Study of 2,4-dienoyl-CoA reductase in unsaturated fatty acid beta-oxidation.
Item
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Title
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Study of 2,4-dienoyl-CoA reductase in unsaturated fatty acid beta-oxidation.
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Identifier
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AAI9224844
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identifier
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9224844
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Creator
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Nada, Mohamed Abou El-Yazeid.
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Contributor
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Adviser: Horst Schulz
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Date
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1992
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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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A disorder in the beta-oxidation of polyunsaturated fatty acids is described that appears to be caused by a deficiency of 2,4-dienoyl-CoA reductase. Further studies of this disorder, with small samples of human tissues or human fibroblasts, require a more sensitive assay of 2,4-dienoyl-CoA reductase than is currently used. A radioactive method for assaying 2,4-dienoyl-CoA reductase is described. This assay is at least 30-times more sensitive than the spectrophotometric assay, even though rates determined by the radioactive method are 10 times lower than rates obtained spectrophotometrically due to a primary kinetic isotope effect. The spectrophotometric assay of 2,4-dienoyl-CoA reductase was modified to improve the sensitivity and linearity of this method. A new substrate, 5-phenyl-2,4-pentadienoyl-CoA, was introduced which has an absorbance maximum at 340 nm with an extinction coefficient of 44,338 M{dollar}\sp{lcub}-1{rcub}{dollar}cm{dollar}\sp{lcub}-1{rcub}{dollar}. This assay is more linear and two times more sensitive than the currently used spectrophotometric assay.;Activities of carnitine palmitoyl transferase I and mitochondrial respiration rates were measured to determine whether the reaction catalyzed by CPT I is a rate-limiting step in the mitochondrial oxidation of long chain unsaturated fatty acids in heart. It is concluded from these studies that the CPT I catalyzed reaction may not be the rate-limiting step in beta-oxidation in heart mitochondria unless the intracellular concentration of malonyl-CoA is sufficiently high to cause a significant inhibition of CPT I activity.;The mitochondrial metabolism of 5-enoyl-CoAs, which are formed during the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms, was studied with mitochondrial extracts and purified enzymes of beta-oxidation. 5-cis-Octenoyl-CoA, a putative metabolite of linolenic acid, was efficiently dehydrogenated by medium-chain acyl-CoA dehydrogenase to 2-trans,5-cis-octadienoyl-CoA, which was isomerized to 3,5-octadienoyl-CoA either by mitochondrial delta{dollar}\sp3{dollar},delta{dollar}\sp2{dollar}-enoyl-CoA isomerase or by the peroxisomal trifunctional enzyme. Further isomerization of 3,5-octadienoyl-CoA to 2-trans-4-trans-octadienoyl-CoA in the presence of soluble extracts of either rat liver or rat heart mitochondria was observed and attributed to a novel delta{dollar}\sp{lcub}3,5{rcub}{dollar},delta{dollar}\sp{lcub}2,4{rcub}{dollar}-dienoyl-CoA isomerase. It is concluded that odd-numbered double bonds, like even-numbered double bonds, can be reductively removed during the beta-oxidation of polyunsaturated fatty acids.
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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.
