A new lectin from tulip (Tulipa) bulbs

A lectin was isolated from tulip (Tulipa) bulbs by affinity chromatography on fetuin-agarose and partially characterized. The tulip lectin is a tetrameric protein composed of four identical subunits of M_r 28 000, which are not held together by disulphide bonds. It is not glycosylated and has an amino-acid composition typified by a high content of asparagine-aspartic acid, leucine, glycine and serine. Tulip lectin agglutinates human red blood cells, but has a much higher specific activity with rabbit erythrocytes. In hapten-inhibition assays with the latter type of red blood cell the lectin exhibits a complex specificity, whereas its agglutination with human erythrocytes is readily inhibited by N-acetylgalactosamine, lactose, fucose and galactose.Abbreviations DEAE diethylaminoethyl - PBS phosphate-buffered saline - TL Tulipa lectin - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

Purification and properties of diaminopimelate decarboxylase ofMicrococcus glutamicus

Diaminopimelate decarboxylase (EC 4.1.1.20) ofMicrococcus glutamicus ATCC 13059 was purified to homogeneity. The enzyme had an apparent molecular weight of 191,000 as determined by gel filtration on Sephadex G-200. At protein concentrations of 20 and 10 μg per ml and in the absence of pyridoxal-5′-phosphate, it dissociated into a species of molecular weight 94,000. The polypeptide chain molecular weight as determined by sodium dodecyl sulphate Polyacrylamide gel electrophoresis was 100,000. TheK _m formeso diaminopimelate was 0.5 mM and that for pyridoxal-5′-phosphate was 0.6 μI. Sulphydryl groups and pyridoxal-5′-phosphate were essential for activity and stability. The enzyme was inhibited significantly by L-lysine and DL-aspartic β-semialdehyde.     

Isolation and partial characterization of a lectin from ground elder (Aegopodium podagraria) rhizomes

A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high M_r (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different M_r (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.Abbreviations APA Aegopodium podagraria agglutinin - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Purification and properties of a carboxylesterase from germinated finger millet (Eleusine coracana Gaertn.)

A carboxylesterase (EC 3.1.1.1) was purified from germinated finger millet by ammonium sulphate fractionation, diethylaminoethyl-cellulose chromatography and Sephadex G-200 filtration. The homogeneity of the enzyme was established by Polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme has a single polypeptide chain with a molecular weight of 70,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to, basic amino acid residues. The isoelectric pH of the enzyme was found to be 5·1. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was more sensitive to organophosphate inhibitors than carbamates. The rate constantsk _i andl ₅₀ for different inhibitors were calculated. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear noncompetitive inhibition with 1-naphthol     

Purification of iron superoxide dismutase from the cyanobacterium Anabaena cylindrica Lemm. and localization of the enzyme in heterocysts by immunogold labeling

Iron superoxide dismutase (Fe-SOD; EC 1.15.1.1) was isolated from the nitrogen-fixing cyanobacterium Anabaena cylindrica Lemm. Polyacrylamide gel electrophoresis separated the purified protein into three closely running, enzymatically active bands. The molecular weight of the enzyme was estimated by gel filtration to be about 40 kDa. Polyclonal antibodies were produced by immunization of rabbits with the isolated enzyme, and were purified on a column of protein A-Sepharose. The Fe-SOD antibody reacted with the purified Fe-SOD and also specifically recognized the protein in extracts of A. cylindrica. In the extracts, anti-Fe-SOD did not cross-react with Mn-SOD, an enzyme which belongs to an SOD class displaying high homology of primary and three-dimensional structure with respect to Fe-SOD. Iron superoxide dismutase was localized in heterocysts by immunogold labeling and transmission electron microscopy. These results are the first in-situ evidence for the presence of SOD in the cells specialized for nitrogenase activity.Abbreviations ELISA enzyme-linked immunosorbent assay - SDS sodium dodecyl sulfate - SOD superoxide dismutase - PAGE polyacrylamide gel electrophoresis - pI isoelectric point This work was supported by a C.N.R. grant. We are grateful to Dr. A. De Martino for technical assistance.     

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.     

The molecular organization of the lysostaphin gene and its sequences repeated in tandem

Summary The gene encoding lysostaphin of Staphylococcus staphylolyticus was cloned in Escherichia coli and its DNA sequence was determined. The complete coding region comprises 1440 base pairs corresponding to a precursor of 480 amino acids (molecular weight 51669). It was shown by NH₂-terminal amino acid sequence analysis of the purified extracellular lysostaphin from S. staphylolyticus that the mature lysostaphin consists of 246 amino acid residues (molecular weight 26926). Polyacrylamide gel electrophoresis revealed a similar molecular weight for the most active form. By computer analysis the secondary protein structure was predicted. It revealed three distinct regions in the precursor protein: a typical signal peptide (ca. 38 aa), a hydrophilic and highly ordered protein domain with 14 repetitive sequences (296 aa) and the hydrophobic mature lysostaphin. The lysostaphin precursor protein appears to be organized as a preprolysostaphin.Abbreviations aa amino acid(s)     

野花生豆凝集素的纯化和性质

用猪胃粘蛋白-Sepharose 4B作亲和吸附剂,可从野花生豆(Crotalarta mucronata)的种子中分离纯化出对人类A型血专一凝集的凝集素。该凝集素可用pH30.,Gly-HCl-1mol/L NaCl溶液解吸附。纯化的凝集素在PAGE或SDS-PAGE中均显示单一蛋白带,表明凝集素分子内只有一种亚基。用SDS-PAGE测得其亚基分子量为49,000。氨基酸组成分析表明,该凝集素富含甘氨酸和谷氨酸,不合甲硫氮酸。纯化的野花生豆凝集素(简称CML)含有4.11％的中性糖。它对人A型血细胞有强烈凝集作用,对AB型血有弱凝集作用,但对B型和O型血均不凝集。其对A型血细胞的凝集作用可被N-乙酰半乳糖胺抑制,但对AB型血则无抑制作用。CML是一个促有絲分裂原,对人外周血中淋巴细胞有促有絲分裂作用。     

Affinity purification, physicochemical and immunological characterization of a galactose-specific lectin from the seeds of Dolichos lablab (Indian lablab beans)

A galactose-specific lectin has earlier been isolated from the seeds of Dolichos lablab in our laboratory by conventional protein purification methods. We now established conditions to bind the lectin on Sepharose-galactose gel in the presence of 1.5 M ammonium sulfate in Tris-buffered saline, pH 7.4. It can be specifically eluted with 0.3 M galactose. The purified lectin is a glycoprotein, binds to Con A, agglutinates erythrocytes, and has an apparent native molecular weight of 120 +/- 5 kDa. In SDS-PAGE under reducing conditions, it dissociates into two subunits of molecular mass (Mr) 31 and 29 kDa. Among a number of sugars tested for inhibitory activity of the lectin, galactose was found to be a potent inhibitor. Rabbit polyclonal antibody to the purified lectin specifically reacted with the lectin subunits in Western blot analysis and additionally, an antibody raised to the isolated 31 kDa subunit show reactivity with both the subunits. Amino terminal sequences of both the subunits are identical. The purified lectin is stable up to 40 degrees C with a pH optimum of 7.4. The lectin has a high content of acidic amino acids and lacks sulfur-containing amino acids. Chemical modification of the lectin with group-specific reagents indicates the possible role of histidine, lysine, and tyrosine residues in lectin activity.     

A lectin from Bryonia dioica root stocks

An N-acetylgalactosamine-specific lectin has been isolated from root stocks of Bryonia dioica by affinity chromatography on fetuin-agarose. It is a dimeric protein composed of two different subunits of relative molecular masses 32,000 and 30,000, held together by intermolecular disulphide bonds. Although most abundant in root stocks, the lectin occurs in all vegetative parts of the plant but not in seeds. Bryony lectin differs from other Cucurbitaceae lectins and from all known N-acetylgalactosamine-specific lectins.Abbreviations BDA Bryonia dioica agglutinin - M_r relative molecular mass - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Purification and Characterization of a Novel β-<Emphasis Type="SmallCaps">d</Emphasis>-Galactosides-Specific Lectin from <Emphasis Type="Italic">Clitoria ternatea</Emphasis>

A lectin present in seeds of Clitoria ternatea agglutinated trypsin-treated human B erythrocytes. The sugar specificity assay indicated that lectin belongs to Gal/Gal NAc-specific group. Hence the lectin, designated C. ternatea agglutinin (CTA), was purified by the combination of acetic acid precipitation, salt fractionation and affinity chromatography. HPLC gel filtration, SDS-polyacrylamide gel electrophoresis and mass spectrometry indicated that the native lectin is composed of two identical subunits of molecular weight 34.7 kDa associated by non covalent bonds. The N-terminal sequence of CTA shared homology with Glycine max and Pisum sativum. Complete sequence was also found to be homologous to S-64 protein of Glycine max, suggesting that CTA probably exhibits both hemagglutination and probably sugar uptake activity. The carbohydrate binding specificity of the lectin was investigated by quantitative turbidity measurements, and percent inhibition assays. Based on these assays, we conclude that CTA binds β-d-galactosides, and also may has an extended specificity towards non-reducing terminal Neu5Acα2,6Gal.     

A novel method for the purification of recombinant subunit I of theDolichos biflorus seed lectin

Lectins are carbohydrate-binding proteins that are ubiquitous in nature. Their ability to specifically bind carbohydrates has been used as a means of purification mainly through affinity chromatography techniques. Plant lectins are one of the most thoroughly studied class of lectins, however, details of theirin situ function remains elusive. Recent advances in recombinant DNA techniques have been used in several laboratories to study the function of these lectins by heterologous over-expression. The larger subunit of theDolichos biflorus seed lectin was described by Chao et al. in 1994 and purification through affinity chromatography techniques was described. Here we report on a new method for the purification of this recombinant protein with techniques that are not dependent on the ability of the lectin to bind sugars. This method may have uses in the purification of mutant proteins that may not bind carbohydrates. Characterization of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization (MALDI) mass spectroscopy shows that the lectin is over 99% pure with a molecular weight of 27,090±16.17 Da, and hemagglutination assays confirm that the lectin retains its biological activity.     

Polymorphisme et caractère glycoprotéique des lectines de Ricinus communis purifiées par chromatographie d'affinité

Two lectin fractions (S^o_{20 w} = 6,8 and 4,9 S) were purified from Ricinus communis seeds. The purification was carried out in four steps : ammonium sulfate fractionation, affinity chromatography on Sepharose 4 B, gel filtration on Sephadex G 150 and chromatography on CM cellulose. The purified lectins were glycoproteins whose chemical composition was determined. Amino terminal analysis of the two fractions revealed glycine and serine. Polyacrylamide gel electrophoresis of the higher molecular weight fraction allowed the separation of several components with different affinity for PAS staining.     

雪山豆(Phaseolus sp.)凝集素的亲和层析纯化与其部分性质

 用猪甲状腺球蛋白-对氨基苯砜乙基-交联琼脂作亲和吸附剂,可对雪山豆(Phaseolus sp.)凝集素进行亲和层析纯化。纯化后的凝集素在聚丙烯酰胺凝胶电泳中显示单一蛋白质带。Sephadex G-100凝胶层析法测得分子量为65,000道尔顿,SDS-聚丙烯酰胺凝胶电泳表明该凝集素分子仅有一个分子量为65,000道尔顿的亚基,酚-硫酸法测得总糖含量为4.6％,氨基酸组成分析表明雪山豆凝集素富含门冬氨酸,而甲硫氨酸含量甚少。该凝集素是强促有丝分裂原,对人外周血中淋巴细胞的转化率大于90％,细胞分裂比率达12％。雪山豆凝集素不仅能凝集多种动物红细胞,还能凝集人精细胞。经体外实验表明,雪山豆凝集素对人肝癌细胞有抑制作用。     

Characterization of a wheat germ agglutinin-like lectin from adult wheat plants

Radioimmuno-and enzyme-linked immunosorbent assays show that a substantial amount of wheat germ agglutinin(WGA)-like protein is present at the base of the shoot and in the roots of adult wheat (Triticum aestivum L.) plants. The protein can be purified by hapten-and antibody-mediated affinity procedures. It forms an arc of identity with the embryo lectin upon Ouchterlony double-diffusion and is an active lectin that agglutinates trypsinized erythrocytes in an N-acetylglucosamine-and chitin-inhibitable manner. Reduced and carboxyamidated protein comigrates with the 18-kdalton subunits of embryo lectin on sodium dodecyl sulfate-polyacrylamide gels. Invivo labeling of 9-d-old, hydroponically grown plants with ³⁵S-labeled sulfate demonstrates that at least some of the WGA-like protein is synthesized de novo. Immunocytochemistry with rabbit anti-WGA and colloidal-gold-conjugated second antibody shows that cross-reactive protein is present at the tips of new adventitious roots. In reactive cells, the lectin is localized near the inner surface of the vacuole membrane. Wheat plants contain up to 100 ng of WGA-like protein after the first week of growth, but the level fluctuates thereafter. Since most of the lectin is present at the base of the shoot and much less is found in older roots, these fluctuations may be the consequence of changes in the initiation of new advantitious roots.Abbreviations ELISA enzyme-linked immunosorbent assay - GlcNAc N-acetylglucosamine - PBS phosphate-buffered saline - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - WGA wheat germ agglutinin     

不吸水链霉菌葡萄糖异构酶的物化表征

葡萄糖异构酶是一种催化葡萄糖异构为果糖的酶。本文用紫外吸收光谱、红外光谱、氨基酸组分分析、聚丙烯酰胺凝胶梯度电泳、SDS-聚丙烯酰胺凝胶电泳、超薄 层聚丙烯酰胺凝胶等电聚焦电泳技术研究了不吸水链霉菌嗜热亚种M1033菌株产生的葡萄糖异构酶的一些物化性质。结果表明由本实验室制备的均一葡萄糖异构酶的A₂₈₀与A₂₆₀的比值是1.76。它是由一个亚单位组成的酶分子。最小分子量是49000。pI值是5.2。氨基酸组分与其它来源的葡萄糖异构酶的氨基酸组分相比较有一些差异,其中Glu,Gly,ALa和Leu的含量都此其它异构酶的高,而Met,Trp,Asp,Thr则比其它葡萄糖异构酶的低。     

Purification and characterisation of a carboxylesterase from the latex ofSynadenium grantii Hook, ‘f’

The latex ofSynadenium grantii was found to contain esterolytic activity. Polyacrylamide gel electrophoretic study coupled with substrate and inhibitor specificity studies revealed the presence of multiple forms of carboxylesterases and cholinesterases in the latex. One of the carboxylesterases of the latex was purified by acetone fractionation, carboxymethyl-Sephadex chromatography and Sepharose-6B gel filtration. The homogeneity of the enzyme was established by polyacrylamide gel electrophoresis, isoelectric focussing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme consists of a single polypeptide chain with a molecular weight of 14,000. The amino acid analysis of the purified enzyme revealed that it contained a greater number of neutral and acidic, compared to basic amino acid residues. The isoelectric pH of the enzyme was found to be 4.0. The enzyme was a glycoprotein as revealed by periodic acid Schiff-staining technique. Studies with different organophosphate and carbamate inhibitors showed that this enzyme was sensitive to organophosphates. The product inhibition studies with this enzyme showed linear competitive inhibition with acetate and linear non-competitive inhibition with 1-naphthol.     

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.     

Sexual agglutinin of mating-type minus gametes inChlamydomonas reinhardtii

The sexual agglutinin from the mating-type minus gametes ofChlamydomonas reinhardtii was purified by gel filtration and hydroxyapatite chromotography. The minus agglutinin was identified as a single glycopolypeptide termed Agg(-) of very high molecular weight by SDS-poly-acrylamide gel electrophoresis. It was also observed as a glycoprotein with agglutinin activity on non-SDS polyacrylamide gels. The native agglutinin appeared to be composed of a complex of Agg(-) subunits. It consisted of about 60% protein and 40% carbohydrate and the activity was diminished by a mild periodate oxidation. When the plus agglutinin was also purified and compared with the minus agglutinin, it was found that both agglutinins migrate in the same position by SDS-polyacrylamide gel electrophoresis, whereas their behaviors on gel filtration and hydroxyapatite chromatography are different.Abbreviations mt ^+/– mating-tape plus or minus - SDS sodium dodecyl sulfate - V_e elution volume - V_o void volume - kDa kilodalton     

Purification and study of carbohydrate specificity of lectin from Sarcoscypha coccinea (Fr.) Lambette

A lectin that revealed affinity for lactose, N-acethylactosamine, 4-nitrophenyl-beta-D-gluco- and galactopyranosides from fruiting bodies of Sarcoscypha coccinea (Fr.) Lambette was purified by affinity chromatography on immobilized ovomucoid. According to electrophoresis data in 15% SDS-PAGE the lectin contains two very low-differing components with molecular weight 32 kDa. Molecular weight of the lectin is 128 kDa according to gel-chromatography on sephadex G-200. The lectin agglutinates rabbit erythrocytes and slightly weaker agglutinates human erythrocytes. After dialysis against 1% EDTA sodium salt solution the lectin loses hemaglutinating activity, but after the next dialysis against CaCl2 solution it is restored.