Abstract
α-6 L -Fucosyltransferase (α1,6FucT; EC 2.4.1.68) from human platelets, the enzyme that is released into serum during coagulation of blood, was purified 100,000-fold. The purification required three sequential chromatographic steps: chromatofocusing, affinity column chromatography on GnGn-Gp(asialo-aglacto-transferrin glycopeptide)-CH-Sepharose, and gel filtration of Sephadex G-200. The final preparation contained a protein that migrated as a single discrete band Mr of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak Mr of 58,000 in gel filtration. Although the purified enzyme utilized the biantennary GnGn-Gp as substrate, it was twice as active with the triantennary oligosaccharide when the Man α1,3 antenna was substituted with GlcNacβ1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The Km values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP L -fucose were 29 and 28 μ M, respectively. The optimum pH of the enzyme was 6.0. The activity of α1,6FucT was abolished in the presence of β-mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.
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Kaminska, J., Glick, M.C. & Koscielak, J. Purification and characterization of GDP L -Fuc: N-acetyl β D -glucosaminide α1→6fucosyltransferase from human blood platelets. Glycoconj J 15, 783–788 (1998). https://doi.org/10.1023/A:1006959915435
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DOI: https://doi.org/10.1023/A:1006959915435