Purification and some properties of UDP-xylosyltransferase of rat ear cartilage.
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.
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Institute of Physiological Chemistry, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Karl-Marx-Strasse 3, D-01109 Dresden, Germany.
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D000595:Amino Acid Sequence; D000818:Animals; D002356:Cartilage / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004423:Ear; D004591:Electrophoresis, Polyacrylamide Gel; D010430:Pentosyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats; D000097608:UDP Xylose-Protein Xylosyltransferase
D010430:Pentosyltransferases; D000097608:UDP Xylose-Protein Xylosyltransferase