Purification and partial characterization of an alpha-2,8-sialyltransferase from human erythroleukemia K562 cells

An alpha -2,8-sialyltransferase (ST8), the enzyme involved in the biosynthe sis of polysialic acid chains, has been purified and partly characterized f rom undifferentiated human erythroleukemia K562 cells. Purification, based on a key step of affinity chromatography utilizing immobilized colominic ac id, was greater than 1000-fold. The enzyme molecular weight determined by S DS-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), fetui n turned out to be the unique substrate acceptor. In fact, other compounds such as asialofetuin, transferrin, alpha1 -acid glycoprotein, and G(M3), ro utinely used to explore the different ST8 isoforms' activities, did not ser ve as substrate acceptors. In all cases, contrary to the routinely adopted protocol where a radioactive substrate donor is employed, for our purpose a non-radioactive, fluorescent substrate donor such as cytidine-5'-monophosp ho-9-(3-fluoresceinylthioureido)-9-deoxy-N-acetyl-neuraminic acid (CMP-9-fl uoresceinyl-NeuAc) was used. Thus, under our experimental conditions, by us ing fetuin. data reported in a typical Lineweaver-Burk plot gave a V-max va lue of about 4 nkatal/mg of protein and a K-M value around 0.61 mM. Just as with the estimated molecular weight, these kinetic data were also in good agreement with those already reported for the ST8 purified from human neuro blastoma 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 cell s and 4.35 nkatal/mg of protein for ST8 purified from CHP-134 cells); conve rsely, 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 w as lower than that obtained by Stoykova and Glick(1) (1080-fold vs. 2910-fo ld), the enzyme we purified showed a greater apparent affinity.