concanavalin-a and metaperiodate

Mycobacterium tuberculosis, the primary causative agent of tuberculosis, infects macrophages and transforms the hostile intracellular environment into a permissive niche. M. tuberculosis infects macrophages using a variety of microbial ligand/cell receptor systems. In this study, binding assays with biotin-labelled mycobacterial cell wall proteins revealed five Concanavalin A-reactive proteins that bind macrophages. Among these proteins, we identified PstS-1, a 38-kDa M. tuberculosis mannosylated glycolipoprotein, and characterized it as an adhesin. Inhibition assays with mannan and immunoprecipitation demonstrated that PstS-1 binds the mannose receptor. We purified PstS-1 to 95.9% purity using ion exchange chromatography. The presence of mannose in purified PstS-1 was demonstrated by Concanavalin A interaction, which was abolished in the presence of sodium m-periodate and α-D-mannosidase. Gas chromatography revealed that purified PstS-1 contained 1% of carbohydrates by weight, which was mainly mannose. Finally, we used fluorescent microbeads coated with purified PstS-1 in phagocytosis assays and discovered that microbead uptake was inhibited by the pre-incubation of cells with GlcNAc, mannan and α-methyl mannoside. The interaction of PstS-1 coated beads with the mannose receptor was confirmed by confocal colocalization studies that showed high Pearson and Manders's colocalization coefficients. Our findings contribute to a better understanding of the strategies M. tuberculosis uses to infect host cells, the critical first step in the pathogenesis of tuberculosis.

Topics: Acetylglucosamine; Acyltransferases; Adhesins, Bacterial; alpha-Mannosidase; Animals; Antigens, Bacterial; ATP-Binding Cassette Transporters; Bacterial Adhesion; Bacterial Proteins; Cell Line, Tumor; Cell Wall; Concanavalin A; Immunoprecipitation; Lectins, C-Type; Macrophages; Mannans; Mannose; Mannose Receptor; Mannose-Binding Lectins; Membrane Proteins; Methylmannosides; Mice; Mycobacterium tuberculosis; Periodic Acid; Phagocytosis; Protein Binding; Receptors, Cell Surface; Tuberculosis, Pulmonary

The synthesis of small glycoclusters with high affinity toward lectins is one of the important subjects in glycotechnology. Although cyclic α-(1→6)-d-octaglucoside (CI8) is an attractive scaffold on which to put glycosyl pendants, the compound has only secondary hydroxyl groups, which are relatively unreactive for substitution reactions. The oxidation of the vicinal diols of CI8 and reductive amination of the resultant dialdehydes with 2-aminoethyl mannoside gave mannose-CI8 conjugates with a variety of average mannose incorporation numbers (2-7). The average numbers were deduced from MALDI-TOF mass and (1)H NMR spectroscopy. The binding ability of mannose-CI8 conjugates to concanavalin A increased with the increasing numbers of average mannose incorporation, reaching a plateau at tetravalence, as estimated from a latex bead-based agglutination lectin assay. Toxicity tests demonstrated the biocompatibility of mannose-CI8 conjugates.

Topics: Concanavalin A; Glucosides; Glycoconjugates; Lectins; Molecular Structure; Oxidation-Reduction; Periodic Acid

Concanavalin A and anti-alpha-D-glucose antibodies form precipitin complexes with antigens having alpha-D-glucose as terminal units. The sedimentation rates, molecular weights, gel electrophoretic mobilities, isoelectric points, and immunoglobulin type of Con A and alpha-Ab have been determined. The interactions of the compounds with antigens in the presence of potential inhibitors have been compared. The data show that the interaction of Con A with glucose units occurs with hydrogen bonding at hydroxyl groups at C1,3,4, and 6 and van der Waals bonding at the pyranose ring oxygen. In the alpha-Ab complex with glucose units, in addition to the above bond types, a hydrogen bond at the hydroxyl at C2 occurs and this bond is essential for interaction.

Topics: Antibodies; Antigen-Antibody Complex; Binding Sites; Centrifugation, Density Gradient; Concanavalin A; Electrophoresis, Polyacrylamide Gel; Glucose; Glycated Serum Albumin; Glycation End Products, Advanced; Glycogen; Hydrogen Bonding; Immunodiffusion; Periodic Acid; Serum Albumin

Starches, isolated from rapidly expanding tobacco leaves four times during the day and night and once from fully expanded leaves, were fractionated with concanavalin A. From an examination of the amounts and properties of amylose, the diurnal decrease in iodine absorption of the starches on illumination appeared to be due to an increase in its branched character, and possibly the presence of unbranched polymer of low dp, combined with a decrease in the proportion of amylose fraction. The increase in apparent amylose content with age was due to an increase in the proportion of amylose. The amylose fractions at different times had only small differences in average mol size in solution and relative mol wt (M(r) near 4 x 10(5)) which were lower than those of storage organs. The average mol size in solution and relative mol wt of the amylopectins decreased during illumination, increased in darkness, and were lower (M(r) 2-2.7 x 10(6)) at all times than those from storage organs. Debranching followed by size-exclusion chromatography [on Fractogel TSK 50(S)] gave similar proportions of long, medium, and short chains for all amylopectin samples, and these proportions differed from those for debranched amylopectin from n-maize seed starch. On debranching and chromatography of the amylopectin beta-limit dextrins (which gives an estimate of the proportions of core chains) differences persisted. Structural characteristics of amylopectin from tobacco leaf starch were similar to those of normal genotypes from storage organs. The proportion of glucosyl units in core chains, the external-to-core chain ratio, and indices of compactness were calculated for a number of (1-->4)(1-->6) alpha-glucans. A plot of the index of compactness for glycogens and amylopectins showed that the decrease in compactness and the increase in total average chain length that occurs from glycogen to normal and then to amylose extender amylopectins involves a proportionate increase in average internal, external, and core chain lengths and not a selective increase in one type of chain.

Topics: Amylopectin; Amylose; Borohydrides; Cell Fractionation; Chromatography, Gel; Circadian Rhythm; Concanavalin A; Dextrins; Nicotiana; Periodic Acid; Plant Lectins; Plants, Toxic; Starch

The effect of the monocyte locomotion inhibitory factor (MLIF) produced by Entamoeba histolytica is diminished, if not cancelled, when human monocytes are pre-exposed to concanavalin A or sodium periodate, respectively, but not when MLIF is pretreated with sodium periodate. When the MLIF-inhibited monocyte locomotion assays were performed in the presence of 12 different carbohydrates, only the runs containing D-mannose, 4-O-beta-galactosyl-mannoside or mannan revealed a significant reduction in the inhibitory effect. Finally, the MLIF activity was virtually absorbed out with mannan-coupled Sepharose 4B beads. This suggests that D-mannose constitutes an essential part of the receptor for MLIF on the human monocyte membrane.

Topics: Animals; Biological Factors; Cell Movement; Chemotaxis, Leukocyte; Concanavalin A; Entamoeba histolytica; Humans; Mannose; Monocytes; N-Formylmethionine Leucyl-Phenylalanine; Periodic Acid

Two different methods of detecting electroblotted glycoproteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Tris-buffer soluble barley seed proteins were examined for their applicability for barley cultivar discrimination. These are the highly specific, lectin-based concanavalin A/peroxidase method and the more general periodate/danyslhydrazine method. The results of the periodate/dansylhydrazine method enabled us to divide the 20 examined cultivars into three groups, whereas the more sensitive concanavalin A/peroxidase method revealed six different glycoprotein patterns. In comparison, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining of the alcohol-soluble barley seed proteins (hordeins) gave nine different banding patterns. A combination of hordein electrophoresis together with glycoprotein staining by the concanavalin A/peroxidase method made it possible to classify the cultivars into twelve groups, the largest of which contained four cultivars. The qualitative expression of the glycoprotein patterns seemed to be independent of growth conditions, whereas the band intensities obviously were not. As a whole, glycoprotein blotting is a valuable supplement to sodium dodecyl sulfate-polyacrylamide gel electrophoresis of hordeins in barley cultivar discrimination.

Topics: Concanavalin A; Dansyl Compounds; Electrophoresis, Polyacrylamide Gel; Glutens; Glycoproteins; Hordeum; Horseradish Peroxidase; Hydrazines; Periodic Acid; Plant Lectins; Plant Proteins; Seeds; Staining and Labeling

In this paper we present first the results of our most recent investigations on gravitational effects on the activation of human lymphocytes: by immunoenzymatic staining and by using concanavalin A (Con A) coated to red blood cells (RBC) we demonstrate that the increase of activation measured at 10xg is due to a simultaneous activation of T- and B-lymphocytes whereas at 1xg only T-cells are stimulated. Conversely, activation of T-cells by chemical modification of the membrane with sodium periodate is depressed at 10xg. Secondly, experiments performed in the centrifuge as well as in the clinostat with Friend, K-562, and hybridoma cells show that each cell line develops its own adaptation reaction to gravitational stress.

Topics: B-Lymphocytes; Cells, Cultured; Centrifugation; Concanavalin A; Dimethyl Sulfoxide; Erythrocytes; Friend murine leukemia virus; Hemin; Humans; Hybridomas; Hypergravity; Leukemia, Erythroblastic, Acute; Lymphocyte Activation; Mitogens; Periodic Acid; Rotation; T-Lymphocytes; Tumor Cells, Cultured

The production of bovine IL 2 was studied and IL 2 was partially characterized. PMA at 5 ng/ml + Concanavalin A at 5 micrograms/ml treatment of peripheral blood mononuclear cells gave a greater yield of IL 2 activity in the supernatants than Con A, PMA or sodium periodate treatments alone. Macrophage depletion increased yields as did the addition of indomethacin, a prostaglandin E2 inhibitor. Bovine IL 2 was sensitive to trypsin, relatively stable at pH 2-9, 2-ME resistant and sensitive to increasing molar concentrations of urea. The activity of bovine IL 2 was reduced by over 45% at 70 degrees C for 30 min and 95% at 90 degrees C for 30 min. Bovine IL 2 was more stable at 4 degrees C than at room temperature and the stability at room temperature could be improved by inclusion of 1% BSA. Bovine IL 2 eluted from DEAE-Sephadex as a broad peak with 0.1-0.2 M NaCl. Peak activity corresponded to a molecular weight of approximately 16,000 daltons on Sephadex G-100.

Topics: Animals; Cattle; Cell Adhesion; Cell Separation; Concanavalin A; Cross Reactions; Drug Stability; Guinea Pigs; Hot Temperature; Humans; Hydrogen-Ion Concentration; Interleukin-2; Periodic Acid; Sheep; Species Specificity; Tetradecanoylphorbol Acetate; Trypsin

Highly purified rat Ia-negative (OX-6-) and Ia-positive (OX-6+) T cells were employed to examine the requirement for accessory cells (AC) and/or soluble factors in the activation of resting T cells with Con A, PHA, sodium periodate, or antigen. A variety of cells were employed as AC, including Ia-positive and Ia-negative macrophages (M phi), gamma-irradiated (2000 rad) or non-irradiated OX-6+ T cells, and several Ia-negative adenovirus-transformed rat embryo fibroblast cell lines. Our results suggested that for the expression of IL-2 receptors (IL-2R) and proliferation of OX-6- T cells in response to Con A, PHA, or antigen, there was an obligatory requirement for the presence of AC which could not be overcome by the addition of IL-1 and/or IL-2. Activation of OX-6- T cells with antigen required the presence of Ia+ AC, while activation with mitogens could be initiated with Ia- AC. M phi were efficient in AC function in all responses tested, while the AC function of OX-6+ T cells (TAPC) proved discriminatory under different conditions. The optimal response to PHA required much higher concentrations of TAPC as AC than for the Con A response. TAPC failed to stimulate sodium periodate-treated T cells under any conditions tested. Furthermore, when TAPC were employed as AC, their antigen-presenting ability was radiosensitive, while their AC function for Con A and PHA was radioresistant. These results suggest that molecules involved in T cell-AC interactions may differ, depending on the source of AC and/or type of the proliferative stimulus provided to T cells. This data has been discussed in the context of T-cell activation.

Topics: Animals; Antigen-Presenting Cells; Concanavalin A; Histocompatibility Antigens Class II; Interleukin-1; Interleukin-2; Lymphocyte Activation; Macrophages; Periodic Acid; Phytohemagglutinins; Rats; Rats, Inbred F344; Receptors, Immunologic; Receptors, Interleukin-2; T-Lymphocytes

Plastic-embedded bone marrow biopsies from four patients with Gaucher's disease have been studied histochemically. Concanavalin A (ConA) was found to bind to cytoplasmic inclusions of Gaucher cells; the binding was prevented by lipid extraction or beta-glucosidase digestion. This suggests that glucocerebrosides stored in Gaucher cells are responsible for ConA binding; ConA staining combined with lipid extraction and beta-glucosidase digestion tests may be taken as a tool for the demonstration of Gaucher's cerebrosides of possible practical importance in diagnosis and investigation of Gaucher's disease. An excess of vic-glycol groups with respect to ConA binding-sugar residues and not extractable by lipid solvents are demonstrable in Gaucher cells. Vic-glycols appear to be regularly arranged at the electron microscopy level within Gaucher cell lysosomes along typical Gaucher "tubules", where some kind of interaction between lipid and protein should occur. Acid phosphatase might be one protein species involved in such interaction.

Topics: Acid Phosphatase; beta-Glucosidase; Bone Marrow; Concanavalin A; Gaucher Disease; Glucosylceramides; Histocytochemistry; Humans; Inclusion Bodies; Lysosomes; Methenamine; Microscopy, Electron; Periodic Acid; Silver; Staining and Labeling

Topics: Animals; Carbohydrates; Chemotaxis, Leukocyte; Concanavalin A; Entamoeba histolytica; Humans; Leukocyte Migration-Inhibitory Factors; Lymphokines; Monocytes; Periodic Acid

Complex carbohydrates in secretory granules and at the apical cell surface of mouse gastric mucoid cells were studied during embryogenesis and in the early postnatal period by various cytochemical methods; the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) and tannic acid-uranyl acetate (TA-UA) procedures made neutral mucosubstances (NMS) visible, whereas the hexose residues of glycoconjugates were identified using WGA-, RCA II- and ConA-ferritin. The glycocalyx was stained with ruthenium red (RR). During differentiation of the embryonic mucoid cells the number of secretory granules increased in parallel to the increase in their carbohydrate component. NMS-stainable parts in secretory granules also had binding sites for the conjugates RCA II- and WGA-ferritin, but the binding of ConA could not be identified. The increasing quantity of NMS in secretory granules was correlated with the increased amount of PA-TCH-SP and TA-UA positive substances in the apical glycocalyx only in 14- and 18-day-old embryos. The observed uniform affinity for RR and lectin conjugates in all analysed developmental stages remains to be explained.

Topics: Animals; Animals, Newborn; Cell Membrane; Concanavalin A; Cytoplasmic Granules; Embryo, Mammalian; Ferritins; Gastric Mucosa; Glycosaminoglycans; Histocytochemistry; Horseradish Peroxidase; Hydrazines; Hydrolyzable Tannins; Lectins; Mice; Organometallic Compounds; Periodic Acid; Plant Lectins; Ruthenium Red; Silver Proteins; Staining and Labeling; Uranium; Wheat Germ Agglutinins

Attempts to target antibody-ricin conjugates (immunotoxins) to designated cell types in vivo may be thwarted by their rapid clearance by hepatic reticuloendothelial cells which have receptors that recognise oligosaccharide side chains on the toxin. The B-chain of ricin contains high mannose type oligosaccharides and the A-chain contains a complex unit (GlcNAc)2-Fuc-Xyl-(Man)4-6, all of which potentially could be recognised by the reticuloendothelial system. Treatment of ricin with a mixture of sodium metaperiodate and sodium cyanoborohydride at pH 3.5 resulted in oxidative cleavage of the carbohydrates and reduction of the aldehyde groups thus formed to primary alcohols. By conducting the modification procedure at acidic pH, both the possibility of Schiff's base formation between the aldehyde groups and amino groups in the protein and the possibility of non-specific oxidation of amino acids were minimised. The extent of the carbohydrate modification depended on the duration of treatment, resulting maximally in the destruction of 13 of the 18 mannose residues and of all xylose and fucose. The toxicity of the modified toxin to cells in culture declined by up to 90% as the carbohydrate was destroyed. This was not due to a reduced ability of the B-chain to bind to cells or of the A-chain to inactivate ribosomes. In contrast to the in vitro results, the toxicity of the modified toxin to mice and rats was elevated by up to fourfold. The modification greatly reduced the clearance of the toxin by non-parenchymal cells in the liver and prevented the damage to hepatic Kupffer and sinusoidal cells and to the red pulp of the spleen that is inflicted by the native toxin. The elevated toxicity to animals appears to be because the modified toxin evades the reticuloendothelial system and persists in the bloodstream for longer periods, thus resulting in lethal damage to vital tissues in the animal at lower dosage. The results suggest that immunotoxins prepared from modified ricin would not be readily cleared by the reticuloendothelial system and so be more effective at killing their target cells.

Topics: Amino Acids; Animals; Borohydrides; Carbohydrates; Cells, Cultured; Chemical Phenomena; Chemistry; Concanavalin A; Leucine; Liver; Male; Mice; Mice, Inbred AKR; Mice, Inbred CBA; Oxidation-Reduction; Periodic Acid; Rats; Rats, Inbred Strains; Ricin; Tissue Distribution

Sialic acid residues of plasma membrane glycoproteins of rabbit corneal endothelial cells were radiolabeled by oxidation with sodium periodate and reduction with sodium borotritide. Surface-labeled glycoproteins were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The major surface labeled glycoproteins were designated GP 1-8 in order of their increasing mobility on the gel (M.W. = 220K (GP-1), 200K (GP-2), 170K (GP-3), 135K (GP-4), 110K (GP-5), 95K (GP-6), 80K (GP-7), and 44K (GP-8). On the basis of the behavior of these glycoproteins on various carrier-bound lectins, preliminary information concerning their saccharide moieties was obtained. All 8 components bound to agarose-linked wheat germ agglutinin; GP 4-6 bound to concanavalin A and GP 6-7 bound to Ricinus communis agglutinin. No component bound to Bandeiraea simplicifolia I, Bandeiraea simplicifolia II, Ulex europeus or soybean agglutinin. These data suggest that in addition to the presence of sialic acid/N-acetylglucosamine residues in all the eight glycoproteins, oligosaccharides with terminal beta-galactose residues occur in GP-6 and GP-7 while mannose (glucose) residues occur in GP 4-6.

Topics: Animals; Borohydrides; Chromatography, Affinity; Concanavalin A; Cornea; Culture Techniques; Electrophoresis, Polyacrylamide Gel; Endothelium; Lectins; Membrane Proteins; Periodic Acid; Rabbits; Sialoglycoproteins

Rat thymus cells were fractionated by centrifugation on a discontinuous bovine serum albumin gradient into two subpopulations: one of high density that accounted for greater than 90% of the recovered cells, and a minor low-density subpopulation containing 4 to 10% of the total cells. The high-density subpopulation consisted mainly of uniform small-sized thymocytes, whereas the low-density subpopulation contained mostly larger-sized cells. High-density thymus cells did not function either as stimulators in a mixed leukocyte reaction or as accessory cells required for T-cell response to mitogens, Con A and sodium periodate, as determined by 3H-thymidine incorporation. Dense thymus cells also responded poorly to allogeneic and to mitogenic stimulation, even when accessory cells were added. In contrast, the low-density thymus cells responded well to allogeneic stimulation and to both mitogens. In addition, low-density thymus cells possessed stimulatory activity in mixed leukocyte cultures, as well as accessory activity for mitogenic responses. Both activities were found to reside in dendritic cells that were purified extensively (70-90% of the preparation) with good yield. When tested as accessory cells for T-cell responses to periodate, thymic dendritic cells were as potent as lymph node dendritic cells on a per cell basis. A small number of thymic dendritic cells was able to cause marked enhancement in T-lymphocyte proliferation in response to stimulation. By immunofluorescence thymic dendritic cells were shown to be Ia-positive, but Thy 1.1 -negative.

Topics: Animals; Antigens, Surface; Cell Separation; Concanavalin A; Lymph Nodes; Lymphocyte Activation; Lymphocyte Cooperation; Lymphocyte Culture Test, Mixed; Male; Membrane Proteins; Periodic Acid; Rats; Rats, Inbred BUF; Rats, Inbred Lew; T-Lymphocytes; Thy-1 Antigens; Thymus Gland

Rat spleen cells were mitogenically stimulated with concanavalin A (Con A) or sodium periodate and cultured for 14-20 hr with trypsin. When these trypsin-activated cells were co-cultured with fresh mitogenically stimulated cells, [3H]-thymidine incorporation into the fresh cells was suppressed. Artifactual and trivial effects of trypsin (e.g. increased release of cold thymidine, decreased cell viability, and a change in thymidine transport) could not account for the suppressor effect. Suppressor activity was not affected by removing B cells or macrophages before trypsin activation nor by treating the trypsinized cells with mitomycin C. Suppressor activity could only be generated when the spleen cells were stimulated with Con A or periodate during culture with trypsin, and supernates from the trypsin-activated cells did not have suppressor activity. The physiological significance of the results and possible mechanisms of action of the suppressor cells are discussed.

Topics: Animals; Cells, Cultured; Concanavalin A; Interleukin-2; Lymphocyte Activation; Male; Periodic Acid; Rats; Rats, Inbred Strains; Spleen; T-Lymphocytes; T-Lymphocytes, Regulatory; Thymidine; Trypsin

Suppressor cells were assayed by numerical and functional tests in adults on chronic hemodialysis. Peripheral blood mononuclears (PBM) were classified as total T-cells by E-rosettes and by the monoclonal antibody OKT3, as T-cell subsets by OKT4 (inducer/helper T-cells) and OKT8 (cytotoxic/suppressor T-cells) and as B-cells by the presence of surface immunoglobulin. The suppressive effect of PBM pretreated with either Concanavalin A (Con A), sodium periodate, or serum rich in immune complexes, on normal homologous phytohemagglutinin (PHA) lymphocyte transformation, was determined. Usual tests of T-cell function were not done. T lymphopenia was due to significant diminution (P less than 0.002) in numbers of OKT4+ cells in patients (516 +/- 44 cells/mm3, mean +/- sem) as compared to controls (906 +/- 96 cells/mm3). The number of OKT8+ cells in patients was not different from normal although their percentage (45 +/- 4%) was slightly higher than controls (36 +/- 5%) (P less than 0.10). Suppressor activity using only a suboptimal dose of Con A (5 micrograms/ml), was significantly lower (P less than 0.002) in uremic patients (36 +/- 12%) than in controls (67 +/- 7%). An important finding was that no significant correlations were detected between the numerical and functional assays of suppression used or between any of these immunological tests and biochemical parameters studied. The implications of these results for immunoparesis in uremia are discussed with particular reference to the discordance between marker and functional assays of suppressor cells.

Topics: Adolescent; Adult; Antibodies, Monoclonal; Antigen-Antibody Complex; Concanavalin A; Female; Humans; Kidney Failure, Chronic; Lymphocyte Activation; Male; Middle Aged; Periodic Acid; Phytohemagglutinins; Rosette Formation; T-Lymphocytes, Regulatory; Uremia

Microsomal and lysosomal beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) of monkey brain were differentially eluted from Con A-Sepharose when subjected to chromatography and linear gradient elution with methyl alpha-glucoside at 28+/-1 degree C. The lysosomal enzyme was eluted as a sharp peak in the first few fractions, while the microsomal enzyme was eluted as a broad peak extending over several fractions. This differential pattern of elution was dependent only on the temperature of elution and the concentration of methyl alpha-glucoside used. The lysosomal and microsomal glucuronidases were purified to apparent homogeneity and their neutral sugar analysed. Both of them contained glucose, mannose and fucose but the microsomal enzyme contained about 3-times as much of all these sugars as the lysosomal enzyme. Sodium periodate treatment of the microsomal enzyme resulted in a shift in its elution pattern, similar to the lysosomal enzyme when subjected to Con A-Sepharose chromatography. The content of neutral sugars and the structural features of the oligosaccharide units in the microsomal glucuronidase might be responsible for its elution pattern. A processing of the carbohydrate units of the microsomal glucuronidase might be envisaged to take place if it were to act as a precursor of the lysosomal glucuronidase.

Topics: Animals; Brain; Carbohydrates; Chromatography, Affinity; Concanavalin A; Glucuronidase; Lysosomes; Macaca radiata; Methylglucosides; Microsomes; Periodic Acid

Several agents that react with plasma membranes, namely the native lectins concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin, the modified lectin succinyl concanavalin A, and sodium meta-periodate, inhibited the ecto-5'-nucleotidase of intact guinea pig granulocytes. Stimulation of the enzyme was not observed at any lectin concentration. Inhibition by native lectins could be blocked or reversed by appropriate competing hapten sugars. In the case of concanavalin A, reversal could be achieved at 37 degrees C, but not at 5 degrees C. When lectins were used in combination with each other, the effects were found to be largely independent. However, when concanavalin A and R. communis agglutinin were applied together, complications arose because the former lectin binds to the latter as well as to the cell surface. To avoid some of the complexities inherent in studying intact cell 5'-nucleotidase and to gain additional information about the system, two broken cell enzyme preparations were also examined. The enzyme of plasma membrane-enriched fractions was inhibited by all five agents mentioned above. 5'-Nucleotidase solubilized in sodium deoxycholate was inhibited by the four lectins but stimulated by periodate. The effects of the surface modifiers on kinetic data for all three enzyme preparations are consistent with the hypothesis that direct interactions with the enzyme molecule give rise to changes in Vmax; interactions at membrane sites other than 5'-nucleotidase itself could cause increases in apparent Km values. Effects of interactions of ectoenzymes with plant lectins may serve as models for phenomena that result from cell-cell interactions or from interactions of animal cells with lectin-like components of the cellular environment.

Topics: 5'-Nucleotidase; Animals; Cell Membrane; Concanavalin A; Granulocytes; Guinea Pigs; Haptens; Kinetics; Lectins; Nucleotidases; Periodic Acid; Plant Lectins; Plant Proteins; Wheat Germ Agglutinins