Purification and characterization of GDP-L-Fuc:: N-acetyl β-D-glucosaminide α1→6-fucosyltransferase from human blood platelets

alpha-6-L-Fucosyltransferase (alpha 1,6FucT; 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 M-r of 58,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions, and as a single enzymatically active peak M-r 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 alpha 1,3 antenna was substituted with GlcNac beta 1,4. On the other hand the tetraantennary oligosaccharide was not a preferred substrate. The K-m values for the substrate asialo-agalactotransferrin-glycopeptide, and GDP-L-fucose were 29 and 28 mu M, respectively. The optimum pH of the enzyme was 6.0. The activity of alpha 1,6FucT was abolished in the presence of beta-mercaptoethanol. Divalent cations such as Mg2+ and Ca2+ activated, but Cu2+, Zn2+ and Ni2+ strongly inhibited the activity.