Glycoprotein Biosynthesis: Purification and Properties of Glycoprotein

A glycoprotein :N‐acetylglucosamine transferase utilizing endogenous acceptors and a fetuin: N‐acetylglucosamine transferase utilizing a defined exogenous fetuin acceptor from which sialic acid, galactose and N‐acetylglucosamine had been removed were characterized from guinea pig liver. Both glycoprotein :N‐acetylglucosamine systems used UDP‐N‐acetylglucosamine as substrate, required Mn++ and had optimum pH of 5.4. Both enzyme systems were solubilized by extraction of the liver with 0.1% Triton X‐100. The fetuin :N‐acetylglucosamine transferase was separated from endogenous acceptors and purified more than 24‐fold on Sephadex G‐200. The endogenous system had a Km of 0.6 μM and the exogenous fetuin :N‐acetylglucosamine transferase had a Km of 3.8 μM with respect to N‐[14C]acetylglucosamine. The fetuin :N‐acetylglucosamine transferase was highly specific; 11 glycoproteins and 20 mono‐ and disaccharides and amino acids were negative when tested for acceptor activity. The fetuin :N‐acetylglucosamine transferase did not transfer any other nucleotide diphosphate sugar onto the acceptor; even UDP‐N‐acetylgalactosamine was not transferred. It appeared that the exogenous and endogenous systems may well be identical except that the endogenous acceptor was removed during purification of the enzyme.