In vivo evidence for the essential function of 2,4-dienoyl-CoA reductase in the beta-oxidation of unsaturated fatty acids.
Item
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Title
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In vivo evidence for the essential function of 2,4-dienoyl-CoA reductase in the beta-oxidation of unsaturated fatty acids.
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Identifier
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AAI9000743
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identifier
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9000743
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Creator
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You Yi, Seh-Yoon.
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Contributor
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Adviser: Horst Schulz
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Date
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1989
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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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E. coli mutants able to grow on oleate (9-cis-octadecenoic acid) but unable to utilize petroselinic acid (6-cis-octadecenoic acid) were isolated for the purpose of demonstrating in vivo the essential function of 2,4-dienoyl-CoA reductase (EC 1.3.1.34) in the {dollar}\beta{dollar}-oxidation of unsaturated fatty acids. One mutant (fadH) exhibited 12% of the 2,4-dienoyl-CoA reductase activity present in the parental strain with other {dollar}\beta{dollar}-oxidation enzymes being essentially unaffected.;Anti-reductase antibodies were used to prove the presence of a fadH gene product at a level similar to that observed in the parental strain. Thus, the mutation seems to have resulted in the synthesis of a fadH gene product with lower specific activity.;The mutation was mapped in the 71 to 75 min region of the E. coli chromosome where no other gene for {dollar}\beta{dollar}-oxidation enzymes has so far been located. A transconjugant of the mutant complemented with F{dollar}\sp\prime{dollar}141 which carries the 67 to 75.5 min region of the E. coli genome, exhibits 80% of the 2,4-dienoyl-CoA reductase activity of parental Pet{dollar}\sp{lcub}+{rcub}{dollar} strain. Measurements of respiration rates of the mutant, the transconjugant and parental Pet {dollar}\sp{lcub}+{rcub}{dollar} strain on petroselinic acid as the sole carbon source demonstrated a linear relationship between rates of respiration and levels of 2,4-dienoyl-CoA reductase activity.;Although the mutant was unable to grow on petroselinic acid, it grew on oleate at a rate indistinguishable from that of its parental strain. 2,4-Dienoyl-CoA reductase, like other enzymes of {dollar}\beta{dollar}-oxidation, was induced when E. coli was grown on oleate as the sole carbon source. Glucose repressed its synthesis while cAMP partially relieved the repression. It is concluded that 2,4-dienoyl-CoA reductase is required in vivo for the {dollar}\beta{dollar}-oxidation of unsaturated fatty acids with double bonds extending from even-numbered carbon atoms.;Additionally, the gene for 2,4-dienoyl-CoA reductase was isolated by complementation of the mutant with a genomic library of E. coli for the purpose of sequencing the gene and purifying 2,4-dienoyl-CoA reductase.
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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.
