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|Title:||Glycosyl Transferases in Chondroitin Sulfate Biosynthesis: Subcellular Location and Elongation Reaction|
|Author(s):||Johnson, Jane Cutler|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||The subcellular location and polymerization reaction of the glucuronosyl transferase and the N-acetylgalactosaminyl transferase involved in the synthesis of chondroitin sulfate were examined in cultured embryonic chick tibial chondrocytes. The chondrocytes were subjected to subcellular fractionation by differential centrifugation and sucrose density gradient centrifugation. The glucuronosyl transferase, the N-acetylgalactosaminyl transferase, and also the sulfotransferases co-fractionated with the Golgi marker enzyme, galactosyl transferase. The specific activities and recoveries of the two glycosyl transferases in each fraction paralleled each other almost exactly.
An in vitro elongation assay for the glycosyl transferases was developed using chondroitin oligosaccharides as glycosyl acceptors. The radiolabeled oligosaccharides allowed exact quantification of the amount of substrate used and products formed. Elongation of up to eight or ten sugar residues occurred under optimal conditions. Brij-58 stimulated the elongation reaction by two- to threefold. The optimum pH for elongation was pH 7.0. The structure of the non-reducing terminal varied with pH according to the results seen with the assays for the separate glycosyl transferase activities. At pHs below pH 6.5, there were approximately equal amounts of N-acetylgalactosamine and glucuronic acid on the non-reducing terminal, but at higher pHs, there was one and a half- to threefold more N-acetylgalactosamine. Concentrations of the UDP-sugar substrates ranging from 20 to 40 mM did not inhibit the elongation reaction. Disruption of the membrane structure during the preparation of the microsomal fraction used in the elongation reaction reduced the polymerization activity of the glycosyl transferases. Crude microsomal preparations appeared to have the most elongation activity. Several attempts were made to examine possible involvement of glycosyl lipids in the synthesis of the chondroitin sulfate chains. The results obtained here support the theory that elongation occurs by single, direct, glycosyl transfers from the appropriate sugar donors to the acceptor molecules.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1987.
|Date Available in IDEALS:||2014-12-15|