BIOCHEMICAL AND SOMATIC CELL GENETIC STUDIES OF HUMAN AND FELINE MUCOPOLYSACCHARIDOSES I.
Item
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Title
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BIOCHEMICAL AND SOMATIC CELL GENETIC STUDIES OF HUMAN AND FELINE MUCOPOLYSACCHARIDOSES I.
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Identifier
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AAI8312372
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identifier
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8312372
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Creator
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SCHUCHMAN, EDWARD HOWARD.
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Contributor
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Prof. R.J. Desnick
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Date
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1983
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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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Biochemical and somatic cell genetic studies have been undertaken to initiate investigations into the molecular pathology of human and feline mucopolysaccharidoses I ((alpha)-L-iduronidase deficiency diseases). Towards this end a new, continuous, monodimensional cellulose acetate electrophoretic system was developed for the resolution of six major mammalian glycosaminoglycans. Using this micro-method, an extensive survey of the stored glycosaminoglycans in the tissues of homozygotes with human or feline mucopolysaccharidoses I was undertaken characterizing, for the first time, the distribution of the stored glycosaminoglycans in these disorders.;In addition, two major fractions containing (alpha)-L-iduronidase activity have been identified in normal human tissues and purified over 150,000-fold to homogeneity. The more electronegative form of the enzyme is membrane-associated and taken up rapidly by cultured (alpha)-L-iduronidase deficient fibroblasts ("high uptake"), while the more soluble form is "low uptake" enzyme. Isoelectric focusing studies of crude tissue homogenates revealed that a broad region (pI 5.85-7.35) stained for (alpha)-L-iduronidase activity. Two distinct, major bands (pI values of 6.00 and 6.85) were observed.;SDS polyacrylamide gel electrophoresis under denaturing conditions revealed that the low uptake fraction was composed of a 72,000 dalton doublet and a diffuse 43,000 component, while the high uptake fraction had a consistent 90,000 band in addition to the 72,000 and 43,000 molecular weight species. Further purification using high performance liquid chromatography, combined with these data, indicated that the homogeneous enzyme fractions contained a number of discrete polymeric and proteolytic intermediates, each of which retained catalytic activity.;Kinetic studies using artificial and natural substrates revealed that the K(,m) and apparent V(,max) values for both enzyme forms were nearly identical. The thermal stabilities of the two enzyme forms at 45(DEGREES)C, 52(DEGREES)C and 60(DEGREES)C and the pH optima using three different substrates were also nearly identical. Heparin was a potent non-competitive inhibitor of the 4-methylumbelliferyl (alpha)-L-iduronide activity for both the high and low uptake enzymes (K(,i) values of 0.13 mg/ml and 0.09 mg/ml, respectively). Interestingly, the inhibitory effect of each glycosaminoglycan was significantly less for the high uptake than the low uptake form despite their similar catalytic properties. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI.
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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
