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

摘要: Galactosyltransferase (UDPgalactose:glycoprotein galactosyltransferase EC 2.4.1.22) was isolated from swine mesentary lymhromatography on Sepharose 4B colums containing covalently bound p-aminophenyl-beta-D-N-acetylglucosamine. The homogenous enzyme showed a single band disc gel electrophoresis and had specific activity of 35 nmol min-1 (mg protein)-1 at 37 degrees C. A molecular weight 57 000 obtained by exclusion chromatography, sucrose density centrifugation, sodium dodecyl sulfate-gel electrophoresis. same after reduction alkylation which indicates that the is composed only polypeptide chain. catalyzed formation beta1 leads to 4 bonds between galactose free terminal N-acetylglucosaminyl residues soluble preparations porcine IgG immunoglobulin heavy chain, fetuin, ovalbumin, ovomucoid. An endogenous glycoprotein, present in particulate subcellular preparations, also very good substrate for enzyme, it identified as incomplete Km purified 2.9 x 10(-5) M 5.4 2.0 immlnoglobulin 2.2 UDP-galactose. About 20% total lymph node homogenates cytosol fraction, 80% microsomal Golgi fractions. kinetic properties galactosyltransferases were similar,and both required Mn2+ maximal activity. However, addition detergent, lysolecithin, GDP-mannose, UDP-N-acetylglucosamine maximum These compounds did not influence transferase. membrane transfer UDP-galactose N-acetylglycosamine oligosaccharide chains these particles. labeled products reactions isolated, structures their determined compared with those chain immunoglobulin. glycopeptide prepared acceptor major proteolytic digestion has identical structures. following structure carbohydrate sequential enzymatic hydrolysis methylation studies.