Con A-Sepharose 4B Research Articles

Isolation and identification of a new type of .BETA.-conglycinin in soybean globulins.

A new type of β-conglycinin was found in precipitates of a crude glycinin fraction at pH 6.4 and low ionic strength. The protein was purified by ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography and ion-exchange chromatography on DEAE-Sepharose CL-6B. The purity check and identification of the protein were performed by gel electrophoresis, gel filtration, N-terminal amino acid analysis, immunochemical analysis and ultra-centrifugal analysis. This protein (designated as B0-conglycinin) was composed of three identical subunits which were identical with the β-subunit in β-conglycinins at 0.5 ionic strength, but differed from the other β-conglycinins in that B0-conglycinin formed an insoluble aggregate at ionic strengths less than 0.2.

Isolation of α-fetoprotein in milligram quantities from human cord serum

A new procedure for the isolation of milligrams of AFP from a large volume of human cord serum is described. The purification steps include: 1. 1. batchwise chromatography on DEAE-cellulose, 2. 2. DEAE-cellulose chromatography, 3. 3. Sephadex G-200 column chromatography, 4. 4. Con A-Sepharose 4B column chromatography, 5. 5. Blue Sepharose column chromatography, 6. 6. immunoadsorbent column chromatography on columns filled with anti-cord serum-coupled Sepharose and anti-cord serum-coupled Sepharose but with a majority of anti-albumin being depleted. The procedure does not require an anti-AFP immunoadsorbent column. Usually more than 10 mg AFP may be obtained from 500 ml of cord serum. The purified AFP contains no measurable albumin and shows a single band after polyacrylamide gel electrophoresis. The purified AFP is suitable for raising anti-AFP and for the development of sensitive AFP radioimmunoassay.

Isolation, partial characterization and specificity of glycoprotein elicitors from culture filtrates, mycelium and cell walls of Cladosporium fulvum (syn. Fulvia fulva)

Cell-free culture filtrates, mycelium extracts and cell walls of Cladosporium fulvum (syn. Fulvia fulva ) contained high molecular weight components which elicited rishitin accumulation in tomato fruit tissue. Elicitors from mycelium extracts (MEE) and cell walls (CWE) appeared to be 5 to 10 times as active as those isolated from culture filtrates (CFE). Elicitors isolated from two races of Cladosporium fulvum were not host-specific. Of the four non-hosts of Cladosporium fulvum which were assayed for phytoalexin accumulation after treatment with elicitor, pea and soybean reacted by accumulation of pisatin and glyceollin, respectively, while under the conditions used potato and jack bean did not accumulate rishitin and medicarpin, respectively. The rishi tin-inducing activity of CFE, MEE and CWE was sensitive to NaIO 4 , α-mannosidase, Pronase, Proteinase K and also to NaOH, which suggest that the elicitors are glycoproteins. The rishitin-inducing activity of CFE was not bound to DEAE-Sephadex and could be purified threefold by chromatography on Con A-Sepharose 4B. CFE contained mainly glucose and some mannose, galactose and protein. MEE and CWE contained 5 to 10 times more mannose, galactose and protein than CFE. The protein part of all elicitors was especially rich in asparagine/aspartic acid, threonine, serine, gluta-mine/glutamic acid and proline.

Purification and partial characterization of three forms of alpha-glucosidase from the fruit fly Drosophila melanogaster.

Three forms of alpha-glucosidase, I, II, and III, have been purified from the whole body extract of adult flies of Drosophila melanogaster in yields of 2.1, 5.3, and 6.7%, respectively. The purification procedures involved ammonium sulfate fractionation, Con A-Sepharose 4B affinity chromatography, DEAE-Sepharose CL-6B ion exchange chromatography, Sephacryl S-200 gel filtration, and preparative gel electrophoresis. Each purified enzyme showed a single band on polyacrylamide gel on both protein and enzyme activity staining. The molecular weights of alpha-glucosidases I, II, and III were estimated to be 200,000, 56,000, and 76,000, respectively, by gel filtration. SDS gels indicated that alpha-glucosidases II and III were each composed of a single polypeptide chain, whereas alpha-glucosidase I was composed of two identical subunits. Both alpha-glucosidases II and III hydrolyzed sucrose and p-nitrophenyl-alpha-D-glucoside (PNPG), but alpha-glucosidase I hydrolyzed PNPG to a much lesser extent than sucrose. For sucrose the pH optima of alpha-glucosidases I, II, and III were pH 6.0, 5.0, and 6.0 and the Km values were 13.1, 8.9, and 10 mM, respectively. For PNPG the pH optima of alpha-glucosidases II and III were pH 5.5 and 6.5 and the Km values were 0.77 and 0.21 mM, respectively.

Enzymatically active rat liver microsomal antigens with concanavalin A-binding properties

By a combination of affinity chromatography and immunoelectrophoretic methods, it was shown that antigens exhibiting several different enzyme activities (nucleoside di- and triphosphatase, NADH-tetrazolium reductase and also in some cases alkaline phosphodiesterase activities), solubilized by detergents from rat liver microsomes, carry sugar moieties that bind to Concanavalin A (Con A) coupled to Sepharose 4B. Similarly, some enzyme active antigens showing only one type of enzyme activity, bind to Con A-Sepharose 4B and can be specifically eluted with α-methyl- d-mannoside. However, two esterase- and two NADH-reductase-active antigens distinct from the multienzyme complexes do not interact with Con A. Thus, Con A binding not only provides a means of characterizing enzyme active antigens as glycoproteins containing certain sugar moieties, but it also gives a simple method for the separation of antigens with similar activities.

Activité 5′-nucléotidase des membranes plasmiques de lymphocytes. Effet de la concanavaline A

The 5'-nucleotidase properties of isolated lymphocyte plasma membranes from young pig mesenteric nodes are described; nucleosides-5'-monophosphates are the substrates of this specific enzyme. Concanavalin A inhibits this enzyme; on the same membranes this mitogen does not affect alkaline phosphatase and activates the membrane bound (Ca2+) ATPase. The 5'-nucleotidase inhibition is due to a specific interaction of Con A with carbohydrate groups of the membrane; its high positive cooperativity suggests that the lectin promotes reorganization of the membrane bound 5'-nucleotidase. Solubilization of the 5'-nucleotidase does not prevent the effect of Con A and the solubilized enzyme is firmly bound by Con A-Sepharose 4B; these results suggest that Con A inhibits the enzyme by a direct interaction and that 5'-nucleotidase can be considered as an eventual receptor for the lectin.

Leishmania donovani: Surface membrane carbohydrates of promastigotes

Cloned promastigote forms of Leishmania donovani, a human protozoan pathogen, were agglutinated specifically with the plant lectins concanavalin A (Con A), fucose binding protein, phytohemagglutinin-M and -P, soybean agglutinin (SBA), and wheat germ agglutinin (WGA). Cell agglutination with lectins was inhibited, and reversed in the presence of specific lectin-binding monosaccharides. Promastigotes also were agglutinated by viable influenza virions. Neuraminidasetreated cells were not agglutinated by the virions. Cells were agglutinated randomly with all lectins and the influenza virions suggesting a uniform distribution in the promastigote cell surface of the lectin and influenza virion-binding saccharide ligands. Both living and glutaraldehyde-fixed cells gave similar agglutination results with the various lectins and influenza virus. Promastigotes were also agglutinated with low concentrations of cationized ferritin indicative of a negative surface charge. Noncationized ferritin did not induce cell agglutination. Living L. donovani promastigotes were also specifically adsorbed onto the surface of Con A-Sepharose beads but not onto heat-denatured Con A-Sepharose or Sepharose 4B. No cell adsorption to Con A-Sepharose was observed in controls treated with α-methyl- D-mannoside. The lectin-binding kinetics of the cell surface α- D-mannose receptor ligand was assessed using 3H-labeIed Con A. At saturation, 2.78–4.86 × 10 −4 μmole of [ 3H]Con A were bound per 10 8 promastigotes with a mean of 3.73 × 10 −4 μmole/10 8 cells. Membrane receptor sites sterically available for binding [ 3H]Con A were calculated from kinetic data, and ranged from 1.67–2.93 × 10 6 sites/cell with a mean of 2.24 × 10 6 ligand sites/promastigote. Cell-surface-bound Con A was visualized at the ultrastructure level with an iron-dextran (Fe-Dex) conjugate. The dense Fe-Dex particles were distributed randomly on the pellicular and flagellar membrane outer lamina. Con A bound at the cell surface was also visualized using horseradish peroxidase (HRPO) and diaminobenzidine (DAB) coupled reactions. Dense Con A-HRPO-DAB reaction product was deposited uniformly on the cell pellicular and flagellar membranes. SBA and WGA were chemically conjugated to HRPO and these were used in DAB coupled reactions with the L. donovani promastigotes. The fine structure cell surface localization and distribution of these HRPO-conjugated lectins was similar to that obtained with the Con A-HRPO-DAB preparations. Influenza virions visualized at the fine structure level were bound randomly to the outer lamina of the cell pellicular and flagellar membranes. At the ultrastructure level, cationized ferritin was uniformly distributed over the entire cell surface. The cumulative results demonstrate that ligands similar or identical to α- d-mannose, N-acetylgalactosamine, N-acetylglucosamine, α- l-fucose, and an N-acetylated neuraminic acid are constituents of the L. donovani pellicular and flagellar membranes. These carbohydrates probably represent terminal moieties of cell membrane structural and functional glycoproteins. Further, the polyanionic carbohydrates impart a net negative charge to the L. donovani surface membrane.

Affinity chromatography studies of the de novo glucan synthesizing phosphorylase isozyme of blue-green algae

The non-primer requiring phosphorylase isozyme of the Cyanophyte, Oscillatoria princeps, binds to Con A-Sepharose 4B matrices. This electrophoretically homogeneous isozyme was eluted by borate buffer from such columns in two distinct and separate peaks, while the primer-requiring isozyme was not retained. Both fractions of the primer-independent isozyme contained bound glucan and were active in forming glucan from the Cori ester. The later eluted fraction showed more rapid synthesis from the substrate than the first eluted fraction. From the retention of the later fraction by the matrix, it is inferred that this fraction may possibly contain bound glucan which is more branched than the bound glucan of the first fraction.

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

(1974). The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B. Agricultural and Biological Chemistry: Vol. 38, No. 5, pp. 1083-1085.

The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B

(1974). The Purification of Soybean 11S Globulin with ConA-Sepharose 4B and Sepharose 6B. Agricultural and Biological Chemistry: Vol. 38, No. 5, pp. 1083-1085.