Triazine dyes as inhibitors and affinity ligands of glycosyltransferases

Abstract

The triazine dyes: Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Green 19, Reactive Blue 4, Reactive Brown 10 inhibited the activity of a purified preparation of α1,6fucosyltransferase (GDP-L-fucose:N-acetyl β-glucosaminide 6-α-L-fucosyltransferase, EC 2.4.1.68) from human blood platelets. Cibacron Blue 3GA and Reactive Red 120 were examined for the nature of the inhibition and both were found to be competitive inhibitors of the enzyme, with K_i=11[emsp4 ]μM and 2[emsp4 ]μM, respectively. The two dyes inhibited also serum glycosyltransferases: α1,2fucosyltransferase (GDP-L-fucose: β-D-galactosyl-R2-α-L-fucosyltransferase, EC 2.4.1.69), β1,4galactosyltransferase (UDP-galactose: N-acetyl-D-glucosamine 4-β-D-galactosyltransferase, EC 2.4.1.90) and β1,3N-acetylglucosaminyltransferase (UDP-GlcNAc: 4-β-D-galactosyl-D-glucose). Cibacron Blue 3GA was a more effective inhibitor of the glycosyltransferases that use UDP-linked sugar donors than Reactive Red 120 while the latter was a stronger inhibitor of the fucosyltransferases that use GDP-linked donor. All four glycosyltransferases could be affinity purified on Cibacron Blue 3GA-Agarose columns. The order of elution of glycosyltransferases from the columns with solutions of 0.25–1.0[emsp4 ]M potassium iodide also depended upon the structure of nucleotide sugar donor, i.e. whether it contained UDP or GDP. Thus, triazine dyes should interact with the sugar donor binding sites of glycosyltransferases. The main advantages of the use of triazine dyes as affinity ligands for isolation of glycosyltransferases are their universal applicability regardless of enzyme specificity, low cost, and insensitivity to high concentration of other proteins present in the solution.