An electrophoresis-based assay for glycosyltransferase activity.
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.
Lee
K B
KB
Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
Desai
U R
UR
Palcic
M M
MM
Hindsgaul
O
O
Linhardt
R J
RJ
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D002240:Carbohydrate Sequence; D004586:Electrophoresis / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005647:Fucosyltransferases / Q000378:metabolism; D016695:Glycosyltransferases / Q000032:analysis*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008895:Milk, Human / Q000201:enzymology; D008969:Molecular Sequence Data; D016339:Spectrometry, Mass, Fast Atom Bombardment
C033153:amido-G-acid; D015081:2-Naphthylamine; D016695:Glycosyltransferases; D005647:Fucosyltransferases