STUDIES ON THE CHARACTERIZATION OF DELTA-AMINOLEVULINIC ACID SYNTHASE AND AN ASSOCIATED AMIDASE.
Item
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Title
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STUDIES ON THE CHARACTERIZATION OF DELTA-AMINOLEVULINIC ACID SYNTHASE AND AN ASSOCIATED AMIDASE.
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Identifier
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AAI8222960
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identifier
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8222960
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Creator
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LIEN, LI-FEN ELIZABATH.
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Contributor
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Diana S. Beattie
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Date
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1982
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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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Biology, General
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Abstract
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The colorimetric assay and radiochemical assay for determining the activity of (delta)-aminolevulinic acid synthase (ALAS, E.C.4.2.1.24) were compared. A rapid and sensitive radioactive method for the assay of soluble ALAS has been developed based on the single column procedure of Ebert et al. It was concluded that the major contaminant is the cysteamine thioester of succinate. This contaminant was shown to be formed by the enzymatic cleavage of the amide bond between (beta)-mercaptoethylamine and pantothenic acid of succinyl CoA by an amidase.;The product of the amidase is optimally formed in the absence of glycine and is isolated by stopping the reaction with acidic SDS. Optimal conditions for the measurement of this amidase activity are described.;The submitochondrial localization was compared for the (delta)-aminolevulinic acid synthetase (ALAS) and this amidase. It was found that the amidase is localized on the outer surface of the inner membrane while ALAS is localized in the matrix.;The protease inhibitors, PMSF, TLCK or TPCK were without effect on the amidase, however, it was inhibited by o-phenantheoline and the inhibition was reversed by addition of either Co('2+) or Mn('2+) suggesting a metal requirement at the active site of the amidase.;Optimal conditions were established to assay both liver ALAS and skeletal muscle ALAS by using the improved radiochemical procedure. The enzymatic activities of both enzyme were investigated under conditions known to affect the heme biosynthetic pathway. Compared with controls, streptozotocin induced diabetic rats had a 56% decrease in ALAS activity in skeletal muscle mitochondria, while no change was found in ALAS activity in liver mitochondria. Administration of insulin to the diabetic animals partially reversed the effect of diabetes on skeletal muscle ALAS; however, administration of insulin to control animals caused a 21% decrease in skeletal muscle ALAS activity. By contrast, treatment with inducers of hepatic ALAS such as allylisopropyacetamide or 3,5-dicarbethoxy-1,4-dihydrocollidine had no effect on skeletal muscle ALAS. These results confirmed that ALAS activity is regulated in a tissue-specific manner.
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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.
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Program
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Biomedical Sciences
