Complete Purification and Characterization of α-3-N-Acetylgalactosaminyltransferase Encoded by the Human Blood Group A Gene1
| Autor: | Osamu Hosomi, Masahiro Ishiura, Akira Takeya |
|---|---|
| Rok vydání: | 1990 |
| Předmět: |
chemistry.chemical_classification
Chromatography biology Stereochemistry General Medicine Oligosaccharide Biochemistry Sepharose chemistry.chemical_compound Enzyme chemistry Affinity chromatography Enzyme inhibitor Galactose biology.protein Transferase Uridine diphosphate galactose Molecular Biology |
| Zdroj: | The Journal of Biochemistry. 107:360-368 |
| ISSN: | 1756-2651 0021-924X |
| DOI: | 10.1093/oxfordjournals.jbchem.a123051 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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