Purification and characterization of GDP-L-Fuc: N-acetyl beta-D-glucosaminide alpha1-->6fucosyltransferase from human blood platelets.
c-6-L-Fucosyltransferase (alpha1,6FucT; 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 alpha1,3 antenna was substituted with GlcNacbeta1,4. 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 microM, respectively. The optimum pH of the enzyme was 6.0. The activity of alpha1,6FucT 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.
Kamińska
J
J
Department of Biochemistry, Institute of Hematology and Blood Transfusion, Warsaw, Poland.
Glick
M C
MC
Kościelak
J
J
D001792:Blood Platelets / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D002240:Carbohydrate Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D005647:Fucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D019216:Metals, Heavy / Q000494:pharmacology; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity
D002413:Cations, Divalent; D019216:Metals, Heavy; D009844:Oligosaccharides; D008623:Mercaptoethanol; D005647:Fucosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase; D008274:Magnesium; D002118:Calcium