PROTEIN GLYCONEOGENESIS IN TETRAHYMENA PYRIFORMIS.
Item
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Title
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PROTEIN GLYCONEOGENESIS IN TETRAHYMENA PYRIFORMIS.
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Identifier
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AAI8112765
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identifier
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8112765
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Creator
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WAJNBERG, EWA F.
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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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Well aerated shallow cultures of Tetrahymena pyriformis, strain GL, grown in proteose peptone/liver extract medium (PPLE) and in the exponential phase of growth, responded to heat shock treatment by the synthesis of additional glycogen.;When washed cell suspensions obtained from such cultures were shaken in the air, the glycogen content of the cells increased significantly during the incubation time (4 hours). With casein hydrolysate added to the cells, the extra glycogen synthesized by the cells was on average about three-fold higher than the endogenous increase. Under these experimental conditions, acetate had no effect on glycogen production.;A series of amino acid substrates was subsequently tested for glyconeogenesis. Among amino acids tested the best stimulant of glyconeogenesis was L-proline, but L-threonine, L-asparagine and L-leucine were also effective. L-methionine caused strong inhibition of the endogenous glyconeogenesis.;In an attempt to overcome a permeability barrier some dipeptides were tested for glyconeogenesis. Proline dipeptides with N-terminal proline were equal to or better than the C-terminal proline dipeptides. Prolyl-valine and valyl-proline both stimulated glyconeogenesis efficiently with prolyl-valine being a slightly better substrate. Prolyl-phenylalanine stimulated glycogen synthesis efficiently. In contrast phenylalanyl-proline was a poor substrate for glyconeogenesis. The efficient dipeptide substrates (pro-val, val-pro, pro-phe) stimulated glyconeogenesis better than the mixtures of the corresponding amino acids. The use of dipeptides caused increased utilization of L-valine and L-phenylalanine by the protozoan cells and thus these two amino acids must be added to the list of the efficient substrates.;Prolyl-methionine added to the cell suspension exhibited inhibitory action that overcame the stimulatory effect of proline. However, when a mixture of proline and methionine was used, the inhibitory effect on glyconeogenesis of methionine disappeared.;Among lipid substrates tested, Tween 40 stimulated glyconeogenesis significantly. Tween 80 and lecithin had no effect on the glycogen content of the cells.;The stimulation of glyconeogenesis by L-proline is accompanied by a significant increase of NH(,3) production. However, careful quantitative experiments proved that the increase cannot be accounted for by L-proline disappearance only. The incorporation of label from {lcub}U-('14)C{rcub}-L-proline into the glycogen fraction was much lower than the value predicted from the balance study.;The conversion of proline to glutamate was confirmed by the chromatography of the protein hydrolysate of washed cells incubated for 4 hours with radioactive proline. In the hydrolysates, labelled glutamate and aspartate could be detected, that summed up to about 50% of the total radioactivity present.;The alcohol extract of the cells contained glutamate, aspartate and asparagine, that accounted for 4.5% of total radioactivity available to the cells. Radioactive glutamine was not detected inside the cells. The incubation medium contained glutamine, asparagine, aspartate and glutamate, that accounted for 17.9% of the total radioactivity available. Thus during proline degradation amino acids accumulate and are excreted to the incubation medium. A possible mechanism for the stimulatory action of proline is suggested.;The stimulation of glyconeogenesis caused by leucine was accompanied by an increased ammonia production. The incorporation of label into the glycogen fraction {lcub}U-('14)C{rcub}-L-leucine was in agreement with the value predicted from the balance studies. Thus this ketogenic amino acid appears to be glucogenic in Tetrahymena. When washed cells were incubated with a mixture of L-proline and L-leucine, both the glycogen and ammonia increments were exactly additive.
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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
