A quantitative method for GDP-l-Fuc:N-acetyl-β-d-glucosaminide α1→6fucosyltransferase activity with lectin affinity chromatography

Judith A. Voynow, Thomas F. Scanlin, Mary Catherine Glick

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

Abstract

A quantitative method for the activity of GDP-l-Fuc:N-acetyl-β-d-glucosaminide α1→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 α1→6 to the asparagine-linked N-acetylglucosamine of the substrate, bound to lentil lectin-Sepharose and was eluted with 0.4 m methyl α-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.

Original languageEnglish (US)
Pages (from-to)367-373
Number of pages7
JournalAnalytical Biochemistry
Volume168
Issue number2
DOIs
StatePublished - Feb 1 1988
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Keywords

  • affinity chromatography
  • bioassays
  • carbohydrate structure
  • enzymes
  • lectins

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