Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

Winged bean acidic lectin was purified by DEAE-Sephadex A-50 and affinity chromatography on N-acetylgalactosamine-agarose gel. The purified lectin was a glycoprotein homogeneous on polyacrylamide gel electrophoresis, isoelectric focusing, and gel filtration. The molecular weight of the lectin was 52,000 by gel filtration, and SDS-polyacrylamide gel electrophoresis gave a single component of molecular weight of 27,000. Its isoelectric point was 5.5. The acidic lectin was rich in acidic amino acids, and contained 2mol of methionine but no cystine. It also agglutinated both trypsinized and untreated human erythrocytes (types A, B, AB and O), but not rabbit erythrocytes. The hemagglutination was inhibited by d-galactose and related sugars. Modification of the acidic lectin with N-bromosuccinimide caused a concomitant loss of the hemagglutinating activity with oxidation of tryptophan residue. The acidic lectin was immunologically different from the purified winged bean basic lectin by double immunodiffusion using antiserum raised against the basic lectin.     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.     

Purification and characterization of an α-d-galactosyl-binding lectin from Artocarpus lakoocha seeds

A lectin from Artocarpus lakoocha seeds has been purified by affinity chromatography on a melibiose-agarose column. The homogeneity of the purified lectin was tested by several criteria, viz., poly(acrylamide)-gel electrophoresis, ultracentrifugal analysis, and gel filtration. The molecular weight of the lectin was estimated to be ∼70,000 as determined by Sephadex gel filtration. SDS-poly-(acrylamide)-gel electrophoresis gave a single component of molecular weight 18,000, suggesting that the lectin is a tetramer composed of four apparently identical subunits. The lectin agglutinated human erythrocytes, regardless of blood group. Artocarpus lakoocha lectin is a glycoprotein, and contains 11.7% of carbohydrates, in which d-xylose (6%) is the main sugar, with smaller proportions of d-galactose, d-glucose, d-mannose, N-acetyl-d-glucosamine, and N-acetyl-d-mannosamine. Amino acid analysis of the lectin revealed a high content of acidic and hydroxylic amino acids, a relatively low proportion of basic amino acids, and a trace of cysteine and methionine. In hapten-inhibition assays with simple sugars, glycosides of α-d-galactopyranose and N-acetyl-d-galactosamine were potent inhibitors of the purified lectin.     

Heparin-inhibitable lectins: Marked similarities in chicken and rat

Extracts of young rat lung contain a heparin-inhibitable lectin that closely resembles one recently purified from chicken liver. Both lectins interact with heparin and N-acetyl-D-galactosamine, and were purified by gel filtration on Sepharose CL-2B followed by affinity chromatography on heparin-Sepharose. They both behave as high molecular weight aggregates that can be dissociated into two peptides with apparent molecular weights of 13,000 and 16,000 by gel electrophoresis in SDS. Samples of purified lectin contained up to 20% DNA by weight, and the degree of lectin aggregation and hemagglutination activity was greatly reduced by treatment with micrococcal nuclease without inhibiting heparin-binding activity. Association of lectin with DNA is an artifact of homogenization in high salt, since only 2% of the lectin is found associated with a purified nuclear fraction.     

A lectin from the thigh muscle of Rana tigerina

Lectin activity has been detected in the thigh muscle extracts of Rana tigerina, which was found to agglutinate both trypsinized and untrypsinized rabbit erythrocytes. The lectin has been purified to homogeneity by MEPBS (0.01 M phosphate-buffered saline (pH 7.2) with 4 mM beta-mercaptoethanol) buffer extraction of the tissue and affinity chromatography on acid-treated Sepharose 6B. The molecular weight (Mr) of the purified lectin was determined by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-75, which gave values of 15,500 +/- 1000 and 32,000 +/- 1000, respectively, suggesting that the lectin is a dimer. Amino acid composition data of the lectin has revealed that it contains a high proportion of glycine and alanine, and low amounts of sulphur-containing amino acids. Hapten-inhibition study of this lectin has shown that it is galactose-specific. Hemagglutination activity of the lectin can also be inhibited by beta-galactoside containing oligosaccharides.     

Purification and characterization of N-acetyl-D-galactosamine-binding lectin from Falcata japonica

An N-acetyl-D-galactosamine-binding lectin from Falcata japonica seeds was purified by affinity column chromatography of N-acetyl-D-galactosamine coupled to epoxy-activated Sepharose 6B. A 1000-fold purification of lectin was obtained from the crude extracts. The purified lectin agglutinated blood group A red cells, but neither blood group B nor O red cells. Polyacrylamide gel electrophoresis of the lectin showed one diffuse band. Molecular weights of 125,000 and 117,000 were estimated by gel filtration and ultracentrifugal analysis, respectively. SDS-polyacrylamide gel electrophoresis of the lectin also showed a single band which has a molecular weight of 34,000. Therefore, the lectin molecule was estimated to be a tetramer composed of four identical non-covalently bound subunits. F. japonica lectin was a glycoprotein containing 5% total carbohydrate, and the amino acid composition was characterized by a high content of aspartic acid, serine and glycine, a low content of methionine and the absence of cysteine.     

Isolation and properties of a lectin from the seeds ofMimosa invisa L.

A lectin has been purified from the seeds ofMimosa invisa L. by gel filtration and preparative Polyacrylamide gel electrophoresis. The purified lectin was homogeneous as judged by analytical Polyacrylamide gel electrophoresis, immunodiffusion and Immunoelectrophoresis. The apparent molecular weight is 100,000; the protein is a tetramer with two types of subunits (molecular weight 35,000 and 15,000). The lectin is a glycoprotein with approximately 21% carbohydrate and interacts with Sephadex and concanavalin A-Sepharose. It agglutinates erthrocytes non-specifically, does not agglutinate leucocytes and is not mitogenic, agglutinates Mimosa-nodulatingRhizobium and is a panagglutinin; the agglutination is not inhibited by several simple sugars. It is thermo-stable and has no metal ions.     

Purification and characterization of a lectin from rice bran

A rice bran lectin was purified to homogeneity by precipitation with ammonium sulfate and chromatography on ovomucoid-Sepharose and CM-cellulose. The molecular weight of the dimer lectin was estimated to be around 37,000 by ultracentrifugation studies. The sedimentation coefficient was 3.8S. On Sepharose 6B gel filtration in the presence of 6 M guanidine-HCl, the lectin showed a molecular weight of 19,000. On reduction and carboxymethylation, the lectin further dissociated into two nonidentical subunits, with molecular weights of about 11,000 and 8,000. These subunits did not show hemagglutinating activity. Equilibrium dialysis experiments using N-acetyl-[1-14C]glucosamine indicated that about 1.8 mol of the sugar was bound to 19,000 g of the lectin. The lectin was mitogenic against mouse splenic lymphocytes and human peripheral lymphocytes. The lectin enhanced the rate of glucose oxidation and inhibited epinephrine-stimulated lipolysis in mouse adipocytes. Some characteristics of the lectin are compared with those of wheat germ agglutinin.     

Isolation and characterization of a lectin from edible mushroom, Volvariella volvacea

A lectin was purified from edible mushroom, Volvariella volvacea by extraction with 5% cold acetic acid in the presence of 0.1% 2-mercaptoethanol, followed by ammonium sulfate fractionation, and DEAE-C-52 and CM-C-52 column chromatographies. The molecular weight was measured to be 26,000, and the lectin consisted of two non-identical subunits as demonstrated by gel filtration and polyacrylamide gel electrophoresis. The lectin does not contain half-cystine, methionine, or histidine. The LD50 of the lectin is 17.5 mg per kg body weight of mice. The lectin has a moderate inhibitory effect on the growth of tumor cells.     

番茄凝集素的分离纯化及某些性质的研究

番茄成熟果实经抽提上鸡卵类粘蛋白(OM)-Sepharose 4B亲和层析柱分离纯化制得番茄凝集素。制品在SDS—聚丙烯酰胺凝胶电泳上呈一条带,表观分子量为205,000。Sephadex G200凝胶过滤行为呈单一吸收峰,分子量为180,000。等电聚焦测得等电点为7.6和9.4。它能使人和多种动物红细胞,以及某些培养细胞凝集,但效价不同。N—乙酰葡萄糖胺寡聚糖是其专一性结合糖。     

A chitotetrose specific lectin fromLuffa acutangula: Physico-chemical properties and the assignment of orientation of sugars in the lectin binding site

A chitooligosaccharide specific lectin (Luffa acutangula agglutinin) has been purified from the exudate of ridge gourd fruits by affinity chromatography on soybean agglutinin-glycopeptides coupled to Sepharose-6B. The affinity purified lectin was found homogeneous by polyacrylamide gel electrophoresis, in sodium dodecyl sulphate-polyacrylamide gels, by gel filtration on Sephadex G-100 and by sedimentation velocity experiments. The relative molecular weight of this lectin is determined to be 48,000 ±1,000 by gel chromatography and sedimentation equilibrium experiments. The sedimentation coefficient (S₂₀, w) was obtained to be 4.06 S. The Stokes’ radius of the protein was found to be 2.9 nm by gel filtration. In sodium dodecyl sulphate-polyacrylamide gel electrophoresis the lectin gave a molecular weight of 24,000 in the presence as well as absence of 2-mercaptoethanol. The subunits in this dimeric lectin are therefore held by non-covalent interactions alone. The lectin is not a glycoprotein and circular dichroism spectral studies indicate that this lectin has 31% α-helix and no β-sheet. The lectin is found to bind specifically to chitooligosaccharides and the affinity of the lectin increases with increasing oligosaccharide chain length as monitored by near ultra-violet-circular dichroism and intrinsic fluorescence titration. The values of ΔG, ΔH and ΔS for the binding process showed a pronounced dependence on the size of the oligosaccharide. The values for both ΔH and ΔS show a significant increase with increase in the oligosaccharide chain length showing that the binding of higher oligomers is progressively more favoured thermodynamically than chitobiose itself. The thermodynamic data is consistent with an extended binding site in the lectin which accommodates a tetrasaccharide. Based on the thermodynamic data, blue shifts and fluorescence enhancement, spatial orientation of chitooligosaccharides in the combining site of the lectin is assigned.     

Properties of Lectins in the Root and Seed of Lotononis bainesii

A lectin was purified from the root of Lotononis bainesii Baker by affinity chromatography on Sepharose-blood group substance A + H. The molecular weight of the lectin was estimated by gel filtration to be 118,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the lectin was a tetramer composed of two slightly different subunits with respective molecular weights of 32,000 and 35,000. The lectin had a hexose content of 12% (w/w) and contained the sugars fucose, glucosamine, mannose, and xylose. Root lectin hemagglutination was preferentially inhibited by disaccharides with terminal nonreducing galactose residues. Antigens capable of cross-reaction with root lectin antibody were not detected in the seed of L. bainesii.

A lectin from the seed of L. bainesii was partially purified by adsorption to pronase-treated rabbit erythrocytes. The lectin preparation had a molecular weight of approximately 200,000. Galactose and galactono-1,4-lactone inhibited seed lectin hemagglutination but lactose was ineffective. There was no evidence that the root of L. bainesii contained material antigenically related to the seed lectin.

     

Isoiation and Characterization of Vicia FABA Lectin Affinity Purified on Chitin Column

Abstract

A lectin from early long pod var. of Vicia faba seed has been purified to homogeneity on chitin. The purified lectin is shown to be homogeneous in nature by Bio Gel P - 150 gel filtration, fast protein liquid chromatography and polyacrylamide gel electrophoresis. The lectin is a glycoprotein with molecular weight of 51,000. The lectin molecule is possibly composed of two types of subunits devoid of any covalent linking through sulfhydryl groups, with molecular weights 9,000 and 15,000 respectively in the ratio 2:2. The purified lectin shows a high affinity for N-acetyl-D-glucosamine (GlcNAc).

Amino acid analyses show that cysteine and methionine are absent, and a high proportion of aspartic acid and glutamic acid are present in the protein molecule. The extinction coefficient of the purified lectin is 7.22. The lectin behaves as a, cold agglutinin displaying stronger agglutination than the naturally occurring ABO agglutinin in the cold.     

格拉姆柱花草(Stylosanthes gracilis H.B.K.cv.Graham)凝集素的纯化与性质研究

为了探讨凝集素在豆科植物与根瘤菌的识别过程中的作用,用ＤＥＡＥ－３２离子交换层析和ＳｅｐｈａｄｅｘＧ－１５０凝胶过滤分离、纯化格拉姆柱花草种子凝集素（ＳＧＬ）,其分子量约为４５ｋＤ,由两个相同的亚基组成,等电点约ｐＨ５．８,它是一种糖蛋白,含糖量约为２．６％．ＳＧＬ的热稳定性强．ＳＧＬ的血凝活性能被甘露糖所抑制．ＳＧＬ对红细胞的凝集作用可能具有种属专一性;ＳＧＬ具有强的促有丝分裂作用;荧光标记实验显示：９株能与格拉姆柱花草植株结瘤的菌株有７株能与ＳＧＬ结合,６株不能与之结瘤的菌株,只有１株能与ＳＧＬ结合,这表明不同根瘤菌菌株对ＳＧＬ的结合能力,和它们在格拉姆柱花草上结瘤能力之间可能具有一定的生物学相关性．     

Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes.

The Thy-1-molecule, which was identified by its antigenic activities, has been purified from rat thymocytes. The purification involved preparation of crude membranes and solubilization in deoxycholate, followed by gel filtration and affinity chromatography on antibody or lectin columns. In all cases the purified molecule was a glycoprotein that did not form higher polymers and was not associated with other polypeptide chains. The Thy-1 glycoprotein could be found in two forms, one binding to lentil lectin, the other not. Both forms had the same detectable antigens and were of a similar but not identical size. After sodium dodecyl sulphate-polyacrylamide-gel electrophoresis the apparent molecular weight of Thy-1 binding to lentil lectin was 25 000, whereas that not binding to the lectin was 27 000, with heterogeneity towards forms of apparently higher molecular weight.     

Isolation of an N-acetyl-d-galactosamine-binding protein from winged bean (Psophocarpus tetragonolobus)

A lectin has been purified to homogeneity by affinity chromatography on a Sepharose-N-caproyl-d-galactosamine column from the local variety of winged bean (Psophocarpus tetragonolobus). The lectin agglutinated native erythrocytes of all blood groups. This hemagglutination was inhibited best by N-acetyl-d-galactosamine. A molecular weight of 41,000 was obtained for the lectin by gel filtration on Bio-Gel P-100. Sodium dodecyl sulfate-polyacryl-amide gel electrophoresis of the same lectin showed a single M_r 35,000 polypeptide.     

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.)

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.     

Mucin binding mitogenic lectin from freshwater Indian gastropod Belamyia bengalensis: purification and molecular characterization

A lectin was purified from the hemolymph of the freshwater Indian gastropod Belamyia bengalensis. The purification involved successive ion-exchange chromatography on Resource Q and gel filtration on Superose 12 column in FPLC system. Homogeneity of the protein was confirmed by polyacrylamide gel electrophoresis. Belamyia bengalensis lectin (BBL) was a monomeric protein with a molecular weight of 33 kDa as demonstrated by gel filtration and SDS-PAGE. It is a glycoprotein containing 6% total sugar and its activity is highly dependent on Ca(2+). BBL agglutinated human erythrocytes and is a blood group non-specific lectin. It agglutinated animal erythrocytes also. Hapten inhibition studies indicated that BBL shows binding specificity only for N-acetyl-D-glucosamine and N-acetyl-D-galactosamine at a high concentration among the mono- and oligosaccharides tested. Among the glycoproteins used for hemagglutination-inhibition assay, porcine submaxillary mucin was found to be the best inhibitor. Chemical modification studies indicated that Lys, Arg, and Trp are essential for the sugar-binding activity of BBL. Circular dichroism spectra revealed high content of alpha-helical structure in the lectin. BBL is a potent mitogen as it stimulated the T-lymphocyte proliferation, specifically the Th1 subset.     

黑色菜豆(Phaseolus sp.)凝集素的纯化和性质

黑色菜豆(phaseolussp.)种子中含有对人A型血专一凝集的凝集素。用猪胃粘蛋白-Sepharose 4B作亲和吸附剂和Sephadex G-200凝胶过滤,可以纯化这种凝集素。纯化的凝集素在pH8.9,Tris-EDTANa_2-borate缓冲液的PAGE中,呈现单一蛋白带;酚-硫酸法测得总糖含量为3.22％。在SDS-PAGE中发现其分子由两种亚基所组成,亚基分子量分别为38,000和35,000。当凝集素浓度分别为0.98μg/ml和1.95μg/ml时能强烈地凝集人A型和AB型血细胞。在凝集素浓度高达500μg/ml时,B型血细胞能发生弱凝集反应,但对O型血和兔红细胞则完全不发生凝集反应。其凝集活性可被GalNAC、L-Fuc、猪甲状腺球蛋白和卵粘蛋白所抑制。该凝集素对人外周血中淋巴细胞的转化率达80％,细胞分裂比率高达37.1％;氨基组成分析表明,凝集素分子中Asp和Glu含量较高,而cys和Met含量很低。     

A sialic acid-binding lectin from ovine placenta: purification,specificity and interaction with actin

A sialic-acid-specific lectin from ovine placental cotyledons was purified by affinity chromatography on bovine submaxillary mucin-agarose followed by gel filtration, and it showed a molecular weight of 65 000 by sodium dodecylsulfate-polyacrylamide gel electrophoresis. This lectin has the capacity to interact with actin, since in binds to actin-F in a cosedimentation assay and it acts as a mediator in the binding of action to the affinity column. The lectin agglutinated rabbit and rat erythrocytes, but not human A, B or O erythrocytes. Haemagglutination inhibition assays of different saccharides, glycoproteins and glycolipids indicate that this lectin has affinity for sialic acid, which is enhanced by its O-acetylation. The N-terminal sequence of the protein shows 92% identity with rabbit and porcine uterine calreticulin.