Purification of the beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase from human serum.
A beta-N-Acetylglucosaminide alpha 1----3-fucosyltransferase was purified from human serum by ammonium sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose, ion-exchange chromatography on sulfopropyl-Sepharose, affinity chromatography on GDP-hexanolamine-Sepharose, and finally high pressure liquid chromatography gel filtration. Gel filtration chromatography of the native enzyme revealed a Mr of 45,000. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified protein also appeared as a single molecular species of Mr 45,000. In contrast to the multisubunit beta-galactoside alpha 1----2-fucosyltransferases with an apparent Mr of 150,000, present in human serum, the native beta-N-acetylglucosaminide alpha 1----3-fucosyltransferase is a monomer with a Mr of 45,000. The enzyme is glycosylated, as revealed by wheat germ agglutinin binding properties. The alpha 1----3 linkage formed by the enzyme between alpha-L-fucose and the penultimate beta-N-acetylglucosamine by the purified enzyme was confirmed by 1H NMR homonuclear cross-irradiation analysis of the oligosaccharide product. The specificity of the purified enzyme is restricted to type 2 structures, as revealed by its reactivity with different substrates and from the Km values calculated from the initial rate data using various oligosaccharide acceptors. The enzyme has the ability to utilize the N-acetyl-beta-lactosamine determinant (Gal beta 1----4GlcNAc) and the sialylated (NeuAc alpha 2----3Gal beta 1----4GlcNAc) and fucosylated (Fuc alpha 1----2Gal beta 1----4GlcNAc) derivatives of N-acetyl-beta-lactosamine and thus is distinct from both the human Lewis gene-encoded enzyme and the alpha 1----3-fucosyltransferase of the myeloid cell type.
Sarnesto
A
A
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Köhlin
T
T
Hindsgaul
O
O
Vogele
K
K
Blaszczyk-Thurin
M
M
Thurin
J
J
D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005647:Fucosyltransferases / Q000097:blood / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008623:Mercaptoethanol / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism
D009844:Oligosaccharides; D008623:Mercaptoethanol; D005647:Fucosyltransferases; C031692:galactoside 3-fucosyltransferase