Triazine dyes as inhibitors and affinity ligands of glycosyltransferases

The triazine dyes: Cibacron Blue 3GA, Reactive Red 120, Reactive Yellow 86, Reactive Green 19, Reactive Blue 4, Reactive Brown 10 inhibited the activi ty of a purified preparation of alpha 1,6fucosyltransferase (GDP-L-fucose:N -acetyl beta-glucosaminide 6-alpha-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 inh ibitors of the enzyme, with K-i=11 mu M and 2 mu M, respectively. The two d yes inhibited also serum glycosyltransferases: alpha 1,2fucosyltransferase (GDP-L-fucose: beta-D-galactosyl-R2-alpha-L-fucosyltransferase, EC 2.4.1.69 ), beta 1,4galactosyltransferase (UDP-galactose: N-acetyl-D-glucosamine 4-b eta-D-galactosyltransferase, EC 2.4.1.90) and beta 1,3N-acetylglucosaminylt ransferase (UDP-GlcNAc: 4-beta-D-galactosyl-D-glucose). Cibacron Blue 3GA w as a more effective inhibitor of the glycosyltransferases that use UDP-link ed sugar donors than Reactive Red 120 while the latter was a stronger inhib itor of the fucosyltransferases that use GDP-linked donor. All four glycosy ltransferases could be affinity purified on Cibacron Blue 3GA-Agarose colum ns. The order of elution of glycosyltransferases from the columns with solu tions of 0.25-1.0 M potassium iodide also depended upon the structure of nu cleotide sugar donor, i.e. whether it contained UDP or GDP. Thus, triazine dyes should interact with the sugar donor binding sites of glycosyltransfer ases. The main advantages of the use of triazine dyes as affinity ligands f or isolation of glycosyltransferases are their universal applicability rega rdless of enzyme specificity, low cost, and insensitivity to high concentra tion of other proteins present in the solution.