Purification and Partial Characterization of an #alpha#,-2,8-Sialyltransferase From Human Erythroleukemia K562 Cells

摘要

An #alpha#,-2,8-sialytransferase (ST8), the enzyme involved in the biosynthesis of polysialic acid chains, has been purified and partly characterized from undifferentiated human erythroleukemia K562 cells. Purification, based on a key step of affinity chromatography utilizing immobilized colominic acid, was greater than 1000-fold. The enzyme molecular weight determined by SDS-PAGE was estimated to be about 40 kDa, in good agreement with literature data. For the determination of the main kinetic parameters (V_max and K_M), fetuin turned out to be the unique substrate acceptor. In fact, other compounds such as aiisalofetuin, transferin, #alpha#1-acid glycoprotein, and G_M3, routinely used to explore the different ST8 isoforms' activities, did not serve as substrate acceptors. In all cases, contrary to the routinely adopted protocol where a radioactive substrate donor is employed, for our purporse a non-radioctive, fluoroescent substrate donor such as cytidine-5'-monophospho-9-(3-fluoresceinylthioureido)-9-deoxy-N-acetyl-neuraminic acid (CMP-9-fluoresceinyl-NeuAc) was used. Thus, under our experimental conditions, by using fetuin, data reported in a typical Linewweaver-Burk plot gave a V_max value of about 4 nkatal/mg of porotein and a K_M value around 0.61 mM. Just at with the estimated molecular weight, these kinetic data were also in good agreement with those already reported for the ST8 purified from human neuroblatoma CHP-134 cells. (1). In particular, in both cases, V_max values were almost similar (4 nkatal/mg of protein for our ST8 purified from K562 cells and 4.35 nkatal/mg of protein for ST8 purified from CHP-134 cells); conversely, the K_M value we found was about 3.25-fold lower than that found by Stoykova and Glick (1) (0.61 mM vs. 2 mM). Then, although our purification was lower than that obtained by Stoykova and Glick(1) (1080-fold vs. 2910-fold), the enzyme we purified showed a greater apparent affinity.