Purification and characterization of a GDP-fucose:polypeptide fucosyltransferase from Chinese hamster ovary cells.
A GDP-fucose:polypeptide fucosyltransferase was purified 5000-fold to homogeneity from Chinese hamster ovary cell extracts in the absence of detergent. The purification procedure included two affinity chromatographic steps using the acceptor substrate, a recombinant factor VII EGF-1 domain, and the donor substrate analog, GDP-hexanolamine, as ligands. The purified enzyme migrates as a single band of 44,000 daltons on SDS-polyacrylamide gel electrophoresis and is itself a glycoprotein with more than one high mannose type oligosaccharide chain with a total molecular weight of 4000. The Km values for factor VII EGF-1 domain and GDP-fucose are 15 and 6 microM, respectively. The Vmax is 2.5 micromol.min-1.mg-1. The presence of 50 mM Mn2+ increased the enzyme activity 17-fold, but Mn2+ was not absolutely required, since the enzyme exhibited some activity even in the presence of EDTA. The acceptor substrate specificity was studied using site-directed mutagenesis of human factor IX EGF domain. Only one of several differently folded species could serve as acceptor substrate, although they all had the same molecular weight as determined by liquid chromatography on-line with mass spectrometry. This indicates that the enzyme requires proper folding of the epidermal growth factor domain for its activity.
Wang
Y
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Department of Pharmacokinetics and Metabolism, Genentech, Inc., South San Francisco, California 94080, USA.
Spellman
M W
MW
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000201:enzymology*; D006224:Cricetinae; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005167:Factor VII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005647:Fucosyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D005167:Factor VII; D005647:Fucosyltransferases; C105148:polypeptide fucosyltransferase