Differential binding characteristics and applications of DGal beta 1----3DGalNAc specific lectins

The binding properties of Arachis hypogaea (PNA), Bauhinia purpurea alba (BPL), Maclura pomifera ( MPL ) and Sophora japonica (SJL) lectins were studied by quantitative precipitin and precipitin inhibition assays, demonstrating them to be most specific for DGal beta 1---- 3DGalNAc residues. Additionally, each lectin had its own binding characteristic such as different binding activities to DGal beta 1---- 4DGlcNAc or DGal beta 1---- 3DGlcNAc beta 1----linked oligosaccharides, and/or DGalNAc alpha 1----linked to the Ser or Thr of the protein moiety. These differential binding characteristics can be used for investigating fine differences of the carbohydrate structure of the glycoconjugates, especially those having DGal beta 1---- 3DGalNAc residues as terminal non-reducing ends.     

Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues. These results indicate that the minimal structural requirement for glycoprotein precipitation or cell agglutination by the lectin includes any of the three trisaccharides (fucosylated or nonfucosylated) derived from the blood group B tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin in as much as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA, Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding. Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers [Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can. J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the beta-side of these oligosaccharides. These sides, on both blood group H type 1 and blood group H type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. The only difference found between Lotus and Ulex I lectins in precipitating ability was that only Lotus precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)     

A monoclonal IgM lambda macroglobulin with specificity for lacto-N-tetraose in a patient with bronchogenic carcinoma

The serum of a patient with bronchogenic carcinoma was found to contain a monoclonal IgM lambda (IgMwoo) that precipitated with a precursor blood group glycoprotein containing I and i determinants. IgMwoo did not agglutinate O cells in the cold or at room temperature, and by quantitative precipitin and precipitin inhibition assay its specificity was shown not to be to the I and i determinants. IgMwoo reacted best with lacto-N-tetraose, DGal beta 1 leads to 3DGlcNAc beta 1 leads to 3DGal beta 1 leads to 4DGlc, and was specific for the non-I or non-i determinant dGal beta 1 leads to 3DGlcNAc beta 1 leads to 3DGal-moiety present as a distinct chain on precursor glycoproteins containing I and i determinants. Human ovarian cyst blood group A and B glycoproteins were inactive, but removal of the outer tier of sugars involved in A, B, and H specificity exposed this non-I or non-i determinant as well as the determinant reacting with anti-I Ma. IgMwoo was neither a cold agglutinin nor a cryoglobulin. It precipitated with precursor blood group glycoproteins somewhat less at 37 degrees C than at 0 degrees C, the differences being ascribable to solubility.     

木菠萝凝集素结合部位糖的特异性

用定量免疫沉淀法和定量免疫沉淀抑制法研究了从木菠萝(Arthrocarpus integrifolia)种子提取的凝集素(jacalin)结合部位糖的特异性。Jacalin最强烈地沉淀含有DGalβ1→3DGalNAc结构的无活性抗冻糖蛋白,不同程度非特异地沉淀各种血型物质。研究发现凝集素结合部位对DGalβ→3DGalNAc有最高特异性。最强抑制剂是DGalβ1→3DGalNAcal→φNO_2,其抑制活性分别比DGalNAc和DGal高380倍和1000倍。对于各种甲基化或对硝基酚化的糖苷以及寡糖,除methylaDGalNAc_f外,仅α-构型表现出抑制活性,所有β-构型的糖苷均无抑制活性,jacalin结合部位对糖的结合是构型依赖性的。     

Immunochemical studies on the specificity of soybean agglutinin

The specificity of the purified soybean agglutinin has been studied immunochemically by quantitative precipitin and quantitative precipitin inhibition assays. The lectin is precipitated by human A and Le^a blood-group substance, by the products of the second, third, fourth, and fifth stages of periodate oxidation of a human H blood-group substance (JS), and by precursor blood-group substances, as well as by a pig-submaxillary mucin having blood-group A activity, by partially hydrolyzed blood-group B substances (Pl fraction), and by group C streptococcal polysaccharide. The activity is attributable to terminal α-linked 2-acetamido-2-deoxy-d-galactopyranosyl or to α- or β-d-galactopyranosyl residues. The lectin did not precipitate with human blood-group H substances, with the product of the first stage of periodate oxidation (JS), with streptococcal group A polysaccharide, or with pig-submaxillary mucin devoid of blood-group A activity, and is poorly precipitated by blood-group B substances. Inhibition of precipitation with various monosaccharides indicated that the lectin is strongly specific for 2-acetamido-2-deoxy-d-galactose and for its oligosaccharides, and to a lesser extent for d-galactose and its oligosaccharides; the α-glycosides of both sugars were slightly more reactive than the β-glycosides of 2-acetamido-2-deoxy-d-galactose, and both α- and β-glycosides were more active than the free monosaccharides. Aromatic α- and β-glycosides of 2-acetamido-2-deoxy-d-galactose and d-galactose were better inhibitors than the corresponding methyl or ethyl compounds. The blood-group A trisaccharide α-d-GalNAcp-(1→3)-β-d-Galp-(1→3)-d-GlcNAc was more active than the disaccharide lectins by the use of precipitation with polysaccharides, as well as inhibition reactions, is essential to the understanding of their reactivity with cell-surface receptors.     

Structures of oligosaccharides cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein

Oligosaccharides were cleaved by base-borohydride from an I, H and Lea active ovarian cyst glycoprotein and purified by Bio-Gel P-6 and paper chromatography. The structures of five oligosaccharides, determined by compositional analyses, quantitative periodate oxidation, chronic acid oxidation, methylation analyses and enzymatic degradations, were as follows: oligosaccharide I, beta DGal1----3DGalNAc-ol; II, beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; III, alpha LFuc1----2 beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGalNAc-ol; IV, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3beta DGal1----4 beta DGlcNAc1----6(beta DGal1----3)DGal1NAcol; and V, beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----4 beta DGlcNAc1----6[beta DGal1----3(alpha LFuc1----4)beta DGlcNAc1----3 beta DGal1----3 beta DGal1----3]DGalNAc-ol. Of the oligosaccharides 60% had a molecular size of a decasaccharide or smaller, the tetra- and pentasaccharides II and III predominating. Oligosaccharides I through IV have been previously isolated from several glycoproteins by other laboratories; the decasaccharide, V, is a new structure.     

A study of the specificity of Bandeiraea simplicifolia lectin I by competitive-binding assay with blood-group substances and with blood-group A and B active and other oligosaccharides

The specificity of Bandeiraea simplicifolia lectin I (BS I) has been studied by competitive-binding assays (CBA) using tritium-labeled human B and hog A substances. Blood-group B substances isolated from horse gastric mucosae and from human ovarian-cyst fluids were much better inhibitors of binding of tritiated blood-group B substance to insoluble BS I-Sepharose 2B than were human blood-group A substances from saliva and ovarian-cyst fluid. A and B active blood-group substances showed the same range of potency in inhibiting binding of tritium-labeled hog A substance to BS I-Sepharose 2B. CBA with BS I-Sepharose 2B, labeled human blood-group B substance, and human blood-group A and B active aligosaccharides separated the haptens into two groups differing in slope. Group 1, containing methyl alpha-D-GalNAcp, D-GalNAcp, and an A active pentasaccharide ARL 0.52, with 3, 19, and 25 nmol respectively needed for 50% inhibition of binding, has a lower slope than group 2, which contains alpha-D-GalNAcp-(1 leads to 3)-2-acetamido-2-deoxy-D-galactitol and p-nitrophenyl alpha-D-GalNAcp, with 3 nmol of each required for 50% inhibition of binding, as well as ten glycosides with terminal, nonreducing, alpha-linked D-Galp. The most potent inhibitors of this group were p-nitrophenyl alpha-D-Galp, alpha-D-Galp-(1 leads to 3)-D-Galp, alpha-D-Galp-(1 leads to 6)-D-Glcp, and methyl alpha-D-Galp, with 5, 7.4, 9.6, and 11 nmol respectively needed to inhibit binding by 50%. The difference in slopes was explainable in terms of a recent finding that BS I exists as a mixture of five isolectins composed of two subunits having different specificities; subunit A is most specific for alpha-linked, terminal, nonreducing D-GalNAcp, but it also reacts with alpha-linked, terminal, nonreducing D-Galp, whereas subunit B tends to be more specific for terminal, nonreducing, alpha-linked D-Galp.     

Purification of a lectin from fruit bodies of <Emphasis Type="Italic">Lactarius pergamenus</Emphasis> (Fr.) Fr. and studies of its properties

A lectin was purified from fruit bodies of the milk mushroom Lactarius pergamenus (Fr.) Fr. by a combination of ethanol precipitation, affinity chromatography on copolymer of polyvinyl alcohol and human blood B-group-specific sub-stance, and ion-exchange chromatography on DEAE-Toyopearl. The lectin yield was 3 mg/kg of fresh mushrooms. Considerable loss of primary activity was observed during its purification, which, presumably, could be explained by disin-tegration of the lectin molecule, which consisted of six subunits, first to two molecules of three subunits, and then to individual subunits. There was a reverse tendency to aggregation during concentration of lectin solutions. Similar processes can take place in nature because of considerable individual variations of the lectin activity during growth of mushroom fruit bodies. The lectin weakly interacts with DGalNAc, while DGalβ1-3DGalNAc and DGalβ1-3DGlcNAc are the most probable candidates for ligands, with which the L. pergamenus lectin interacts at disaccharides level. The purified lectin may find application in histochemical research.     

The binding site of chicken hepatic lectin

The binding site of the chicken hepatic lectin involved in the clearance of N-acetylglucosamine-terminated serum glycoproteins was explored by a competitive binding assay using 3H-labeled agalacto-orosomucoid and various glycoproteins, polysaccharides, monosaccharides, and glycosides as inhibitors. The binding site is relatively small, involving a terminal nonreducing DGlcNAc structure with an equatorial N-acetamido group on carbon 2 and an equatorial hydroxyl group on carbon 4. Among the mono- and oligosaccharides tested, benzyl alpha DGlcNAc was the best inhibitor, being three times as effective as DGlcNAc; and in general, all alpha-anomeric glycosides were better than beta-glycosides. All oligosaccharides with terminal nonreducing beta DGlcNAc have almost the same inhibitory power, whereas those with nonreducing DGlc or DGal were relatively inactive. Among the serum and blood group glycoproteins, a Smith degraded human H substance with several exposed terminal nonreducing beta DGlcNAc residues was the most active and twice as effective as agalacto-orosomucoid and an A substance, Hog 75 10% precipitate. Almost all hog preparations, some with A or with H activity, were equally effective. A glycopeptide with terminal DGlcNAc was twice as active as one with terminal nonreducing DMan and DGlcNAc residues and almost three times as potent as one with terminal nonreducing DGal; a glycopeptide with terminal sialic acid was inactive. The slopes of the inhibition lines differed, reflecting the heterogeneity of the various determinant groups on the glycoproteins.     

Isolation and characterization of amaranthin, a lectin present in the seeds of Amaranthus caudatus, that recognizes the T- (or cryptic T)-antigen

A lectin (Amaranthin) present in the seeds of Amaranthus caudatus has been isolated by fractionation on DEAE-cellulose followed by affinity chromatography on Synsorb-T beads (Gal beta 1,3GalNAc alpha-O-R-Synsorb). The lectin appeared homogeneous by gel electrophoresis at pH 4.3 and gave a single protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr = 33,000-36,000. A native Mr = 54,000 was determined by gel filtration suggesting that amaranthin exists as a homodimer. Compositional analysis revealed high amounts of acidic and hydroxyamino acids and relatively large amounts of lysine, methionine, and tryptophan for a plant protein. Amaranthin formed a precipitate with asialo-bovine submaxillary mucin, asialo-ovine submaxillary, porcine submaxillary mucin, asialo-fetuin and asialoglycophorin. Hapten inhibition of precipitate formation between amaranthin and asialo-ovine submaxillary indicated that the T-disaccharide and its alpha-linked glycosides (Gal beta 1,3GalNAc alpha-O-R; R = OH, methyl, -(CH2)8-COOCH3, allyl, o-nitrophenyl, or benzyl) were the best inhibitors. N-Acetylgalactosamine, the only monosaccharide which inhibited precipitation, was 350-fold less effective than Gal beta 1,3GalNAc alpha-O-R. Hapten inhibition with derivatives of the T-disaccharide suggested that the C'-4 axial hydroxyl group of the galactosyl moiety, and the C-4 axial hydroxyl group, and the C-2 acetamido group of the GalNAc unit are the most important loci for lectin interaction. NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-(CH2)8CO2CH3 was as potent an inhibitor as Gal beta 1,3GalNAc alpha-O-(CH2)8CO2-CH3, and amaranthin was precipitated by NeuAc alpha 2,3Gal beta 1,3GalNAc alpha-O-BSA (where BSA is bovine serum albumin), indicating that the amaranthin-combining site tolerates substitutions at the C'-3 hydroxyl group. Amaranthin was precipitated by a Gal beta 1,3GalNAc alpha-O-BSA glycoconjugate but not by the anomeric Gal beta 1,3GalNAc beta-O-BSA glycoconjugate illustrating that the disaccharide must be linked alpha in order to interact with the lectin. Metal ions do not appear to be required for lectin activity. A study of pH dependence showed significant precipitate formation between pH 4 to 9 with a maximum at pH 5. Hapten inhibition and glycoconjugate precipitation assays were also conducted for peanut (Arachis hypogaea) agglutinin. A comparison between the carbohydrate-binding specificities of amaranthin and peanut (Arachis hypogaea) agglutinin is discussed.     

Properties of the human erythrocyte membrane receptors for peanut and Dolichos biflorus lectins.

Neuraminidase treatment of blood type A and B human erythrocytes, which is required for the agglutination of these cells by peanut (Arachis hypogaea) lectin, increased the number of receptor sites for the lectin from about 5 × 10⁴ to 1.8 × 10⁶ sites/ cell for both blood types. The same treatment also increased the agglutinability of type A cells by the blood group A-specific Dolichos biflorus lectin, but the number of receptor sites for this lectin (~6 × 10⁵ sites/cell) did not change. D. biflorus lectin binding and agglutination of blood type B cells were negligible both before and after neuraminidase treatment. To isolate the peanut agglutinin receptor from the membrane of these cells, washed type A erythrocytes were incubated with neuraminidase and galactose oxidase and then treated with NaB³H₄, thus labeling the galactose residues on the membrane. For measuring peanut agglutinin receptor activity, a radioaffinity assay was developed based on the displacement of [¹⁴C]asialofetuin from peanut agglutinin by receptor and precipitation of the complex in the presence of polyethyleneglycol. Membranes were isolated by hypotonic lysis and were solubilized in 0.5% Empigen BB, a zwitterionic detergent, which was found to be superior to Triton X-100 for this purpose. The cell extract, after centrifugation, was subjected to affinity chromatography on peanut agglutinin-polyacrylhydrazido-Sepharose. Elution with lactose afforded a peak of radioactivity (32% yield) containing 70% of the applied receptor activity. The eluting sugar and the receptor were separated by chromatography on Bio-Gel P-2 with subsequent dialysis against 80% acetone to remove the detergent. The bulk of the isolated receptor radioactivity (91%) precipitated with peanut agglutinin. The amino acid composition, the glucosamine and galactosamine content and the electrophoretic mobility, on polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the peanut receptor were similar to those of asialoglycophorin. In addition, the peanut receptor coprecipitated with asialoglycophorin and with isolated erythrocyte T antigen on Ouchterlony double-diffusion plates against peanut agglutinin and the Ricinus communis lectin, but not with D. biflorus lectin, suggesting that the receptor for the latter lectin is distinct from the peanut agglutinin receptor.     

Defining the carbohydrate specificities of Abrus precatorius agglutinin as T (Gal beta 1----3GalNAc) greater than I/II (Gal beta 1----3/4GlcNAc).

The combining site of the nontoxic carbohydrate binding protein (Abrus precatorius agglutinin, APA) purified from the needs of Abrus precatorius (Jequirity bean), was studied by quantitative precipitin and precipitin-inhibition assays. Of 26 glycoproteins and polysaccharides tested, all, except sialic acid-containing glycoproteins and desialized ovine salivary glycoproteins, reacted strongly with the lectin, and precipitated over 70% of the lectin added, indicating that APA has a broad range of affinity and recognizes (internal) Gal beta 1----sequences of carbohydrate chains. The strong reaction with desialized porcine and rat salivary glycoproteins as well as pneumococcus type XIV polysaccharide suggests that APA has affinity for one or more of the following carbohydrate sequences: Thomsen-Friedenreich (T, Gal beta 1----3GalNAc), blood group precursor type I and/or type II (Gal beta 1----3/4GlcNAc) disaccharide determinants of complex carbohydrates. Among the oligosaccharides tested, the T structure was the best inhibitor; it was 2.4 and 3.2 times more active than type II and type I sequences, respectively. The blood group I Ma-active trisaccharide, Gal beta 1----4GlcNAc beta 1----6Gal, was about as active as the corresponding disaccharide (II). From the above results, we conclude that the size of the combining site of the A. precatorius agglutinin is probably as large as a disaccharide and most strongly complementary to the Gal beta 1----3GalNAc (T determinant) sequence. The carbohydrate specificities of this lectin will be further investigated once the related oligosaccharide structures become available.     

A method of selective histochemical analysis of sialoglycoproteins using lectins from elder (Sambucus nigra L.)]

Good potentialities in application of elderberry (Sambucus nigra L.) bark lectin for selective histochemical identification of sialylated glycoconjugates has been demonstrated using lectin-peroxidase technique. In order to omit this lectin binding to D-galactose and N-acetyl-D-galactosamine residues, preincubation of tissue sections with non-marked PNA and SBA (or other lectins with similar carbohydrate specificity) is proposed. By means of neuraminidase digestion it has been ascertained, that oligosaccharide chains of secretory glycopolymers, synthesised in ovine submandibular gland mucocytes, contain DGal and DGalNAc residues penultimate to terminal sialic acids.     

Distribution of lectins in membranes of soybean and peanut plants

The distribution of lectin activity in soybean and peanut plants has been investigated. In both plants activity is found in all tissues examined (roots, shoots and leaves) at all stages of development from seedling to maturity (7 weeks). The cellular location of the lectins differs between soybean and peanut: in soybean the lectins are generally membrane-associated, whereas in peanut plants lectin activity is present also in the soluble cytoplasmic fraction. The membrane-associated lectins appear to differ from the seed lectins of the respective plants. The function of membrane-associated lectins is discussed.Abbreviations RCA lectin of castor bean - SBA soybean agglutinin - PNA peanut agglutinin - HEPES 2-[4-(2-Hydroxyethyl)-piperazinyl-(1)]ethanesulphonic acid - MES morpholinoethane sulphonic acid - PBS phosphate-buffered saline     

Analysis of saccharide binding to Artocarpus integrifolia lectin reveals specific recognition of T-antigen (beta-D-Gal(1----3)D-GalNAc)

The binding of Artocarpus integrifolia lectin to N-dansylgalactosamine (where dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) leads to a 100% increase in dansyl fluorescence with a concomitant blue shift in the emission maximum by 10 nm. This binding is carbohydrate-specific and has an association constant of 1.74 X 10(4) M-1 at 20 degrees C. The lectin has two binding sites for N-dansylgalactosamine. The values of -delta H and -delta S for the binding of N-dansylgalactosamine are in the range of values reported for several lectin-monosaccharide interactions, indicating an absence of nonpolar interaction of the dansyl moiety of the sugar with the combining region of the protein. Dissociation of the bound N-dansylgalactosamine from its complex with the lectin and the consequent change in its fluorescence on addition of nonfluorescent sugars allowed evaluation of the association constant for competing ligands. The thermodynamic parameters for the binding of monosaccharides suggest that the OH groups at C-2, C-3, C-4, and C-6 in the D-galactose configuration are important loci for interaction with the lectin. The acetamido group at C-2 of 2-acetamido-2-deoxygalactopyranose and a methoxyl group at C-1 of methyl-alpha-D-galactopyranoside are presumably also involved in binding through nonpolar and van der Waals' interactions. The T-antigenic disaccharide Gal beta 1----3GalNAc binds very strongly to the lectin when compared with methyl-beta-D-galactopyranoside, the beta(1----3)-linked disaccharides such as Gal beta 1----3GlcNAc, and the beta(1----4)-linked disaccharides, N-acetyllactosamine and lactose. The major stabilizing force for the avid binding of T-antigenic disaccharide appears to be a favorable enthalpic contribution. The combining site of the lectin is, therefore, extended. These data taken together suggest that the Artocarpus lectin is specific toward the Thomsen-Friedenreich (T) antigen. There are subtle differences in the overall topography of its combining site when compared with that of peanut (Arachis hypogaea) agglutinin. The results of stopped flow spectrometry for the binding of N-dansylgalactosamine tot he Artocarpus lectin are consistent with a simple single-step bimolecular association and unimolecular dissociation rate processes. The value of K+1 and K-1 at 21 degrees C are 8.1 X 10(5) M-1 s-1 and 50 s-1, respectively. The activation parameters indicate an enthalpy-controlled association process.     

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

The glycosylation status of murin postnatal thymus: a study by histochemistry and lectin blotting

During the intrathymic development, the fate of the thymocytes depends largely on variable expression of CD4/CD8 markers and T cell receptor protein expressions. In addition, changes of cell surface glycosylation status also affect the thymocyte maturation. In this study the glycosylation alterations in thymic tissues from 1, 9, 13 and 16 days old mice were evaluated by histochemical and lectin blotting techniques. With alcian blue (AB) at pH 5.7/periodic acid-Schiff (PAS) stainings, it was shown that thymic microenvironments contained carboxlylated and sulfated glycosaminoglycans (GAGs). Strong positivity to AB at pH 2.5, which specific for sialomucins, was seen in some medullary thymocytes. Similarly, it was shown that with Maackia amurensis agglutinin (MAL) medullary thymocytes, but not cortical ones, contained alpha(2 --> 3) linked sialic acid structures. On the other hand, while reaction with peanut agglutinin (PNA), which specific for core disaccharide galactose beta(1 --> 3) N-acetylgalactosamine, was only seen in cortical thymocytes, reaction with Galanthus nivalis agglutinin (GNA), which specific for terminal mannose residues, was seen in both cortex and medulla. However, Datura stramonium agglutinin (DSA), which recognizes galactose beta(1 --> 4) N-acetylglucosamine, was not only cell-specific, but it was bound some thymic vessels. With lectin blotting studies, five glycoprotein bands of molecular weights ~39, ~54, 100, ~110 and ~212 were found which reacted with MAL, PNA and DSA as well as GNA. These results suggest that glycosylation patterns of cell surface glycoconjugates are modified during thymocyte selection processes of postnatal days.     

Studies on Allergen of Fasciola hepatica

Two allergenic substances (C 5 and P 4), which consisted of ribonucleic acid as their main component, were isolated from the heated extract of liver fluke (Fasciola hepatica) by means of precipitation by ammonium sulphate and phenol extraction, followed by extraction with potassium acetate and ethanol fractionation.

One of these substances, C 5, which was extracted with phenol was electrophoretically homogeneous, but ultracentrifugally was shown to contain one other substance in a small quantity.

The other substance, P 4, which was isolated from the phenol insoluble fraction was electrophoretically and ultracentrifugally homogeneous.

Both of these were almost the same in biological activity, and their solutions, which were diluted 1 : 100,000 with Ringer’s solution, were still active in the intradermal reaction to the fascioliasis of cattle. Judging from their activity and yield, it seems that all the allergenic substances which are in the heated extract of liver fluke, are represented by the protein allergen reported already, as well as by C 5 and P 4 described here.     

Studies on the effect of 3,4-dehydroproline on collagen metabolism in carbon tetrachloride-induced hepatic fibrosis.

The lectin from Euonymus europeus seeds was purified by adsorption onto insoluble polyleucyl hog A + H blood group substance and subsequent elution with lactose. The isolated lectin formed three lines in immunoelectrophoresis against rabbit antisera to the crude seed extract and showed three components on electrophoresis in acrylamide gel at pH 9.4. In analytical isoelectric focusing the purified lectin had six closely spaced bands with pI from 4.3 to 4.7. It sedimented as two peaks: a big symmetrical peak with s_20,w⁰ of 7.8 and another small, diffuse moving peak. The intrinsic viscosity was 0.057 dl/g and the M_r calculated from the sedimentation coefficients, intrinsic viscosity, and V? of 0.71 was about 166,000. In sodium dodecyl sulfate, it gives subunits of M_r 17,000 and 35,000; 20% of the 35,000 subunit resists reduction by dithiothreitol in 7 m guanidine-HCl. The Euonymus lectin is a glycoprotein containing 4.8% d-galactose, 2.9% d-glucose, and 2.8% N-acetyl-d-glucosamine. The purified lectin precipitated well with B and H blood group substances and with the P1 fraction of blood group B substance but not with A₁ substances. It precipitated poorly with Le^a and Le^b and precursor I blood group substances. Inhibition of precipitation with milk and blood group oligosaccharides showed the lectin to be most specific for blood group B oligosaccharides having the structure: dGalα1 → 3[lFucα1 → 2]dGalβ1 → 3 or 4dGlcNAcβ→. It is also inhibited by blood group H oligosaccharides but to a lesser degree. For 50% inhibition of precipitation, 3.5, 850, and 290,000 nmol of B and H oligosaccharides and lactose, respectively are required. The B and H specificities are an intrinsic property of a single lectin site since absorption and elution from an H immunoadsorbent gave material with B as well as H specificity. Millipore-filtered crude extracts of Euonymus europeus preserved with 0.02% sodium azide are stable in the refrigerator for many months and can be used for quantitative precipitin and for quantitative inhibition assays, results being the same as with purified lectin.     

Glycopeptides from murine teratocarcinoma cells: structure of the determinants recognised by Griffonia simplicifolia agglutinin I and by sera from patients with ovarian germ cell tumors

High-molecular-weight glycopeptides synthesised by teratocarcinoma OTT6050 bear the binding site for Griffonia simplicifolia agglutinin I and are recognised by antibodies in the sera of patients with ovarian germ cell tumors. Digestion of the glycopeptides with endo-beta-D-galactosidase C abolished the lectin binding activity and the antigenic activity. Since the product of the enzymic digestion is alpha-D-Gal-(1----3)-D-Gal, it is concluded that the disaccharide structure is involved in the lectin binding site and the antigenic site.