An Electrophoresis-Based Assay For Glycosyltransferase Activity

Lee, K.B.
Desai, U.R.
Palcic, M.M.
Hindsgaul, O.
Linhardt, Robert J.
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Biology , Chemistry and chemical biology , Chemical and biological engineering , Biomedical engineering
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An Electrophoresis-Based Assay For Glycosyltransferase Activity, K.B. Lee, U.R. Desai, M.M. Palcic, O. Hindsgaul, R.J. Linhardt, Analytical Biochemistry, 205, 108-114 (1992).
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.
Analytical Biochemistry, 205, 108-114
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The Linhardt Research Labs.
The Shirley Ann Jackson, Ph.D. Center for Biotechnology and Interdisciplinary Studies (CBIS)
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Rensselaer Polytechnic Institute, Troy, NY
Analytical Biochemistry