Characterization of a lectin from the seeds of Erythrina vespertilio

A lectin was isolated from the seeds of Erythrina vespertilio by affinity chromatography on lactose-Sepharose 6B. The lectin has an M, of 59 000 and consists of two non-covalently associated subunits (M, ∼ 30 000). The lectin is devoid of cysteine but has six methionine residues/mol and a neutral sugar content of 9.7% The carbohydrate composition was mannose, N-acetylglucosamine, fucose, xylose and galactose in amounts of 15.0, 4.0, 1.0, 5.0 and 25 mol/59 000 g, respectively. Alkaline gel electrophoresis and isoelectric focusing showed that the affinity purified lectin consists of a family ofisolectins. Valine was the only N-terminal amino acid found and the N-terminal sequence was homologous with that found for other legume lectins. The lectin was inhibited by galactosyl containing carbohydrates; p-nitrophenyl-β-galactoside was the best inhibitor and the lectin showed a slight preference for β-galactosides. Comparison of its properties with those of other Erythrina lectins shows that most of the lectins of this genus are closely related.     

Biosynthesis of Tetrapyrrole Pigment Precursors : Pyridoxal Requirement of the Aminotransferase Step in the Formation of delta-Aminolevulinate from Glutamate in Extracts of Chlorella vulgaris

The aminotransferase that catalyzes the formation of δ-aminolevulinic acid from glutamate-1-semialdehyde or from glutamate in a reconstituted enzyme system was isolated and partially purified from Chlorella vulgaris. The apparent molecular weight of the aminotransferase was determined by Sephadex G-100 and Ultrogel AcA 54 gel filtration to be 60,000 ± 5,000. Catalytic activity of the aminotransferase required pyrixodal phosphate (PALP). The cofactor could not be removed by gel filtration after exposure of the enzyme to PALP. Aminotransferase was inhibited by gabaculine (3-amino-2,3-dihydrobenzoic acid). The concentration of gabaculine required for half maximal inhibition was about 0.05 micromolar. Aminotransferase activity could be regained upon the removal of gabaculine by gel filtration and supplementing the assay medium with PALP. Neither the inhibitory action of gabaculine nor its reversibility was affected by preincubation of the enzyme with the keto acids levulinate and δ-aminolevulinic acid.     

Comparative studies of seed proteins of the genusArtocarpus with respect to lectins

Proteins from two species of the genusArtocarpus (A. integrifolia L. andA. incisa L.) were compared by ammonium sulphate fractionation, molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis, with special attention to the lectins. The protein content and hemagglutinating activity were markedly different in the two seeds. The protein pattern obtained by both molecular sieve chromatography and SDS-polyacrylamide gel electrophoresis were quite different. The only similarities found were the elution volume of the lectins in the Sephadex G-100 column and the lectin bands (11 500 and 15 000 daltons) in SDS-polyacrylamide gel electrophoresis.     

The human LT system. III. Characterization of a high molecular weight LT class (complex) composed of the various smaller MW LT classes and subclasses in association with Ig-like molecules.

Cytotoxic activity (lymphotoxin (LT)) present in supernatants from lectin stimulated human lymphocytes in vitro is composed of a heterogeneous system of biological macromolecules which can be separated into multiple classes and subclasses on the basis of their molecular weight and charge. These studies further characterize a large molecular weight human LT class, termed complex (MW >200,000 d), which elutes in the void volume off Sephadex G-150 or Ultrogel AcA 44. Immunological studies on the complex, employing various rabbit anti-LT class and subclass antisera, revealed this material is a macromolecular assemblage of the smaller MW α, β, γ LT classes and subclasses. Furthermore, the reactivity of this material with anti-human Fab′₂ (IgG) indicates these smaller molecular weight LT components can associate with immunoglobulin or Ig-like molecules. The materials present in the LT complex class appear to be noncovalently associated, since conditions of high ionic strength dissociate certain small MW LT components, while low ionic strength buffers may cause these components to reaggregate with the complex. When subjected to velocity sedimentation on sucrose gradients or gel filtration on Ultrogel AcA 22, LT complex activity elutes as several discrete peaks of activity in the 200,000 to 1,000,000 MW range. These findings suggest the concept that LT molecules can form discrete and specific macromolecular structures which contain the smaller MW LT classes. Moreover, these structures can also associate with immunoglobulin-like molecules to form secondary LT-Ig complexes. This may have important biological significance in explaining how nonspecific cell toxins could play a role in specific or nonspecific cell lytic reactions in vitro.     

Purification,Chemical, and Immunochemical Properties of a New Lectin from Mimosoideae (Parkia Discolor)

ABSTRACT

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P.discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.     

Purification and characterization of lectins from Vicia hirsuta

The presence of three lectins in the seeds of Vicia hirsuta (L.) S. F. Gray, a wild-growing vetch, was shown. The main lectin was purified to homogeneity by buffer extraction, ammonium sulfate precipitation, affinity chromatography on Sephadex G-100 and isoelectric focusing in granulated gel. By chromatofocusing instead of isoelectric focusing the yield was increased 5-fold. The lectin has a pi of 6.4. It is composed of large β-subunits (M_r 19.200) and small α-subunits (M_r 12.800) in a 1:1 ratio. The subunits can be separated on Sephadex G-75 when equilibrated with 6 M guanidine-HCl. The amino acid composition of the two different subunits has been determined. No sulfur-containing amino acids are present. The lectin resembles the lectins from legumes from the same cross-inoculation group, i.e. Lens culinaris, Lens esculenta, Pisum satiyum and several Vicia spp. by the same type of sugar specificity and amino acid composition.     

Purification and some properties of a lectin from the seeds ofTrichosanthes anguina

A galactose-binding lectin was isolated in electrophoretically pure form from the seeds of the snake gourd,Trichosanthes anguina, by affinity chromatography on an immobilised lactose column, as well as on a cross-linkedGuar Gum column. The lectin agglutinates native erythrocytes of human A, B and 0 phenotypes and of rabbit, rat and mouse. The molecular mass of the lectin, as estimated bySephadex G-200 gel chromatography, is 49 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis, after reduction with β-mercaptoethanol, revealed two polypeptide chains linked by disulphide bonds in the lectin molecule. It contains no covalently linked sugars. Amino acid analysis of the lectin revealed a high content of acidic amino acids, relatively lower proportion of basic amino acids and traces of cysteine and methionine. The lectin has good thermal stability, and is inactivated when oxidised by metaperiodate.     

Purification, chemical, and immunochemical properties of a new lectin from Mimosoideae (Parkia discolor)

A glucose/mannose-binding lectin was isolated from seeds of Parkia discolor (Mimosoideae) using affinity chromatography on Sephadex G-100 gel. The protein presented a unique component in SDS-PAGE corresponding to a molecular mass of 58,000 Da, which is very similar to that of a closely related lectin from Parkia platycephala. Among the simple sugars tested, mannose was the best inhibitor, but biantennary glycans, containing the trimannoside core, present in N-glycoproteins, also seem to be powerful inhibitors of the haemagglutinating activity induced by the purified lectin. The protein was characterised by high content of glycine and proline and absence of cysteine. Rabbit antibodies, anti-P. platycephala seed lectin, recognised the P. discolor lectin. However, no cross-reaction was observed when a set of other legume lectins from sub-family Papilionoideae and others from families Moraceae and Euphorbiaceae were assayed with the Parkia lectins. This suggests that Parkia lectins comprise a new group of legume lectins exhibiting distinct characteristics.     

Purification and characterization of a β-galactoside-binding soluble lectin from rat and bovine brain

A β-galactoside-binding activity has been detected in mammalian brain extracts using a hemagglutination test and a nerve cell aggragation assay. Inhibition studies suggested the involvement of lectin-carbohydrate interactions in these processes. In an attempt to explore further the biological role of brain lectins, the β-galactoside-binding activity has been purified to apparent homogeneity from bovine and rat brain by salt extraction of the brain tissue and affinity chromatography on asialofetuin-agarose. The molecular weights determined by gel filtration, under native conditions on Ultrogel AcA-34, were 30 000 for the bovine brain lectin and 32 000 for the rat brain lectin; polyacrylamide gel electrophoresis in SDS gave molecular weights of 15 000 and 16 000, respectively, suggesting that the two brain lectins are dimers. Both lectins have an isoelectric point of 3.9. Amino acid composition data indicate that both lectins contain high proportions of glycine and acidic amino acids. The lectins are specific for β-D-galactosides and related sugars and the configuration of carbon atoms 1, 2 and 4 seems of primary importance. Moreover, the nerve cell aggregation-promoting activity of the purified lectin is 300-fold that of the crude extracts.     

Purification and characterization of a Corynebacterium ulcerans bacteriocin (ulceracin 378)

Corynebacterium ulcerans strain 378 produces a bacteriocin (ulceracin 378) and a toxin when grown on semi-solid medium. Ulceracin 378 was purified 360-fold by dialysis and chromatography on DEAE-cellulose and Sephadex G-200. On the basis of Ultrogel AcA22 gel filtration its molecular weight was about 900 000. It could be dissociated by 2-mercaptoethanol and sodium dodecyl sulphate into smaller subunits of 25 000. The bactericidal activity was associated with this subunit which contained no carbohydrate or lipid. Ulceracin 378 was thermostable and stable over a wide pH range. Purified ulceracin 378 did not have a toxic effect (lethal) on guinea-pigs and rabbits and was immunologically distinct from the toxin.     

Purification and some properties of a trypsin inhibitor from carp (Cyprinus carpio) muscle

A trypsin inhibitor was purified from carp muscle to apparent homogeneity by the successive chromatographies of DEAE-cellulose, DEAE-Sepharose CL-6B, Con A-Sepharose, Ultrogel AcA 44 and hydroxylapatite. The mol. wt of the inhibitor was estimated to be 58,000 by SDS-polyacrylamide gel electrophoresis or 50,000 by gel filtration. The inhibitor seemed to form a 1:1 stoichiometric complex with trypsin, alpha-chymotrypsin and elastase, respectively. Carp muscle trypsin inhibitor was likely to be identical with serum alpha 1-proteinase inhibitor judging from its glycoprotein nature, mol. wt and the inhibition stoichiometry.     

Lectin fromCanavalia brasiliensis (MART.). isolation,characterization and behavior during germination

A lectin was isolated fromCanavalia brasiliensis Mart. seeds by combining solubility fractionation with affinity chromatography on Sephadex G-50. The lectin showed a carbohydrate specificity for D-mannose (D-glucose) binding and a requirement for Ca²⁺ and Mn²⁺. All the hemagglutinating activity was found in the cotyledons and the presence of the lectin was followed during the first 15 days of plant germination, through the activity against rabbit erythrocytes, the presence of the “lectin peak” in Sephadex G-50 affinity chromatography, presence of the “lectin bands” in SDS-polyacrylamide gel electrophoresis and the “lectin arcs and rockets” in immunoelectrophoresis in agarose gel. On application of all these methods the lectin showed a differentiated metabolism, disappearing more slowly than the other high molecular weight proteins of the seed.     

Single-chain, low molecular weight kininogen in the blood plasma of rabbits: isolation and fragmentation by porcine pancreatic kallikrein

A highly purified preparation of low molecular weight kininogen (LMrK) was isolated from the plasminogen-free rabbit blood plasma, using chromatography on DEAE-Sepharose CL-6B, gel filtration on Ultrogel AcA 34 and Sephadex G-100 as well as gradient chromatography on a hydroxylapatite column. The yield of the 320-fold purified LMrK was 16%. Trypsin released 13-14 micrograms-eq. of bradykinin (BK) from 1 mg of LMrK or 0.85-0,95 mol of BK per mol of kininogen. Rabbit LMrK consists of one polypeptide chain of Mr 69 000 and pI 4.63. Porcine pancreatic kallikrein splits off kinin from the LMrK polypeptide chain by disrupting two peptide bonds resulting in the formation of S-S-bound two chain molecule. After reduction of the S-S bonds by dithioerithritol the latter is separated into a heavy (Mr 61 000) and light (Mr 6 800) chains. A biologically active peptide was isolated from the products of CNBr cleavage of LMrK. This peptide consists of Lys-BK elongated from the C-terminal with several amino acid residues. Rabbit LMrK closely resembles human LMrK in terms of Mr, pI and location of the kinin fragment in the protein molecule.     

The major sialoglycoprotein of human T-lympocytes

Human, peripheral-blood T-lymphocytes and human, T-lymphoblastoid cells of a MOLT 4B cell-line were surface-labeled by lactoperoxidase-catalyzed iodination, periodate and sodium borotritide, and galactose oxidase and sodium borotritide, and analyzed by dodecyl sodium sulfate-polyacrylamide gel-electrophoresis. Both types of cells were found to show a major, cell-surface sialoglycoprotein with an apparent mol. wt. of 95 000. After neuraminidase treatment, this glycoprotein showed a higher mol. wt. of 120 000. The major sialoglycoprotein of both types of cells bound to wheat-germ agglutinin and concanavalin A and, after neuraminidase treatment, to Arachis hypogaea agglutinin. The glycopeptides obtained from these glycoproteins by Pronase digestion gave similar elution-profiles on Sephadex G-50 gel filtration. These results suggest that the major sialoglycoprotein of normal T cells and that of MOLT 4B cells are very similar, if not identical.     

Erythro- and lymphoagglutinins of Phaseolus acutifolius

A potent lymphoagglutinin which had low affinity for red cells or fetuin and another lectin which reacted strongly with red cells and fetuin but was a poor agglutinin for lymphocytes were isolated from seeds of Phaseolus acutifolius. A number of other lectin components with intermediate activity towards these cells was also isolated. All the lectins had very similar amino acid and carbohydrate composition, sedimentation patterns, partial specific volume and molecular weight values of about 116 600 and were thus smaller than the related Phaseolus vulgaris lectins (M_r = 119 000). The lectins contained four subunits with only minor size and charge differences between the lympho- and erythroagglutinating subunits and their electrophoretic mobility in SDS gel electrophoresis was anomalously high. The existence of lympho- and erythroagglutinating subunits in two members of the genus Phaseolus supports their close morphological similarity.     

Studies on lectins: XLIII. Isolation and characterization of the lectin from restharrow boots (Ononis hircina Jacq.)

From 1 kg of dried Ononis hircina Jacq. roots 36 mg of a lectin were isolated by affinity chromatography on O-β-lactosyl polyacrylamide gel. The lectin is homogeneous as judged by ultracentrifugal analysis (s_20,w = 6.2 S), polyacrylamide disc electrophoresis at pH 8.9 or 4.5, gel filtration on thin layers of Sephadex G-200 (M_r = 110 000) and dodecyl sulfate electrophoresis (M_r of sub-units 31 000, both in presence and absence of mercaptoethanol) and disc dodecyl sulfate electrophoresis (pH 9.5). The lectin contains much aspartic and glutamic acids, serine and threonine and also 7.2% of neutral sugar. It is relatively specific for human type O erythrocytes that are agglutinated at a minimal lectin concentration 0.3 μg/ml. The erythroagglutinating activity is not stimulated by Ca²⁺, Zn²⁺, Mg²⁺, Mn²⁺, Co²⁺, or Ni²⁺ salts; it is inhibited most effectively by N-acetyl-D-galactosamineandanumberofD-galactosederivatives. Dissociation constants of several lectin · sugar complexes were estimated by affinity electrophoresis. The lectin is not mitogenic in rabbit lymph nodes lymphocytes.     

Characterization of the acidic lectins from winged bean seed (Psophocarpus tetragonolobus(L.)DC)

The seeds of winged bean, Psophocarpus tetragonolobus(L.)DC, contain two distinct groups of lectins characterized by different erythrocyte hemagglutinating specificities and isoelectric points. Three acidic lectins (I, II, and III) (pI approximately 5.5) were purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP-Sephadex C-25. These lectins are glycoproteins with relative molecular mass of 54,000. The total carbohydrate content of the acidic lectins was 7% and was comprised of mannose, N-acetylglucosamine, fucose, and xylose in amounts corresponding to 9.2, 4.8, 1.6, and 7.0 mol/54,000 g, respectively. Electrophoresis in dodecyl sulfate, in the presence and absence of 2-mercaptoethanol, gave a single subunit of apparent relative molecular mass 30-32,000, somewhat higher than expected from the native relative molecular mass. On isoelectric focusing in 8 M urea the subunits of the acidic lectins did not show any significant charge heterogeneity as found for the winged bean basic lectins. The acidic lectins have very similar amino acid compositions. They contain essentially no half-cystine, 1-2 methionine residues, and are rich in acidic and hydroxy amino acids. The amino-terminal sequences of lectins II and III were identical while the amino-terminal sequence of lectin I contained five differences in the first 25 residues; the acidic lectins showed extensive sequence homology with the winged bean basic lectins, the other one-chain subunit lectins and the beta subunit of the two-chain subunit legume lectins. The acidic lectins agglutinated trypsinized human (type A, B, AB, and O) erythrocytes but not trypsinized rabbit erythrocytes. They were inhibited by various D-galactose derivatives and D-galactose-containing disaccharides and trisaccharides. N-Acetylgalactosamine was the best inhibitor, and the specificity appears to be directed to beta-D-galactosides. However, compared with winged bean basic lectins and soybean lectin, the winged bean acidic lectins show a low affinity for the inhibitory sugars.     

Purification and partial characterization of a lectin from Canavalia grandiflora benth. seeds

A D-glucose/D-mannose specific lectin from seeds of Canavalia grandiflora (ConGF) was purified by affinity chromatography on Sephadex G-50. By SDS-PAGE ConGF yielded three protein bands with apparent molecular masses of 29-30 kDa (alpha chain), 16-18 kDa (beta fragment) and 12-13 kDa (gamma fragment), like other related lectins from the genus Canavalia (Leguminosae). ConGF strongly agglutinates rabbit erythrocytes, has a high content of ASP and SER, and its N-terminal sequence (30 residues) is highly similar to the sequences of other related lectins from subtribe Diocleinae.     

Mannose Binding Lectins of Vicia tetrasperma Seed and Their Immunological Relationship to Other Legume Lectins of Similar Specificity

Mannose specific lectins of Vicia tetrasperma were purifiedby affinity chromatography with Sephadex G-100, and ion exchangechromatography. Chromatofocusing using PBE-94 gel was successfullyemployed to separate the major isolectins, lectin I and II.Both lectins had the same molecular weight of 78,000 and weretetramers composed of a uniform subunit with a molecular weightof 18,700. Amino acid compositions of these lectins were quitesimilar to each other, rich in aspartic acid (and/or asparagine)and hydroxyl amino acids, and lacking methionine and cysteine.Agar gel double diffusion using anti V. tetrasperma lectin antiserumrevealed that lectins from V. cracca, Pisum sativum, and Lensculinaris, all of which have mannose binding properties, wereantigenically identical. The antiserum reacted with the analogouslectins from V.faba, V. hirsuta, and V. angustifolia, but formationof a spur in the diffusion assay showed that they were slightlydifferent from V. tetrasperma lectin. (Received December 24, 1985; Accepted March 12, 1986)     

Studies on lectins XLIV. The pH dependence of lectin interactions with sugars as determined by affinity electrophoresis

The pH dependence of association constants of the lectin-sugar complexes was determined by means of affinity electrophoresis. All the lectins studied (from the seeds of Dolichos biflorus, Glycine soja, Lens esculenta and Vicia cracca and of the fruiting body of Marasmius oreades) were characterized by a similar course of pH dependence of the association constants, with the maximum values at pH 7–9. For concanavalin A and the l-fucose binding Ulex europaeus lectin only the association constants at three selected pH values were determined. Concanavalin A does not interact with immobilized α-d-mannosyl residues at pH 2.3. The association constants vs. pH curves measured for lectins isolated from two different varieties slightly differ in accordance with the differences observed in the interaction of these lectins with the Sephadex gel.