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

UDP-xylosyltransferase (UDP-D-xylose:proteoglycan core protein beta-D-xylos yltransferase EC 2.4.2.26) initiates the formation of chondroitin sulfate i n the course of proteoglycan biosynthesis, The enzyme catalyzes the transfe r of D-xylose from UDP-D-xylose to specific serine residues in the core pro tein. A procedure for purification of xylosyltransferase from rat ear carti lage was developed which includes ammonium sulfate fractionation, chromatog raphy on heparin-agarose, on Sephacryl S300 and finally a substrate affinit y chromatography applying the dodeca peptide Q-E-E-E-G-S-G-G-G-Q-G-G. The s pecific activity of the purified enzyme was about 420 mU per mg protein. Th e purification factor was about 26.000 with 27 % yield. In SDS-polyacrylami de gel electrophoresis, the highly purified enzyme is homogeneous and yield s only a single distinct band of 78 KDa, An apparent molecular mass of 71 k Da was determined for the native enzyme. These data suggest a monomeric str ucture for the enzyme. Xylosyltransferase activity was found to depend esse ntially on the presence of divalent metal ions. The K-m value for UDP-D-xyl ose was determined to 6.5 mu mol/l and for the dodeca peptide Q-E-E-E-G-S-G -G-G-Q-G-G as xylose acceptor to 8 mu mol/l.