Purification and characterization of three novel enzymes of mitochondrial fatty acid beta-oxidation.
Item
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Title
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Purification and characterization of three novel enzymes of mitochondrial fatty acid beta-oxidation.
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Identifier
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AAI9521294
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identifier
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9521294
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Creator
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Luo, Ming Jiang.
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Contributor
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Adviser: Horst Schulz
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Date
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1995
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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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The trifunctional beta-oxidation complex and its relationship to the known long-chain enoyl-CoA hydratase (EC 4.2.1.74) from pig heart mitochondria were investigated. Both partially purified long-chain hydratase and pure complex contained long-chain specific activities of enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase and 3-ketoacyl-CoA thiolase but were virtually inactive toward short-chain (C4) substrates. The molecular weights of the two subunits of the pig heart complex were estimated to be 81,000 and 45,000, respectively. It is concluded that long-chain enoyl-CoA hydratase (EC 4.2.1.74) is a component enzyme of the trifunctional beta-oxidation complex which is associated with the inner membrane of pig heart mitochondria.;Mitochondrial {dollar}\it{lcub}\Delta\sp{lcub}3,5{rcub},\Delta\sp{lcub}2,4{rcub}{rcub}{dollar}-dienoyl-CoA isomerase, which catalyzes the conversion of 3,5-octadienoyl-CoA to 2,4-octadienoyl-CoA, was purified from rat liver 370-fold at almost 30% yield by a six-step purification procedure. The molecular weights of the native enzyme and its subunit(s) were estimated to be 126,000 and 32,000, respectively. The purification of {dollar}\it{lcub}\Delta\sp{lcub}3,5{rcub},\Delta\sp{lcub}2,4{rcub}{rcub}{dollar}-dienoyl-CoA isomerase completes the characterization of the enzymes functioning in the NADPH-dependent pathway for the {dollar}\beta{dollar}-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms.;Short-chain L-3-hydroxy-2-methylacyl-CoA dehydrogenase (SC-HMAD), a soluble mitochondrial enzyme, was purified 6000-fold from rat liver in 6% yield by a six-step purification procedure. The subunit molecular weight of this enzyme was estimated to be 28,000. The enzyme seems to be a dimer composed of two identical subunits. SC-HMAD and L-3-hydroxyacyl-CoA dehydrogenase are immunologically unrelated proteins. The apparent Km values for L-3-hydroxy-2-methybutyryl-CoA and L-3-hydroxybutyryl-CoA are 5 {dollar}\mu{dollar}M and 19 {dollar}\mu{dollar}M, respectively. It is concluded that SC-HMAD catalyzes the second dehydrogenation step during the beta-oxidation of the isoleucine metabolite 2-methylbutyryl-CoA.
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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.
