An electrophoresis-based assay for glycosyltransferase activity. |
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Authors: | K B Lee U R Desai M M Palcic O Hindsgaul R J Linhardt |
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Institution: | Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242. |
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Abstract: | Polyacrylamide gel electrophoresis (PAGE) and capillary zone electrophoresis (CZE) were used to measure the activity of glycosyltransferases. Acceptor molecules were prepared by reductive amination of the monopotassium 7-amino-1,3-naphthalenedisulfonic acid (AGA) Schiff base with sugars. The resulting sugar conjugates were purified by gradient PAGE and recovered using semidry electrotransfer into a positively charged nylon membrane. The beta(1----4)galactosyltransferase was shown, by PAGE analysis, to transfer a beta-galactosyl residue to the AGA conjugate of beta-D-GlcNAc-(1----4)-beta-D-GlcNAc-(1----4)-D-GlcNAc (compound 4). Similarly, alpha(1----2)fucosyltransferase isolated from porcine submaxillary glands was shown to transfer fucose from GDP-fucose to the AGA conjugate of beta-D-Gal-(1----4)-beta-D-GlcNAc-(1----6)-D-Gal (compound 5). This conjugate (compound 5) was also an acceptor for the alpha(1----3/4)fucosyltransferase partially purified from human milk. The latter reaction was followed by both gradient PAGE and CZE, having sensitivities of 200 pmol and 80 fmol, respectively. |
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