Assay of alpha 1,3 N-acetyl-D-galactosaminyl transferase by affinity chromatography.
A high-performance liquid affinity chromatography column that contains immobilized anti-A monoclonal antibody specifically retards blood group A-active oligosaccharides and can be used to detect the product(s) of the reaction catalyzed by alpha-1,3-N-acetyl-D-galactosaminyltransferase: [formula: see text] After a brief incubation (15 min) of an assay mixture containing 1-100 microliters human serum, the sugar nucleotide donor UDP-GalNAc, and radiolabeled oligosaccharide acceptors 2'-fucosyllactose and/or lacto-N-fucpentaose I blood group A-active products are isolated and quantitated in a single affinity chromatographic step that takes less than 30 min. Kinetic studies to determine the pH optima for serum alpha-3-GalNAc transferase from individuals of blood groups A1 and A2 and the Km value for UDP-GalNAc for the A1 transferase agree with previous determinations. As monoclonal antibodies against many different complex carbohydrate antigens are now available, the method described could be adapted to give rapid, inexpensive assays for a variety of glycosyltransferases.
Dakour
J
J
Department of Clinical Chemistry, University Hospital, Lund, Sweden.
Zopf
D
D
Lundblad
A
A
D002240:Carbohydrate Sequence; D002846:Chromatography, Affinity / Q000379:methods*; D005700:Galactosyltransferases / Q000032:analysis* / Q000097:blood; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D017350:N-Acetylgalactosaminyltransferases; D009844:Oligosaccharides / Q000737:chemistry; D013379:Substrate Specificity
D009844:Oligosaccharides; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase