Heme biosynthesis in Escherichia coli: Regulation of 5-aminolevulinic acid synthesis and the purification and characterization of 5-ALA dehydratase.
Item
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Title
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Heme biosynthesis in Escherichia coli: Regulation of 5-aminolevulinic acid synthesis and the purification and characterization of 5-ALA dehydratase.
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Identifier
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AAI9130296
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identifier
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9130296
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Creator
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Brathwaite, Ormond Dennis.
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Contributor
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Adviser: Charlotte S. Russell
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Date
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1991
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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 hemA mutant of E. coli, containing a multi-copy plasmid which complemented the mutation, excreted ALA into medium and accumulated uroporphyrinogen III when grown under aerobic or anaerobic conditions. The synthesis of ALA by cell-free extracts was dependent on glutamate, ATP, NADPH, glu-tRNA{dollar}\sp{lcub}\rm glu{rcub}{dollar} and PLP. Glu-tRNA{dollar}\sp{lcub}\rm glu{rcub}{dollar} stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase lowered this stimulation. Superaerobic growth inhibited ALA excretion and the cell-free extract failed to synthesize ALA from glutamate and ATP. These results confirmed that E. coli synthesizes ALA from glutamate.;E. coli RP523, a 5-aminolevulinic acid dehydratase (ALA D) mutant accumulated ALA under aerobic and anaerobic growth conditions. Less ALA accumulated in the medium when glucose or hemin was added to the medium. The glucose effect was reversed in strain C600 by cAMP. RP523 had more hemA specific mRNA when RP523 was grown anaerobically than aerobically or superaerobically. Strain JL1268 synthesized hemA mRNA under all growth conditions. The glutamyl tRNA synthetase (gltX), mRNA was highest in RP523 grown anaerobically; JL1268 grown aerobically and anaerobically. These results and an analysis of the upstream region of the hemA gene suggest that the glucose effect is positively mediated by cAMP-CRP binding to a promoter region of hemA. In vivo high expression of the hemA gene product may stimulate gltX gene expression.;E. coli ALA D was purified 1000-fold by a four step procedure including ammonium sulfate precipitation, ion exchange-, hydrophobic- and affinity-chromatography. The native molecular weight of 275,000 daltons and the subunit molecular weight was 39,000 daltons were determined. This data suggest that the E. coli ALA D is heptameric.;ALA D was activated by divalent cations and thiol reagents. The K{dollar}\sb{lcub}\rm m{rcub}{dollar} for ALA was 0.82 {dollar}\pm{dollar} 0.06 mM. ALA D was inhibited by lead, sulfhydryl-directed reagents, metal ion chelators, porphyrins, porphyrinogens and hemin. Succinyl-acetone, succinlyacetone-amino-levulinic acid pyrrole and levulinic acid were competitive inhibitors with K{dollar}\sb{lcub}\rm i{rcub}{dollar} of 1.38, 0.04 and 16.34 mM respectively.;The spectra of hemin dispersed in Tween-80 and dextran suggest that hemin is monomeric and polymeric respectively. The spectra of a solution of hemin arginate suggest that it is monomeric. ALA D was not inhibited by hemin arginate.
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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.
