BIOCHEMICAL AND GENETIC STUDIES OF FUCOSYL- AND SIALYLTRANSFERASES.
Item
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Title
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BIOCHEMICAL AND GENETIC STUDIES OF FUCOSYL- AND SIALYLTRANSFERASES.
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Identifier
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AAI8112744
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identifier
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8112744
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Creator
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MARGOLIES, RITA T.
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Contributor
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Robert J. Desnick
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Date
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1981
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Genetics
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Abstract
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A major result of this study is the first report of (alpha)l (--->) 6 fucosyltransferase in a tissue of human origin. This enzyme was characterized in human lymphoid lines and detected in human skin fibroblasts, kidney, spleen, adult and fetal intestine, and liver. An optimized assay method was developed that used asialo, agalactofetuin as acceptor. Lymphoid line (alpha)l (--->) 6 fucosyltransferase had a pH optimum of 7.0 and was stimulated by Mg('2+) and Ca('2+) in the presence of Triton X-100. Apparent K(,m)'s for GDP-fucose and asialo, agalactofetuin were 66.6 (mu)M and 0.24 mM, respectively.;Optimized assays for lymphoid line (alpha)l (--->) 2 fucosyltransferase and sialyltransferase were also developed. (alpha)l (--->) 2 fucosyltransferase had a pH optimum of between 6.5 and 7.0 when HEPES buffer was used. This enzyme was stimulated slightly by Mg('2+) and Ca('2+) and had an apparent K(,m) of 7.7 (mu)M, for GDP-fucose and 0.326 mM for the acceptor, asialofetuin.;Sialyltransferase was assayed using asialofetuin as an acceptor. The enzyme had a strict requirement for divalent metal ion, 10 mM Mg('2+) being optimal; however, enzyme activity also appeared to be stimulated by 20 mM EDTA and EGTA. It had an apparent K(,m) of 25 (mu)M for CMP-sialic acid and a pH optimum of 6.;All three enzymes were membrane bound. When lymphoid line crude membrane preparations were treated with a combination of 0.3% deoxycholate and 0.1% Triton X-100, over 90% of fucosyltransferase activity was released into the soluble phase. Detergent treatment also activated these enzymes to a considerable extent.;None of the detergents tested in this study, including Triton X-100, deoxycholate, Brij 35, Cutscum, and octylglucoside were able to solubilize sialyltransferase activity, and with the exception of Cutscum and octylglucoside, detergent treatment resulted in inhibition of enzyme activity.;The differential effects of sulfhydryl reagents, including mercaptoethanol, dithiothreitol, N-ethylmaleimide, iodoacetate, glutathione (reduced), and p-chloromercuribenzoate on fucosyl- and sialyltransferases from lymphoid lines, fibroblasts, and serum were examined. (alpha)l (--->) 2 fucosyltransferase from all three sources was generally stimulated by sulfhydryl reagents with the exception that the serum enzyme was inhibited by N-ethylmaleimide. (alpha)l (--->) 6 fucosyltransferase and sialyltransferase were unaffected or inhibited by sulfhydrylreagents. para-Chloromercurdbenzoate was inhibitory to all three enzymes.;Dithiothreitol almost totally inhibited lymphoid lines (alpha)l (--->) 6 fucosyltransferase while stimulating the (alpha)l (--->) 2 enzyme twofold. This provides a way of distinguishing between these two cellular enzymes.;In light of proposals that cystic fibrosis is the result of abnormal glycosylation mechanisms, the fucosyl- and sialyltransferases were examined in lymphoid lines derived from obligate heterozygotes and homozygotes and compared withthose from normal individuals with respect to enzyme activities and their ratios. No differences among the groups could be detected. Fibroblasts from patients suffering from cystic fibrosis were also examined, and again no differences could be detected.;These enzymes were also assayed in lymphoid lines from individuals with a variety of metabolic defects, including fucosidosis, cystathioninuria, homocystinuria, hypercholesterolemia, and Sanfillipo syndrome. No striking differences were found with respect to enzyme activities or their ratios.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
