Endogenous lectins from cultured soybean cells: isolation of a protein immunologically cross-reactive with seed soybean agglutinin and analysis of its role in binding of Rhizobium japonicum

Incubation of Rhizobium japonicum with the cultured soybean cell line SB-1, originally derived from the roots of Glycine max, resulted in specific adhesion of the bacteria to the plant cells. This binding interaction appears to be mediated via carbohydrate recognition, since galactose can inhibit the heterotypic adhesion but glucose cannot. Affinity chromatography, on a Sepharose column derivatized with N-caproyl-galactosamine, of the supernatant fraction of a SB-1 cell suspension after enzymatic removal of cell wall yielded a single polypeptide (Mr approximately 30,000) on immunoblotting analysis with rabbit antibodies directed against seed soybean agglutinin. Fluorescently labeled rabbit anti-seed soybean agglutinin also yielded specific immunofluorescent staining on the cell wall and plasma membrane of the SB-1 cells. These results suggest that one likely candidate that may mediate the recognition between the Rhizobium and the soybean cells is the endogenously produced SB-1 lectin. This notion is supported by the observation that rabbit anti-seed soybean agglutinin blocked the Rhizobium-soybean cell adhesion, whereas control antibodies did not.     

Carbohydrate binding activities of Bradyrhizobium japonicum. I. Saccharide-specific inhibition of homotypic and heterotypic adhesion

Bradyrhizobium japonicum (R110d) exhibited four saccharide-specific binding activities: (a) adsorption to Sepharose beads containing covalently coupled lactose; (b) homotypic agglutination through one pole of the cell (star formation); (c) heterotypic adhesion to the cultured soybean cell line, SB-1; and (d) attachment to roots of soybean plants. Each of these binding activities can be inhibited by the addition of galactose or lactose, but not by derivatives such as N-acetyl-D-galactosamine or melibiose. Treatment of wild-type bacteria with N-methyl-N'-nitro-N-nitrosoguanidine followed by selection on the basis of reduced binding to SB-1 cells, resulted in two specific mutants, designated N4 and N6. Compared to wild type, these two mutants also exhibited decreased binding activity in: (a) adsorption to lactose-Sepharose beads; (b) homotypic star formation; and (c) heterotypic attachment to roots of soybeans plants. These results suggest that all four of the saccharide-inhibitable binding activities of Bradyrhizobium japonicum may be mediated by the same mechanism(s) or molecular component(s).     

Bradyrhizobium japonicum glnB, a putative nitrogen-regulatory gene, is regulated by NtrC at tandem promoters.

The glnB gene from Bradyrhizobium japonicum, the endosymbiont of soybeans (Glycine max), was isolated and sequenced, and its expression was examined under various culture conditions and in soybean nodules. The B. japonicum glnB gene encodes a 12,237-dalton polypeptide that is highly homologous to the glnB gene products from Klebsiella pneumoniae and Escherichia coli. The gene is located directly upstream from glnA (encoding glutamine synthetase), a linkage not observed in enteric bacteria. The glnB gene from B. japonicum is expressed from tandem promoters, which are differentially regulated in response to the nitrogen status of the medium. Expression from the downstream promoter involves the B. japonicum ntrC gene product (NtrC) in both free-living and symbiotic cells. Thus, glnB, a putative nitrogen-regulatory gene in B. japonicum, is itself Ntr regulated, and NtrC is active in B. japonicum cells in their symbiotic state.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of N-acetyl-D-galactosamine residue on B151K12-derived T cell-replacing factor (B151-TRF) molecule in B cell-receptor binding and -stimulating activity

The role of sugar moiety on T cell-replacing factor molecule derived from a monoclonal T cell hybridoma B151K12 (B151-TRF) was analyzed with respect to the interaction with receptor on B cells. The induction of B cell differentiation into Ig-secreting cells by B151-TRF was specifically inhibited by addition of N-acetyl-D-galactosamine (GalNAc) to culture. Such inhibition appeared to be attributed to the interference of GalNAc in the interaction of TRF with its receptor, because absorption of TRF activity with B cells was notably inhibited by the presence of GalNAc. To substantiate this point further, we established binding assay of B151-TRF molecule to the receptor on B cells by using 125I-labeled TRF fraction enriched by reversed-phase high-performance liquid chromatography and gel filtration. The results revealed that the binding of 125I-TRF molecule to the B cells was almost completely blocked by GalNAc. Moreover, the existence of GalNAc residue(s) on B151-TRF molecule was evidenced by the facts that 1) the TRF activity was eluted from lectin gels with specificity for GalNAc as revealed by the functional assay, and 2) the 125I-TRF molecule specifically bound to such lectin gels. Thus, the GalNAc residue(s) on B151-TRF molecule plays an important role in binding of TRF molecule to the receptor and in the stimulation of B cells. The molecular properties of B cell-stimulatory B151-TRF and its mode of interaction with corresponding receptor on B cells were discussed in the context of B151-TRF as a glycosylated lymphokine molecule and B151-TRF receptor as a carbohydrate-binding protein (animal lectin).     

O-antigen from Bradyrhizobium japonicum lipopolysaccharide inhibits intercellular (symplast) communication between soybean (Glycine max) cells

The technique of fluorescence redistribution after photobleaching was utilized to measure intercellular movement of low molecular weight fluorescent hydrophilic substances across the cell wall/membrane interface between contiguous soybean (Glycine max (L.) Merr. cv. Mandarin) root cells (SB-1 cell line) in tissue culture. Lipopolysaccharide (LPS) purified from Bradyrhizobium japonicum R110d, a Gram-negative bacterium that normally infects and induces nodulation in soybean roots in vivo, inhibits intercellular communication between the soybean cells in a dose-dependent manner. In contrast, LPS from noninfecting strains failed to yield the same effect. The inhibitory activity of the LPS was localized to the O-antigen region of the LPS.     

In Vitro Binding of Agrobacterium tumefaciens to Plant Cells from Suspension Culture

In vitro binding experiments were carried out using (32)P-labeled cells of the virulent Agrobacterium tumefaciens strain B6 and Datura innoxia cells from suspension culture. Binding kinetics showed that adherence of bacteria to Datura cells increased gradually during the first 60 minutes and attained a maximum level within 120 minutes of incubation. Maximum binding occurred at pH 6.0. The presence of Ca(2+) and Mg(2+) reduced binding slightly and EDTA had little effect at concentrations of 0.1 to 10 millimolar. The binding of bacteria to Datura cells was temperature-dependent. Escherichia coli, Salmonella typhimurium, Rhizobium japonicum, and Micrococcus lysodeikticus did not compete with virulent A. tumefaciens strain B6 for binding to Datura cells. The admixture of avirulent A. tumefaciens strain IIBNV6 enhanced adherence of virulent A. tumefaciens strain B6 to Datura cells. Octopine had no effect on the binding of virulent A. tumefaciens strain B6 to Datura cells, but 10 millimolar canavanine was inhibitory. Arginine enhanced the adherence of the bacteria at concentrations higher than 0.1 millimolar. Incubation with DNase, RNase, and lipase did not affect the binding, but protease stimulated the adherence of bacteria to Datura cells. Concanavaline A and soybean lectin had little effect whereas lecithin and lysolecithin enhanced binding slightly. Poly-l-lysine markedly stimulated the bacteria-plant cell adherence. Cells from suspension cultures of pea, vetch, and soybean had a 2- to 3-fold higher binding capacity than Datura cells, whereas cells from wheat, corn, rice, and sorghum had a considerably lower affinity for binding with virulent A. tumefaciens strain B6. Bacterial adherence to plant cells was confirmed by autoradiography and electron microscopy. Autoradiographic analysis showed that bacteria were associated with the cell wall, and that often binding of bacteria was localized. Electron micrographs clearly illustrated a tight association of virulent A. tumefaciens strain B6 cells to the Datura cell wall.     

Catabolism of human low density lipoproteins by human hepatoma cell line HepG2

The mechanism of hepatic catabolism of human low density lipoproteins (LDL) by human-derived hepatoma cell line HepG2 was studied. The binding of 125I-labeled LDL to HepG2 cells at 4 degrees C was time dependent and inhibited by excess unlabeled LDL. The specific binding was predominant at low concentrations of 125I-labeled LDL (less than 50 micrograms protein/ml), whereas the nonsaturable binding prevailed at higher concentrations of substrate. The cellular uptake and degradation of 125I-labeled LDL were curvilinear functions of substrate concentration. Preincubation of HepG2 cells with unlabeled LDL caused a 56% inhibition in the degradation of 125I-labeled LDL. Reductive methylation of unlabeled LDL abolished its ability to compete with 125I-labeled LDL for uptake and degradation. Chloroquine (50 microM) and colchicine (1 microM) inhibited the degradation of 125I-labeled LDL by 64% and 30%, respectively. The LDL catabolism by HepG2 cells suppressed de novo synthesis of cholesterol and enhanced cholesterol esterification; this stimulation was abolished by chloroquine. When tested at a similar content of apolipoprotein B, very low density lipoproteins (VLDL), LDL and high density lipoproteins (HDL) inhibited the catabolism of 125I-labeled LDL to the same degree, indicating that in HepG2 cells normal LDL are most probably recognized by the receptor via apolipoprotein B. The current study thus demonstrates that the catabolism of human LDL by HepG2 cells proceeds in part through a receptor-mediated mechanism.     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

Studies on the binding and degradation of human very-low-density lipoproteins by human hepatoma cell line HepG2

The regulation of the hepatic catabolism of normal human very-low-density lipoproteins (VLDL) was studied in human-derived hepatoma cell line HepG2. Concentration-dependent binding, uptake and degradation of 125I-labeled VLDL demonstrated that the hepatic removal of these particles proceeds through both the saturable and non-saturable processes. In the presence of excess unlabeled VLDL, the specific binding of 125-labeled VLDL accounted for 72% of the total binding. The preincubation of cells with unlabeled VLDL had little effect on the expression of receptors, but reductive methylation of VLDL particles reduced their binding capacity. Chloroquine and colchicine inhibited the degradation of 125I-labeled VLDL and increased their accumulation in the cell, indicating the involvement of lysosomes and microtubuli in this process. Receptor-mediated degradation was associated with a slight (13%) reduction in de novo sterol synthesis and had no significant effect on the cellular cholesterol esterification. Competition studies demonstrated the ability of unlabeled VLDL, low-density lipoproteins (LDL) and high-density lipoproteins (HDL) to effectively compete with 125I-labeled VLDL for binding to cells. No correlation was observed between the concentrations of apolipoproteins A-I, A-II, C-I, C-II and C-III of unlabeled lipoproteins and their inhibitory effect on 125I-labeled VLDL binding. When unlabeled VLDL, LDL and HDL were added at equal contents of either apolipoprotein B or apolipoprotein E, their inhibitory effect on the binding and uptake of 125I-labeled VLDL only correlated with apolipoprotein E. Under similar conditions, the ability of unlabeled VLDL, LDL and HDL to compete with 125I-labeled LDL for binding was a direct function of only their apolipoprotein B. These results demonstrate that in HepG2 cells, apolipoprotein E is the main recognition signal for receptor-mediated binding and degradation of VLDL particles, while apolipoprotein B functions as the sole recognition signal for the catabolism of LDL. Furthermore, the lack of any substantial regulation of beta-hydroxy-beta-methylglutaryl-CoA reductase and acyl-CoA:cholesterol acyltransferase activities subsequent to VLDL degradation, in contrast to that observed for LDL catabolism, suggests that, in HepG2 cells, the receptor-mediated removal of VLDL proceeds through processes independent of those involved in LDL catabolism.     

Formation and function of the bean-rhizobium symbiosis in soy plants upon introduction of strains of Azotobacter and Bacillus species

The effects of bacteria belonging to the genera Azotobacter and Bacillus in a mixed culture with Bradyrhizobium japonicum strains on formation and function of the legume-rhizobium symbiosis of soybean plants were studied. The data showed that the bacterial compositions B. japonicum 634b + B. subtilis 5, B. japonicum 634b + A. chroococcum 20, and B. japonicum 10k + A. vinelandii 56 with a cell ratio of 1:0.1 increased the number and weight of root nodules as well as the height and weight of the aboveground plant parts in almost all the cases by 22-105% compared with the control variants. These binary microbial cultures may be used for development of combined bacterial preparations for soybean.     

Binding site for chitin oligosaccharides in the soybean plasma membrane.

Affinity cross-linking of the plasma membrane fraction to an (125)I-labeled chitin oligosaccharide led to the identification and characterization of an 85-kD, chitin binding protein in plasma membrane-enriched fractions from both suspension-cultured soybean cells and root tissue. Inhibition analysis indicated a binding preference for larger (i.e. degrees of polymerization = 8) N-acetylated chitin molecules with a 50% inhibition of initial activity value of approximately 50 nM. N-Acetyl-glucosamine and chitobiose showed no inhibitory effects at concentrations as high as 250 microM. It is noteworthy that the major lipo-chitin oligosaccharide Nod signal produced by Bradyrhizobium japonicum was also shown to be a competitive inhibitor of ligand binding. However, the binding site appeared to recognize the chitin portion of the Nod signal, and it is unlikely that this binding activity represents a specific Nod signal receptor. Chitooligosaccharide specificity for induction of medium alkalinization and the generation of reactive oxygen in suspension-cultured cells paralleled the binding activity. Taken together, the presence of the chitin binding protein in the plasma membrane fraction and the specificity and induction of a biological response upon ligand binding suggest a role for the protein as an initial response mechanism for chitin perception in soybean (Glycine max).     

Endogenous lectin from cultured soybean cells. Chemical characterization of the lectin of SB-1 cells

A lectin has been identified in the cell line, SB-1, originally derived from the roots of Glycine max. This lectin, which we shall refer to as SB-1 lectin, was isolated on the basis of its carbohydrate-binding activity (affinity chromatography on Sepharose column derivatized with N-caproyl-galactosamine) and its immunological cross-reactivity (immunoblotting with rabbit antibodies directed against seed soybean agglutinin (SBA]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis of SB-1 lectin revealed a major polypeptide (Mr approximately equal to 30,000) which co-migrated with seed SBA. This form of the lectin was observed in fractions purified from culture medium of SB-1 cells or supernatant fraction of SB-1 cell suspension after enzymatic removal of cell wall. Extracts of SB-1 cells under some other conditions yielded a major band (Mr approximately equal to 60,000) as revealed by SDS-PAGE and immunoblotting with rabbit anti-seed SBA; prolonged incubation of these samples in the presence of SDS resulted in the appearance of the 30-kDa polypeptide. It appears that the 60-kDa band represented a highly stable, even under SDS-PAGE conditions, dimeric form of the 30-kDa subunit. The SB-1 lectin derived from the culture medium was compared with seed SBA by gel filtration and by peptide mapping after limited proteolysis; no difference between the lectins from the two sources was found. Extracts of soybean roots fractionated on N-caproyl-galactosamine-Sepharose affinity columns yielded, upon elution with galactose, polypeptides of Mr 30,000 and 60,000. These results suggest that soybean roots contain a lectin whose polypeptide composition corresponds to that of seed SBA and SB-1 lectin.     

Analysis of lectin binding by Bradyrhizobium japonicum strains grown on nitrocellulose filters using peroxidase-labeled lectin

A procedure was developed to assess the ability of wild-type and mutant strains of Bradyrhizobium japonicum to bind soybean lectin. The lectin-binding ability of bacteria grown on nitrocellulose filters was determined using peroxidase-labeled soybean lectin. The assay produced clear differences between strains known to be unable to bind soybean lectin and those which can. The assay gave results identical to those of the fluorescein isothiocyanate-soybean lectin-binding assay of T. V. Bhuvaneswari, S. G. Pueppke, and W. D. Bauer (1977, Plant Physiol. 60, 486-491) with regard both to the ability of particular B. japonicum strains to bind lectin and to the inhibition caused by N-acetyl-D-galactosamine. The method was used to screen Tn5-induced mutants of B. japonicum 2143 for their inability to bind soybean lectin. The procedure provides a sensitive and convenient method to screen Bradyrhizobium strains for the ability to bind soybean lectin.     

Solubilization of the 17 alpha-ethinyl estradiol-stimulated low density lipoprotein receptor of male rat liver

Pharmacological doses of 17 alpha-ethinyl estradiol induce a low density lipoprotein (LDL) receptor in the liver of male rats. Our aim was to solubilize this receptor. Isolated liver membranes (8,000-100,000 g fraction) from male rats treated with 17 alpha-ethinyl estradiol and from control rats were solubilized in 1% (w/v) Triton X-100. Using Amberlite XAD-2, more than 90% of the detergent was then removed. Liposomes were prepared by precipitating the solubilized proteins with acetone in the presence of phosphatidylcholine. The receptor activity of these liposomes was assayed using human 125I-labeled LDL. Filtration was used to separate bound from free 125I-labeled LDL. The assay was optimized; 0.25 mM CaCl2, 25 mM NaCl, pH 8.0, were chosen as the standard conditions. Binding of 125I-labeled LDL was dependent on Ca2+. Liposomes containing solubilized membrane proteins from treated rats displayed Ca2+-dependent binding which was 11 times higher than for control rats. The specific binding of 125I-labeled LDL was saturable with a Kd = 18 micrograms/ml. 125I-Labeled LDL was displaced by unlabeled lipoproteins containing apolipoproteins B and E and by dimyristoylphosphatidylcholine liposomes containing purified apolipoprotein E, but not by HDL3. The binding was abolished by pronase and was inhibited by suramin. Ligand blotting with 125I-labeled LDL revealed one band of protein with an apparent molecular weight of 133,000 daltons. These properties are characteristic of the low density lipoprotein receptor.     

Binding and degradation of human high-density lipoproteins by human hepatoma cell line HepG2

The catabolism of human HDL was studied in human hepatoma cell line HepG2. The binding of 125I-labeled HDL at 4 degrees C was time-dependent and reached completion within 2 h. The observed rates of binding of 125I-labeled HDL at 4 degrees C and uptake and degradation at 37 degrees C indicated the presence of both high-affinity and low-affinity binding sites for this lipoprotein density class. The specific binding of 125I-labeled HDL accounted for 55% of the total binding capacity. The lysosomal degradation of 125I-labeled HDL was inhibited 25 and 60% by chloroquine at 50 and 100 microM, respectively. Depolymerization of microtubules by colchicine (1 microM) inhibited the degradation of 125I-labeled HDL by 36%. Incubation of cells with HDL caused no significant change in the cellular cholesterol content or in the de novo sterol synthesis and cholesterol esterification. Binding and degradation of 125I-labeled HDL was not affected by prior incubation of cells with HDL. When added at the same protein concentration, unlabeled VLDL, LDL and HDL had similar inhibitory effects on the degradation of 125I-labeled HDL, irrespective of a short or prolonged incubation time. Reductive methylation of unlabeled HDL had no significant effect on its capacity to inhibit the 125I-labeled HDL degradation. The competition study indicated no correlation between the concentrations of apolipoproteins A-I, A-II, B, C-II, C-III, E and F in VLDL, LDL and HDL and the inhibitory effect of these lipoprotein density classes on the degradation of 125I-labeled HDL. There was, however, some association between the inhibitory effect and the levels of apolipoprotein D and C-I.     

Expression of IL-1 receptors on human peripheral B cells

The expression of IL-1R on human peripheral B cells was analyzed by the binding assay with 125I-labeled human rIL-1 alpha and by the flow cytofluorometry by the use of FITC-conjugated IL-1 alpha. The proliferation and the differentiation of B cells stimulated with Staphylococcus aureus Cowan I (SAC) in the presence of T cell-derived factors were dependent on IL-1. By the binding experiment with 125I-labeled IL-1 alpha, B cells expressed only few IL-1R without any stimulations. When they were stimulated with SAC, IL-1R on B cells began to increase by only 1 h, reached the maximum level at 6 h. The binding of 125I-labeled IL-1 alpha to B cells was inhibited by the addition of either cold IL-1 alpha or IL-1 beta suggesting that IL-1R on B cells reactive for IL-1 alpha and IL-1 beta were identical. By Scatchard plot analysis, the existence of two classes of IL-1R on B cells was found. A major class of IL-1R (320 molecules/cell) has a lower affinity (Kd = 3.8 x 10(-10) M) and a minor class of IL-1R (70 molecules/cell) has a higher affinity (Kd = 4.4 x 10(-12) M). When B cells were stimulated with SAC, both lower and higher affinity IL-1R were increased to 1960 molecules/cell and 300 molecules/cell, respectively. Furthermore, IL-1R on B cells were also detected with FITC-conjugated IL-1 alpha by a flow cytofluorometer. Only 3 to 5% of B cells expressed IL-1R without any stimulations. When B cells were stimulated with SAC, IL-1R-positive B cells were increased to 20%. The addition of anti-class II antibodies inhibited B cell proliferation and differentiation induced with SAC, IL-1, and T cell-derived factors. Anti-class II antibodies also inhibited the number of IL-1R on B cells. These results suggest that the expression of IL-1R was induced as the initial stage of B cell activation and that class II Ag play an important role for the expression of IL-1R on B cells.     

Role of lectins in plant-microorganism interactions: I. Binding of soybean lectin to rhizobia

Highly purified soybean lectin (SBL) was labeled with fluorescein isothiocyanate (FITC-SBL) or tritium ((3)H-SBL) and repurified by affinity chromatography. FITC-SBL was found to bind to living cells of 15 of the 22 Rhizobium japonicum strains tested. The lectin did not bind to cells of the other seven R. japonicum strains, or to cells of any of the nine Rhizobium strains tested which do not nodulate soybean. The binding of the lectin to the SBL-positive strains of R. japonicum was shown to be specific and reversible by hapten inhibition with d-galactose or N-acetyl-d-galactosamine.The lectin-binding properties of the SBL-positive R. japonicum strains were found to change substantially with culture age. The percentage of cells in a population exhibiting fluorescence after exposure to FITC-SBL varied between 0 and 70%. The average number of SBL molecules bound per cell varied between 0 and 2 x 10(6). While most strains had their highest percentage of SBL-positive cells and maximum number of SBL-binding sites per cell in the early and midlog phases of growth, one strain had a distinctly different pattern. The SBL-negative strains did not bind lectin at any stage of growth.Quantitative binding studies with (3)H-SBL indicated that the affinity constant for binding of SBL to its receptor sites on R. japonicum is approximately 4 x 10(7)m(-1). Many of the binding curves were biphasic. An inhibitor of SBL binding was found to be present in R. japonicum culture filtrates.     

Identification of the insulin receptor in undifferentiated and differentiated NB-15 mouse neuroblastoma cells by affinity labeling

Plasma membranes prepared from clonal NB-15 mouse neuroblastoma cells were sequentially incubated with 125I-labeled insulin (10 nM) and the bifunctional cross-linking agent disuccinimidyl suberate. This treatment resulted in the cross-linking of 125I-labeled insulin to a polypeptide that gave an apparent Mr of 135 000 on a sodium dodecyl sulfate-polyacrylamide gel electrophoresed in the presence of 10% beta-mercaptoethanol. Affinity labeling of this polypeptide was inhibited by the presence of 5 microM unlabeled insulin, but not by 1 microM unlabeled nerve growth factor. Using the same affinity labeling technique, 125I-labeled nerve growth factor (1 nM) did not label any polypeptide appreciably in the plasma membranes of NB-15 cells but labeled an Mr 145 000 and an Mr 115 000 species in PC-12 rat pheochromocytoma cells. The number of insulin binding sites per cell in the intact differentiated NB-15 mouse neuroblastoma cells was approx. 6-fold greater than that in the undifferentiated NB-15 mouse neuroblastoma cells as measured by specific binding assay, suggesting an increase of the number of insulin receptors in NB-15 mouse neuroblastoma cells during differentiation.     

Localization and partial characterization of soybean lectin-binding polysaccharide of Rhizobium japonicum.

Immunoelectron microscopy was combined with partial characterization of isolated exopolysaccharide to study binding of soybean lectin by Rhizobium japonicum strain USDA 138. Lectin-binding activity resided in two forms of exopolysaccharide produced during growth: an apparently very high-molecular-weight capsular form and a lower-molecular-weight diffusible form. At low-speed centrifugation, the capsular form cosedimented with cells to form a viscous, white, cell-gel complex which was not diffusible in 1% agar, and the diffusible form remained in the cell-free supernatant. Electron microscopic observation of the cell-gel complex after labeling with soybean lectin-ferritin conjugate revealed that capsular polysaccharides, frequently attached to one end of the cells, were receptors for lectin. The outer membrane of the cell bound no lectin. Various preparations of exopolysaccharide isolated from the culture supernatant were tested for lectin binding, interaction with homologous somatic antigen, and the presence of 2-keto-3-deoxyoctonate and were chromatographed in Sepharose 4B and 6B gel beds. Lectin binding was restricted to a polysaccharide component designated as lectin-binding polysaccharide. This polysaccharide, as present in the cell-free culture supernatant, was a diffusible acidic polysaccharide devoid of 2-keto-3-deoxyoctonate, with a molecular weight of 2 X 10(6) to 5 X 10(6). It was concluded that the soybean lectin-binding component of R. japonicum is an extracellular polysaccharide and not a lipopolysaccharide and that the diffusible lectin-binding polysaccharide probably differs from the very high-molecular-weight lectin-binding polysaccharide of the loose capsule (slime) only in the degree of polymerization.