Enzymological studies of mitochondrial fatty acid beta-oxidation.
Item
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Title
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Enzymological studies of mitochondrial fatty acid beta-oxidation.
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Identifier
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AAI9720154
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identifier
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9720154
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Creator
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Yao, Kuo-Wei.
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Contributor
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Adviser: Horst Schulz
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Date
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1997
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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 | Biology, Molecular
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Abstract
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Mitochondrial enzymes of fatty acid {dollar}\beta{dollar}-oxidation have been studied with the aim of elucidating the regulation of this process in both liver and heart tissues and to develop specific assay methods for diagnostic purposes. Three projects are described. The first one describes a specific assay for the diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) which is absent in patients with MCAD deficiency. This assay measures spectrophotometrically at 308 nm the formation of cinnamoyl-CoA from 3-phenylpropionyl-CoA in the presence of phenazine methosulfate as an electron acceptor. Since absorbance changes at 308 nm caused by other reactions are less than 5% of the absorbance change due to cinnamoyl-CoA formation catalyzed by medium-chain acyl-CoA dehydrogenase, the assay can be used to measure the activity of this enzyme in crude tissue homogenates.;The second project addresses the relationship between mitochondrial activation and toxicity of some substituted carboxylic acids. The activation of 4-bromocrotonic acid, 4-bromo-2-octenoic acid, valproic acid, and 3-methylglycidic acid by conversion to their CoA thioesters and the effects of these carboxylic acids on palmitoylcarnitine-supported respiration were studied with rat liver and rat heart mitochondria. The results showed that the substituted-carboxylic acids that were activated also inhibited palmitoylcarnitine-supported respiration. This leads to the conclusion that substituted medium-chain carboxylic acids, which enter the mitochondria directly, may inhibit {dollar}\beta{dollar}-oxidation as long as they are activated. Liver is more susceptible to inhibition due to the broader substrate specificity of its mitochondrial medium-chain acyl-CoA synthetase.;The last project is a kinetic study of the recently discovered membrane-bound trifunctional {dollar}\beta{dollar}-oxidation complex enzyme having enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase in one complex. The degradation of 20 {dollar}\mu{dollar}M 2-trans-hexadecenoyl-CoA to myristoyl-CoA and acetyl-CoA catalyzed by the {dollar}\beta{dollar}-oxidation complex was kinetically analyzed. Quantification of intermediates by HPLC revealed that the steady-state concentrations of 3-hydroxyhexadecanoyl-CoA and 3-ketohexadecanoyl-CoA were lower than their calculated concentrations necessary to sustain the observed rate of the overall reaction. The observed lower-than-expected concentrations of intermediates are indicative of a channeling mechanism which would explain the virtual absence of {dollar}\beta{dollar}-oxidation intermediates from the mitochondrial matrix.
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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.
