Caracterización enzimática de la α-1,3- galactosiltransferasa bovina. Validación de un ensayo radiométrico y mecanismo cinético

α3-Galactosyltransferase (α3GT) transfers galactose from UDP-Gal (sugar nucleotide donor) to the N-acetyllactosaminyl or lactosyl terminal groups of glycoproteins and glycolipids, catalyzing the formation of an α-1,3 glycosidic bond. The terminal saccharide Galα3NAcGalβ 4Glu-R is the main antigenic determinant responsible of the hyperaccute rejection in xenotransplantation. A radiometric assay for the determination of α3GT activity is implemented and validated. The recombinant enzyme (catalytic domain) expressed in Eschericia coli follows a bi bi sequential ordered kinetic mechanism with binding of donor substrate (UDP-Gal) first and acceptor substrate to form a productive ternary complex. K _M values are 30 μM for UDP-Gal, and 1.2 mM for lactose.