Biosynthesis of chondroitin sulfate. Solubilization of chondroitin sulfate glycosyltransferases and partial purification of uridine diphosphate-D-galactose:D-xylose galactosyltrans.
UDP-D-Galactose:D-xylose galactosyltransferase, a membrane-bound enzyme which catalyzes the second glycosyl transfer reaction in the biosynthesis of chondroitin sulfate chains, has been solubilized and partially purified from embryonic chick cartilage. Solubilization was effected by treatment of a particulate fraction of a homogenate (sedimenting between 10,000 and 100,000 times g) with the nonionic detergent Nonidet P-40 (0.5%) and KCl (0.5 M) or by the alkali-detergent method described previously (Helting, T. (1971) J. Biol. Chem. 246, 815-822). The applicability of the salt-detergent procedure as a general method for solubilization of membrane-bound glycosyltransferases was tested by assay of four other glycosyltransferases involved in chondroitin sulfate synthesis (UDP-D-xylose:core protein xylosyltransferase, UDP-D-galactose:4-O-beta-D-galactosyl-D-xylose galactosyltransferase, UDP-D-glucuronic acid: 3-O-beta-D-galactosyl-D-galactose glucuronosyltransferase, and UDP-N-acetyl-D-galactosamine: (GlcUA-GalNAc-4-sulfate)4 N-acetylgalactosaminyltransferase). In each case, greater than 70% of the activity was solubilized and, on gel chromatography on Sephadex G-200, the enzymes appeared largely in included positions and partially separated from each other. After partial purification by gel chromatography on Sephadex G-200, UDP-D-galactose:D-xylose galactosyltransferase was purified further by chromatography on one of several affinity matrices, i.e. xylosylated core protein of cartilage proteoglycan coupled to CNBr-activated Sepharose, a core protein matrix saturated with UDP-D-xylose:core protein xylosyltransferase or UDP-D-xylose:core protein xylosyltransferase covalently bound to Sepharose. The specific activities of the enzyme preparations obtained by these procedures were approximately 1000-fold greater than that of the crude homogenate.
D000818:Animals; D002250:Carbon Radioisotopes; D002642:Chick Embryo; D002807:Chondroitin / Q000096:biosynthesis*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003902:Detergents / Q000494:pharmacology; D004838:Epiphyses / Q000201:enzymology; D005269:Femur; D005964:Glucosyltransferases / Q000378:metabolism*; D006602:Hexosyltransferases / Q000378:metabolism*; D010430:Pentosyltransferases; D011136:Polysorbates / Q000494:pharmacology; D011509:Proteoglycans; D013464:Sulfuric Acids; D013977:Tibia; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism; D014536:Uridine Diphosphate N-Acetylgalactosamine / Q000378:metabolism; D014540:Uridine Diphosphate Xylose / Q000378:metabolism; D014994:Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase
D002250:Carbon Radioisotopes; D002807:Chondroitin; D003902:Detergents; D005964:Glucosyltransferases; D006602:Hexosyltransferases; D010430:Pentosyltransferases; D011136:Polysorbates; D011509:Proteoglycans; D013464:Sulfuric Acids; D014531:Uridine Diphosphate Galactose; D014535:Uridine Diphosphate Glucuronic Acid; D014536:Uridine Diphosphate N-Acetylgalactosamine; D014540:Uridine Diphosphate Xylose; D014994:Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase