Purification and characterization of a Fuc alpha 1-->2Gal beta 1--> and GalNAc beta 1-->-specific lectin in root tubers of Trichosanthes japonica.
A prominent lectin in the root tubers of Trichosanthes japonica was purified by affinity chromatography on a porcine stomach mucin-Sepharose column and termed TJA-II. The molecular mass of the native lectin was determined to be 64 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and TJA-II was separated into two different subunits of 33 and 29 kDa in the presence of 2-mercaptoethanol. The respective subunits contained mannose, N-acetylglucosamine, fucose, and xylose. It was determined by equilibrium dialysis to have two equal binding sites per molecule, the association constant toward tritium-labeled Fuc alpha 1-->2Gal beta 1-->3GlcNAc beta 1-->3Gal beta 1-->4GlcOT being K alpha = 3.05 x 10(5) M-1. The precise carbohydrate binding specificity of immobilized TJA-II was studied using various tritium-labeled oligosaccharides. A series of oligosaccharides possessing Fuc alpha 1-->2Gal beta 1--> or GalNAc beta 1--> groups at their nonreducing terminals showed stronger binding ability than ones with Gal beta 1-->GlcNAc (Glc) groups, indicating that TJA-II fundamentally recognizes a beta-galactosyl residue and the binding strength increases on substitution of the hydroxyl group at the C-2 position with a fucosyl or acetylamino group. This lectin column is useful for fractionating oligosaccharides or glycoproteins containing blood group type 1H, type 2H, and Sd antigenic determinants.
Yamashita
K
K
Department of Biochemistry, Sasaki Institute, Tokyo, Japan.
Ohkura
T
T
Umetsu
K
K
Suzuki
T
T
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D005260:Female; D006384:Hemagglutination; D006801:Humans; D037102:Lectins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D046911:Macromolecular Substances; D008895:Milk, Human / Q000737:chemistry; D008969:Molecular Sequence Data; D009077:Mucins; D009844:Oligosaccharides / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D013552:Swine
D000596:Amino Acids; D002241:Carbohydrates; D037102:Lectins; D046911:Macromolecular Substances; D009077:Mucins; D009844:Oligosaccharides