Studies on the Method for the Determination of beta1-4 Galactosyltransferase and Its Kinetics

A fluorescence assay method for beta1-4galactosyltransferase (beta1-4GT) has been developed involving a pyridylaminated sugar as an acceptor substrate, a fluorescent sugar chain, with the reducing end of the Gnbeta1 - 2Malpha1 - 6(Gnbeta1-2Malpha1-3)Mbeta1 - 4Gnbeta1 - 4Gn - PA aminated with 2-aminopyridine. Microsome was prepared from the liver of normal male rats as an enzyme sample. Then the fluorescent reaction product was separated by reverse-phase HPLC. The kinetic experiments were carried out using crude enzyme extracts of the Golgi complex from the rat liver. The enzyme has a pH optimum of 6.5,and optimal concentration of Triton X-100 of 0.5%. The K(m) and V(max) values for the sugar acceptor substrates were found to be 2.31x10(-3)M(-1) and 5.75x10(-2) &mgr;mol/(min.mg) respectively. Furthermore, our research revealed that beta1-4GT had branch specificity. The Gn of alpha1-3 mannose branch of the acceptor substrate was more susceptible to galactosylation than that of the alpha1-6 branch by 2.10 times.