A quantitative method for GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase activity with lectin affinity chromatography.
A quantitative method for the activity of GDP-L-Fuc:N-acetyl-beta-D-glucosaminide alpha 1----6fucosyltransferase has been developed using a well-characterized substrate to which other fucosyltransferases fail to transfer and lentil lectin-Sepharose, which will bind this substrate only after fucosylation of the asparagine-linked N-acetylglucosamine. The enzyme was extracted from human skin fibroblasts and incubated with GDP-[14C]fucose and a specific substrate, asialo-agalactotransferrin glycopeptide. The product of the enzyme reaction, [14C]fucose alpha 1----6 to the asparagine-linked N-acetylglucosamine of the substrate, bound to lentil lectin-Sepharose and was eluted with 0.4 M methyl alpha-D mannopyranoside. The method was shown to be specific after characterization of the lentil lectin-bound glycopeptides by enzyme degradation and affinity chromatography. Quantitation of the method was shown by several parameters, including the linearity of product formed with respect to time, GDP-[14C]fucose concentration and enzyme concentration.
Voynow
J A
JA
Children's Hospital of Philadelphia, Pennsylvania 19104.
Scanlin
T F
TF
Glick
M C
MC
D002846:Chromatography, Affinity / Q000379:methods; D005647:Fucosyltransferases / Q000032:analysis*; D006602:Hexosyltransferases / Q000032:analysis*; D037102:Lectins; D037121:Plant Lectins
D037102:Lectins; D037121:Plant Lectins; C033619:lentil lectin; D005647:Fucosyltransferases; D006602:Hexosyltransferases; C020559:Glycoprotein 6-alpha-L-fucosyltransferase