Purification and some properties of UDP-xylosyltransferase of rat ear cartilage

• Published in: Glycobiology (Oxford) 2000-08, Vol.10 (8), p.803-807
• Main Authors: Pfeil, U, Wenzel, K W
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cartilage - enzymology; Chromatography, Affinity; Chromatography, Gel; Ear; Electrophoresis, Polyacrylamide Gel; Pentosyltransferases - chemistry; Pentosyltransferases - isolation & purification; Pentosyltransferases - metabolism; Rats; UDP Xylose-Protein Xylosyltransferase
• ISSN: 0959-6658; 1460-2423
• DOI: 10.1093/glycob/10.8.803

UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core protein beta-D-xylosyltransferase EC 2.4.2.26) initiates the formation of chondroitin sulfate in the course of proteoglycan biosynthesis. The enzyme catalyzes the transfer of D-xylose from UDP-D-xylose to specific serine residues in the core protein. A procedure for purification of xylosyltransferase from rat ear cartilage was developed which includes ammonium sulfate fractionation, chromatography on heparin-agarose, on Sephacryl S300 and finally a substrate affinity chromatography applying the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G. The specific activity of the purified enzyme was about 420 mU per mg protein. The purification factor was about 26.000 with 27% yield. In SDS-polyacrylamide gel electrophoresis, the highly purified enzyme is homogeneous and yields only a single distinct band of 78 kDa. An apparent molecular mass of 71 kDa was determined for the native enzyme. These data suggest a monomeric structure for the enzyme. Xylosyltransferase activity was found to depend essentially on the presence of divalent metal ions. The K(m) value for UDP-D-xylose was determined to 6.5 micromol/l and for the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G as xylose acceptor to 8 micromol/l.