Galactosyltransferase catalyzes the transfer of galactose from UDP-galactose to either glucose or ^-acetylgalactosamine or to N-acetylglucosamine when this is a terminal residue of complex oligosaccharides. The activity occurs in tissues and body fluids of many different types.
Most assays for this activity either determine the amount of radioactive product formed from radioactive UDP-Gal or measure the amount of UDP present using a coupled assay in which the UDP formed is coupled to the formation of NAD or NADH.
The HPLC assay developed for this activity is based on the isocratic separation of UDP, UDP-Gal, and UMP on an amino-bonded column (¿¿Bondapak NH2 column) eluted with 0.167 M KH2P04 (pH 4.0). The separation is shown in Figure 9.67. Detection was at 260 nm.
The reaction mixture contained N-acetylglucosamine, UDP-Gal, MnCl2, and buffer at pH 8.0. The reaction was started by the addition of the enzyme. Samples were transferred at intervals to cacodylate buffer on ice (pH 6.5) to terminate the reaction. Samples (10 ¡xL) were analyzed by HPLC. The conversion of UDP-Gal to UDP is shown in Figure 9.68. Each panel represents a different time point, from 0 to 60 minutes. During the incubation, the disappearance of the substrate and the formation of the two products is seen. The enzyme was obtained from commercial sources and human serum.
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