Studies on the method for the determination of beta 1-4 galactosyltransferase and its kinetics

A fluorescence assay method for beta 1 - 4galactosyltransferase (beta 1 - 4GT) has been developed involving a pyridylaminated sugar as an acceptor substrate, a fluorescent sugar chain, with the reducing end of the Gn beta 1 - 2M alpha 1 - 6(Gn beta 1 - 2M alpha l - 3)M beta 1 - 4Gn beta 1 - 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 2.31 x 10(-3)M(-1) and 5.75 x 10(-2) mu mol/(min.mg) respectively. Furthermore, our research revealed that beta 1 - 4GT had branch specificity. The Gn of alpha 1 - 3 mannose branch of the acceptor substrate was more susceptible to galactosylation than that of the alpha 1 - 6 branch by 2,10 times.