Isolation and characterization of a lectin from the fruit of Clerodendron trichotomum

An agglutinin of Clerodendron trichotomum fruit (CTA), found to be specific for N-acetyl-D-galactosamine and D-galactose, was isolated and characterized. The fruit extract was decolorized first by passage through a Toyopearl column and a phenyl-Sepharose column. Then the lectin activity was adsorbed on p-aminophenyl N-acetyl-alpha-D-galactosaminide- or p-aminophenyl beta-D-galactoside-Sepharose, and eluted as a sharp peak with 0.2 M lactose. The purified CTA was found to be homogeneous by SDS-polyacrylamide gel electrophoresis, gel chromatography and ultracentrifugal analysis, and was determined to be a glycoprotein homodimer with a molecular weight of 56,000 daltons. Hemagglutination-inhibition assay indicated that CTA is most specific for N-acetyl-D-galactosaminide with a hydrophobic aglycon.     

Purification of basic FGF receptors from rat brain

Receptor molecules for basic fibroblast growth factor (bFGF) were isolated from rat brain by a novel and rapid procedure and characterized. Purification was performed by wheatgerm agglutinin (WGA) gel affinity chromatography in combination with bFGF gel affinity chromatography, utilizing a novel elution method involving heparin. The eluted proteins were active in binding bFGF and were separated as two bands with respective molecular masses of 140 kDa and 110 kDa on SDS-PAGE. More than half of this bFGF-binding activity was lost after 16 h at 4 degrees C. Thus, bFGF receptors were purified as labile glycoconjugates.     

Purification of Phytohaemagglutinins from Phaseolus vulgaris and their Interaction with Plant Pathogenic Bacteria

Erythrocytes and different strains of plant pathogenic bacteria agglutinated with aqueous and saline 1M NaCl extracts from Phaseolus vulgaris L. seeds, which showed similar gel electrophoretic patterns of proteins. Haemagglutinating activity in aqueous extracts was inhibited by some carbohydrates, especially N-acetyl-D-galactosamine, D-galactose and lactose. The agglutinin in this extract was purified by utilizing its ability to bind to Pseudomonas pisi cells followed by its removal from the bacteria by 0.3M galactose. Mitogenic activity of purified agglutinin was calculated as incorporation of ¹²⁵I-iododesoxy-uridine into DNA of lymphocytes in vitro, which gave values between 25 and 50 μg/ml. The molecular weight of the subunits was found to be 31,500, estimated by mobility in SDS-PAGE. A solid phase competition-binding radioimmunoassay for bean agglutinin was developed in order to determine the affinity of this lectin in bean plants to phytopathogenic bacteria. The highest level of affinity was associated with the system formed by, Ps. pisi and lectin from fresh seeds.     

Peanut Agglutinin Receptor Is a Marker of Myelin in Rat Brain. Developmental Changes in Its Distribution

Peanut agglutinin receptor, which is a glycoconjugate with terminal Gal(beta 1-3)GalNAc residues, was found to be a specific biochemical marker of the myelin in rat brain. The localization of peanut agglutinin receptor in rat brain was studied by use of fluorescein-conjugated peanut agglutinin. In adult rat brain, receptors were found only on myelin. In contrast, the receptors in fetal brain were not found in the white matter, but only in the ependyma. Peanut agglutinin receptors were extracted from adult and fetal rat brains with 2% Triton X-100, subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gel, blotted onto nitrocellulose filters, and detected by their reaction with peroxidase-conjugated peanut agglutinin. A major receptor of Mr 130,000 and a minor one of Mr 200,000 were found in both adult and fetal rat brains.     

Intrinsic inhibitor of inositol 1,4,5-trisphosphate binding

Rat brain cytosol was applied to a heparin column and eluted with 0.9 M-NaCl. The total binding activity of [3H]inositol 1,4,5-trisphosphate to the eluate was increased about 6-fold compared with the original cytosol. When the eluate was mixed with a flow-through fraction from the heparin column, however, the activity returned to the original level, suggesting that the flow-through fraction contained an inhibitory factor(s) which prevented the binding. The factor(s) was purified by sequential column chromatography using gel permeation, a hydrophobic gel, and finally, a hydroxylapatite gel. Silver staining of sodium dedecyl sulfate gel electrophoresis of the sample thus purified showed a broad band located between the authentic molecular weight markers of 580 and 390 k. A carbohydrate staining method showed that the factor is a glycoprotein.     

Further characterization of the cold agglutinin from the snail Achatina fulica.

The cold agglutinin from the albumin gland of the snail Achatina fulica was purified to homogeneity by using sheep gastric mucin-Sepharose 4B as affinity column followed by gel filtration on Bio-Gel P-300. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. The purified cold agglutinin is a glycoprotein of native M2 220,000 consisting of three non-covalently bound subunits of Mr 84,000, 74,000 and 62,000 and having a pI value of 4.5. The predominant amino acids are aspartic acid and glutamic acid (or amides) and serine, which account for 39% of the residues. About 3% of the residues are half-cystine. The lectin is a glycoprotein with about 30.7% carbohydrate, the most abundant sugars being galactose, N-acetylgalactosamine and N-acetylglucosamine. Mannose, xylose and fucose are also present. The inhibition of agglutination of human umbilical-cord erythrocytes by the cold agglutinin is specific for methyl beta-D-galactoside and also for glycolipids present on cord erythrocytes. The c.d. data show only negative ellipticity values in the far-u.v. region for the protein at various concentrations and temperatures and also in the presence of the hapten lactose (at different concentrations), indicating the presence of a random-coil conformation in the agglutinin that varies according to temperature.     

Occurrence of heparin inhibitable hemagglutinating activity in leaves of fava bean (Vicia faba)

A hemagglutinating activity which is inhibitable by heparin was extracted from leaves of fava bean. The activity was partially purified by ammonium sulfate fractionation followed by affinity chromatography of heparin-agarose. The purified activity was inhibited only by heparin but not by simple monosaccharides or glycosaminoglycans tested. These results showed that this activity was very different from its well known seed lectin, Vicia faba agglutinin.     

Physicochemical Properties and Oxidative Inactivation of Soluble Lectin from Water Buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) Brain

Lectins are carbohydrate-binding proteins present in a wide variety of plants and animals, which serve various important physiological functions. A soluble β-galactoside binding lectin has been isolated and purified to homogeneity from buffalo brain using ammonium sulphate precipitation (40–70%) and gel permeation chromatography on Sephadex G_50–80 column. The molecular weight of buffalo brain lectin (BBL) as determined by SDS-PAGE under reducing and non-reducing conditions was 14.2 kDa, however, with gel filtration it was 28.5 kDa, revealing the dimeric form of protein. The neutral sugar content of the soluble lectin was estimated to be 3.3%. The BBL showed highest affinity for lactose and other sugar moieties in glycosidic form, suggesting it to be a β-galactoside binding lectin. The association constant for lactose binding as evidenced by Scatchard analysis was 6.6 × 10³ M⁻¹ showing two carbohydrate binding sites per lectin molecule. A total inhibition of lectin activity was observed by denaturants like guanidine HCl, thiourea and urea at 6 M concentration. The treatment of BBL with oxidizing agent destroyed its agglutination activity, abolished its fluorescence, and shifted its UV absorption maxima from 282 to 250 nm. The effect of H₂O₂ was greatly prevented by lactose indicating that BBL is more stable in the presence of its specific ligand. The purified lectin was investigated for its brain cell aggregation properties by testing its ability to agglutinate cells isolated from buffalo and goat brains. Rate of aggregation of buffalo brain cells by purified protein was more than the goat brain cells. The data from above study suggests that the isolated lectin may belong to the galectin-1 family but is glycosylated unlike those purified till date.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Lectins as membrane components of mitochondria from Ricinus communis.

1. Mitochondria were isolated from developing endosperm of Ricinus communis and were fractionated into outer membrane and inner membrane. The relative purity of the two membrane fractions was determined by marker enzymes. The fractions were also examined by negative-stain electron microscopy. 2. Membrane fractions were sequentially extracted in the following way. (a) Suspension in 0.5M-potassium phosphate, pH7.1; (b)suspension in 0.1M-EDTA (disodium salt)/0.05M-potassium phosphate, pH7.1; (c) sonication in 0.05M-potassium phosphate, pH7.1;(d)sonication in aq. Triton X-100 (0.1%). The membranes were pelleted by centrifugation at 100 000g for 15 min, between each step. Agglutination activity in the extracts was investigated by using trypsin-treated rabbit erythrocytes. 3. The addition of lactose to inner mitochondrial membrane resulted in the solubilization of part of the lectin activity, indicating that the protein was attached to the membrane via its carbohydrate-binding site. Pretreatment of the membranes with lactose before tha usual extraction procedure showed that lactose could extract lectins that normally required more harsh treatment of the membrane for solubilization. 4. Lectins extracted from inner membranes were purified by affinity chromatography on agarose gel. Polyacrylamide-gel electrophoresis of purified samples in sodium dodecyl sulphate indicated that at least part of the lectin present in inner mitochondrial membrane was identical with the R. communis agglutinin of mol.wt. 120 000.     

Chemical modification studies on Ricinus communis (Castor Bean) agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.     

Chemical modification studies on Abrus agglutinin. Involvement of tryptophan residues in sugar binding.

The galactose-binding lectin from the seeds of the jequirity plant (Abrus precatorius) was subjected to various chemical modifications in order to detect the amino acid residues involved in its binding activity. Modification of lysine, tyrosine, arginine, histidine, glutamic acid and aspartic acid residues did not affect the carbohydrate-binding activity of the agglutinin. However, modification of tryptophan residues carried out in native and denaturing conditions with N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide led to a complete loss of its carbohydrate-binding activity. Under denaturing conditions 30 tryptophan residues/molecule were modified by both reagents, whereas only 16 and 18 residues/molecule were available for modification by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide respectively under native conditions. The relative loss in haemagglutinating activity after the modification of tryptophan residues indicates that two residues/molecule are required for the carbohydrate-binding activity of the agglutinin. A partial protection was observed in the presence of saturating concentrations of lactose (0.15 M). The decrease in fluorescence intensity of Abrus agglutinin on modification of tryptophan residues is linear in the absence of lactose and shows a biphasic pattern in the presence of lactose, indicating that tryptophan residues go from a similar to a different molecular environment on saccharide binding. The secondary structure of the protein remains practically unchanged upon modification of tryptophan residues, as indicated by c.d. and immunodiffusion studies, confirming that the loss in activity is due to modification only.     

Identification of glycoprotein components of alpha-agglutinin, a cell adhesion protein from Saccharomyces cerevisiae.

Several glycoproteins which inhibit the agglutinability of Saccharomyces cerevisiae mating type a cells were partially purified from extracts of mating type alpha cells. These proteins, called alpha-agglutinin, were labeled with 125I-Bolton-Hunter reagent. The labeled alpha-agglutinin showed specific binding to a cells. Such specific binding approached saturation with respect to agglutinin or cells and was inhibited in the presence of excess unlabeled alpha-agglutinin. Nonspecific binding was similar in a and alpha cells, was neither saturable nor competable, and was three- to fourfold less than the specific binding to a cells at maximum tested agglutinin concentrations. The major a-specific binding species had a low electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels and had an apparent molecular weight of 155,000 by rate zonal centrifugation. Endo-N-acetylglucosaminidase H digestion of the purified glycoprotein complex converted the low-mobility material to four major and several minor bands which were resolved by polyacrylamide gel electrophoresis. All but two minor peptides bound specifically to a cells. Analyses of agglutinin from mnn mutants confirmed the deglycosylation results in suggesting that the N-linked carbohydrate portion of alpha-agglutinin was not necessary for activity.     

Activation of heparin cofactor II by heparin oligosaccharides

Heparin was partially depolymerized with heparinase or nitrous acid. The resulting oligosaccharides were fractionated by gel filtration chromatography and tested for the ability to stimulate inhibition of thrombin by purified heparin cofactor II or antithrombin. Oligosaccharides containing greater than or equal to 18 monosaccharide units were active with antithrombin, while larger oligosaccharides were required for activity with heparin cofactor II. Intact heparin molecules fractionated on a column of immobilized antithrombin were also tested for activity with both inhibitors. The relative specific activities of the unbound heparin molecules were 0.06 with antithrombin and 0.76 with heparin cofactor II in comparison to unfractionated heparin (specific activity = 1.00). We conclude that heparin molecules much greater than 18 monosaccharide units in length are required for activity with heparin cofactor II and that the high-affinity antithrombin-binding structure of heparin is not required.     

A homotetrameric agglutinin with antiproliferative and mitogenic activities from haricot beans

A homotetrameric agglutinin with a molecular mass of 130 kDa was isolated from seeds of the haricot bean. The agglutinin was isolated using a procedure that involved ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel and gel filtration by fast protein liquid chromatography on Superdex 200. Haricot bean agglutinin was adsorbed on DEAE-cellulose and Affi-gel blue gel. The hemagglutinating activity of the agglutinin was stable up to 40 degrees C. It underwent a 40% decline when the temperature was raised to 50 degrees C and a complete loss when the temperature was further increased to 80 degrees C. The hemagglutinating activity exhibited a time-dependent loss in activity when the agglutinin was incubated at 100 degrees C for different durations. No activity was discernible when the agglutinin was left at 100 degrees C for 1 min. The activity also underwent a decline in the presence of 500 mM FeCl(3) and CaCl(2). Haricot bean agglutinin manifested a weaker mitogenic activity than concanavalin A toward mouse splenocytes. It exhibited antiproliferative activity toward the tumor cell lines M1 [leukemia], HepG2 [hepatoma] and L1210 [leukemia] cells.     

泥蚶(Tegillarca granosa Linnaeus)血淋巴液凝集素的分离纯化及其性质研究

泥蚶是一种重要的海产经济贝类,其血淋巴液经硫酸铵二步分级沉淀后,再经Sephadex G- 100凝胶过滤和Sepharose 4B亲和层析纯化制得泥蚶血淋巴液凝集素。经测定,该凝集素分子量约为123Kda,为两个亚基的蛋白质,其相对分子量分别为15 KDa和16 KDa,分子中含5．02％的糖。在氨基酸组成中,天门冬氨酸(Asp)含量最高,其次是谷氨酸(Glu)和组氨酸(His),不含蛋氨酸(Met)。泥蚶血淋巴液凝集素对多种天然或经酶修饰的人或动物红细胞具有不同的凝集作用,其中对兔红细胞的凝集活性最大。半乳糖和乳糖对其凝集活性具有抑制作用。凝集活性依赖于Ca2 ,在pH7．0较稳定,热稳定性不高,在30℃-70℃时凝集效价由原来的25下降为21,当温度超过80℃以后,凝血活性完全丧失。     

Partial purification of galactocerebroside sulfotransferase in the rat brain

We describe a method of partial purification of rat brain 3'-phosphoadenosine-5'-phosphosulfate galactocerebroside sulfotransferase (EC 2.8.2.11, CST). The first steps consist of a Triton X-100 extraction of the enzyme from delipidated microsomes and the fractionation of this extract by precipitation with ammonium sulfate, followed by successive chromatography on hydrophobic gel (phenyl sepharose), heparin, gel filtration (Trisacryl GF2000), hydroxy-apatite and cation exchange resin (SP trisacryl). The denaturation of the enzyme and its instability account for the low enrichment achieved in terms of specific activity, but an evident simplification of the electrophoretic pattern was obtained.     

Biochemical and immunological properties of alkaline invertase isolated from sprouting soybean hypocotyls.

Alkaline invertase from sprouting soybean (Glycine max) hypocotyls was purified to apparent electrophoretic homogeneity by consecutive use of DEAE-cellulose, green 19 dye, and Cibacron blue 3GA dye affinity chromatography. This protocol produced about a 100-fold purification with about a 11% yield. The purified protein had a specific activity of 48 mumol of glucose produced mg-1 protein min-1 (pH 7.0) and showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (58 kDa) and in native PAGE, as indicated by both protein and activity staining. The native enzyme molecular mass was about 240 kDa, suggesting a homotetrameric structure. The purified enzyme exhibited hyperbolic saturation kinetics with a Km (sucrose) near 10 mM and the enzyme did not utilize raffinose, maltose, lactose, or cellibose as a substrate. Impure alkaline invertase preparations, which contained acid invertase activity, on contrast, showed biphasic curves versus sucrose concentration. Combining equal activities of purified alkaline invertase with acid invertase resulted in a biphasic response, but there was a transition to hyperbolic saturation kinetics when the activity ratio, alkaline: acid invertase, was increased above unity. Alkaline invertase activity was inhibited by HgCl2, pridoxal phosphate, and Tris with respective Ki values near 2 microM, 5 microM, and 4 mM. Glycoprotein staining (periodic acid-Schiff method) was negative and alkaline invertase did not bind to two immobilized lectins, concanavalin A and wheat germ agglutinin; hence, the enzyme apparently is not a glycoprotein. The purified alkaline invertase, and a purified soybean acid invertase, was used to raise rabbit polyclonal antibodies. The alkaline invertase antibody preparation was specific for alkaline invertase and cross-reacted with alkaline invertases from other plants. Neither purified soybean alkaline invertases nor the crude enzyme from several plants cross-reacted with the soybean acid invertase antibody.     

A new agglutinin from the Tulipa gesneriana bulbs

Two agglutinins with different agglutinating activity exist in Tulipa gesneriana bulbs. One is the T. gesneriana lectin which agglutinates yeasts as reported previously [Oda, Y. and Minami, K. (1986) Eur. J. Biochem. 159, 239-245]. The other agglutinin is a new one which agglutinates animal erythrocytes and was purified from the tulip bulbs using affinity chromatography on thyroglobulin-Sepharose 4B. The agglutinin agglutinated mouse and rat erythrocytes at a minimum concentration of 2 micrograms/ml and 30 micrograms/ml respectively, but did not agglutinate erythrocytes from other animals and yeasts even at a concentration of 1000 micrograms/ml. The agglutinin appeared homogeneous by disc gel electrophoresis at pH 4.3 and gel filtration. Its relative molecular mass was determined by gel filtration to be approximately 40,000. It was suggested that the agglutinin was composed of two different subunits of 26 kDa and 14 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis. Binding of radioiodinated agglutinin to mouse erythrocytes indicated that the presence of a high-affinity site with a dissociation constant of 2.00 X 10(-9) M. In inhibition experiments thyroglobulin glycopeptides were the most potent inhibitors; thyroglobulin was also a potent inhibitor. Orosomucoid and mucin showed weak inhibition. The other glycoproteins, glycopeptides and sugars examined showed no inhibition.     

Purification and characterization of a galactan-reactive agglutinin from the clam Tridacna maxima (R?ding) and a study of its combining site.

1. A beta-galactosyl-binding lectin was purified from the haemolymph of the clam Tridacna maxima by affinity chromatography using polylecyl larch galactan, D-galactosamine coupled to epoxy-activated Sepharose or acid-treated Sepharose. Elution with N-acetyl-D-galactosamine or lactose displaced the bound lectin, which appeared homogeneous by sedimentation analysis. On immunoelectrophoresis at pH8.6 and against rabbit antisera to crude T. maxima haemolymph, the lectin gave one precipitin arc in the alpha-region. 2. On a alkaline polyacrylamide disc gels, one lightly stained band and a broad diffuse band were seen close to the cathode. Ioselectric focusing in solution revealed two peaks of pI4.05 and 4.25 and a shoulder, pI4.0, whereas at least three bands close together (pI3.9-4.3) were seen after electrofusing in gel. 3. The agglutinin is a glycoprotein with a mol.wt. of 470300 +/- 20000. Amino acid analysis revealed no methionine and a significant amount of half-cystine residues. 4. Tridacna lectin is a metalloprotein requiring Ca2+ for its haemagglutinating and precipitating activities. 5. In haemagglutination studies the agglutinin exhibited a broad pH optimum (4.8-10.6). 6. Polysaccharides and glycoproteins with terminal non-reducing beta-D-galactosyl residues reacted with the lectin to form precipitates both in gel and in solution. Inhibition experiments showed that N-acetyl-D-galactosamine was the best inhibitor of the agglutinin combining sites, followed by p-nitrophenyl beta-D-galactoside, methyl beta-D-galactoside, D-galactosamine and 60O-beta-D-galactopyranosyl-D-galactopyranose. On a molar basis, N-acetyl-D-galactosamine was 20-fold more active than D-galactose and nearly 10-fold more inhibitory than D-galactosamine. 7. Circular-dichroism studies showed that the lectin contains a relatively high proportion of beta-structure. 8. Mercaptoethanol treatment of the agglutinin followed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed subunits with approx. mol.wts. of 10000, 20000 and 40000.