Sulfation of chondroitin oligosaccharides in vitro. Analysis of sulfation ratios.
Microsomal preparations from cultured chick embryo chondrocytes were incubated with 3'-phosphoadenosine 5'-phosphosulfate and oligosaccharides prepared from chondroitin. Rates of 4- and 6-sulfation were measured at pH 6 and 8 in the presence of MnCl2 and Brij 58. Ratios of the overall 6-sulfation to 4-sulfation rates ranged from 40-200 at pH 8 and from 6-35 at pH 6, depending upon the composition of the assay mixture. When saturating concentrations of 3'-phosphoadenosine 5'-phosphosulfate and the oligosaccharide acceptors were used, the resulting products were mixtures of monosulfated oligosaccharides. The compositions of the mixtures formed from oligosaccharides with degrees of polymerization from 4-12 at pH 6 and 8 were analyzed. Sulfate substituents were found at all N-acetyl-D-galactosamine (GalNAc) residues in the acceptors but were not evenly distributed along the oligosaccharide chains. For oligosaccharides with nonreducing terminal D-glucuronic acid (GlcUA) residues, sulfation at the nonreducing terminal GlcUA----GalNAc occurred exclusively at the C6 of the GalNAc residue. However, for oligosaccharides with nonreducing terminal GalNAc residues the rate of 6-sulfation of the nonreducing terminal GalNAc was markedly reduced and was similar to the rate of 4-sulfation at the same position. The rates of sulfation at the reducing ends of the oligosaccharides were relatively high for the shorter oligosaccharide acceptors but decreased with increasing length of the acceptor, suggesting that the sulfotransferases recognized primarily the GalNAc residues in the nonreducing terminal regions.
D000818:Animals; D002240:Carbohydrate Sequence; D002241:Carbohydrates / Q000032:analysis; D002356:Cartilage / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D002807:Chondroitin / Q000031:analogs & derivatives* / Q000378:metabolism*; D002809:Chondroitin Sulfates / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D013431:Sulfates / Q000378:metabolism; D015238:Sulfotransferases; D013462:Sulfur Radioisotopes; D013466:Sulfurtransferases / Q000378:metabolism
D002241:Carbohydrates; D009844:Oligosaccharides; D013431:Sulfates; D013462:Sulfur Radioisotopes; D002807:Chondroitin; D002809:Chondroitin Sulfates; D013466:Sulfurtransferases; D015238:Sulfotransferases; C019893:chondroitin 4-sulfotransferase