Purification and characterization of chick corneal β-D-glucuronyltransferase involved in chondroitin sulfate biosynthesis

beta-(D)-Glucuronyltransferase, which transfers D-glucuronic acid (GlcA) from UDP-G1cA to N-acetyl-D-galactosamine (Ga1NAc) at the nonreducing end of chondro-pentasaccharide-PA (pyridylamino-), Ga1NAcbeta1- (4GlcAbeta1-3GaINAcbeta1)(2)-PA, was purified 339-fold with an 11.0% yield from 2-d-old chick corneas by chromatography on DEAE-Sepharose, WGA-agarose, heparin-Sepharose, and 1st and 2nd UDP-G1cA-agarose (in the presence of Gal) columns. The activity was detected by fluorescence of PA residues of the product. The purified enzyme has an optimum pH of 7.0 (Mes buffer), and much higher activity toward chondro-heptasaccharide-PA than toward the chondro-pentasaccharide-PA, but no activity toward p-nitrophenyl-beta-Ga1NAc. The enzyme activity was almost completely inhibited by Ga1NAc (20 mm). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme fraction showed one band of 38 kDa with many other bands. The amino acid sequence was determined for the tryptic digests of the 38 kDa band protein. The sequences determined showed no homology to those of several beta-glucuronyltransferases reported previously. It seems that the enzyme is involved in the elongation of chondroitin sulfate chains in vivo.