Measurement of alpha(1-3) fucosyltransferase activity using scintillation proximity

Analytical Biochemistry
C M HoodC J Britten

Abstract

The alpha 3 fucosyltransferases are a family of glycosyltransferases involved in the addition of fucose onto glycoproteins and glycolipids. One of the best defined roles for the alpha 3 fucosyltransferases is in the biosynthesis of the carbohydrate antigen sialyl Lewis X, the minimal ligand for the selectin family of adhesion molecules. We describe here the development of a single-step assay for the measurement of alpha 3 fucosyltransferase activity based on the principle of scintillation proximity. The fucosyltransferase catalyses the transfer of [3H]fucose, from GDP-[3H]fucose, onto the sugar chains of a glycoprotein acceptor noncovalently bound to a scintillant-impregnated microsphere (SPA bead). The resultant signal can be used as a measure of enzyme activity. Due to the nature of this assay no steps are required to separate unused substrate from product. Kinetic data from the assay compare favorably with those obtained from assays currently used for the alpha 3 fucosyltransferases. This SPA-based assay appears generic for the alpha 3 fucosyltransferases and readily adaptable for other glycosyltransferases. The particular advantage of the assay is anticipated to be found in the simple, routine testing of a large number of s...Continue Reading

References

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Citations

Jul 3, 2008·Assay and Drug Development Technologies·J Fraser GlickmanSandrine Ferrand
Oct 10, 2007·Analytical Biochemistry·Oliver von AhsenKarsten Parczyk
Apr 15, 2004·Archives of Biochemistry and Biophysics·Xiaoda NiuDaniel Lundell
Dec 22, 2006·Experimental Dermatology·Thomas M ZollnerMichael P Schön
Jul 31, 2010·Chembiochem : a European Journal of Chemical Biology·Gerd K Wagner, Thomas Pesnot
Jan 6, 2006·BioDrugs : Clinical Immunotherapeutics, Biopharmaceuticals and Gene Therapy·Shaogui Wu, Bailing Liu
Apr 23, 2008·Current Protocols in Neuroscience·Steven D Kahl, Christian C Felder
Apr 10, 2013·Chembiochem : a European Journal of Chemical Biology·Sonia SernaNiels-Christian Reichardt

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