Identification and characterization of Sarcophaga lectin receptor on the surface of murine macrophages by use of monoclonal antibodies

The structure of Sarcophaga lectin receptor on the surface of murine macrophages was analyzed using monoclonal antibodies. This receptor was found by gel filtration to have a molecular weight of 460 kDa. SDS-polyacrylamide gel electrophoresis showed that this receptor consists of two subunits of 170 kDa and 110 kDa. The results indicated that it is probably a heterotetramer of two molecules of each subunit. Two monoclonal antibodies recognized epitopes in the 110 kDa subunit, and one of them specifically inhibited the binding of Sarcophaga lectin to macrophages and the cytotoxic reaction mediated by this lectin in the presence of macrophages. Therefore, it is likely that the 110 kDa protein in the receptor plays a role in activation of macrophages by this lectin.     

Xanthosoma sagittifolium tubers contain a lectin with two different types of carbohydrate-binding sites

An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (alpha(2)beta(2)) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal alpha1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Galbeta1,4GlcNAc-) or lacto-N-biose (i.e. Galbeta1,3GlcNAc-) groups at its three nonreducing termini.     

Purification of a Sperm Lectin Extracted from Spermatozoa of the Sea Urchin Hemicentrotus pulcherrimus

Hemagglutinating activity for human type A erythrocytes was detected in a sperm extract obtained by treatment with Triton X-100 of spermatozoa from the sea urchin Hemicentrotus pulcherrimus. Among tested sugars only N-acetyl-D-galactosamine had any inhibitory effect on the hemagglutinating activity of the sperm extract. The lectin was purified by a combination of affinity chromatography and ion-exchange chromatography. A single band was obtained after SDS-polyacrylamide gel electrophoresis of the purified lectin, corresponding to an apparent molecular weight of 15,000 daltons. Trypsin-generated fragments of the surface of eggs significantly inhibited hemagglutination of erythrocytes by the purified lectin. The biological role of the sperm lectin is discussed.     

Purification and Characterization of the Acidic Lectin from Winged Bean Seeds

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

A lectin from the thigh muscle of Rana tigerina

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

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

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

Purification and characterization of a lectin from the beetle, Allomyrina dichotoma

A lectin was purified from the hemolymph of Allomyrina dichotoma larvae by affinity chromatography on acid-treated Sepharose 4B. The purified lectin showed two protein bands on polyacrylamide gel electrophoresis. These two lectin bands (allo A-I and -II) were separated by DEAE-Cellulofine column chromatography. By gel filtration on Sephadex G-100, the molecular weights of allo A-I and -II were estimated to be 65,000 and 66,500, respectively. On the other hand, by SDS-polyacrylamide gel electrophoresis after cross-linking of subunits with glutaraldehyde, they are estimated to be 38,000 and 39,000, respectively. On SDS-polyacrylamide gel electrophoresis, it was proved that both allo A-I and -II lectin consisted of two subunits, respectively. The molecular weights were 17,500 and 20,000 for allo A-I, and 19,000 and 20,000 for allo A-II. The isoelectric points of allo A-I and -II were estimated to be 6.4 and 5.9, respectively. On double immunodiffusion, allo A-I and -II gave single precipitin lines, which fused completely with each other, against the antibody to crude allo A. The hemagglutinating activity of allo A-I and -II was inhibited only by beta-linked D-galactose such as lactose and lactulose.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

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.     

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.     

Isolation of lectin from horse chestnut (Aesculus hippocastanum L.) seeds and study of its interaction with carbohydrates and glycoproteins]

The lectin from horse chestnut seeds was obtained by affinity chromatography on a sorbent prepared from the egg white, 95 mg of lectin per 1 kg of fresh seeds being obtained. Molecular weight was determined by gel-filtration on tojopearl HW-55 and it composed 132 kDa. SDS-polyacrylamide gel electrophoresis revealed the presence of one component with molecular weight of 33 kDa. One band has been revealed by means of disc-electrophoresis in acidic (pH 4.5) and alkaline system (pH 8.9). Sugar was not detected in the lectin. Amino acid composition of the lectin has been determined. The lectin agglutinated horse erythrocytes in minimal concentration of 9.5 ngml, to the less extent rabbit (4.9 mkg/ml), rat (62 mkg/ml), human (73 mkg/ml), but did not agglutinate erythrocytes of a sheep and cow. Purified lectin did not interact with monosaccharides, but interacted with O-glycans.     

Chemical and physicochemical characterization of the sialic acid-specific lectin from Cepaea hortensis

A sialic acid-specific lectin was isolated from the albumin glands of the garden snail Cepaea hortensis by affinity chromatography on fetuin-Sepharose following gel filtration on Superdex 200. The purified native lectin showed a molecular mass of about 95 kDa by gel filtration and 100 kDa by SDS electrophoresis. It was cleaved by boiling in buffer containing SDS in three serological identical bands corresponding to molecular masses of about 24, 20 and 16 kDa, respectively. From these three fragments, only the 24- and the 20-kDa bands were found to be glycosylated. Only the three sugars mannose, galactose and N-acetylglucosamine could be detected in a molar ratio of 3:8.6:2. The oligosaccharide moieties seem to be N- and partially O-glycosidic bound. Isoelectric focusing (IEF) of the purified lectin revealed a heterogeneous pattern with bands in the pH range of 4.3-5.0. Isolated bands of different isoelectric points showed in SDS electrophoresis the same three fragments with molecular masses of 24, 20 or 16 kDa. The heterogeneity of the lectin was revealed either by IEF or amino acid sequencing of internal tryptic peptides.     

Isolation of the major glycoprotein from plasma membranes of an ascites hepatoma AH 66.

Plasma membranes were isolated from AH 66 cells, some of which had been labeled with [14C]glucosamine, by the following procedure: homogenization of cells which had been hardened by treatment with Zn ions, fractionation of the homogenate by sucrose density gradient centrifugation and purification of the membranes by partition in an aqueous two-phase polymer system. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) of the plasma membranes and subsequent staining of the gel for protein and carbohydrate, and determination of radioactivity on the gel eluates indicated the presence of at least 10 bands of glycoproteins. The major band contained 27% of the total radioactivity incorporated into the plasma membranes and was most heavily stained with the periodate-Schiff reagent. To isolate the major glycoprotein, the membranes were solubilized with 0.6 M lithium diiodosalicylate containing 0.5% Triton X-100, then the solution was treated with phenol. The major glycoprotein, obtained in the aqueous phase, was further purified mainly by repeated chromatographies on Sepharose 6B. The purified preparation was practically homogeneous on SDS-polyacrylamide gel electrophoresis, as judged by radioactivity determination and by carbohydrate staining, but contained small amounts of carbohydrate-free proteins. The major glycoprotein had an apparent molecular weight of 160,000, as determined by SDS-polyacrylamide gel electrophoresis. The final preparation contained about 44% carbohydrate on a weight basis, and the carbohydrate moiety was composed of glucosamine, galactosamine, galactose, mannose, fucose, and sialic acid. This composition indicates that the major glycoprotein contains both N- and O-glycosidically linked oligosaccharide moieties.     

Purification and molecular characterization of rat intestinal brush border membrane dipeptidyl aminopeptidase IV

Dipeptidyl aminopeptidase IV (EC 3.4.14.-) was solubilized from a particulate membrane fraction of rat intestinal mucosa with Triton X-100. The solubilized enzyme was purified to homogeneity following ammonium sulfate fractionation, chromatography on DEAE-Sepharose and hydroxyapatite, gel filtration and preparative polyacrylamide gel electrophoresis. The final enzyme preparation had a specific activity of 55 units/mg protein representing a 1373 fold purification over the starting material. Purity was judged by polyacrylamide gel electrophoresis and double immunodiffusion. The molecular weight of the native undenatured enzyme was estimated to be 230000 by gel filtration and polyacrylamide gel electrophoresis. Electrophoresis under denaturing conditions (sodium dodecyl sulfate) indicated that the protein consists of two identical 98 kDa subunits. Dipeptidyl aminopeptidase IV is a glycoprotein containing approx. 8% carbohydrate by weight. A detailed analysis of the individual sugar components demonstrated that fucose, galactose, glucose, mannose, sialic acid and hexosamine sugars were present. The nature of the constituent asparagine linked oligosaccharide side chains was further examined following cleavage from the peptide backbone by hydrazinolysis. Following high voltage paper electrophoresis approx. 80% of the isolated oligosaccharide was found with the neutral fraction while the remaining 20% consisted of a single acidic component. Gel filtration of the neutral oligosaccharide fraction indicated that it contains approx. 19 sugar residues.     

Isolation and amino terminal sequencing of a novel melanoma-associated antigen

The biochemical characteristics are described for a melanoma-associated glycoprotein antigen, whose expression depends on stage of tumor progression and melanocytic differentiation. This antigen, identified using a monoclonal antibody which specifically stains melanoma cells in immunoperoxidase assay of fixed tissue sections, is synthesized as a 30,000-Da precursor and then processed to a 30,000- to 60,000-Da sialylated glycoprotein. Two-dimensional gel electrophoresis of the antigen resolved more than 20 forms, heterogeneous in both charge and molecular weight. The kinetics of post-translational processing, the sensitivity of processing to tunicamycin, and the molecular weight of the oligosaccharide chains indicate that the oligosaccharides are N-linked. Amino acid sequencing of the antigen purified by immunoaffinity chromatography and by high-pressure liquid chromatography or polyacrylamide gel electrophoresis allowed the assignment of the first 20 acids.     

Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium

In human unbilical artery and vein endothelial cell conditioned medium fibrinolytic inhibitors have been detected by two different techniques. A fast-acting inhibitor of tissue-type plasminogen activator (t-PA)_and urokinase has been detected and quantified by its capacity to neutralize the above-mentioned plasminogen activators in a kinetic assay. By reverse fibrin autography after SDS-polyacrylamide gel electrophoresis a fibrinolytic inhibitor can be detected with a molecular mass of 52 kDa. The mutual relationship between these two inhibitors was studied. Neutralization of the fast-acting inhibitor by t-PA results in the formation of a complex with a molecular mass of 100 kDa. The t-PA added to endothelial cell conditioned medium in excess of the fast-acting inhibitor is fully stable. However, the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography is not affected by complete neutralization of the fast-acting inhibitor, and removal of the formed complexes by immune adsorption with immobilized anti-t-PA IgG. This suggests that the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography does not react with t-PA. Moreover, endothelial cell conditioned medium that is depleted of the fast-acting inhibitor does not show lysis resistance when directly applied to the reverse fibrin autography indicator gel (without previous electrophoresis), although the inhibitor is still present in the zymogram after SDS-polyacrylamide gel electrophoresis. This suggests that the inhibitor is induced by the SDS treatment. Heating the endothelial cell conditioned medium for 15 min at 70°C fully destroys the fast-acting inhibitory activity, but leaves the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography unaffected. Moreover, at least one additional fibrinolytic inhibitor is detected in the zymogram after SDS-polyacrylamide gel electrophoresis. We conclude that the fast-acting inhibitor is not the same as the inhibitor that is detected by SDS-polyacrylamide gel electrophoresis and reverse fibrin autography; the latter inhibitor is not operational in endothelial cell conditioned medium, but is induced by SDS-polyacrylamide gel electrophoresis.     

Purification and characterization of a lectin (plant hemagglutinin) with N blood group specificity from Vicia graminea seeds.

A lectin with N blood group specificity was isolated from Vicia graminea seeds. This lectin was purified from a crude extract by precipitation with ammonium sulfate, DEAE-cellulose chromatography and Sephadex G-150 gel filtration. Purification steps were followed by increase of specific activity. Its homogeneity was demonstrated by polyacrylamide gel electrophoresis, immunoelectrophoresis, electrofocusing and ultracentrifugation. This lectin is an acid glycoprotein with 7.3% carbohydrate, a high percentage of serine and contains no sialic acid. The native lectin has a molecular weight about 100 000 and dissociates into four subunits of 25 000 as shown by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Preliminary hemagglutination inhibition has shown that the lectin was not inhibited by any of the monosaccharides contained in N blood group substances; however it was inhibited by the erythrocyte membrane major glycoprotein and the tryptic fragments obtained from erythrocytes.     

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.     

一种新型家蝇蛹凝集素结构的初步探讨*

初步分析了分离出的一种分子量为55kDa,具有免疫活性的新型家蝇蛹D-半乳糖结合凝集素的结构,为深入研究其结构与功能之间的关系提供大量的信息。首先通过凝胶电泳染色确定此新型家蝇蛹凝集素具有糖链结构,借助蛋白质测序仪、分光光度计比色、β-消除反应、红外光谱以及原子力显微镜技术对其结构进行初步分析。此种家蝇蛹凝集素是一种蛋白和糖含量分别为97.36% 和2.1% 的球状单体,直径为75nm左右。肽链与糖链的连接方式为O-糖苷键型,肽链的N-末端封闭,糖环为吡喃型,为进一步分析其结构提供了可靠的依据。     

Existence of associated, non-associated, and oligomeric forms of human chorionic gonadotropin subunits in placental extracts

The molecular sizes of human chorionic gonadotropin (hCG) subunits in the native state in normal first trimester placental extracts were determined by gel filtration on Sephacryl S-300, followed by SDS-polyacrylamide gel electrophoresis, protein blotting, and immunobinding analysis using anti-alpha and - beta antibodies. Mature forms of hCG subunits in the extracts were only found in the same fraction as that which contained standard urinary hCG, indicating an alpha beta dimer. On the other hand, immature forms were detected with a wide range of molecular weights, which were higher than that of standard hCG, suggesting oligomerization of associated or non-associated immature subunits. In order to determine the associated state of these subunits, various forms of associated subunits (hCG alpha beta) in placental extracts were immunoprecipitated with anti-hCG antiserum, which only recognized hCG alpha beta, and Protein A-Sepharose. They were then analyzed by SDS-polyacrylamide gel electrophoresis under reducing and non-reducing conditions, followed by immunobinding assaying. It has been suggested that there are three kinds of hCG alpha beta S (one mature and two immature). To confirm the above results and to clarify the existence of free subunits, placental extracts were subjected to two-dimensional SDS-polyacrylamide gel electrophoresis. With this technique, high molecular weight forms of immature hCG subunits were found to be present in placental cells as an oligomer of not only the alpha beta dimer but of each subunit as well.