Purification and characterization of fetal bovine serum beta-N-acetyl-D-galactosaminyltransferase and beta-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis

beta-N-Acetylgalactosaminyltransferase II and beta-glucuronyltransferase II , involved in chondroitin sulfate biosynthesis, transfer an N-acetylgalacto samine (GalNAc) and glucuronic acid (GlcA) residue, respectively, through b eta-linkages to an acceptor chondroitin oligosaccharide derived from the re peating disaccharide region of chondroitin sulfate. They were copurified fr om fetal bovine serum approximately 2500-fold and 850-fold, respectively, b y sequential chromatographies on Red A-agarose, phenyl-Sepharose, S-Sepharo se and wheat germ agglutinin-agarose. Identical and inseparable chromatogra phic profiles of both glycosyltransferase activities obtained through the a bove chromatographic steps and gel filtration suggest that the purified enz yme activities are tightly coupled, which could imply a single enzyme with dual transferase activities; beta-N-acetylgaIactosarninyltransferase and be ta-glucuronyltransferase, reminiscent of the heparan sulfate polymerase rea ction. However, when a polymerization reaction was performed in vitro with the purified serum enzyme preparation under the polymerization conditions r ecently developed for the chondroitin-synthesizing system, derived from hum an melanoma cells, each monosaccharide transfer took place, but no polymeri zation occurred. These results may suggest that the purified serum enzyme p reparation contains both beta-N-acetylgalactosaminyltransferase II and beta -glucuronyltransferase Il activities on a single polypeptide or on the resp ective polypeptides forming an enzyme complex, but is different from that o btained from melanoma cells in that it transfers a single GalNAc or GlcA re sidue but does not polymerize chondroitin.