A quantitative method for GDP-l-Fuc:N-acetyl-β-d-glucosaminide α1→6fucosyltransferase activity with lectin affinity chromatography
✍ Scribed by Judith A. Voynow; Thomas F. Scanlin; Mary Catherine Glick
- Publisher
- Elsevier Science
- Year
- 1988
- Tongue
- English
- Weight
- 604 KB
- Volume
- 168
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.