ENZYMATIC ASSAY OF GALACTOSYLTRANSFERASE BY CAPILLARY-ELECTROPHORESIS

The kinetic parameters of a galactosyltransferase-catalyzed reaction w ere determined for the first time using capillary zone electrophoresis (CZE) using the methylumbelliferyl (MU) glycoside of N-acetylglucosam ine as the acceptor molecule. The CZE was performed using berate buffe r and the enzymatic transformations were monitored at 214 nm. The kine tic parameters obtained for MU-GlcNAc were K-m = 35.9 mu M and V-max = 7.5 mu mol/min/mg, and those for UDP-Gal were K-m = 115.3 mu M and V- max = 12.4 mu mol/min/mg. A representative inhibition assay was also c arried out using UDP as an inhibitor to give the K-i value of 83.9 mu M against MU-GlcNAc. The structure of the synthetic product was also c onfirmed using H-1 NMR spectroscopies after isolation by simple chroma tography. (C) 1998 Academic Press.