Agarose Derivatives of Uridine Diphosphate and N-Acetylglucosamine for the Purification of a Galactosyltransferase

Abstract

Abstract The applicability of affinity chromatography for the purification of UDP-galactose: N-acetylglucosamine galactosyltransferase has been evaluated. Three different types of specific adsorbents have been synthesized by reaction of a ligand structurally related to portions of the substrates of the transferase with cyanogen bromide-treated agarose. The synthesis of the ligands, P1-(6-amino-1-hexyl)-P2-(5'-uridine)-pyrophosphate, 6-amino-1-hexyl-2-acetamido-2-deoxy-β-d-glucopyranoside and P1-(6-amino-1-hexyl)-P2-(β-d-galactopyranosyl)pyrophosphate is described. The galactosyltransferase from bovine milk has been found to bind to two of the adsorbents and some of the parameters influencing the binding have been evaluated. The affinity of the transferase for the UDP-hexanolamine-agarose adsorbent was enhanced by manganous ions whereas the affinity was decreased by EDTA, urea, borate, or magnesium ions. The affinity of the enzyme for N-acetylglucosamine hexanolamine-agarose was enhanced considerably by UDP or UMP and was decreased by borate, urea or N-acetylglucosamine. N-Acetylglucosamine-agarose also served as an acceptor substrate for the transferase. The galactosyl pyrophosphate-agarose was found to have a low capacity and specificity of binding for the galactosyltransferase. On the basis of the properties of the adsorbents, it has been possible to purify the galactosyltransferase from bovine milk to constant specific activity soley by affinity chromatography. The enzyme from the whey of bovine milk was first adsorbed on UDP-agarose and the eluate from this adsorbent then chromatographed on either N-acetylglucosamine-agarose or α-lactalbumin-agarose. The properties of the transferase are very similar to those reported earlier although at least three species of enzyme differing slightly in molecular weight have been found. The three species are glycoproteins and their amino acid compositions are very similar. The UDP-agarose adsorbent may be useful for purification of other UDP-glycosyltransferases as shown in preliminary studies with rabbit muscle glycogen synthetase. It is also possible that other nucleotide-agarose or glycosyl-agarose adsorbents may be used together for the purification of many nucleotide-sugar transferases.