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An -d-Galactosyl-binding Lectin from Bandeirae simplicifolia Seeds

ISOLATION BY AFFINITY CHROMATOGRAPHY AND CHARACTERIZATION

From the ¹ From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48104

An -d-galactosyl binding lectin was purified from Bandeiraea simplicifolia seeds by affinity chromatography. Melibiose was oxidized to the corresponding aldonic acid, and condensed with aminoethyl-Bio-Gel P-300 in the presence of a water-soluble carbodiimide to afford a specific adsorbent which retained B erythrocyte hemagglutinating activity from the seed extract. Elution with galactose-containing buffer displaced the bound lectin which appeared homogeneous by disc gel electrophoresis, pH 4.3, immunoelectrophoresis, gel filtration, and sedimentation analysis. Multiple bands were obtained upon electrophoresis at pH 9.5 and isoelectric focusing.

The lectin (mol wt 114,000) is composed of four apparently identical subunits and contains 6.7% by weight carbohydrate (mannose, glucosamine, fucose, and xylose). A metalloprotein, the lectin requires Ca²⁺ for hemagglutinating activity. Amino acid analysis revealed a high content of acidic and hydroxylic amino acids, only trace amounts of methionine, and one cysteine per subunit. Modification of cysteine with 5,5'-dithiobis(2-nitrobenzoic acid) resulted in complete loss of hemagglutinating activity. Inactivation was partially reversible upon reduction with dithiothreitol.

The lectin agglutinates B and AB erythrocytes very strongly, A₁ cells weakly, and does not agglutinate A₂ or O cells.

Polysaccharides and glycoproteins with terminal, nonreducing -d-galactosyl residues reacted with the lectin to form precipitin bands by Ouchterlony double diffusion, whereas those with terminal ß-d-galactosyl residues did not (the galactomannan of Torulopsis gropengiesseri excepted).

Polysaccharide precipitating activity of the lectin exhibited an unusually broad pH optimum (6.0 to 11.0), was inhibited by EDTA, destroyed by dialysis of the lectin against acetic acid to remove bound metal, and restored by addition of a divalent metal cation. Glycosides of -d-galactopyranose were the best inhibitors examined by the hapten inhibition technique. Interestingly, UDP-galactose was shown to inhibit polysaccharide precipitation. Lactose, l-arabinose, d-fucose, d-glucose, and d-mannose were noninhibitory.

We suggest that Bandeirae simplicifolia lectin, the first -d-galactopyranosyl binding lectin (anti-B lectin) to be purified and characterized, will be valuable not only as a serological reagent, but in studies of polysaccharides, glycoproteins, and cell membrane structure.

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