PEROXISOMAL FATTY ACID OXIDATION IN TETRAHYMENA AND IN RAT LIVER.
Item
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Title
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PEROXISOMAL FATTY ACID OXIDATION IN TETRAHYMENA AND IN RAT LIVER.
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Identifier
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AAI8103934
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identifier
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8103934
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Creator
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HRYB, DANIEL JOSEPH.
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Contributor
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James F. Hogg
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Date
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1980
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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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Chemistry, Biochemistry
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Abstract
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This thesis reports the presence of a new enzyme for fatty acid oxidation, the fatty acyl-CoA oxidase, in Tetrahymena and rat liver. The oxidase activity (a fatty acyl-CoA-dependent H(,2)O(,2) producing activity) was assayed by using a modified and much improved method to assay H(,2)O(,2)-producing oxidases. The oxidase assay involves the continuous spectrophotometric measurement of the reaction product, H(,2)O(,2), by coupling it to the oxidation of a chromogen (o-dianisidine or a p-hydroxybenzoic acid and 4-aminoantipyrine mixture) by horseradish peroxidase. There was no catalase inhibition of the oxidase activity as long as the catalase/peroxidase ratio of activities was lower than 10.;The oxidase in Tetrahymena was extracted from lyophilized cells with 50% (v/v) glycerol, which was required to stabilize the enzyme, and characterized. The oxidase from Tetrahymena is a FAD-dependent enzyme, has a pH maximum of 8.0-9.0, low affinity for acyl-CoA's, and high affinity for oxygen. The oxidase from cells grown on the standard medium (proteose-peptone-glucose-acetate) showed a chain length specificity for short-chain length acyl-CoA substrates (C(,4)-C(,8)) and it was not able to oxidize any acyl-CoA compound of chain length greater than C(,8). However, if Tetrahymena cells are grown in an oleate-rich medium (proteose-peptone-glucose-Tween 80), the chain length specificity of the oxidase becomes much broader (C(,4)-C(,20)) and their activity increases by several fold over the cells grown in the standard medium. Assay of subcellular fractions indicate the oxidase to be a peroxisomal enzyme. The fatty acyl-CoA dehydrogenase was shown to be present in the mitochondria. (beta)-oxidation also occurs in the peroxisome.;The (beta)-oxidation system for fatty acids and the novel fatty acyl-CoA oxidase activity of rat liver peroxisomes were characterized in terms of assay requirements and of chain length specificity of substrate. The rat liver oxidase is a FAD-dependent enzyme, has low affinity for acyl-CoA, high affinity for oxygen, and it also is a membrane-bound enzyme. The oxidase, as well as the peroxisomal (beta)-oxidation system showed a specificity for medium to long-chain length acyl-CoA's (C(,10)-C(,16)) as substrates, with a well defined peak of activity at C(,12). Their specific activities were similar and, in each case, oleoyl-CoA was a better substrate than stearoyl-CoA. The mitochondrial fatty acyl-CoA dehydrogenase activity showed a broader chain length specificity (C(,4)-C(,16)), with the shorter-chain length substrates being more active and no preference being shown for oleoyl-CoA over stearoyl-CoA. Using palmitoyl-CoA as substrate, the total enzymatic activity for the first step of (beta)-oxidation in the mitochondrion was 7.7-fold greater than for the corresponding step in the peroxisome (11% of the total oxidation by peroxisomes).;A hypothesis is presented which postulates that the energy of peroxisomal oxidations (respiration) is conserved (or used by the cell) by the establishment of a thermal-gradient in such a way that it can do several different kinds of work: (1) heating of the cell and/or organism (thermogenesis), (2) the movement of metabolites and organized structures within the cytosol (cytoplasmic streaming), and the mechanical work of conformational changes in the peroxisomal membrane or intrinsic proteins.
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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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Biochemistry
