Biosynthesis of the carbohydrate units of immunoglobulins. 1. Purification and properties of galactosyltransferases from swine mesentary lymph nodes

Publisher Website
Google Scholar
Abstract
Galactosyltransferase (UDP-galactose:glycoprotein galactosyltransferase, EC 2.4.1.22) was isolated from swine mesentary lymph node homogenates using procedures which included affinity chromatography on Sepharose 4B columns containing covalently bound p-aminophenyl-.beta.-D-N-acetylglucosamine. The homogenous enzyme showed a single band on disc gel electrophoresis and had a specific activity of 35 nmol min-1 (mg of protein)-1 at 37.degree. C. MW of 57,000 was obtained by exclusion chromatography, sucrose density centrifugation and sodium dodecyl sulfate-gel electrophoresis. The same MW was obtained after reduction and alkylation which indicates that the enzyme is composed of only a single polypeptide chain. The enzyme catalyzed the formation of .beta.1 .fwdarw. 4 bonds between galactose and free terminal N-acetylglucosaminyl residues of soluble preparations of porcine IgG immunoglobulin heavy chain, fetuin, ovalbumin and ovomucoid. An endogenous glycoprotein, present in particulate subcellular preparations, was also a very good substrate for the enzyme, and it was identified as incomplete IgG immunoglobulin heavy chain. The Km of the purified enzyme was 2.9 .times. 10-5 M for fetuin, 5.4 .times. 10-5 M for ovalbumin, 2.0 .times. 10-5 M for IgG immunoglobulin heavy chain and 2.2 .times. 10-5 M for UDP-galactose. About 20% of the total galactosyltransferase activity in lymph node homogenates was present in the cytosol fraction and 80% was in the microsomal and Golgi fractions. The kinetic properties of the bound and soluble galactosyltransferases were similar, and both required Mn2+ for maximal activity. The bound enzyme required the addition of detergents, lysolecithin, GDP-mannose and UDP-N-acetylglucosamine for maximum activity. These compounds did not influence the activity of the soluble transferase. The membrane preparations catalyzed the transfer of galactose from UDP-galactose and N-acetylglucosamine from UDP-N-acetylglucosamine, to incomplete oligosaccharide chains of endogenous IgG immunoglobulin bound to these particles. The labeled products of these reactions were isolated, and the structures of their oligosaccharide chains were determined and compared with those isolated from the heavy chain of porcine IgG immunoglobulin. The glycopeptide prepared from the endogenous acceptor and the major glycopeptide prepared by proteolytic digestion of the heavy chain of porcine IgG immunoglobulin had identical structures. The structure for the carbohydrate chains of porcine IgG immunoglobulin was determined by sequential enzymatic hydrolysis and methylation studies.