The assay of xylosyltransferase in cartilage extracts. A modified procedure for preparation of Smith-degraded proteoglycan.
A modification of the published method [Baker, Rodén & Stoolmiller (1972) J. Biol. Chem. 247, 3838--3847] for preparation of Smith-degraded proteoglycan is described. The new method is based on the finding that most of the chondroitin sulphate is cleaved from proteoglycan core protein by periodate oxidation. The borohydride reduction procedure was modified because the periodate-oxidized core protein is extensively degraded under the highly alkaline conditions previously used. The new method involves the separation of periodate-oxidized core protein from chondroitin sulphate by gel filtration on Sepharose 6B, and the reduction of the former in H3BO3/NaBH4 at pH 8.5 to produce the reduced species. Smith-degraded proteoglycan prepared by this method exhibited high acceptor activity for xylosyltransferase from embryonic-chick cartilage and had an apparent Km of 160 microgram/ml or 45 micrometer on a serine basis. In this assay system an apparent Km of 19 micrometer was obtained for UDP-xylose. The intermediate products periodate-oxidized core protein and reduced proteoglycen were inactive as xylosyltransferase acceptor substrates.
D000818:Animals; D001894:Borohydrides; D002356:Cartilage / Q000201:enzymology*; D002642:Chick Embryo; D008722:Methods; D010430:Pentosyltransferases / Q000378:metabolism*; D010504:Periodic Acid; D011509:Proteoglycans / Q000138:chemical synthesis*; D014540:Uridine Diphosphate Xylose / Q000378:metabolism; D000097608:UDP Xylose-Protein Xylosyltransferase
D001894:Borohydrides; D010430:Pentosyltransferases; D010504:Periodic Acid; D011509:Proteoglycans; D014540:Uridine Diphosphate Xylose; D000097608:UDP Xylose-Protein Xylosyltransferase