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PubMedDate not supplied

Affinity adsorption of Saccharomyces cerevisiae on concanavalin A perflurocarbon emulsions.

Perfluorocarbon affinity emulsions have been generated by immobilizing concanavalin A onto the surface of triazine-activated perfluorocarbon droplets. Immobilized concanavalin A densities of 0.1, 0.7 and 2.1 mg/ml were obtained by varying the concentration of cyanuric chloride used for activation. The affinity emulsions were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of 1 x 10(9) cells, 4.6 x 10(9) and 6 x 10(9) cell/ml, respectively. Optimal conditions for the elution of bound cells were obtained by studying inhibition curves of concanavalin A-mannan precipitation using simple sugars. Methyl alpha-D-mannopyranoside showed the greatest inhibitory power with 50 per cent inhibition displayed at a concentration of 0.05 mM. Experiments carried out examining the concentrations of methyl alpha-D-mannopyranoside necessary to dissociate a concanavalin A-mannan precipitate demonstrated that at least a seven-fold higher concentration of methyl alpha-D-mannopyranoside was required for dissociation than that required for inhibition of its formation. The efficiency of elution of bound yeast cells was found to be dependent on the concentration of methyl alpha-D-mannopyranoside used, the time of elution and the immobilized ligand density. Thus, 100 per cent elution was obtained with a concanavalin A affinity emulsion (0.1 mg/ml) by incubation with 500 mM methyl alpha-D-mannopyranoside for 1 h.

Adsorption↗
PubMed1984

Endocytosis in nonciliated epithelial cells of the ductuli efferentes in the rat.

The nonciliated cells lining the ductuli efferentes presented three distinct cytoplasmic regions. The apical region contained, in addition to cisternae of endoplasmic reticulum and mitochondria, two distinct membranous elements. The tubulovesicular system consisted of dilated tubules connected to the apical plasma membrane and subjacent distended vesicular profiles. The apical tubules, not connected to the cell surface, consisted of numerous densely stained tubules of small size which contain a compact, finely granulated material. The supranuclear region, in addition to a Golgi apparatus and ER cisternae, contained dilated vacuoles, pale and dense multivesicular bodies, as well as numerous dense granules identified cytochemically as lysosomes. The basal region contained the nucleus and many lipid droplets. The endocytic activity of these cells was investigated using cationic ferritin (CF) and concanavalin-A-ferritin (Con-A-ferritin) as markers of adsorptive endocytosis; and native ferritin (NF), concanavalin-A-ferritin in the presence of alpha-methyl mannoside, and horseradish peroxidase or albumin bound to colloidal gold for demonstrating fluid-phase endocytosis. These tracers were injected separately into the rete testis, and animals were sacrificed at various time intervals after injection. At 1 min, CF or Con-A-ferritin were seen bound to the apical plasma membrane, to the membrane of microvilli, and to the membrane delimiting elements of the tubulovesicular system. Between 2 and 5 min, these tracers accumulated in the densely stained apical tubules and at 15 min in the dilated vacuoles. Between 30 min and 1 hr, the tracers appeared in multivesicular bodies of progressively increasing density, whereas at 2 hr and later time intervals, many dense lysosomal elements became labeled. The tracers for fluid-phase endocytosis showed a distribution similar to that for CF or Con-A-ferritin except that they did not bind to the apical plasma membrane, microvilli, or membrane delimiting the tubulovesicular system. At no time interval were any of the tracers observed in the abluminal spaces. Thus, the nonciliated epithelial cells of the ductuli efferentes are actively involved in fluid-phase and adsorptive endocytosis, both of which result in the sequestration of endocytosed material within the lysosomal apparatus of the cell.

Animals↗
PubMed2007

Complexation of fatty acids and fatty acid-CoAs with synthetic O-methylated polysaccharides.

An experimentally simple, but highly reproducible and reliable method has been developed to follow the complexation event of fatty acid (FA) and FA-CoA with polysaccharides. This method was based on the recent discovery of the unique blue-shifted UV absorption associated with the aggregation of tetraenoic fatty acids (TE-FAs) in aqueous solution. Complexation was monitored by recording the intensity of UV absorption at 250 nm ([free TE-FA]) and 303 nm ([complexed TE-FA]), and the K aggregate of C(20) t,t,t,t-TE-FA exhibited the ideal property for this purpose. Synthetic 3-O-methyl-D-mannose- and 6-O-methyl-D-glucose-containing lipopolysaccharides were found to exhibit a broad range of the binding affinities with C(20) t,t,t,t-TE-FAs as well as saturated FAs/FA-CoAs.

Acyl Coenzyme A↗
PubMed1984

T-dependent activation of resting B cells mediated by concanavalin A.

In cultures containing long-term cultured lines of antigen-specific helper T (Th) cells, normal unprimed B cells and concanavalin A (Con A), induction of B cells to immunoglobulin secretion and DNA synthesis was observed. The plaque-forming cell (PFC) response was large (frequently greater than 75 000 PFC/10(6) input B cells) demonstrating the polyspecific nature of the response. Con A-mediated maturation and induction to DNA synthesis of responding B cells was completely Th cell dependent and inhibited with methyl-alpha-D-mannoside. Both resting and blasted B cells, separated by Percoll density centrifugation, were induced to DNA synthesis and immunoglobulin secretion. Responses were completely unrestricted by the B cell major histocompatibility complex, even at the level of the resting B cell. The polyclonal nature of the response taken together with the Con A-mediated bypassing of T cell specificity and restricting haplotype indicates that this response is analogous to lectin-mediated cytotoxicity.

Animals↗
PubMed1989

The structure of the saccharide-binding site of concanavalin A.

A complex of concanavalin A with methyl alpha-D-mannopyranoside has been crystallized in space group P212121 with a = 123.9 A, b = 129.1 A and c = 67.5 A. X-ray diffraction intensities to 2.9 A resolution have been collected on a Xentronics/Nicolet area detector. The structure has been solved by molecular replacement where the starting model was based on refined coordinates of an I222 crystal of saccharide-free concanavalin A. The structure of the saccharide complex was refined by restrained least-squares methods to an R-factor value of 0.19. In this crystal form, the asymmetric unit contains four protein subunits, to each of which a molecule of mannoside is bound in a shallow crevice near the surface of the protein. The methyl alpha-D-mannopyranoside molecule is bound in the C1 chair conformation 8.7 A from the calcium-binding site and 12.8 A from the transition metal-binding site. A network of seven hydrogen bonds connects oxygen atoms O-3, O-4, O-5 and O-6 of the mannoside to residues Asn14, Leu99, Tyr100, Asp208 and Arg228. O-2 and O-1 of the mannoside extend into the solvent. O-2 is hydrogen-bonded through a water molecule to an adjacent asymmetric unit. O-1 is not involved in any hydrogen bond and there is no fixed position for its methyl substituent.

Binding Sites↗
PubMed1995

Dual mechanism of protein-tyrosine phosphorylation in concanavalin A-stimulated platelets.

Treatment of human platelets with the lectin Concanavalin A (Con A) resulted in the tyrosine phosphorylation of several proteins with molecular masses 65, 80, 85, 95, 120, 135, and 150 kDa. These proteins were divided in two groups: the first group included the 65-, 85-, 95-, and 120-kDa bands, which were tyrosine phosphorylated also in thrombin-stimulated platelets; the second group (80-, 135-, and 150-kDa bands) included proteins whose tyrosine phosphorylation was exclusively promoted by Con A, but not by thrombin. Members of the second group were rapidly dephosphorylated when the lectin was displaced from the cell surface by methyl alpha-D-mannopyranoside. Pretreatment of intact platelets with the prostacyclin analog iloprost, inhibited Con A-induced tyrosine phosphorylation of the first group of proteins, but had no effect on the tyrosine phosphorylation of the proteins of the second group. Succinyl-Con A, a dimeric derivative of the lectin, which binds to the platelet surface but does not promote clustering of the receptor, did not induce tyrosine phosphorylation of the second group of proteins, although phosphorylation of some members of the first group was observed. Our results demonstrate the presence of two different mechanisms leading to protein-tyrosine phosphorylation in Con A-stimulated platelets, and identify a new signal transduction pathway, promoted by the clustering of membrane glycoproteins, that produces tyrosine phosphorylation of specific substrates. This new pathway may be activated by platelet interaction with multivalent ligands, such as adhesive proteins, during adhesion, spreading, and aggregation.

Blood Platelets↗
PubMed1975

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization, cellular ATP and associated membrane changes in D+ and D- cells.

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D+ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D- myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D+ than in D- cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D- than in D+ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D+ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D+ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D- cells, were found before most of the D+ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D- cells to differentiate and the acquistion of the metabolic properties of normal granulocytes by differentiating D+ cells, were associated with differences in the organization of the cell surface membrane.

Adenosine Triphosphate↗
PubMed1976

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.

Animals↗
PubMed1977

Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.

Azides↗
PubMed1979

Enhanced sterol synthesis in concanavalin A-stimulated lymphocytes: correlation with phospholipid synthesis and DNA synthesis.

Incorporation of (14C)choline and (3H)myo-inositol into the total lipid fraction, incorporation of (14C)acetate into the sterol fraction and incorporation of (3H)thymidine into DNA were studied in human lymphocyte cultures. Concanavalin A induced an increase in the incorporation of these labels with the following features: (a) Phospholipid synthesis was increased promptly. The lag time for the increase in sterol synthesis and DNA synthesis were 5 hours and 27 hours respectively; (b) The increase in phospholipid synthesis and sterol synthesis was proportional to ConA concentration initially. Cells treated with a high concentration of ConA showed very low levels of DNA synthesis; (c) The increase in phospholipid synthesis could be abolished immediately by alpha-Methyl-Mannoside. alpha-Methyl-Mannoside blunted but did not abolish the increase in sterol synthesis. alpha-Methyl-Mannoside enhanced DNA synthesis of those cells which had been treated by a high concentration of ConA; and (d) Selective inhibition of sterol synthesis with 25-hydroxycholesterol did not prevent the increase in phospholipid synthesis, but it blocked the increase in DNA synthesis. Supplement of LDL, HDL or total lipoproteins to lymphocyte cultures was effective in preventing the inhibition of DNA synthesis by 25-hydroxy-cholesterol. These results suggest that in lymphocyte activation by ConA phospholipid synthesis, sterol synthesis and DNA synthesis were sequentially increased. The rate of cellular commitment to mitogenesis was proportional to ConA concentrations. High concentrations of ConA arrested the cell growth at a postcommitment point in the G1 phase. Enhanced phospholipid synthesis was a precommitment event. Enhanced sterol synthesis was a postcommitment event and reflected the requirement of an increased cholesterol supply for the passage of cell growth through G1.

Acetates↗
PubMed1983

Concanavalin A increases spontaneous beat rate of embryonic chick heart cell aggregates.

The plant lectin concanavalin A (Con A), at concentrations of 5-200 micrograms/ml, induced a twofold to fivefold increase in spontaneous beat rate of cultured aggregates of ventricular cells from seven-day chick embryos. This response was time, dose, and temperature dependent and was accompanied by a decrease in transmembrane potential. It could be blocked or reversed by alpha-methyl-D-mannoside but was not reversed by dilution alone. Binding of the lectin occurred in the cold, but a temperature-dependent process was also necessary to produce the response. Divalent (succinyl) Con A did not cause a beat rate increase. Whole heart aggregates responded similarly but less intensely than ventricular aggregates. Atrial aggregates, and whole heart aggregates treated with 5 microgram/ml of Con A, produced a biphasic chronotropic response, first decreasing then increasing their beat rates. These results suggest that saccharide-bearing macromolecules on the heart cell surface play a role in regulating spontaneous beat rate.

Action Potentials↗
PubMed1975

Surface modifications evoked by antidiuretic hormone in isolated epithelial cells: evidence from lectin probes.

Epithelial cells (80-90% "granular" type) were isolated from urinary bladders of Bufo marinus and Rana catesbiana. The inhibitory effect of alpha-methyl-D-mannoside on fluorescein-labeled concanavalin A (Con A) binding to these cells indicates that they possess specific binding sites for Con A. The lectin also mediates adsorption of erythrocytes to these cells. Both Con A binding and Con A-mediated hemadsorption to epithelial cells are depressed at 4 degrees C, as compared with cells maintained at 22 degrees C. Elevation of temperature to 37 degrees C, however, enhances hemadsorption independently of alterations in lectin binding. Treatment of cells with antidiuretic hormone (ADH) at 22 degrees C followed by 15 min of incubation at 22 degrees or 37 degrees C before exposure of cells to Con A promotes increments in Con A-mediated hemadsorption, but not in lectin binding, at 22 degrees or 37 degrees C. These hormonal effects are not significant when hemadsorption is assayed at 4 degrees C. Treatment of cells with another octapeptide, angiotensin, elicits a small, but significant, increment in hemadsorption to epithelial cells which is likewise uninfluenced by quantitative changes in lectin binding. Collectively, these data and other independent observations suggest that treatment with octapeptide hormones acts to enhance the redistribution and aggregation of lectin-binding proteins in the membranes of granular epithelial cells from amphibian urinary bladder. Such changes, in turn, may contribute to the alterations in membrane transport properties which characterize the hormonal response.

Angiotensin II↗
PubMed1990

Three-dimensional structures of complexes of Lathyrus ochrus isolectin I with glucose and mannose: fine specificity of the monosaccharide-binding site.

The structure of the methyl-alpha-D-mannopyranoside-LOL I complex has been solved by the molecular replacement method using the refined saccharide-free LOL I coordinates as starting model. The methyl-alpha-D-mannopyranoside-LOL I complex was refined by simulated annealing using the program X-PLOR. The final R-factor value is 0.182 [Fo greater than 1 sigma(Fo)]. The isostructural methyl-alpha-D-glucopyranoside-LOL I complex was refined by X-Ray coupled energy minimization using the methyl-alpha-D-mannopyranoside-LOL I structure as a starting model to an R factor of 0.179 (all data). In both crystal forms, each dimer binds two molecules of sugar in pockets found near the calcium ions. The two saccharide moieties, which are in the C1 chair conformation, establish the same hydrogen bond pattern with the lectin. However, the van der Waals contacts are different between the O2, C2, C6, and O6 atoms of the two molecules and the backbone atoms of residues 208-211. Mannose, due to its axial C2 conformation, encloses the backbone atoms of the protein in a clamplike way. Van der Waals energy calculations suggest that this better complementarity of the mannoside molecule with the lectin could explain its higher affinity for isolectin I.

Binding Sites↗
PubMed1995

Determination of the affinity constants of concanavalin A for monosaccharides by fluorescence affinity probe capillary electrophoresis.

Dissociation constants between a legume lectin, concanavalin A (Con A), and unlabeled neutral monosaccharides were determined by affinity probe capillary electrophoresis (APCE). An affinity probe was synthesized by coupling Lissamine rhodamine B and p-amino-phenyl-alpha-D-mannopyranoside, with glutathione as a negatively charged linker. Electrophoresis of the affinity probe was carried out at neutral pH in a fused silica capillary coated with succinylpolylysine on its inner surface, using laser-induced fluorescence detection. In the presence of Con A, the mobility of the affinity probe decreased and this decrement was analyzed as a function of the concentration of Con A. In the presence of a mixture of Con A and an unlabeled monosaccharide, APCE can thus measure the concentration of Con A unoccupied by the unlabeled monosaccharide, and this allows the determination of the dissociation constant of the Con A-monosaccharide complex. The method should be useful for the analysis of a wide range of molecular interactions on a microscale.

Concanavalin A↗
PubMed1995

Stabilization of ribonuclease B activity by concentrated xylose solutions.

Ribonuclease B has become a paradigm as a simple example of an N-linked glycoprotein. We have found that certain affinity-purified preparations of this enzyme demonstrated a pronounced tendency to lose activity if stored as dilute aqueous solutions. Such inactivation is accelerated by the presence of NaCl, but can be counteracted by inclusion of high (1 mol/l) concentrations of xylose. Enzyme activity cannot be restored by addition of xylose after storage of the enzyme. In marked contrast to alpha-methyl-mannoside, xylose does not prevent ribonuclease B from binding to concanavalin A and so may be used to stabilize the enzyme during purification by lectin affinity chromatography.

Chromatography, Affinity↗
PubMed2000

Structural basis of functional mimicry between carbohydrate and peptide ligands of con A.

Crystallographic studies have shown independent binding sites for sugar and peptide ligands of concanavalin A, although they were considered functional mimics based on biochemical experiments. The topological correlation of 12-residue peptide with different carbohydrate ligands of concanavalin A showed similarity between trimannose and the YPY region of the peptide establishing structural mimicry. Molecular docking of trimannose and the YPY motif on the reciprocal binding sites revealed equivalent interactions and energetics implying that the peptide-binding sites may constitute additional sugar-binding subsites of concanavalin A. The binding of a mannose-rich neoglycoprotein with significantly higher affinity compared with that of the methyl alpha-d-mannopyranoside is consistent with this interpretation.

Amino Acid Motifs↗
PubMed1995

A confocal microscopic evaluation of the effects of insulin imprinting on the binding of Concanavalin A by Tetrahymena Pyriformis.

With confocal microscopy it is possible to study the Concanavalin A (Con A) binding characteristics of the surface and interior of a single cell by viewing optical sections. It was observed in Tetrahymena pyriformis that Con A bound both to the plasma membrane and to intracellular structures. Incubation of cells with a competing sugar a-methylmannopyranoside, decreased binding. Hormonal imprinting with insulin resulted in an increase in binding of Con A to the cell surface and a decrease in intracellular binding. It is possible that the intracellular binding sites may migrate to the plasma membrane.

Animals↗
PubMed1996

The mannose-specific bulb lectin from Galanthus nivalis (snowdrop) binds mono- and dimannosides at distinct sites. Structure analysis of refined complexes at 2.3 A and 3.0 A resolution.

Galanthus nivalis agglutinin (GNA, a 50 kDa tetramer) is a mannose-specific lectin of the Amaryllidaceae family of bulb lectins. Crystal structures of GNA complexed with methyl-alpha-D-mannose (MeMan) and mannose-alpha 1,3-D-mannose-alpha-OMe (MeMan-2) have been determined and analyzed in terms of internal structural symmetry and saccharide binding. The final model of the 2.29 A orthorhombic methyl-alpha-Man complex refined with an R-factor of 0.167 (all data) includes 12 bound sugar ligands and 327 water molecules. The four independent subunits (A, B, C and D) of the 222 tetramer and the three four-stranded beta-sheets (I,II and III) that constitute each subunit compare closely (r.m.s. delta = < 1.0 A). The 12 bound methyl-alpha-Man molecules refined with B-factors < 22 A2 and occupancies in the range of 0.5 to 1.0. The highest occupied site is located in beta-sheet I (site 1), where interactions from the dimer-related subunit contribute to complex stabilization. These subunit pairs (A-D and B-C) associate tightly with a buried surface area of 1738 A2 and 33 interchain hydrogen bonds resulting from C-terminal strand exchange. In comparison, the A-B and C-D subunit pairs have narrow interfaces (476 A2) and no direct H-bond contacts. The 3.0 A structure of the cubic Man-alpha 1,3-Man-OMe complex, determined by molecular replacement and refined with X-PLOR using NCS constraints and density modification methods, is less well ordered due to a high crystal solvent content (68%). Complexed disaccharide is responsible for the most crucial lattice contacts, which involve only one of the two independent subunits (A). The second subunit (C) shows a high degree of flexibility (Bav = 41.7 A2). The complete disaccharide molecule is visible in both subunits at site 3, which is the only extended site. The ligand is oriented with its reducing end positioned in the specificity pocket. The non-reducing manose is in contact through hydrogen bonding with a charged subsite (D37-K38) on the 2-fold-related subunit (A-B or C-D interfaces). Bound Man-alpha 1,3-MeMan is also well defined in site 2 of subunit A, as a result of favorable lattice contacts, while only the mannose residue bound in the specificity pocket is visible at site 2 of subunit C and site 1 of both subunits. Together these results suggest that strong binding correlates with the presence of subsidiary contacts coming either from a dimer-related subunit or from lattice interactions. Site 1 is most specific for terminal non-reducing or reducing mannose, while site 3 is extended and complementary to alpha-1,3 linked mannose oligosaccharides.

Amino Acid Sequence↗