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1.
R S Haltiwanger M A Lehrman A E Eckhardt R L Hill 《The Journal of biological chemistry》1986,261(16):7433-7439
A small-scale affinity chromatographic procedure was developed to screen for the presence of fucose and mannose/N-acetylglucosamine-binding lectins in small amounts of rat tissues. Of all tissues examined, only the liver contained the fucose-binding lectin, whereas both liver and blood serum contained the mannose/N-acetylglucosamine lectin. By means of immunocytological methods using antibodies to hepatic lectins, the fucose lectin was shown to be uniquely present in Kupffer cells and absent in all other types of rat macrophages examined. The binding and uptake of different neoglycoproteins by nonparenchymal cell fractions of liver indicated that the fucose-binding lectin was either not responsible for the uptake or that more than one lectin was acting. With the identification of another lectin (Mr = 180,000) by the above screening procedure for hepatic lectins and the results of studies in the following paper (Haltiwanger, R.S., and Hill, R. L. (1986) J. Biol. Chem. 261, 7440-7444) two lectins appear to be involved. A small amount of the hepatic mannose/N-acetylglucosamine lectin was found by the above screening procedure to have a higher affinity for L-fucosyl-bovine serum albumin-Sepharose than the majority of the lectin in hepatocytes. This lectin, called the high affinity form, was purified and its properties examined. On a weight basis the high affinity form bound 7-12 times more ligand than the normal form. Its Ka for L-fucosyl-bovine serum albumin was 2.3 X 10(9) M-1 compared to 3.5 X 10(8) M-1 for the normal form. Moreover, the concentrations of monosaccharides required to inhibit the high affinity form were about 3 times less than those required to inhibit binding of the normal form. The two forms, however, have identical molecular weights (32,000) under reducing and nonreducing conditions, bind anti-lectin antibodies in the same way, and give identical peptide maps after V-8 protease digestion. The structural basis for the different binding affinities of the two forms remains unknown. 相似文献
2.
Kawsar SM Fujii Y Matsumoto R Ichikawa T Tateno H Hirabayashi J Yasumitsu H Dogasaki C Hosono M Nitta K Hamako J Matsui T Ozeki Y 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》2008,150(4):349-357
A lectin recognizing both Galbeta1-3GlcNAc and Galbeta1-4GlcNAc was purified from the demosponge Halichondria okadai by lactosyl-agarose affinity chromatography. The molecular mass of the lectin was determined to be 30 kDa by SDS-PAGE under reducing and non-reducing conditions and 60 kDa by gel permeation chromatography. The pI value of the lectin was 6.7. It was found to agglutinate trypsinized and glutaraldehyde-fixed rabbit and human erythrocytes in the presence and absence of divalent cations. The hemagglutinating activity by the lectin was inhibited by d-galactose, methyl-d-galactopyranoside, N-acetyl-d-galactosamine, methyl-N-acetyl-d-galactosaminide, lactose, melibiose, and asialofetuin. The K(d) of the lectin against p-nitrophenyl-beta-lactoside was determined to be 2.76x10(-5) M and its glycan-binding profile given by frontal affinity chromatography was shown to be similar to many other known galectins. Partial primary structure analysis of 7 peptides by cleavage with lysyl endopeptidase indicated that one of the peptides showed significant similarity with galectin purified from the sponge Geodia cydonium. 相似文献
3.
Identification and further characterization of the specific cell binding fragment from sponge aggregation factor 总被引:5,自引:0,他引:5
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M Gramzow M Bachmann G Uhlenbruck A Dorn W E Müller 《The Journal of cell biology》1986,102(4):1344-1349
Monoclonal antibodies (McAbs) were raised against the aggregation factor (AF) from the marine sponge Geodia cydonium. Two clones were identified that secrete McAbs against the cell binding protein of the AF complex. Fab fragments of McAbs: 5D2-D11 completely abolished the activity of the AF to form secondary aggregates from single cells. The McAbs were determined to react with the AF in vitro; this interaction was prevented by addition of the aggregation receptor, isolated and purified from the same species. After dissociation of the AF by sodium dodecyl sulfate and 2-mercaptoethanol, followed by electrophoretical fractionation, a 47-kD protein was identified by immunoblotting which interacted with the McAbs: 5D2-D11. During this dissociation procedure, the sunburst structure of the AF was destroyed. In a second approach, the 47-kD protein was isolated by immunoprecipitation; 12 molecules of this protein species were calculated to be associated with the intact AF particle. The 47-kD AF fragment bound to dissociated Geodia cells with a high affinity (Ka of 7 X 10(8) M-1) even in the absence of Ca++ ions; the number of binding sites was approximately 4 X 10(6)/cell. This interaction was prevented by addition of the aggregation receptor to the 47-kD protein in the homologous cell system. Moreover, it was established that this binding occurs species-specifically. The 47-kD fragment of the AF was localized only extracellularly by indirect immunofluorescence staining in cryostat slices. These data suggest that the 47-kD protein is the cell binding molecule of the AF from Geodia. 相似文献
4.
Diehl-Seifert B Amberger V Gramzow M Batel R Bachmann M Schröder HC Müller WE 《Tissue & cell》1989,21(1):25-36
The migration behavior of cells from the sponge Geodia cydonium was studied in vitro, applying the 'Tissue Culture Slide Chamber' technique. The homologous lectin caused a directed cell migration with a maximal locomotory rate of 1.6 mum/min. Competition experiments using the solubilized lectin receptor (= antiaggregation receptor) revealed that the chemotactic ligand (= lectin) interacts directly with the lectin receptor which-in consequence-functions as the chemotactic receptor. The ability of the lectin to promote cell migration is abolished by coincubation with purified leucine aminopeptidase. Biochemical and immunochemical data revealed that this enzyme is present also on the surface of sponge cells. Furthermore, we present evidence that the chemotactic receptor (= anti-aggregation receptor) on the cell surface is, in an hitherto unknown manner, coupled with the intracellularly present actin filaments. From these data we conclude that the directed migration of Geodia cells is mediated by the interaction between the lectin (= chemotactic ligand) and the lectin receptor (= chemotactic receptor); it is very likely that also intracellular structural elements operate simultaneously and coordinately during cell migration. 相似文献
5.
Galactoside-binding serum lectin of Xenopus laevis. Estrogen-dependent hepatocyte synthesis and relationship to oocyte lectin 总被引:1,自引:0,他引:1
M M Roberson A P Wolffe J R Tata S H Barondes 《The Journal of biological chemistry》1985,260(20):11027-11032
Xenopus laevis serum contains a lectin which binds alpha- and beta-galactosides. It was purified to homogeneity by affinity chromatography and consists of a single subunit with Mr approximately 69,000, associated in a multimer. The lectin is synthesized and secreted by hepatic parenchymal cells, and its synthesis is increased about 2-fold by estrogen treatment, both in vivo and in primary cell cultures. The serum lectin has the same carbohydrate binding properties as an oocyte lectin from X. laevis described previously, is immunologically cross-reactive, and shows similarities in its peptide map. However, marked differences in amino acid composition preclude the possibility that the serum lectin is a precursor of the oocyte lectin. 相似文献
6.
A lectin, which agglutinated specifically the yeast cells of the Saccharomyces genus, was isolated from tulip bulbs (Tulipa gesneriana) using affinity chromatography on mannan-Sepharose 4B. Its relative molecular mass was determined by gel filtration to be approximately 67,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulfate, a relative molecular mass of 17,000 was obtained, suggesting that the lectin is a tetramer. Binding studies performed with iodinated lectin indicated that Saccharomyces cerevisiae cells contained approximately 5.7 X 10(6) binding sites per cell, whereas little binding was observed with yeasts other than the Saccharomyces genus, bacteria and animal erythrocytes. D-Mannose, D-mannose 6-phosphate, L-fucose and L-fucosylamine were potent inhibitors of the lectin binding to S. cerevisiae cells, while, D-glucose, D-galactose and D-mannosamine were inactive, indicating that hydroxyl group at C-2 of D-mannose was essential for the lectin binding. Furthermore, inhibition experiments, using various manno-oligosaccharides, suggested that the lectin recognized (1----6)-linked manno-oligosaccharide units larger than mannobiose. 相似文献
7.
- 1. 1. The cytolytic action of glucocorticoids such as dexamethasone was studied in a murine cultured lymphoma L5178Y cell.
- 2. 2. Dexamethasone-resistant cells, which could grow even in a culture medium containing 2 × 10−5 M dexamethasone, were selected from hormone-sensitive L5178Y lymphoma cells by stepwise increasing concentrations of dexamethasone in culture medium.
- 3. 3. The synthetic activities of macromolecules corresponded closely with the viabilities and with the resistibilities at various concentrations of dexamethasone in both cells.
- 4. 4. Studies of binding of dexamethasone to lymphoma L5178Y cells in vivo demonstrated that (1) equilibrium of binding at 37 °C was established within 5 min in both cells; (2) the specific binding to either whole cell or the subcellular fractions was, in sensitive cells, about double that in resistant cells.
- 5. 5. The cytoplasm of L5178Y lymphoma cells contains specific binding sites for dexamethasone and its binding sites (5.7 × 10−10 mmol/mg protein) were about 2.5-fold more than that of dexamethasone-resistant cells (2.3 × 10−10 mmol/mg protein).
- 6. 6. Studies on the dissociation constant, competition of various steroids for the specific binding with dexamethasone, and the sedimentation constant of steroid-receptor complex, suggested that the nature of cytoplasmic binding in resistant cells might be the same as that in sensitive cells.
- 7. 7. The nuclear binding of dexamethasone was extremely dependent on the affinity of cytoplasm to steroid. Studies suggested an equal level of nuclear association sites for cytoplasmic steroidreceptor complex in the two cell types.
8.
Iodinated fibroblast beta-glucuronidase as a ligand for receptor-mediated endocytosis. 总被引:1,自引:1,他引:0
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Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 X 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 X 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell. 相似文献
9.
A recombinant apoA-1--protein A hybrid reproduces the binding parameters of HDL to its receptor. 总被引:2,自引:0,他引:2
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We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
10.
D Merlet J P Merlet J Cambar 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1990,310(12):565-570
An affinity chromatography technique, using a lectin issued from Lotus tetragonolobus as ligand, and coupled to Sepharose 6MB support, has been chosen to enrich a cellular population in tubular proximal cells. Cell desorption was obtained with a competitive sugar, L Fucose. Intensive enzymatic brush border activity, shown in selected cells compared with initial cellular mixture, evidenced an enrichment in proximal tubular cells. These results prove that lectin use as ligand in affinity chromatography permits the separation of renal cell populations and presents the advantage that the bound cells can be recovered by a gentle competitive elution for further cultures. 相似文献
11.
The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin 总被引:2,自引:0,他引:2
The parameters of the reaction between a rat alveolar macrophage lectin (Mr = 180,000) and its ligands have been examined. The reaction is dependent on Ca2+ over the optimal pH range for binding. The apparent dissociation constant for fucosyl bovine serum albumin, the standard ligand used in these studies, is 1.4 X 10(-10) M. The ligand binding specificity was determined by measurement of the inhibition of binding of fucosyl bovine serum albumin by various glycoproteins and saccharides. D-Mannose, L-fucose, and N-acetyl-D-glucosamine were the most effective inhibitors, and D-galactose was much poorer. The equatorial hydroxyl groups on the C-3 and C-4 of the mannose ring are important in the lectin-ligand interaction, and the axial hydroxyl group on the C-2 contributes to a lesser extent. Immunocytological studies revealed that the lectin isolated from alveolar macrophages is widely distributed in other rat tissues. Hepatocytes are devoid of the lectin, but hepatic Kupffer cells and endothelial cells contain significant amounts. This was confirmed by isolation of the lectin from liver. Spleen and skeletal muscle also contain lectin, but much smaller amounts were found in brain, kidney, and heart muscle. 相似文献
12.
Binding and endocytosis of glycoproteins and neoglycoproteins by isolated rabbit hepatocytes.
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D T Connolly R R Townsend K Kawaguchi M K Hobish W R Bell Y C Lee 《The Biochemical journal》1983,214(2):421-431
Rabbit hepatocytes were isolated by a collagenase perfusion technique, and used to study the binding and endocytosis of the glycoprotein, asialo-orosomucoid, and the neoglycoprotein, Gal39-bovine serum albumin. Both of these proteins contain exposed galactosyl residues, and were avidly bound by the lectin on the hepatic parenchymal cell surface. Steady state and kinetic experiments performed at 2 degrees C and at 37 degrees C revealed the presence of two apparent classes of binding sites totalling 4.7 X 10(5) sites/cell at 2 degrees C, and 6.3 X 10(5) sites/cell at 37 degrees C. At 37 degrees C, both classes of sites participated in internalization of bound ligand. The cells were capable of internalizing about 60 000 molecules/min per cell. The process appeared to be first-order, with a rate constant k = 0.098 min-1 and t1/2 = 7.1 +/- 0.6 min. Binding could be inhibited by galactose-containing compounds, EGTA, and by anti-(hepatic lectin) immunoglobulin G. The inhibition by antibody appeared to be reversible upon removal of antibody-containing medium. 相似文献
13.
免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素 总被引:1,自引:0,他引:1
报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果.哺乳动物红细胞能够特异地吸附凝集素.用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体.再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素. S D S P A G E结果显示,该凝集素的分子量约为73 k D.这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的.为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法. 相似文献
14.
The repair of double-strand DNA breaks correlates with radiosensitivity of L5178Y-S and L5178Y-R cells 总被引:4,自引:0,他引:4
To better understand the basis for the difference in radiosensitivity between the variant murine leukemic lymphoblast cell lines L5178Y-R (resistant) and L5178Y-S (sensitive), the production and repair of DNA damage after X irradiation were measured by the DNA alkaline and neutral elution techniques. The initial yield of single-strand DNA breaks and the rates of their repair were found to be the same in both cell lines by the DNA alkaline elution technique. Using the technique of neutral DNA elution, L5178Y-S cells exhibited slightly increased double-strand breakage immediately after irradiation, most significantly at lower doses (i.e., less than 10 Gy). Nevertheless, even at doses that yielded equal initial double-strand breakage of both cell lines, the survival of L5178Y-S cells was significantly less than that of L5178Y-R cells. When the technique of neutral DNA elution was employed to measure the kinetics of DNA double-strand break repair, both cell lines exhibited biphasic fast and slow components of repair. However, the double-strand repair rate was much lower in the radiosensitive L5178Y-S cells than in the L5178Y-R cells (T1/2 of 60 vs 16 min). This difference was more pronounced in the fast-repair component. These results suggest that the repair of double-strand DNA breaks is an important factor determining the radiosensitivity of L5178Y cells. 相似文献
15.
W E Müller J Conrad C Schr?der R K Zahn B Kurelec K Dreesbach G Uhlenbruck 《European journal of biochemistry》1983,133(2):263-267
A D-galactose-specific lectin I was extracted from the sponge Geodia cydonium and purified by affinity chromatography. The molecular weight of lectin I as determined by high-pressure liquid gel chromatography, was found to be 36500 +/- 1300. Disc gel electrophoresis in the presence and in the absence of sodium dodecyl sulfate showed that lectin I is a trimer composed of three different subunits (Mr: 13800, 13000 and 12200); two of the three subunits are linked by one disulfide bond. Isoelectric focusing gave a pI of 5.6 for the native molecule and a pI of 4.4 and of 7.4 for the subunits. The three subunits carry carbohydrate side chains, composed of D-galactose (94%) and of arabinose (5%). Based on experiments with lectins, the terminal D-galactose residues are bound by beta 1 leads to 6 and/or beta 1 leads to 4 glycosidic linkages. The Geodia lectin I contains, besides two carbohydrate recognition sites, at least one receptor site for a second lectin I molecule. 相似文献
16.
Murine macrophage tumors are a source of a 260,000-dalton acetyl-low density lipoprotein receptor 总被引:8,自引:0,他引:8
Subcutaneous injection of murine macrophage cell line P388D1 into syngeneic DBA/2 produced tumors, which upon solubilization with 40 mM octyl glucoside contained acetylated low density lipoprotein binding activity. The tumor-derived receptor specifically bound acetylated low density lipoprotein with an affinity of approximately 3 X 10(-8) M but did not bind low density lipoprotein or high density lipoprotein. It was identical in binding specificity, affinity, and Pronase sensitivity to the receptor in intact cells or that obtained from solubilized cultured cell membranes. Partial purification of the receptor was achieved by solubilizing tumors with 1% Triton X-100 followed by chromatography on polyethyleneimine cellulose. After elution with a NaCl gradient in the presence of octyl glucoside and association with liposomes, a 287-fold purification of the receptor was achieved. The receptor was identified by specific ligand blotting as a 260,000-dalton protein having a pI of approximately 6.0. Binding to the receptor by acetylated low density lipoprotein, malondialdehyde-modified low density lipoprotein, and maleic anhydride-modified serum albumin was demonstrated by ligand blotting. A single receptor protein can, therefore, account for the binding of multiple types of charge-modified lipoprotein and nonlipoprotein ligands to the macrophage cell surface. 相似文献
17.
J Petryniak T K Huard G D Nordblom I J Goldstein 《Archives of biochemistry and biophysics》1986,244(1):57-66
Purified 125I-labeled lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia (I-B4 isolectin) were used to analyze changes in the expression of carbohydrates on the surface of resident (PC) and thioglycollate-stimulated murine (C57B/6J) peritoneal exudate cells (PEC). The lectins from D. stramonium, E. europaea, and G. simplicifolia I-B4 bind specifically to PEC with relatively high affinity (Kd = 5.65 +/- 1.08 X 10(-7) M, 1.08 +/- 0.12 X 10(-8) M, and 1.33 +/- 0.15 X 10(-7) M, respectively). Assuming a single lectin molecule binds to each cell surface saccharide, the number of receptor sites per cell ranged for different cell samples from 22.3 to 50.0 X 10(6), from 3.8 to 4.8 X 10(6), and from 2.0 to 16.8 X 10(6) for D. stramonium, E. europaea, and G. simplicifolia I-B4 lectins, respectively. There were approximately 3- to 7-fold, 16- to 20-fold, and 2- to 20-fold increases in binding capacity for D. stramonium, E. europaea and G. simplicifolia I-B4, respectively, compared to the binding to resident, peritoneal cells. Scatchard plots of the binding of all three lectins to PEC were linear, suggesting that the receptor sites for these lectins are homogeneous and noninteracting. The binding capacity of these lectins to PEC was unchanged after trypsin digestion of cells. The expression of carbohydrates on the surface of PEC was also monitored by an agglutination assay. PEC were agglutinated by all three lectins whereas PC either were not agglutinated or were agglutinated only at high lectin concentrations. On the basis of our knowledge of the carbohydrate binding specificity of the D. stramonium and G. simplicifolia I-B4 lectins, we postulate that, parallel with thioglycolate stimulation, there is an increase in the number of N-acetyllactosamine residues and terminal alpha-D-galactosyl end groups. The blood group B, and H type 1 determinants--DGa1 alpha 1,3[LFuc alpha 1,2]DGa1 beta 1,3(or 4)DGlcNAc and LFuc alpha 1,2DGa1 beta 1,3DG1cNAc, respectively, as well as DGa1 alpha 1,3DGa1 beta 1,3(or 4)DGlcNAc--may be considered to be possible receptors for the E. europaea lectin. These glycoconjugates, present on the surface of peritoneal exudate cells, provide new chemical markers for studying the differentiation of resident peritoneal cells. 相似文献
18.
The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III. 相似文献
19.
The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin 总被引:2,自引:0,他引:2
The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA. 相似文献
20.
Carbohydrate binding specificity of a lectin, allo A, isolated from a beetle (Allomyrina dichotoma), was investigated by means of lectin affinity chromatography. Sialylated complex-type and hybrid-type oligosaccharides/glycopeptides, and sialyllactose were retained by the column, whereas desialylated ones were retarded but not retained by the column. The association constants of allo A for biantennary oligosaccharides from human serum transferrin, determined by frontal analysis, were 8.0 X 10(5) M-1, 4.5 X 10(5) M-1, and 2.5 X 10(5) M-1 for disialo-, monosialo-, and asialo-oligosaccharides, respectively. Removal of the beta-galactose residues markedly reduced the association constant to 3.5 X 10(3) M-1. Furthermore, allo A was found to have no affinity for mucin-type glycopeptides carrying the sialylated Gal beta 1----3 GalNAc sugar sequence (Ka: 3.5 X 10(3) M-1). The results of this study indicated that allo A strongly binds to the trisaccharide structure, NeuAc alpha 2-3(6)Gal-beta 1-4GlcNAc, and that its binding potency is affected by the inner core structures of oligosaccharides and glycopeptides, because the presence of a bisecting N-acetyl-glucosamine residue and an alpha-fucose residue linked to the innermost N-acetylglucosamine residue reduced the association constants for oligosaccharides and glycopeptides. 相似文献