GLYCOPROTEIN BIOSYNTHESIS . PURIFICATION AND CHARACTERIZATION OF A POLYPEPTIDE . N-ACETYLGALACTOSAMINYL TRANSFERASE FROM BOVINE SUBMAXILLARY GLANDS

An enzyme was found in membranes of bovine submaxillary glands that synthesizes a protein-carbohydrate linkage, the N-acetylgalactosaminyl-O-hydroxyamino acid unit found in submaxillary glycoproteins. Most (90% or more) of the enzyme is normally bound to membranous material. The polypeptide:N-acetylgalactosaminyl transferase was purified 70-fold by gel filtration and ultracentrifugation after solubilization from the 16,000g residue with the detergent Triton X-100. The enzyme was highly purified as judged by polyacrylamide gel electrophoresis and ultracentrifugation; glycoprotein, endogenous receptor, and galactosyl transferase were absent from the purified enzyme. The transfer of N-acetylgalactosamine from UDP-galNAc to protein receptor is highly specific; receptor, nucleotide sugar, and Mn2+ (or Co2+) were required. Receptors were prepared from BSM from which hexosamine had been removed by either hexosaminidase or periodate-acid hydrolysis treatment. It was found that 18-22% of the theoretical receptor sites (N-acetylhexosamine removed) of the receptors could be filled by prolonged incubation. N-Acetylgalactosamine-14C was obtained from the receptor-galNAc-14C product with hexosaminidase; N-acetylgalactosaminitol-14C was identified after alkaline-borohydride treatment. The enzyme gave Km values of 6 × 10-5m and 9.5 × 10-4m, with respect to UDP-galNAc and theoretical receptor sites respectively. A pH optimum existed from 6.5-7.5; maximal activity occurred with 6 mM Mn2+. The role of this enzyme in initiation of assembly of the carbohydrate unit of glycoproteins is discussed. © 1969.