Biosynthesis of Chondroitin Sulfate Proteoglycan: Substrate Specificity of N-Acetylgalactosaminyl Transferase and Glucuronosyl Transferase Ii From Cultured Chick Embryo Chondrocytes
Gundlach, Mary Weiler
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https://hdl.handle.net/2142/70524
Description
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
Issue Date
1983
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biophysics, General
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.
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