Michaelis-Menten kinetics of TS reactions. All reactions were started with 50 ng TS in a final volume of 50 μL and incubated for 30 min at 37°C. (A) 600 μM fetuin-bound Sia was used as donor substrate with varying concentrations of lactose as acceptor substrate. (B) Varying concentrations of fetuin-bound Sia were used as donor substrate with 2 mM lactose as acceptor substrate. (C) Varying amounts Gal-MU were used as acceptor substrate and 50 nmol 3'SL as donor substrate. The amount of 3'SL (in the case of (A) and (B)) or 3'Sia-Gal-MU (in the case of (C)) produced was determined by HPAEC-PAD as described under Methods. Data points are means ± standard deviations of three replicates.