Mitochondrial and cytosolic ATP/ADP ratios in isolated hepatocytes. A comparison of the digitonin method and the non-aqueous fractionation procedure.
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ABSTRACT:
SUBMITTER: Soboll S
PROVIDER: S-EPMC1162423 | biostudies-other | 1980 Dec
REPOSITORIES: biostudies-other
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Project description:The ratio of ATP content/ADP content in livers from unanaesthetized fed rat was 0.9 in the mitochondrial matrix and 6.9 in the cytosol; the values for starved (48 h) animals were 1.0 and 5.9 respectively. The mitochondrial ratios observed in unanaesthetized animals were higher than in haemoglobin-free-perfused liver and lower than in isolated hepatocytes. Possible reasons for these differences may be related to oxygen supply and/or other factors. Further, data from anaesthetized rats with the liver exposed are given: mitochondrial ATP/ADP ratios were decreased with pentobarbital, but less so with ketamine as narcotic agent.
| S-EPMC1163549 | biostudies-other
Project description:Adenosine triphosphate (ATP) is a central metabolite that plays fundamental roles as an energy transfer molecule, a phosphate donor, and a signaling molecule inside the cells. The phosphoryl group transfer potential of ATP provides a thermodynamic driving force for many metabolic reactions, and phosphorylation of both small metabolites and large proteins can serve as a regulatory modification. In the process of phosphoryl transfer from ATP, the diphosphate ADP is produced, and as a result, the ATP-to-ADP ratio is an important physiological control parameter. The ATP-to-ADP ratio is directly proportional to cellular energy charge and phosphorylation potential. Furthermore, several ATP-dependent enzymes and signaling proteins are regulated by ADP, and their activation profiles are a function of the ATP-to-ADP ratio. Finally, regeneration of ATP from ADP can serve as an important readout of energy metabolism and mitochondrial function. We, therefore, developed a genetically encoded fluorescent biosensor tuned to sense ATP-to-ADP ratios in the physiological range of healthy mammalian cells. Here, we present a protocol for using this biosensor to visualize energy status using live-cell fluorescence microscopy.
| S-EPMC4323350 | biostudies-literature
Project description:The effect of thyroid-hormone application on cytosolic and mitochondrial ATP/ADP ratio was investigated in rat liver in vivo and in the isolated perfused organ. In vivo the ATP/ADP ratio in livers from hypothyroid rats was 0.84 +/- 0.08 in the mitochondrial matrix and 5.6 +/- 0.9 in the cytosol, as was observed in euthyroid controls. In contrast, hyperthyroidism was followed by a significant decrease in the mitochondrial and by an increase in the cytosolic ATP/ADP ratio (to 0.34 +/- 0.06 and 11.3 +/- 2.8 respectively). In the perfused liver from hypothyroid animals, addition of L-3,3',5-tri-iodothyronine in the perfusate also provoked, within 2 h, a significant decrease in the mitochondrial ATP/ADP ratio, whereas the cytosolic ratio was unaffected. From these and previous data in the isolated perfused liver and in isolated mitochondria from hypothyroid and tri-iodothyronine-treated rats it is concluded that thyroid hormones increase mitochondrial respiration and ATP regeneration, which is associated with an acceleration of mitochondrial adenine nucleotide transport and significant alterations in the mitochondrial and cytosolic ATP/ADP ratios.
| S-EPMC1144819 | biostudies-other
Project description:The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.
| S-EPMC1153637 | biostudies-other
Project description:Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1 degrees C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.
| S-EPMC1161592 | biostudies-other
Project description:1. A modification of the digitonin method of Zuurendonk & Tager (1974) (Biochim. Biophys. Acta 333, 393-399) (i.e. the 'convaentional' method) was developed that allows the fractionation of isolated hepatocytes at -5 degrees C (i.e. 'low-temperature' method). 2. With respect to compartmentation of adenine nucleotides, glutamate and citrate, the two methods yielded very similar results. 3. In contrast, the mitochondrial amounts of aspartate and malate, as revealed by the low-temperature method, were about twice as high as those found by the conventional procedure. No change in the total cellular content occurred. 4. With n-butylmalonate and glisoxepid present in the conventional digitonin medium, significantly higher amounts of malate and aspartate respectively were found in the mitochondrial pellets. The results obtained by the low-temperature method, however, were not influenced by the these inhibitors. 5. It is concluded that under the conventional conditions of cell fractionation no appreciable redistribution of adenine nucleotides, glutamate and citrate occurs.
| S-EPMC1162557 | biostudies-other
Project description:Single rat hepatocytes microinjected with aequorin generate oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) when stimulated with agonists acting through the phosphoinositide signalling pathway. The duration of these transients has been shown to be characteristic of the stimulating agonist, so that transients of very different duration can be induced in the same individual hepatocyte by different agonists. In a previous study we have shown that ADP and ATP, which are believed to act through a single P2y-purinoceptor species, elicit very different [Ca2+]i responses in most of the hepatocytes. We have interpreted this as evidence for two Ca(2+)-mobilizing purinoceptors. The methylated derivative of ATP, adenosine 5'-[alpha beta-methylene]-triphosphate (pp[CH2]pA), is only a weak P2y-purinoceptor agonist. When 100 microM pp[CH2]pA was supplied to aequorin-injected hepatocytes, there was no effect on [Ca2+]i. However, 25 microM pp[CH2]pA co-supplied with ATP causes a potentiation of the [Ca2+]i response in most of the hepatocytes. The effect was specific for ATP-induced transients; [Ca2+]i transients induced by other agonists, and importantly by ADP, were not affected by addition of pp[CH2]pA. This further illustrates differences in the actions of ADP and ATP, strengthening the argument for separate receptors for these nucleotides.
| S-EPMC1134431 | biostudies-other
Project description:Compartmentation of metabolism in developing seeds is poorly understood due to the lack of data on metabolite distributions at the subcellular level. In this report, a non-aqueous fractionation method is described that allows subcellular concentrations of metabolites in developing barley endosperm to be calculated. (i) Analysis of subcellular volumes in developing endosperm using micrographs shows that plastids and cytosol occupy 50.5% and 49.9% of the total cell volume, respectively, while vacuoles and mitochondria can be neglected. (ii) By using non-aqueous fractionation, subcellular distribution between the cytosol and plastid of the levels of metabolites involved in sucrose degradation, starch synthesis, and respiration were determined. With the exception of ADP and AMP which were mainly located in the plastid, most other metabolites of carbon and energy metabolism were mainly located outside the plastid in the cytosolic compartment. (iii) In developing barley endosperm, the ultimate precursor of starch, ADPglucose (ADPGlc), was mainly located in the cytosol (80-90%), which was opposite to the situation in growing potato tubers where ADPGlc was almost exclusively located in the plastid (98%). This reflects the different subcellular distribution of ADPGlc pyrophosphorylase (AGPase) in these tissues. (iv) Cytosolic concentrations of ADPGlc were found to be close to the published K(m) values of AGPase and the ADPGlc/ADP transporter at the plastid envelope. Also the concentrations of the reaction partners glucose-1-phosphate, ATP, and inorganic pyrophosphate were close to the respective K(m) values of AGPase. (v) Knock-out of cytosolic AGPase in Riso16 mutants led to a strong decrease in ADPGlc level, in both the cytosol and plastid, whereas knock-down of the ADPGlc/ADP transporter led to a large shift in the intracellular distribution of ADPGlc. (v) The thermodynamic structure of the pathway of sucrose to starch was determined by calculating the mass-action ratios of all the steps in the pathway. The data show that AGPase is close to equilibrium, in both the cytosol and plastid, whereas the ADPGlc/ADP transporter is strongly displaced from equilibrium in vivo. This is in contrast to most other tissues, including leaves and potato tubers. (vi) Results indicate transport rather than synthesis of ADPGlc to be the major regulatory site of starch synthesis in barley endosperm. The reversibility of AGPase in the plastid has important implications for the regulation of carbon partitioning between different biosynthetic pathways.
| S-EPMC3295393 | biostudies-literature
Project description:Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes carboxylation of ribulose-1,5-bisphosphate, the first in a series of reactions leading to the incorporation of atmospheric CO₂ into biomass. Rubisco requires Rubisco activase (RCA), an AAA+ ATPase that reactivates Rubisco by remodelling the conformation of inhibitor-bound sites. RCA is regulated by the ratio of ADP:ATP, with the precise response potentiated by redox regulation of the alpha-isoform. Measuring the effects of ADP on the activation of Rubisco by RCA using the well-established photometric assay is problematic because of the adenine nucleotide requirement of 3-phosphoglycerate (3-PGA) kinase. Described here is a novel assay for measuring RCA activity in the presence of variable ratios of ADP:ATP. The assay couples the formation of 3-PGA from ribulose 1,5-bisphosphate and CO₂ to NADH oxidation through cofactor-dependent phosphoglycerate mutase, enolase, PEP carboxylase and malate dehydrogenase. The assay was used to determine the effects of Rubisco and RCA concentration and ADP:ATP ratio on RCA activity, and to measure the activation of a modified Rubisco by RCA. Variations of the basic assay were used to measure the activation state of Rubisco in leaf extracts and the activity of purified Rubisco. The assay can be automated for high-throughput processing by conducting the reactions in two stages.
| S-EPMC3923112 | biostudies-literature
Project description:Single aequorin-injected hepatocytes respond to agonists acting via the phosphoinositide signalling pathway by the generation of oscillations in cytosolic free Ca2+ concentration ([Ca2+]free). The duration of [Ca2+]free transients is characteristic of the stimulating agonist. We have previously reported that ADP and ATP, which are believed to act through a single P(2y)-purinoceptor species, induce very different oscillatory [Ca2+]free responses in the majority of hepatocytes. We have interpreted these data as evidence for two separate Ca(2+)-mobilizing purinoceptors for these nucleotides. We show here that the elevation of intracellular cyclic AMP concentration, by the co-application of either dibutyryl cyclic AMP or 7 beta-desacetyl-7 beta-[gamma-(N-methylpiperazino)butyryl]- forskolin (L858051), exerts different modulatory effects on [Ca2+]free oscillations induced by ADP and ATP in single rat hepatocytes. Elevated intracellular cyclic AMP levels enhance the frequency and peak [Ca2+]free of transients induced by ADP. In contrast, the elevation of intracellular cyclic AMP levels in hepatocytes producing [Ca2+]free oscillations in response to ATP stimulates either an increase in the duration of transients or a sustained rise in [Ca2+]free. The data illustrate a further difference between the oscillatory [Ca2+]free responses of hepatocytes to ADP and ATP, thus further arguing against ADP and ATP acting via a single purinoceptor species.
| S-EPMC1137322 | biostudies-other