Sulfate Transfer Reactions in Chondroitin Sulfate Biosynthesis (Chondrocyte, Sulfotransferase, Microsomes)

216 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1984.Chondroitin oligosaccharides were used as SO(,4) acceptors to determine the substrate-product specificity of chondroitin-4-sulfotransferase and chondroitin-6-sulfotransferase from chick embryo chondrocytes. The oligosaccharide substrates ranged in size from tetra- through dodecasaccharides and each contained N-acetylgalactosamine at their reducing terminus. The oligosaccharides which contained an even number of monosaccharide residues contained D-glucuronic acid at their nonreducing terminus and those that contained an odd number of monosaccharide residues contained N-acetylgalactosamine at their nonreducing terminus. These oligosaccharides were quantitatively reduced with NaB('3)H(,4) and the ('3)H-label was used to follow the purification of the oligosaccharides to homogeneity and to measure the concentrations of the oligosaccharides in the sulfotransferase assays.Saturating levels of the ('3)H-oligosaccharide acceptor and the SO(,4) donor, ('35)SO(,4)-labeled adenosine-3'-phosphate-5'phosphosulfate (PAP('35)S) were incubated with a microsomal enzyme fraction from cultured chick embryo tibial chondrocytes at pH 6.0 and at pH 8.0. The sulfated oligosaccharide products were digested wih chondroitinase and the digestion products were analyzed to determine the individual rates of 4- and 6-sulfation. Both sulfotransferases were stimulated by MnCl(,2) and detergents. Dithiothreitol stimulated the rate of 4-sulfation 2-fold at pH 6.0 but had little effect on the rate of 6-sulfation at either pH 8.0 or pH 6.0. Under optimized assay conditions, the rate of 4-sulfation was reduced by as much as 5-fold when the pH was shifted from 6.0 to 8.0, while the rate of 6-sulfation was relatively unchanged. An analysis of the chondroitinase ABC and/or the chondroitinase AC digestion products from the sulfated chondroitin oligosaccharides was used to determine the rates of 4- and 6-sulfation at the nonreducing terminal, internal, and reducing terminal regions of the oligosaccharides. It was found that both acceptor structure and pH affect the distributions and positions of SO(,4) esters on the oligosaccharide products.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD