Study of the energy-dependent regulation of fatty acid oxidation in rat heart mitochondria.
Item
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Title
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Study of the energy-dependent regulation of fatty acid oxidation in rat heart mitochondria.
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Identifier
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AAI9959154
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identifier
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9959154
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Creator
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Abbas, Azfar Syed.
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Contributor
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Adviser: Horst Schulz
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Date
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2000
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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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The subcellular location of cardiac carnitine acetyltransferase (CAT) was investigated by measuring the release of CAT and of marker enzymes from isolated rat myocytes permeabilized with digitonin. Additionally, the CAT activity exposed to the cytosolic compartment was quantified. The results indicate that soluble CAT is not present in the cytosol and that only 5% of the cellular CAT activity is positioned to catalyze the formation of cytosolic acetyl coenzyme A. This situation makes it unlikely that the energy-linked regulation of cardiac fatty acid oxidation proceeds by mechanisms which require the conversion of acetylcarnitine to acetyl coenzyme A in the cytosol.;The phosphorylation of trifunctional beta-oxidation complex (TOC), a mitochondrial membrane-bound enzyme, was investigated to assess the possible role of such modification in the energy-linked regulation of fatty acid oxidation in heart. Phosphorylation was assayed with partially purified TOC, rat heart mitochondrial membrane vesicles (RHMM), and intact rat heart mitochondria (RHM). Results with partially purified TOC and RHMM in the presence and absence of cAMP-dependent protein kinase and at [acetyl-CoA]\[CoASH] ratios characteristic of high and low energy utilizations did not reveal any phosphorylation of TOC. In addition, different metabolic conditions were generated in intact RHM to determine the phosphorylation of TOC. All these observations led to the conclusion that TOC is not phosphorylated and therefore, can not be regulated by it.;Vesicles prepared from RHMM were used as a system to quickly and efficiently assess the channeling of long-chain intermediates on the membrane bound beta-oxidation system. When the membrane-bound oxidation system present in such vesicles was assayed with hexadecanoyl-CoA as a substrate, the accumulation of all possible intermediates was observed in the incubation mixture. Rates calculated based on the observed concentration of 2-hexadecenoyl-CoA and appropriate kinetic parameters, were higher than those observed with beta-oxidation system A. These observations are indicative of a non-channeling situation, therefore, vesicles are not a suitable system to study of intermediate channeling despite the fact that evidence for channeling of long-chain intermediates has been obtained in whole cell (27).
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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.
