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|Title:||Biosynthesis of Chondroitin Sulfate Proteoglycan: Substrate Specificity of N-Acetylgalactosaminyl Transferase and Glucuronosyl Transferase Ii From Cultured Chick Embryo Chondrocytes|
|Author(s):||Gundlach, Mary Weiler|
|Department / Program:||Biochemistry|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||In vitro assays for N-acetylgalactosaminyl transferase and glucuronosyl transferase II were developed and utilized to determine kinetic parameters which define the substrate specificity of the enzymes toward a series of different ('3)H-oligosaccharide substrates. Chondroitin, chondroitin-4-sulfate, chondroitin-6-sulfate, and hyaluronic acid polymers were treated with hyaluronidase to obtain oligosaccharide products which contain an even number of monosaccharide residues and which contain D-glucuronic acid residues at their non-reducing termini. These oligosaccharides were used as acceptors in N-acetylgalactosaminyl transferase assays. Further treatment with (beta)-glucuronidase generated the corresponding oligosaccharides which contain an odd number of monosaccharide residues and which contain N-acetyl-D-galactosamine residues at their non-reducing termini. These oligosaccharides were utilized as substrates in glucuronosyl transferase II assays. The oligosaccharide substrates were quantitatively reduced and radiolabeled with NaB('3)H(,4) and then purified to homogeneity by high performance liquid chromatography for use in the enzyme assays.
Kinetic parameters of N-acetylgalactosaminyl transferase and glucuronosyl transferase II were quantified by incubating near saturating levels of the ('3)H-oligosaccharide acceptor and saturating levels of the appropriate nucleotide sugar donor with a microsomal enzyme fraction from cultured chick embryo chondrocytes at pH 7.0. A divalent metal cation such as MnCl(,2) is required and polyoxyethylene 20 cetyl ether (Brij-58) was found to stimulate the enzyme up to sixfold.
The data show that all of the ('3)H-tri- and ('3)H-tetrasaccharide substrates are poor substrates for glucuronosyl transferase II and N-acetylgalactosaminyl transferase. As the size of the ('3)H-oligosaccharide acceptor increases, the maximum velocity of the enzyme increases for chondroitin-6-sulfate and chondroitin substrates. This effect is most predominate for increases in the acceptor size from the ('3)H-trito the ('3)H-heptasaccharides. For ('3)H-oligosaccharides that are larger than a heptasaccharide, the maximum velocity is less sensitive to increases in ('3)H-oligosaccharide size. The enzymes are highly specific for the nonreducing terminal monosaccharide residue and slightly less specific for the penultimate monosaccharide residue.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.
|Date Available in IDEALS:||2014-12-15|