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1.
The effects of adenine nucleotides on carbohydrate metabolism in pigeon-liver homogenates 总被引:33,自引:30,他引:3
1. The effects of added AMP on carbohydrate metabolism were investigated in pigeon-liver homogenates, which can degrade glucose and synthesize it from lactate. Suitable experimental conditions were established for studying such effects, including the addition of P(i) (20mm) to stabilize adenine nucleotides and supplementation with NAD(+) (0.5mm). 2. Lactate increased the rate of oxygen consumption and kept the concentration of ATP high and that of AMP relatively low. 3. Added AMP (1.25-5mm) raised the net rate of carbohydrate removal and inhibited the net formation of glucose from lactate, as well as the incorporation of lactate into glucose. These effects were accompanied by a fall in the concentrations of hexose 6-phosphates and a rise in those of fructose diphosphate and triose phosphates. When the activity of glyceraldehyde 3-phosphate dehydrogenase was limited experimentally by a low concentration of NAD(+) or when it was blocked by iodoacetate, the accumulations of fructose diphosphate and triose phosphates were large and accounted for most of the carbohydrate degraded in the presence of AMP. 4. AMP also inhibited the conversion of pyruvate into phosphoenolpyruvate. Data on the concentrations of pyruvate, phosphoenolpyruvate and intermediates of the tricarboxylic acid cycle, as well as on isotope distribution, suggest that the effect was due to inhibition of phosphoenolpyruvate carboxykinase. 5. The results indicate that in the homogenates phosphofructokinase and fructose diphosphatase, controlled in their activity by adenine nucleotides and other cell constituents, are enzymes which regulate the direction of carbohydrate metabolism (degradation or synthesis) in the liver. 6. It is suggested that active transport of adenine nucleotides, citrate, Mg(2+), Ca(2+), P(i) and other cell constituents may play a role in regulating the activity of enzymes which are affected by these substances. 7. A procedure is described for generating alkali in a closed manometer vessel, by mixing mercuric oxide and a solution of sodium iodide, for use in a method for measuring the oxygen consumption at physiological bicarbonate concentrations. 相似文献
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Mathias Ziegler Wolfgang Dubiel Alexander M. Pimenov Yuri V. Tikhonov Ruslan T. Toguzov Wolfgang Henke Gerhard Gerber 《Molecular and cellular biochemistry》1990,93(1):7-12
Summary The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37°C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold.Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [14C]-adenosine yielded no [14C]-inosine ruling out extramitochondrial adenosine deamination.It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested. 相似文献
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Efflux of adenine nucleotides from rat liver mitochondria 总被引:3,自引:0,他引:3
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Phosphorylation state of cytosolic and mitochondrial adenine nucleotides and of pyruvate dehydrogenase in isolated rat liver cells. 总被引:15,自引:13,他引:2
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1. Cytosolic and mitochondrial ATP and ADP concentrations of liver cells isolated from normal fed, starved and diabetic rats were determined. 2. The cytosolic ATP/ADP ratio was 6,9 and 10 in normal fed, starved and diabetic rats respectively. 3. The mitochondrial ATP/ADP ratio was 2 in normal and diabetic rats and 1.6 in starved rats. 4. Adenosine increased the cytosolic and lowered the mitochondrial ATP/ADP ratio, whereas atractyloside had the opposite effect. 5. Incubation of the hepatocytes with fructose, glycerol or sorbitol led to a fall in the ATP/ADP ratio in both the cytosolic and the mitochondrial compartment. 6. The interrelationship between the mitochondrial ATP/ADP ratio and the phosphorylation state of pyruvate dehydrogenase in intact cells was studied. 7. In hepatocytes isolated from fed rats an inverse correlation between the mitochondrial ATP/ADP ratio and the active form of pyruvate dehydrogenase (pyruvate dehydrogenase a) was demonstrable on loading with fructose, glycerol or sorbitol. 8. No such correlation was obtained with pyruvate or dihydroxyacetone. For pyruvate, this can be explained by inhibition of pyruvate dehydrogenase kinase. 9. Liver cells isolated from fed animals displayed pyruvate dehydrogenase a activity twice that found in vivo. Physiological values were obtained when the hepatocytes were incubated with albumin-oleate, which also yielded the highest mitochondrial ATP/ADP ratio. 相似文献
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Carboxyatractyloside-insensitive influx and efflux of adenine nucleotides in rat liver mitochondria 总被引:1,自引:0,他引:1
Unidirectional transport (influx and efflux) of adenine nucleotides in rat liver mitochondria was examined using carboxyatractyloside to inhibit rapid exchange of matrix and external adenine nucleotides via the adenine nucleotide translocase. Influx of adenine nucleotides was concentration-dependent. ATP was the preferred substrate with a Km of 2.67 mM and V of the preferred substrate with a Km of 2.67 mM and V of 8.33 nmol/min/mg of protein. For ADP, the Km was 14.7 mM and V was 10.8 nmol/min/mg of protein. Efflux of adenine nucleotides was also concentration-dependent, varying directly as a function of the matrix adenine nucleotide pool size. Any increase in the influx of adenine nucleotides was coupled to an increase in efflux. However, as the external ATP concentration was increased, influx was stimulated to a much greater extent than was efflux. This imbalance suggested that under certain conditions adenine nucleotide movement might be coupled to the movement of an alternate anion such as phosphate. Adenine nucleotide efflux increased as the external phosphate concentration was varied from 0.5 to 4 mM. Also, increasing the external phosphate concentration caused adenine nucleotide influx to decrease, suggesting competition. In the absence of external adenines and phosphate, no efflux occurred. Both adenine nucleotide influx and efflux were depressed if Mg2+ was omitted. Adenine nucleotide efflux in the presence of external phosphate was inhibited much less by lack of Mg2+ than was efflux in the presence of external ATP. This evidence supports a model in which either adenine nucleotides (probably with Mg2+) or phosphate can move across the mitochondrial membrane on a single carrier. Net adenine nucleotide movements can occur when adenine nucleotide movement is coupled to the movement of phosphate in the opposite direction. 相似文献
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A Gómez-Puyou M Tuena de Gómez-Puyou M Klapp E Carafoli 《Archives of biochemistry and biophysics》1979,194(2):399-404
The effect of Ca2+ on the adenine nucleotide translocase activity of intact rat liver mitochondria has been studied. The results indicate that in mitochondria which have been allowed to accumulate Ca2+, the activity of the translocase is strongly diminished; half-maximal inhibition is attained when approximately 40 nmol of Ca2+ are accumulated/mg of mitochondrial protein. Inhibition of electron transport or uncoupling prevents the Ca2+-induced inhibition of translocase activity; inhibition of Ca2+ uptake by ruthenium red also prevents the inhibition of the exchange. These experiments indicate that internal, but not external Ca2+ is responsible for the inhibition of adenine nucleotide translocase activity. Inhibition of the exchange activity by Ca2+ occurs even in conditions in which external adenine nucleotide concentrations are rate-limiting. 相似文献
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June R. Aprille 《Archives of biochemistry and biophysics》1981,207(1):157-164
In newborn rat liver, the adenine nucleotide content (ATP + ADP + AMP) of mitochondria increases severalfold within 2 to 3 h of birth. The net increase in mitochondrial adenines suggests a novel mechanism by which mitochondria are able to accumulate adenine nucleotides from the cytosol (J. R. Aprille and G. K. Asimakis, 1980, Arch. Biochem. Biophys.201, 564.). This was investigated further in vitro. Isolated newborn liver mitochondria incubated with 1 mM ATP for 10 min at 30 °C doubled their adenine nucleotide content with effects on respiratory functions similar to those observed in vivo: State 3 respiration and adenine translocase activity increased, but uncoupled respiration was unchanged. The mechanism for net uptake of adenine nucleotides was found to be specific for ATP or ADP, but not AMP. Uptake was concentration dependent and saturable. The apparent Km′s for ATP and ADP were 0.85 ± 0.27 mM and 0.41 ± 0.20 mM, respectively, measured by net uptake of [14C]ATP or [14C]ADP. The specific activities of net ATP and ADP uptake averaged 0.332 ± 0.062 and 0.103 ± 0.002 nmol/min/mg protein, respectively. ADP was a competitive inhibitor of net ATP uptake. If Pi was omitted from the incubations, net uptake of ATP or ADP was reduced by 51%. Either mersalyl or N-ethylmaleimide severely inhibited the accumulation of adenine nucleotides. Net ATP uptake was stoichiometrically dependent on MgCl2, suggesting that Mg2+ is accumulated along with ATP (or ADP). Uptake was energy dependent as indicated by the following results: Net AdN uptake (especially ADP uptake) was stimulated by the addition of an oxidizable substrate (glutamate) and inhibited by FCCP (an uncoupler). Antimycin A had no effect on net ATP uptake but inhibited net ADP uptake, suggesting that ATP was able to serve as an energy source for its own accumulation. If carboxyatractyloside was added to inhibit the exchange translocase, thereby preventing rapid access of exogenous ATP to the matrix, net ATP uptake was inhibited; carboxyatractyloside had no effect on ADP uptake. It was concluded that the net uptake of adenine nucleotides from the extramitochondrial space occurs by a specific transport process distinct from the classic adenine nucleotide exchange translocase. The accumulation of adenine nucleotides may regulate matrix reactions which are allosterically affected by adenines or which require adenines as a substrate. 相似文献
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Stimulation of glycogenolysis by adenine nucleotides in the perfused rat liver. 总被引:4,自引:3,他引:1
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Infusion of adenine nucleotides and adenosine into perfused rat livers resulted in stimulation of hepatic glycogenolysis, transient increases in the effluent perfusate [3-hydroxybutyrate]/[acetoacetate] ratio, and increased portal vein pressure. In livers perfused with buffer containing 50 microM-Ca2+, transient efflux of Ca2+ was seen on stimulation of the liver with adenine nucleotides or adenosine. ADP was the most potent of the nucleotides, stimulating glucose output at concentrations as low as 0.15 microM, with half-maximal stimulation at approx. 1 microM, and ATP was slightly less potent, half-maximal stimulation requiring 4 microM-ATP. AMP and adenosine were much less effective, doses giving half-maximal stimulation being 40 and 20 microM respectively. Non-hydrolysed ATP analogues were much less effective than ATP in promoting changes in hepatic metabolism. ITP, GTP and GDP caused similar changes in hepatic metabolism to ATP, but were 10-20 times less potent than ATP. In livers perfused at low (7 microM) Ca2+, infusion of phenylephrine before ATP desensitized hepatic responses to ATP. Repeated infusions of ATP in such low-Ca2+-perfused livers caused homologous desensitization of ATP responses, and also desensitized subsequent Ca2+-dependent responses to phenylephrine. A short infusion of Ca2+ (1.25 mM) after phenylephrine infusion restored subsequent responses to ATP, indicating that, during perfusion with buffer containing 7 microM-Ca2+, ATP and phenylephrine deplete the same pool of intracellular Ca2+, which can be rapidly replenished in the presence of extracellular Ca2+. Measurement of cyclic AMP in freeze-clamped liver tissue demonstrated that adenosine (150 microM) significantly increased hepatic cyclic AMP, whereas ATP (15 microM) was without effect. It is concluded that ATP and ADP stimulate hepatic glycogenolysis via P2-purinergic receptors, through a Ca2+-dependent mechanism similar to that in alpha-adrenergic stimulation of hepatic tissue. However, adenosine stimulates glycogenolysis via P1-purinoreceptors and/or uptake into the cell, at least partially through a mechanism involving increase in cyclic AMP. Further, the hepatic response to adenine nucleotides may be significant in regulating hepatic glucose output in physiological and pathophysiological states. 相似文献
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It is shown that in [14C]adenine-labelled thymocytes adenosine increases the content of adenine nucleotides and simultaneously accelerates their catabolism. Papaverine induces acceleration of splitting and a decrease of the specific ATP radioactivity but increases the AMP content and its specific radioactivity. The both effectors intensify considerably the outlet of total radioactive label from cells. If the papaverine effect in the extracellular medium results in accumulation mainly of hypoxanthine in the extracellular medium then the adenosine presence causes accumulation of inosine and hypoxanthine approximately in equal amounts. The release of labelled adenosine from thymocytes in all cases is an insignificant part of extracellular radioactivity. A conclusion is drawn that under conditions of the combined action of the substances under study papaverine removes the adenosine effect caused by its under study papaverine removes the adenosine effect caused by its phosphorylation with the formation of ATP and exerts the dose-depended action on adenine nucleotide metabolism in thymocytes. 相似文献
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1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes. 相似文献
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Evans EA 《The Biochemical journal》1940,34(6):829-837
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W J Vaartjes J N den Breejen M J Geelen S G van den Bergh 《Biochimica et biophysica acta》1980,592(1):28-37
1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, cannot account for the observed changes in the rate of pyruvate transport. 4. It is proposed that the pretreatment of the mitochondria directly modulates the activity of the mitochondrial pyruvate carrier. The possible regulatory role of such a modulation system is discussed. 相似文献
20.
H K Kuramitsu 《Biochemical and biophysical research communications》1966,23(3):329-334