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Bovine galactosyl transferase was found to utilize UDPglucose as a substrate and elicit disaccharide biosynthesis with glucose and N-acetylglucosamine as acceptors. The relative rate of glucosyl transferase with N-acetylglucosamine as acceptor was 0.3%, the rate for N-acetyllactosamine biosynthesis. This activity was also evidenced indirectly from NMR water proton relaxation experiments, and from Mn(II) ESR experiments. In direct experiments with radioactive UDPglucose, paper chromatography showed a product which migrated with cellobiose when glucose was the acceptor and a new, glucose-containing product which resulted when GlcNAc was the acceptor. Despite this marginally expanded specificity of the donor site, spin-label experiments with a covalently bound UDPgalactose analog reaffirmed the restrictive nature of the donor site against this non-glycosyl-like analog.

Bovine galactosyltransferase (UDPgalactose: D-glucose 4beta-galactosyltransferase, EC 2.4.1.22) was covalently coupled to Sepharose 4B by reaction at pH 5.0 with the activated mixed disulfide Sepharose-glutathione-2(5-nitropyridyl)-disulfide. The Sepharose-protein conjugate was presumably coupled via the unique highly reactive cysteine of those thiols on the bovine enzyme. The gel-bound N-acetyllactosamine and lactose synthase activity of about 0.4% was consistent with the affects of diffusion and the 90% activity reduction noted upon thiol modification of the dissolved enzyme. The residual lactose biosynthetic activity of the bound enzyme appeared possible only if the reactive thiol were physically distinct from the active site since the bulky Sepharose-glutathione group must not obscure the alpha-lactalbumin binding region.