PURIFICATION AND CHARACTERIZATION OF UDP-GALNAC-POLYPEPTIDE N-ACETYLGALACTOSAMINYL TRANSFERASE FROM SWINE TRACHEA EPITHELIUM

UDP-GalNac: polypeptide N-acetylgalactosaminyltransferase from swine t rachea epithelium was purified to homogeneity by procedures which incl uded affinity chromatography on Sepharose 4B columns containing bound deglycosylated Cowper's gland mucin. The enzyme, purified 12,000-fold from microsomes with a yield of 40%, showed only a single band on dode cyl sulfate polyacrylamide gel electrophoresis. The homogenous enzyme has an apparent molecular mass of 70,000 Da, as determined by gel elec trophoresis or gel filtration. The transferase has a broad pH optimum between 6.7-7.8 with maximal activity at pH 7.2, and required Mn2+ for activity with maximal activity at 5-7.5 mM. Higher concentrations of Mn2+, inhibited the enzyme. The purified transferase was specific for UDPGalNAc and glycosylated both threonine and serine residues in trypt ic peptides prepared from deglycosylated Cowper's gland and swine and human trachea mucins. The apparent Km of the transferase for UDPGalNAc was 6.3 mu M, and the Km values for deglycosylated Cowper's gland and human and swine trachea mucins were 0.83, 1.12 and 0.94 mg/ml, respec tively. The V-max of the purified enzyme was 2.1 mu mol/min/mg with de glycosylated Cowper's gland mucin, as the glycosyl acceptor. However, the activities with peptides prepared from deglycosylated mucins by li mited acid hydrolysis were 20-fold greater than the intact glycoprotei n under identical conditions. The deglycosylated mucins and larger pep tides aggregated with time of storage and precipitated from solution. Aggregation was accompanied by a corresponding loss of enzymatic activ ity even after dispersion of the aggregate by sonication. The deglycos ylated mucins which were prepared by chemical treatment and periodate oxidation still contained about 20% of the N-acetylgalactosamine prese nt in the intact mucin. When this residual amino sugar was removed by periodate oxidation the completely deglycosylated mucins became very p oor substrates for the purified transferase. Data obtained in the curr ent study indicate that the accessibility of serine and threonine in t he polypeptide chains of mucin glycoproteins significantly influences the rate of glycosylation of these amino acids. The best substrates an d affinity ligand for the enzyme were fragments of incompletely deglyc osylated mucin polypeptide chains.