Concanavalin A — Sepharose

Purified by dissolving in Triton X-100 and deoxycholate, and by affinity chromatography on concanavalin A-Sepharose and AMP-Sepharose [Grondal and Zimmerman Biochem J 245 805 1987]. 

B28. Bladon, P. T., Rowlands, T. E., Whittaker, J. A., and Oakey, R. E., Serum cortisol binding capacity measured with concanavalin A-Sepharose in patients with a recent inflammatory response. Clin. Chim. Acta 253,9-20 (1996). 

Multiple-ion monitoring is, however, of considerable value in structural studies, but only if model compounds of known structure are available for comparison. Such an approach has been used in the study of the carbohydrate structures of glycoproteins from different tissues.50 Separation of glycopeptides obtained from various tissues was performed on columns of concanavalin A-Sepharose. Structural analysis by multiple-ion monitoring of partially methylated, alditol acetates derived from the various fractions indicated that the glycopeptides were separated according to the linkage pattern of mannose (see Fig. 1). 

Instead of dialysis or gel filtration, an affinity chromatography on Concanavalin A Sepharose (cf. Protocol 3.6.2.4) is recommended, because on the one hand, no conjugated antibody is removed, and on the other hand, the sugar used for elution stabilizes the enzyme-antibody conjugate in solution. 

One of the cellobiose oxidoreductases present in S. pulverulentum has been characterised and named cellobiose oxidase (Ander and Eriksson, 1978). The enzyme contains both haem and flavin co factors and binds irreversibly to concanavalin A-Sepharose, suggesting that it is a glycoprotein. Cellobiose oxidase from S. pulverulentum has now been purified to homogeneity by Morpeth (1985). The carbohydrate and amino acid compositions of the enzyme have been determined. The enzyme contains FAD and cytochrome b prosthetic groups and is a monomer with an Mr of 74400 determined by sedimentation equilibrium. 

Removal of unconjugated enzyme wash on funnel gel filtration chromatography or Concanavalin A-Sepharose chromatography... 

Fig. I. Core structures of the carbohydrate units of nervous tissue glycoproteins. The structures are based on analytical data on rat brain glycoproteins and on assumptions of structural similarity with glycan cores from other sources. The main positions of variable or incomplete glycosylation are indicated by arrows. The approximate molar proportions of the glycans in rat brain and the mode of interaction with concanavalin A-Sepharose are indicated (the bisecting GicNAc residue affects the interaction of the diantennary glycans with concanavalin A) [9].

The D. biflorus lectin consists of two isolectins, A and B, separable by fractionation on concanavalin A-Sepharose [150]. Each isolectin consists of distinct subunits lA and IIA in form A and IB and IIB in form B. The first 30 NH2-terminal amino acids of lA and IIA are identical but differ at their carboxyl ends [151]. The D. biflorus lectin has been cloned, thus establishing its primary structure [152]. By equilibrium dialysis, it was established that the D. biflorus lectin has two carbohydrate binding sites per molecule for A -acetylgalactosamine. Similar to other legume lectins, the D. biflorus lectin is a metalloprotein, requiring Ca for its activity. 

Bishayee, S., Farooqui, A. A., and Bachhawat, B. K., Purification of brain lysosomes arylsulfatases by concanavalin A Sepharose chromatography. Indian J. Biochem. Biophys. 10, 1-2 (1973). 

Hexosaminidase A. The enzyme can be purified from normal human liver (Sandhoff et al., 1977), placenta (Lee and Yoshida, 1976 Geiger and Arnon, 1978), kidney (Wiktorowicz et al., 1977), brain (Aruna and Basu, 1976), or urine (Marinkovic and Marinkovic, 1978). The enzyme preparation used does not have to be pure but it is essential to remove 3-hexosaminidase B (or at least to know the proportions of the two enzymes) since only the A isoenzyme interacts with the activator/lipid complex. A preparation suitable for activator determination can be obtained from liver or placenta extract (20% in water) with chromatography on Concanavalin A-Sepharose 4B as described by Sandhoff et al. (1977),... 

The exocellular polysaccharides released by yeast during fermentation and aging on the lees may be isolated by precipitation with ethanol, or membrane ultrafiltration at a cutoff of 10 000. They may be fractionated by two processes precipitation with hexadecyltrimethylammonium bromide (Cetavlon) and affinity chromatography on a Concanavaline A-Sepharose gel column. The composition of the fractions obtained by these two methods is very similar, consisting of the following ... 

Red blood cells bound to Concanavalin A - Sepharose or Lens cull nan s lectin - Sepharose was used in combination with immo-bilized glucose oxidase (E.C. 1.1.3.4) in the thermistor system. The red blood cells operated as oxygen supply in the reactor bed leading to better reaction conditions for the oxidase - thereby widening the practical concentration range for glucose analysis... 

The remaining active groups on the cyanogen bromide activated Sepharose 4B were blocked by treatment with 1 M ethanolamine, pH 7.0 for 2 hr at 4°C. Approximately 90% of the concanavalin A was linked covalently to Sepharose 4B. Immobilized concanavalin A was activated by incubation overnight at 4°C in 40 mM Tris-HCl-40 NaCl-1 Ti MgCl2-l MnCl2-l tiM CaCl2. Before use the concanavalin A-Sepharose 4B preparation was equilibrated with 40 Tris-HCl-40 NaCl-0.24% DOC. 

Fractions containing DOC-soluble glycopeptide obtained from the column of Bio-Gel A-5m were applied to a column of concanavalin A-Sepharose 4B and the column was washed with 40 Tris-HCl-40 Ti NaCl-0.24% DOC until the UV-absorbing material was removed. The DOC-soluble glycopeptide was eluted with 40 Tris-HCl-40 Ti NaCl-0.24% DOC-1% methyl-a-D-mannopyranoside. 

Figure 11. Fractionation of polymer on a concanavalin A-Sepharose 4B column. Galactofuranosyl-containing fractions from a Bio-Gel A-5m column were pooled and applied to a column of concanavalin A-Sepharose 4B. The adsorbent was washed successively with 40mM Tris-HCl-40mM NaCl 0.24% deoxycholate, pH 7.5, followed by the same buffer containing 1% methyl-a-o-mannopyranoside starting with Fraction 27. Fractions containing 100 drops were collected and 0.5 mL samples were assayed for galactofuranosyl residues (211 Fractions containing galactofuranosyl residues are indicated with a bar.

D-Glucose oxidase and peroxidase, when adsorbed on to concanavalin A-Sepharose or to Lens culinaris lectin-Sepharose have been used in an analytical system for the estimation of D-glucose. ... 

Bull seminal-plasm a hyaluronidase has been purified 180-fold by affinity chromatography on concanavalin A-Sepharose , heparin-Sepharose , and chromatography on Sephadex G-200 and Sephacryl S-200. With hyaluronic acid as the substrate, the specific activity and turnover number of purified hyaluronidase were 3.63 /Umol min per mg (10400 National Formulary units mg of protein) and 214min (mol of product formed per mol of enzyme per min), respectively. Polyacrylamide gel electrophoresis indicated that the purified enzyme migrated as a single band at pH 4.3 and 5.3. Bull seminal-plasma hyaluronidase was markedly inhibited by hydroxylamine, phenyl-hydrazine, and semicarbazide. 

Figure 5. Continuous galactosylation of ovalbumin and BSA-GlcNAc in the reactor. The galactosyltransferase was immobilized to Concanavalin A-Sepharose. Efficiency is defined as concentration of galactosylated acceptor in percent of total acceptor concentration. (Reprinted with permission from ref. 24. Copyright 1990 Glycoconjugate Journal.)...

Fig. 2. Concanavalin A-Sepharose chromatography of FPLC Phenyl Superose fraction shown in Fig. 1. The shaded portion represents cytokinin oxidase activity which was collected in 12 4 ml fractions...

In order to study the lipid-protein interaction in the photoreceptor membrane we have changed the micro-environment of the rhodopsin molecules by removal and replacement of lipids. Two different delipidation procedures have been applied 1. treatment of photoreceptor membranes with phospholipase-C (Borggreven et al., 1971), and 2. affinity chromatography over a concanavalin A-sepharose-4B column (van Breugel et al., 1976). 

Fig. 5. Affinity chromatography ofrhodopsin over a concanavalin A"sepharose 4B column in the presence of 100 mM dodecyltrimethyl-ammoniumbromide (from van Breugel et al., 1976).

Two forms of sialyltransferase have been isolated from human placental extracts one (mol. wt. 8 x 10 ) binds to concanavalin A-Sepharose but the other (mol. wt. 8 x 10 ) does not bind. ... 

Ogata S, Muramatsu T, Kobata A (1975) Fractionation of glycopeptides by affinity column chromatography on concanavalin A-Sepharose. J Biochem 78 687-696... 

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