ISOCITRATE DEHYDROGENASE AND ITS RELATIONSHIP TO GLUCONEOGENESIS IN TETRAHYMENA.
Item
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Title
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ISOCITRATE DEHYDROGENASE AND ITS RELATIONSHIP TO GLUCONEOGENESIS IN TETRAHYMENA.
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Identifier
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AAI8203304
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identifier
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8203304
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Creator
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MAY, LESTER THEODORE.
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Contributor
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Prof. James F. Hogg
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Date
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1981
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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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It is reported in this thesis that Tetrahymena pyriformis does have an NAD-linked isocitrate dehydrogenase. This enzyme was assayed in both crude particle and mitochondrial preparations. However, all of its activity was lost upon solubilizing mitochondria in detergents or disruption with sonication. Since solubilization of this enzyme was unsuccessful all kinetic constants reported here were derived from mitochondria that had been separated by isopycnic density centrifugation in a sucrose gradient. Storage of these mitochondria in 50% aqueous-glycerol (v/v) at -20(DEGREES)C showed no loss of activity of the NAD-linked isocitrate dehydrogenase for up to 3 weeks. The NAD-linked isocitrate dehydrogenase from these mitochondria was stimulated by adenine nucleotides (AMP, ADP and ATP), with ADP having the greatest effect. ADP lowered the apparent K(,m) for NAD (from 225 to 45 micromolar) and increased the V(,max) with respect to threo-D(,s)-isocitrate. This ADP stimulation was found to be pH dependent, and was optimal at pH 7.0. This NAD-isocitrate dehydrogenase was inhibited by NADH, but not NADPH.;Homogenates or buffered extracts of freeze-dried cells do show a significant amount of NAD-isocitrate dehydrogenase activity. However, this activity was observed only at high NAD concentrations (greater than 1mM). Attempts to separate this enzyme from a coextracted NADP-isocitrate dehydrogenase activity showed that both coenzyme activities eluted together off Sephadex G-200, DEAE-Sephacel and Blue Dextran-agarose (Affi-Gel Blue) columns. Also, both activities had identical electrophoretic mobilities on a polyacrylamide gel and had equivalent molecular weights (90,000) as determined by gel chromatography. Finally the severe inhibition by NADPH, but not NADH on both coenzyme activities supports the conclusion that this NAD-activity from freeze-dried cells is a non-specific activity of the NADP-linked isocitrate dehydrogenase. The reductive carboxylation of (alpha)-ketoglutarate could be catalyzed by this enzyme with NADH. The physiological importance of this reaction, in regards to gluconeogenesis through the glyoxylate cycle is discussed.;An attempt to physiologically link a decrease in the specific activity of the NAD-linked isocitrate dehydrogenase in T. pyriformis with increasing gluconeogenesis through the glyoxylate cycle is made. Results of these studies do not definitively confirm nor refute this, but suggest a novel set of controls for the activation of gluconeogenesis from fats in T. pyriformis. Biochemical and electron microscopic evidence shows a distinct reorganization of the cytoplasm while T. pyriformis develop the capacity to synthesize glycogen from exogenous acetate or endogenous phospholipids. Specifically enzymes required for gluconeogenesis from fats become sequestered in a fraction that is rich in autophagic vacuoles. Evidence is presented that implicates these autophagic vacuoles in the synthesis of glucose and/or glycogen from fats.
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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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Biochemistry
