Complete purification and characterization of alpha-3-N-acetylgalactosaminyltransferase encoded by the human blood group A gene.
Human alpha-3-N-acetylgalactosaminyltransferase has been purified 27,000,000-fold from A1 plasma by (NH4)2SO4 fractionation and affinity chromatography on Sepharose 4B, anti-human group O plasma antibodies-Sepharose 4B, and Blue Dextran-Sephadex G-25. A modified procedure in the Sepharose 4B step was developed by batch adsorption and desorption experiments. Cibacron Blue F3G-A, the chromophore of Blue Dextran, was found to bind to the enzyme. UDP is an effective inhibitor of this binding. The pure transferase has an apparent molecular weight of 35,000 as judged by SDS-PAGE in the presence of a reducing agent. The specific activity is 16 pmol/min.ng enzyme, which is comparable to that (30 pmol/min.ng enzyme) of alpha-3-N-acetylgalactosaminyltransferase from porcine submaxillary glands [Schwyzer and Hill (1977) J. Biol. Chem. 252, 2338-2355]. The apparent Km values for UDP-GalNAc, 2'-fucosyllactose, and lacto-N-fucopentaose I are 13, 270, and 350 microM, respectively. The reaction velocity was found to fall off again at high concentrations of oligosaccharide acceptor substrates. The apparent Ki values for UDP and UDP-galactose are 8.6 and 6.2 microM, respectively. The pure enzyme also catalyzes the transfer of galactose in alpha-linkage to 2'-fucosyllactose though the transfer rate of galactose is much lower than that of N-acetylgalactosamine.
Takeya
A
A
Department of Legal Medicine, Gunma University School of Medicine.
Hosomi
O
O
Ishiura
M
M
D000017:ABO Blood-Group System / Q000235:genetics*; D002021:Buffers; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006596:Hexosaminidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000032:analysis / Q000302:isolation & purification; D007700:Kinetics; D008345:Manganese / Q000032:analysis; D008970:Molecular Weight; D014530:Uridine Diphosphate / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D048809:alpha-N-Acetylgalactosaminidase
D000017:ABO Blood-Group System; D002021:Buffers; D007074:Immunoglobulin G; D014531:Uridine Diphosphate Galactose; D008345:Manganese; D014530:Uridine Diphosphate; D006596:Hexosaminidases; C482722:NAGA protein, human; D048809:alpha-N-Acetylgalactosaminidase