide new evidence that alpha-glucosidases are not restricted to alpha-D-glucosylic substrates or to reactions providing retention of configuration. They strongly support the concept that product configuration in glycosylase-catalyzed reactions is primarily determined by enzyme structures controlling the direction of approach of acceptor molecules to the reaction center rather than by the anomeric configuration of the substrate.

Aspergillus niger↗

PubMed1990

Isoelectric focusing studies of concanavalin A and the lentil lectin.

Isoelectric focusing (IEF) of metallized and demetallized preparations of concanavalin A (Con A) consisting of either intact or fragmented subunits shows different band patterns. Metallized Con A consisting of intact polypeptide chains (intact Con A) has an isoelectric point (pI) 8.35. Metallized preparations consisting of fragmented chains (fragmented Con A) show three bands with pI values 8.0, 7.8 and 7.7. Demetallized intact Con A (intact apoCon A) has a pI of 6.5, however, it undergoes pH dependent association during IEF under certain conditions, which gives rise to multiple bands. Ampholyte-mediated demetallization of intact and fragmented Con A and subsequent aggregation of the apoprotein results in multiple bands during IEF in the presence of the pH range 3 to 10 ampholytes. However, ampholytes of the pH range 7 to 9 do not demetallize the proteins and show a single band with intact Con A. The pI of intact Con A remains essentially the same in the presence of inhibitory sugar. Furthermore, different moleculars forms of Con A, including locked and unlocked conformers of intact apoCon A, and the dimeric and tetramic states of both intact Con A and intact apoCon A have been identified and their pI values determined. IEF of the lentil isoelectins, LcH-A and LcH-B, shows single bands of pI 8.5 and 9.0, respectively. However, the native lectin mixture gives rise to an additional band of pI 8.8 due to a hybrid protein formed by ampholyte-mediated subunit exchange between the isolectins.

Ampholyte Mixtures↗

PubMed1989

Steric course of the hydrolysis of alpha,alpha-trehalose and alpha-D-glucosyl fluoride catalyzed by pig kidney trehalase.

We are unable to confirm the report of Labat et al.3 that pig kidney trehalase hydrolyzes alpha,alpha-trehalose to form solely alpha-D-glucose. Highly purified trehalase from pig renal cortex was found, in reactions monitored by 1H-n.m.r. spectra, to hydrolyze alpha,alpha-trehalose with the formation of both alpha- and beta-D-glucose. That the beta anomer constitutes the enzymically mobilized glucosyl residue is indicated by the further finding that beta-D-glucose is the product formed on hydrolysis of alpha-D-glucosyl fluoride by the enzyme. Present results show the stereochemical behavior of pig kidney trehalase in hydrolyzing alpha,alpha-trehalose to be indistinguishable from that reported by ourselves and others for trehalase preparations from a range of biological sources including rabbit renal cortex.

Animals↗

PubMed1989

Formation of highly ordered cross-linked lattices between asparagine-linked oligosaccharides and lectins observed by electron microscopy.

The interaction of asparagine-linked carbohydrates (N-linked) with carbohydrate binding proteins called lectins has been demonstrated to be involved in a variety of cellular recognition processes. Certain N-linked carbohydrates have been shown to be multivalent and capable of binding, cross-linking, and precipitating lectins (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1987) J. Biol. Chem. 262, 1294-1299; Bhattacharyya, L., Haraldsson, M., and Brewer, C. F. (1988) Biochemistry 27, 1034-1041). Recent data have further suggested that certain oligomannose and bisected hybrid-type N-linked glycopeptides form homogeneous cross-linked lattices with concanavalin A (Bhattacharyya, L., Khan, M. I., and Brewer, C. F. (1988) Biochemistry 27, 8762-8767). In the present study, evidence has been obtained from electron microscopy for the formation of highly ordered and distinct lattices for two bivalent complex type oligosaccharides cross-linked with soybean lectin (Glycine max) and isolectin A from Lotus tetragonolobus, respectively. The results indicate a new source of specificity for interactions of N-linked carbohydrates with lectins, namely their ability to form highly ordered homogeneous aggregates.

Asparagine↗

PubMed1989

Alpha-secondary tritium kinetic isotope effects for the hydrolysis of alpha-D-glucopyranosyl fluoride by exo-alpha-glucanases.

alpha-Secondary tritium kinetic isotope effects ranging from 1.17 to 1.26 were measured for the hydrolysis of alpha-D-glucopyranosyl fluoride (forming beta-D-glucose) catalyzed by several glucoamylases and a glucodextranase. These results indicate that cleavage of the C-F bond is slow and that the enzymic transition state has significant oxo-carbonium ion character. Strong support for this conclusion is provided by the agreement found in the case of Rhizopus niveus glucoamylase (alpha-TV/K 1.26; Km 26 mM) between measured values of the alpha-secondary deuterium kinetic isotope effects (alpha-DV/K 1.16; alpha-DV 1.20) and those calculated from the tritium isotope effect. The data are consistent with the promotion of an intramolecular elimination of fluoride by the present exo-alpha-glucanases based on their ability to stabilize, perhaps with a counter ion, the development of a carbonium ion-like transition state. Although the oxo-carbonium ion is formally denoted as an intermediate it could represent a transition state along a reaction pathway to a covalent glucosyl intermediate.

Glucose↗

PubMed1989

Interactions of concanavalin A with asparagine-linked glycopeptides. Structure/activity relationships of the binding and precipitation of oligomannose and bisected hybrid-type glycopeptides with concanavalin A.

We have recently demonstrated that certain oligomannose and bisected hybrid-type glycopeptides are bivalent for concanavalin A (ConA) binding and that they can precipitate the lectin [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P & Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293]. Two protein-binding sites on each glycopeptide were identified: one on the alpha(1-6) arm of the core beta-mannose residue which binds with high affinity (primary site); the other on the alpha(1-3) arm of the core beta-mannose residue which binds with lower affinity (secondary site). In the present study, we have investigated the relationship between the structures of the primary sites of oligomannose-type glycopeptides and their affinities for ConA. Two mechanisms of binding at the primary sites of oligomannose-type glycopeptides have been identified which account for the 3000-fold increase in affinity of a Man9 glycopeptide relative to that of methyl alpha-D-mannopyranoside. Changes in the structures and affinities of both the primary and secondary sites are observed to influence the precipitation activities of the glycopeptides. These findings have important consequences for the specificity of ConA binding in solutions containing mixtures of the carbohydrates.

Asparagine↗

PubMed1988

Synthesis of (Z)-3,7-anhydro-1,2-dideoxy-2-deuterio-D-gluco-oct-2- enitol, a prochiral substrate for probing the catalytic functioning of of glucosylases.

Synthesis of the title compound provides a prochiral, glycosyl-donor substrate well suited for use as a probe of the catalytic functioning of D-glucosyl-mobilizing enzymes, because the full stereochemistry of enzymic reactions at its double bond may be unambiguously determined by examining the reaction products. The starting material for the synthesis was 2,6-anhydro-D-glycero-D-gulo-heptonic acid, from which 3,7-anhydro-4,5,6,8-tetra-O-benzyl-1-deoxy-D-glycero-D-gulo-2- octulose was prepared in eight steps. Reduction with lithium aluminum deuteride, and conversion of the resulting diastereomeric alcohols into (Z)-3,7-anhydro-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-2-deuterio-D- gluco-oct-2-enitol (11) and 3,7-anhydro-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-2-deuterio-D- glycero-D-gulo-oct-1-enitol (16), was carried out. By-products were 3,7-anhydro-2-O-benzoyl-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-2-deuterio -D-erythro-L-galacto-octitol and 3,7-anhydro-2-O-benzoyl-4,5,6,8-tetra-O-benzyl-1,2-dideoxy-2-deuterio -D-erythro-L-talo-octitol, which could, like compound 16, be recycled. On debenzylation the oct-2-enitol 11 yielded (Z)-3,7-anhydro-1,2-dideoxy-2-deuterio-D-gluco-oct-2-enitol.

Acetylation↗

PubMed1988

Interactions of concanavalin A with asparagine-linked glycopeptides: formation of homogeneous cross-linked lattices in mixed precipitation systems.

We have previously shown that certain oligomannose and bisected hybrid type glycopeptides are bivalent for binding to concanavalin A (Con A) [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C. F. (1987) J. Biol. Chem. 262, 1288-1293]. Each glycopeptide gives a quantitative precipitation profile with the protein which consists of a single peak that corresponds to the binding stoichiometry of glycopeptide to protein monomer (1:2). We have shown that the affinities of the primary and secondary sites of the glycopeptides influence their extent of precipitation with the lectin [Bhattacharyya, L., & Brewer, C. F. (1988) Eur. J. Biochem. (in press)]. In the present study, we demonstrate that equimolar mixtures of any two of the glycopeptides result in a quantitative precipitation profile which shows two protein peaks. Using radiolabeled glycopeptides, the precipitation profiles of the individual glycopeptides were determined. The results show that each glycopeptide forms its own precipitation profile with the protein which is independent of the profile of the other glycopeptide. For mixtures containing an equimolar ratio of two glycopeptides, the glycopeptide with lower affinity shows a precipitation maximum at a lower concentration than the one with higher affinity. However, this can be reversed by increasing the ratio of the lower affinity glycopeptide in the mixture. Thus, the relative precipitation maxima of the glycopeptides are determined by mass-action equilibria involving competitive binding of the two carbohydrates to the protein. These equilibria, in turn, are sensitive to the relative amounts and affinities of the carbohydrates at both their primary and secondary sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Asparagine↗

PubMed1988

Lectin-carbohydrate interactions. Studies of the nature of hydrogen bonding between D-galactose and certain D-galactose-specific lectins, and between D-mannose and concanavalin A.

The binding of galactose-specific lectins from Erythrina indica (EIL), Erythrina arborescens (EAL), Ricinus communis (agglutinin; RCA-I), Abrus precatorius (agglutinin; APA), and Bandeiraea simplicifolia (lectin I; BSL-I) to fluoro-, deoxy-, and thiogalactoses were studied in order to determine the strength of hydrogen bonds between the hydroxyl groups of galactose and the binding sites of the proteins. The results have allowed insight into the nature of the donor/acceptor groups in the lectins that are involved in hydrogen bonding with the sugar. The data indicate that the C-2 hydroxyl group of galactose is involved in weak interactions as a hydrogen-bond acceptor with uncharged groups of EIL and EAL. With RCA-I, the C-2 hydroxyl group forms two weak hydrogen bonds in the capacity of a hydrogen-bond acceptor and a donor. On the other hand, there is a strong hydrogen bond between the C-2 hydroxyl group of galactose, which acts as a donor, and a charged group on BSL-I. The C-2 hydroxyl group of the sugar is also a hydrogen-bond donor to APA. The lectins are involved in strong hydrogen bonds through charged groups with the C-3 and C-4 hydroxyl groups of galactose, with the latter serving as hydrogen-bond donors. The C-6 hydroxyl group of the sugar is weakly hydrogen bonded with neutral groups of EIL, EAL, and APA. With BSL-I, however, a strong hydrogen bond is formed at this position with a charged group of the lectin. The C-6 hydroxyl groups is a hydrogen-bond acceptor for EIL and EAL, a hydrogen-bond donor for APA and BSL-I, and appears not to be involved in binding to RCA-I. The data with the thiosugars indicate the involvement of the C-1 hydroxyl group of galactose in binding to EIL, EAL, and BSL-I, but not to RCA-I and APA. We have also performed a similar analysis of the binding data of fluoro- and deoxysugars to concanavalin A [Poretz, R. D. and Goldstein, I. J. (1970) Biochemistry 9, 2890-2896]. This has allowed comparison of the donor/acceptor properties and free energies of hydrogen bonding of the hydroxyl groups of methyl alpha-D-mannopyranoside to concanavalin A with the results in the present study. On the basis of this analysis, new assignments are suggested for amino acid residues of concanavalin A [corrected] that may be involved in hydrogen bonding to the sugar.

Concanavalin A↗

PubMed1988

Formation of homogeneous cross-linked lattices between oligomannose type glycopeptides and concanavalin A.

Certain oligomannose type glycopeptides have previously been shown to be bivalent for binding to concanavalin A, and to give quantitative precipitation profiles with the protein that consist of single peaks which correspond to the binding stoichiometry of glycopeptide to protein monomer (1:2) (Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., and Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293). In the present study, equimolar mixtures of two oligomannose type glycopeptides, a Man-6 and a Man-9 glycopeptide, gives a quantitative precipitation profile which shows two protein peaks. Each glycopeptide was radiolabelled with 3H or 14C, and the the precipitation profiles of the individual glycopeptides in the mixture determined. The results show that the radioactivity profile of the Man-6 glycopeptide corresponds to the first protein peak, while the radioactivity profile of the Man-9 glycopeptide corresponds to the second protein peak. The results indicate that each glycopeptide forms a unique homogeneous cross-linked lattice with the lectin which excludes the lattice of the other glycopeptide.

Carbohydrate Conformation↗

PubMed1988

Binding and precipitation of lectins from Erythrina indica and Ricinus communis (agglutinin I) with synthetic cluster glycosides.

We recently reported that tri- and tetraantennary complex type oligosaccharides with nonreducing terminal galactose residues and the triantennary asialofetuin glycopeptide can bind and precipitate certain galactose specific lectins (L. Bhattacharyya, and C.F. Brewer (1986) Biochem. Biophys. Res. Commun. 141, 963-967; L. Bhattacharyya, M. Haraldsson, and C.F. Brewer (1988) Biochemistry 27, 1034-1041). The present study investigates the binding interactions of two of these lectins, those from Erythrina indica and Ricinus communis (Agglutinin I), with mono-, bi-, and triantennary synthetic cluster glycosides, which have little structural resemblance to complex type oligosaccharides other than they possess nonreducing terminal galactose residues (R.T. Lee, P. Lin, and Y.C. Lee (1984) Biochemistry 23, 4255-4261). The enhanced affinities of the bi- and triantennary glycosides relative to the monoantennary glycoside for the two lectins are consistent with an increase in the probability of binding due to multiple binding residues in the multiantennary glycosides. The triantennary glycoside is capable of precipitating the two lectins, and quantitative precipitation data indicate that it is a trivalent ligand. The results show that the binding and precipitation activities of complex type oligosaccharides with these lectins is due solely to the presence of multiple terminal galactose residues and not to the overall structures of the oligosaccharides.

Chemical Precipitation↗

PubMed1988

Steric course of the hydration of D-gluco-octenitol catalyzed by alpha-glucosidases and by trehalase.

Crystalline Aspergillus niger alpha-glucosidase and highly purified preparations of rice alpha-glucosidase II and Trichoderma reesei trehalase were found to catalyze the hydration of [2-(2)H]-D-gluco-octenitol, i.e., (Z)-3,7-anhydro-1,2-dideoxy-[2-2H]-D-gluco-oct-2-enitol, to yield 1,2-dideoxy-[2-2H]-D-gluco-octulose. In each case, the stereochemistry of the reaction was elucidated by examining the newly formed centers of asymmetry at C-2 and C-3 of the hydration product. The C-1 to C-3 fragment of each isolated [2-2H]-D-gluco-octulose product was recovered as [2-2H]propionic acid and identified by its positive optical rotatory dispersion as the S isomer, showing that each enzyme had protonated the octenitol (at C-2) from above its re face. 1H NMR spectra of enzyme/D-gluco-octenitol digests in D2O showed that the alpha-anomer of [2-2H]-D-gluco-octulose was exclusively produced by each alpha-glucosidase, whereas the beta-anomer was formed by action of the trehalase. The trans hydration catalyzed by the alpha-glucosidases was found to be very strongly inhibited by the substrate; the cis hydration reaction catalyzed by the trehalase showed no such inhibition. Special importance is attached to the finding that in hydrating octenitol each enzyme creates a product of the same anomeric form as in hydrolyzing an alpha-D-glucosidic substrate. This result adds substantially to the growing evidence that individual glycosylases create the configuration of their reaction products by a means that is independent of donor substrate configuration, that is, by a means other than "retaining" or "inverting" substrate configuration.

Aspergillus niger↗

PubMed1988

Stereochemical studies of D-glucal hydration by alpha-glucosidases and exo-alpha-glucanases: indications of plastic and conserved phases in catalysis by glycosylases.

Alpha-Glucosidases from Aspergillus niger, pig serum, ungerminated rice, buckwheat, and sugar beet seeds (but not from brewers' yeast or honeybee) were found to catalyze the hydration of D-glucal. Each reactive alpha-glucosidase, incubated with D-glucal in D2O, was shown to protonate (deuteriate) this prochiral substrate from above its re face, i.e., from a direction opposite that assumed for protonating alpha-D-glucosidic substrates. At the same time, D-glucal hydration catalyzed by three of the alpha-glucosidases that acted rapidly enough in D2O to determine product configuration was found to yield 2-deoxy-D-glucose of the same specific (alpha-) configuration as the D-glucose produced from alpha-D-glucosidic substrates. These findings substantially extend those reported earlier for the hydration of D-glucal by one (Candida tropicalis) alpha-glucosidase preparation. Together with other recent results, they suggest that the process of catalysis by alpha-glucosidases (and perhaps glycosylases in general) may comprise two separate and separately controlled parts, namely, a "plastic" phase concerned with substrate protonation and a substrate-unrelated "conserved" phase concerned with the creation of product configuration. In contrast to the alpha-glucosidases, three "inverting" exo-alpha-glucanases (Arthrobacter globiformis glucodextranase; Rhizopus niveus and Paecilomyces varioti glucoamylase) were found to protonate D-glucal from below its si face. Further, whereas the catalysis of D-glucal hydration by the alpha-glucosidases was intensively inhibited by excess substrate, that promoted by the exo-glucanases showed no detectable substrate inhibition.

Animals↗

PubMed1988

Precipitation of galactose-specific lectins by complex-type oligosaccharides and glycopeptides: studies with lectins from Ricinus communis (agglutinin I), Erythrina indica, Erythrina arborescens, Abrus precatorius (agglutinin), and Glycine max (soybean).

We have recently demonstrated that certain oligomannose and bisected hybrid type glycopeptides and bisected complex type oligosaccharides are bivalent for binding to concanavalin A and can precipitate the lectin [Bhattacharyya, L., Ceccarini, C., Lorenzoni, P., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1288-1293; Bhattacharyya, L., Haraldsson, M., & Brewer, C.F. (1987) J. Biol. Chem. 262, 1294-1299]. The present results show that tri- and tetraantennary complex type oligosaccharides containing nonreducing terminal galactose residues, and a related triantennary glycopeptide, precipitate the D-galactose-specific lectins from Ricinus communis (agglutinin I) (RCA-I), Erythrina indica (EIL), Erythrina arborescens (EAL), and Glycine max (soybean) (SBA). Nonbisected and bisected biantennary complex type oligosaccharides can precipitate SBA, which is a tetrameric lectin, but not RCA-I, EIL, or EAL, which are dimeric lectins. The relative affinities of the oligosaccharides and glycopeptide were determined by hemagglutination inhibition measurements and their valencies by quantitative precipitin analyses. The equivalence points of the precipitin curves indicate that the tri- and tetraantennary oligosaccharides are tri- and tetravalent, respectively, for EIL, EAL, and SBA binding. However, the oligosaccharides are all trivalent for RCA-I binding due apparently to the larger size of the monomeric subunit of the lectin. The triantennary glycopeptide was also trivalent for RCA-I and EIL binding. Biantennary oligosaccharides with adequate chain lengths were found to be bivalent for binding to SBA; those with shorter chains did not precipitate the lectin.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗

PubMed1987

Circular dichroism and 1H NMR studies of Co2+- and Ni2+-substituted concanavalin A and the lentil and pea lectins.

Visible absorption, circular dichroism (CD) and magnetic circular dichroism spectra have been recorded for the Ca2+-Co2+ derivatives of the lentil (CCoLcH) and pea (CCoPSA) lectins (Co2+ at the S1 sites and Ca2+ at the S2 sites) and shown to be very similar for both proteins. The visible absorption and magnetic circular dichroism spectra indicate similar octahedral geometries for high spin Co2+ at S1 in both proteins, as found in the Ca2+-Co2+ complex of concanavalin A (CCoPL) (Richardson, C. E., and Behnke, W. D. (1976) J. Mol. Biol. 102, 441-451). The visible CD data, however, indicate differences in the environment around S1 of CCoLcH and CCoPSA compared to CCoPL. 1H NMR spectra at 90 MHz of the Co2+ and Ni2+ derivatives of the lectins show a number of isotropically shifted signals which arise from protons in the immediate vicinity of the S1 sites. Analysis of the spectra of the Co2+ derivatives in H2O and D2O has permitted resonance assignments of the side chain ring protons of the coordinated histidine at S1 in the lectins. Differences are observed in the H-D exchange rate of the histidine NH proton at S1 in concanavalin A compared to the lentil and pea lectins. NMR data of the Ni2+-substituted proteins, together with spectra of the Co2+ derivatives, also indicate that the side chains of a carboxylate ligand and of the histidine residue at S1 are positioned differently in concanavalin A than in the other two lectins. These results appear to account, in part, for the differences observed in the visible CD spectra of the Co2+-substituted proteins. In addition, binding of monosaccharides does not significantly perturb the spectra of the lectins. An unusual feature in the 1H NMR spectra of all three Co2+-substituted lectins is the presence of two exchangeable downfield shifted resonances which appear to be associated with the two protons of a slowly exchanging water molecule coordinated to the Ca2+ ion at S2. T1 measurements of CCoLcH have provided an estimation of the distances from the Co2+ ion to these two protons of 3.7 and 4.0 A.

3-O-Methylglucose↗

PubMed1987

Electron paramagnetic resonance and magnetic susceptibility studies of dimanganese concanavalin A. Evidence for antiferromagnetic exchange coupling.

The double Mn2+ complex of concanavalin A with bound saccharide (SMMPL) was examined by electron paramagnetic resonance (EPR) spectroscopy and magnetic susceptibility measurements. A room temperature X-band (9 GHz) EPR spectrum of SMMPL revealed a relatively weak, broad resonance in contrast to the spectrum with a six-line hyperfine-split pattern observed for the mononuclear, high-spin Mn2+ complex found in Ca2+-Mn2+-concanavalin A with saccharide present (SCMPL). The EPR spectrum of SMMPL at 77 K, however, consisted of a series of overlapping patterns of 11 hyperfine-split lines near g = 2.0 with members of each pattern separated by 47 G, half the value of the hyperfine splitting of SCMPL. These 11-line patterns are preserved at Q-band (35 GHz), indicating that the manganese ions in SMMPL form a spin-coupled, binuclear center. As expected for an exchange-coupled system, the EPR signal of SMMPL at 77 K saturates at a higher microwave power than those for SCMPL or Mn2+ aquoion. There is also a marked loss of EPR signal intensity for SMMPL between 4.2 and 1.4 K, which supports the view that the pair of manganese ions is exchanged-coupled. The temperature dependence of both the magnetic susceptibility and the low-temperature EPR spectral intensity can be explained by a model in which the two high-spin Mn2+ ions of SMMPL are antierromagnetically exchanged-coupled with an isotropic coupling constant J = 1.8 cm-1 (for the spin Hamiltonian Hex = JS1.S2). Zero-field splitting D' was estimated to be 375 G from the EPR spectrum.(ABSTRACT TRUNCATED AT 250 WORDS)

Concanavalin A↗