Energy requirement for the mobilization of vacuolar arginine in Neurospora crassa

The bulk of the intracellular arginine pool in exponentially growing mycelia of Neurospora crassa is sequestered in the vacuoles. Vacuolar arginine effluxes from the vacuoles into the cytosol and is catabolized to ornithine and urea upon nitrogen starvation. The energy requirement for mobilization has been studied by treating nitrogen-starved mycelia with inhibitors or respiration or glycolysis or an uncoupler of respiration. Mobilization was inhibited by the inhibitors or the uncoupler of respiration, but not by the inhibitors of glycolysis. The inhibitors and the uncoupler of respiration reduced the ATP pool and the energy charge of the treated mycelia. The inhibitors of glycolysis reduced the ATP pool but had no effect on the energy charge. The results indicate that mobilization of arginine from the vacuoles requires metabolic energy. The forms of this energy and the mode of its association with the mobilization process are discussed.     

Sphingosine 1-phosphate promotes antigen processing and presentation to CD4+ T cells in Mycobacterium tuberculosis-infected monocytes

It was previously shown that cells die with increased cytosolic ATP after stimulation with apoptotic inducers including staurosporine (STS). To identify the source of apoptotic ATP elevation, we monitored, in real time, the cytosolic ATP level in luciferase-expressing HeLa cells. A mitochondrial uncoupler or a respiration chain inhibitor was found to decrease cytosolic ATP by about 50%. However, even when mitochondrial ATP synthesis was suppressed, STS induced a profound elevation of intracellular ATP. In contrast, the STS-induced ATP increase was prevented by any of three inhibitors of the glycolytic pathway: 2-deoxyglucose, iodoacetamide, and NaF. The STS effect strongly depended on intracellular calcium and was mimicked by a calcium ionophore. We conclude that Ca(2+)-dependent activation of anaerobic glycolysis, but not aerobic mitochondrial oxidative phosphorylation, is responsible for the STS-induced elevation of ATP in apoptotic HeLa cells.     

The metabolic requirements for transcellular movement and secretion of collagen.

Cultures of chick tendon fibroblasts were capable of normal ATP production and protein synthetic activity even though the normally high rate of glycolysis was markedly reduced by substitution of pyruvate for glucose. Iodoacetate and 2-deoxyglucose reduced ATP levels and protein synthesis even in the presence of pyruvate. Under these conditions, both inhibitors were shown to have effects on the energy metabolism of cells which were apparently unrelated to an inhibition of glycolysis. Selective inhibition of either glycolysis, by incubation in glucose-free medium, or of oxidative phosphorylation, by incubation with an uncoupler, was shown to have little effect on cellular ATP levels or intracellular transport and secretion of collagen. However, inhibition of both glycolysis and oxidative phosphorylation resulted in decreased cellular ATP levels and an inhibition of collagen secretion. This effect was not due to a requirement for continued protein synthesis, since inhibition of protein synthesis with cycloheximide or puromycin had little effect on collagen secretion. The ATP requirement for intracellular transport and secretion is discussed in relation to the secretory pathway for collagen.     

Synaptosomal bioenergetics. The role of glycolysis, pyruvate oxidation and responses to hypoglycaemia

The bioenergetic interaction between glycolysis and oxidative phosphorylation in isolated nerve terminals (synaptosomes) from guinea-pig cerebral cortex is characterized. Essentially all synaptosomes contain functioning mitochondria. There is a tight coupling between glycolytic rate and respiration: uncoupler causes a tenfold increase in glycolysis and a sixfold increase in respiration. Synaptosomes contain little endogenous glycolytic substrate and glycolysis is dependent on external glucose. In glucose-free media, or following addition of iodoacetate, synaptosomes continue to respire and to maintain high ATP/ADP ratios. In contrast to glucose, the endogenous substrate can neither maintain high respiration in the presence of uncoupler nor generate ATP in the presence of cyanide. Pyruvate, but not succinate, is an excellent substrate for intact synaptosomes. The in-situ mitochondrial membrane potential (delta psi m) is highly dependent upon the availability of glycolytic or exogenous pyruvate; glucose deprivation causes a 20-mV depolarization, while added pyruvate causes a 6-mV hyperpolarization even in the presence of glucose. Inhibition of pyruvate dehydrogenase by arsenite or pyruvate transport by alpha-cyano-4-hydroxycinnamate has little effect on ATP/ADP ratios; however respiratory capacity is severely restricted. It is concluded that synaptosomes are valuable models for studying the control of mitochondrial substrate supply in situ.     

Soluble adenylyl cyclase regulates the cytosolic NADH/NAD+ redox state and the bioenergetic switch between glycolysis and oxidative phosphorylation

The evolutionarily conserved soluble adenylyl cyclase (sAC, ADCY10) mediates cAMP signaling exclusively in intracellular compartments. Because sAC activity is sensitive to local concentrations of ATP, bicarbonate, and free Ca²⁺, sAC is potentially an important metabolic sensor. Nonetheless, little is known about how sAC regulates energy metabolism in intact cells. In this study, we demonstrated that both pharmacological and genetic suppression of sAC resulted in increased lactate secretion and decreased pyruvate secretion in multiple cell lines and primary cultures of mouse hepatocytes and cholangiocytes. The increased extracellular lactate-to-pyruvate ratio upon sAC suppression reflected an increased cytosolic free [NADH]/[NAD⁺] ratio, which was corroborated by using the NADH/NAD⁺ redox biosensor Peredox-mCherry. Mechanistic studies in permeabilized HepG2 cells showed that sAC inhibition specifically suppressed complex I of the mitochondrial respiratory chain. A survey of cAMP effectors revealed that only selective inhibition of exchange protein activated by cAMP 1 (Epac1), but not protein kinase A (PKA) or Epac2, suppressed complex I-dependent respiration and significantly increased the cytosolic NADH/NAD⁺ redox state. Analysis of the ATP production rate and the adenylate energy charge showed that inhibiting sAC reciprocally affects ATP production by glycolysis and oxidative phosphorylation while maintaining cellular energy homeostasis. In conclusion, our study shows that, via the regulation of complex I-dependent mitochondrial respiration, sAC-Epac1 signaling regulates the cytosolic NADH/NAD⁺ redox state, and coordinates oxidative phosphorylation and glycolysis to maintain cellular energy homeostasis. As such, sAC is effectively a bioenergetic switch between aerobic glycolysis and oxidative phosphorylation at the post-translational level.     

Adenosine induction of rapid catabolism of adenine ribonucleotides and independent elevation of the ATP content in quiescent mouse fibroblasts

The influence of adenosine on the ribonucleotide metabolism in quiescent BALB/c 3T3 cells was studied. The cellular adenine ribonucleotides were labelled by pretreating the cells with [2-3H]-adenine. After addition of adenosine to the cell cultures, the amount and radioactivity of the cellular purine ribonucleotides and the radioactivity of the purine compounds in the medium were determined. It appeared that adenosine gave rise both to rapid catabolism of adenine ribonucleotides with inosine 5'-monophosphate (IMP) as an intermediate and to expansion of the cellular adenosine 5'-triphosphate (ATP) pool. The maximal rates and the apparent activation constants for the two processes have been determined. Experiments with varying concentrations of coformycin (an inhibitor of adenosine 5'-monophosphate [AMP] deaminase and adenosine deaminase) and of 5'-amino-5'-deoxyadenosine (an inhibitor of adenosine kinase), respectively, showed that each compound may almost completely inhibit the adenosine-induced catabolism. This effect can be obtained under conditions where there was little or no effect by the two inhibitors on the rate of expansion of the cellular ATP pool. These results may best be explained by assuming that the process of expansion of the ATP pool is independent of the induced catabolism of adenine ribonucleotides, even though both processes seem to depend on the phosphorylation of adenosine to AMP. The total increase in the pool size of ATP and of guanosine 5'-triphosphate (GTP), both caused by adenosine, seems not to have regulatory effect on adenine ribonucleotide catabolism.     

Study of the regulation of oxidation and CO2 assimilation in intact Nitrobacter winogradskyi cells.

1. Changes of the adenine nucleotides in resting and growing Nitrobacter winogradskyi cells were measured in connection with regulating processes during nitrite oxidation and endogenous respiration. 2. After the addition of nitrite to endogenously respiring cells the ATP pool increased strongly during the first 60 sec at the expense of the ADP pool. At this point the energy charge was approx. 0.55. After the first 90 sec the ATP pool dropped, oscillating, to a lower level. The CO2 assimilation began at this point. 3. Under a nitrogen atmosphere the AMP pool increased and the ATP pool decreased. With a value of approx. 0.17 the energy charge was extremely low. When oxygen was added the Nitrobacter cells began to oxidize stored NADH. The ATP pool increased in a few seconds whereas the AMP pool decreased. The P/O ratio of endogenously respiring cells equaled 0.6 under these conditions. 4. During the changeover from anaerobic to aerobic conditions and in the presence of nitrite the nitrite oxidation and CO2 assimilation, opposed to aerobic conditions, were inhibited at first after the nitrite addition. The changeover of the respiratory chain enzymes from a reduced to an oxidized charge and the ATP increase were delayed in comparison with experiments without nitrite. According to these findings the endogenous respiration must be almost nil while nitrite oxidizing cells are growing.     

Energies of Activation and Uncoupled Growth in Streptococcus faecalis and Zymomonas mobilis

Growing cultures of Streptococcus faecalis at temperatures above 30 C have activation energies for both rates of growth and glycolysis of 10.3 kcal mole(-1), and a constant growth yield; when growth takes place below this temperature, the growth yield decreases and the activation energy for growth increases to 21.1 kcal mole(-1), but the activation energy for glycolysis is unchanged. The adenosine triphosphate pool in the organisms behaves differently above and below 30 C, suggesting that the energetic coupling between anabolism and catabolism is less effective below 30 C. Washed suspensions of S. faecalis have repressed glycolytic activity and an activation energy for glycolysis of 15.6 kcal mole(-1) over the whole temperature range studied. Growing cultures of Zymomonas mobilis below 33 C have a constant growth yield of 8.3 g (dry weight) of organisms per mole of glucose degraded, and activation energies for both glycolysis and growth of 11.1 kcal mole(-1); above this temperature, the growth yield falls, the activation energy for growth changes to -6.9 kcal mole(-1), but the activation energy for glycolysis is unchanged, so that the coupling between anabolism and catabolism is less effective above 33 C. The findings support the view that energy turnover in these bacteria is not well regulated.     

Non-proton-motive-force-dependent sodium efflux from the ruminal bacterium Streptococcus bovis: bound versus free pools.

Growing cells of Streptococcus bovis JB1 had a sodium content of 1,125 nmol/mg of protein and, based on a ratio of cell volume to protein of 4.3 microliters/mg, the apparent intracellular sodium concentration was more than 240 mM. Much of this sodium could not be removed by water washing even if cells were boiled or treated with the pore-forming ionophore, gramicidin, but it could be exchanged for potassium. Stationary cultures had a 2.6-microliters volume per milligram of protein and a total sodium content of 410 mM. When stationary cultures were energized with glucose at pH 6 to 8, sodium (more than 200 mM) was expelled within 2 min, and it appeared that growing cells had a very small pool of free intracellular sodium. Sodium-proton antiport activity could not be demonstrated with a sodium pulse, and the protonophore SF6847, valinomycin, and the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD) had little effect on sodium efflux, even though these inhibitors greatly reduced the proton-motive force. SF6847, valinomycin, and DCCD had little effect on intracellular ATP, but iodoacetate, an inhibitor of glycolysis, decreased ATP as well as sodium efflux. Stationary cells from sodium-deficient medium expelled little sodium after glucose addition and had 35% more ATP than stationary cells which were grown in sodium medium and expelled sodium. An artificial electrochemical gradient of sodium was able to drive ATP synthesis in stationary cells, and this ATP formation was not sensitive to DCCD. These results indicated that bacteria could have a significant pool of bound sodium and that sodium expulsion from S. bovis was directly coupled to ATP hydrolysis.     

Characteristics and energy requirements of an alpha-aminoisobutyric acid transport system in Streptococcus lactis.

Galactose-grown cells of Streptococcus lactis ML3 acculated alpha-aminoisobutyric acid (AIB) by using energy derived from glycolysis and arginine catabolism. The transport system displayed low-affinity Michaelis-Menten saturation kinetics. Using galactose or arginine as energy sources, similar V max and K m values for AIB entry were obtained, but on prolonged incubation the intracellular steady-state concentration of AIB in cells metabolizing arginine was only 65 to 70% that attained by glycolyzing cells. Efflux of AIB FROM PRELOADED CElls was temperature dependent and exhibited the characteristics of a first-order reaction. The rate of AIB exit was accelerated two- to threefold in the presence of metabolizable energy sources. Metabolic inhibitors including p-chloromercuribenzoate, dinitrophenol, azide, arsentate, and N, N'-dicyclohexylcarbodiimide either prevented or greatly reduced AIB uptake. Fluoride, iodoacetate and N-ethylmaleimide abolished galactose-dependent, but not arginine-energized, AIB uptake. K+ and Rb+ reduced the steady-state intracellular AIB concentration by approximately 40%, and these cations also induced rapid efflux of solute from actively transporting cells. Equivalent concentrations (10 mM) of Na+, Li+, or NH4+ were much less inhibitory. The proton-conducting ionophores tetrachlorosalicylanilide and carbonylcyanide m-chlorophenlyhydrazone abolished uptake and induced AIB efflux even though glycolysis and arginine catabolism continued at 60 and 140%, respectively, of control rates. A proton motive force is most likely involved in the active transport of AIB, whereas data from efflux studies suggest that energy is coupled to AIB exit in cells of S. lactis ML3.     

Non-proton-motive-force-dependent sodium efflux from the ruminal bacterium Streptococcus bovis: bound versus free pools

Growing cells of Streptococcus bovis JB1 had a sodium content of 1,125 nmol/mg of protein and, based on a ratio of cell volume to protein of 4.3 microliters/mg, the apparent intracellular sodium concentration was more than 240 mM. Much of this sodium could not be removed by water washing even if cells were boiled or treated with the pore-forming ionophore, gramicidin, but it could be exchanged for potassium. Stationary cultures had a 2.6-microliters volume per milligram of protein and a total sodium content of 410 mM. When stationary cultures were energized with glucose at pH 6 to 8, sodium (more than 200 mM) was expelled within 2 min, and it appeared that growing cells had a very small pool of free intracellular sodium. Sodium-proton antiport activity could not be demonstrated with a sodium pulse, and the protonophore SF6847, valinomycin, and the H+-ATPase inhibitor dicyclohexylcarbodiimide (DCCD) had little effect on sodium efflux, even though these inhibitors greatly reduced the proton-motive force. SF6847, valinomycin, and DCCD had little effect on intracellular ATP, but iodoacetate, an inhibitor of glycolysis, decreased ATP as well as sodium efflux. Stationary cells from sodium-deficient medium expelled little sodium after glucose addition and had 35% more ATP than stationary cells which were grown in sodium medium and expelled sodium. An artificial electrochemical gradient of sodium was able to drive ATP synthesis in stationary cells, and this ATP formation was not sensitive to DCCD. These results indicated that bacteria could have a significant pool of bound sodium and that sodium expulsion from S. bovis was directly coupled to ATP hydrolysis.     

Compartmentation and control of arginine metabolism in Neurospora.

The fate of [14-C]arginine derived from the medium or from biosynthesis has been examined in Neurospora growing in arginine-supplemented medium. In both cases the label enters the cytosol, where it is used efficiently for both protein synthesis and catabolism before mixing with the majority of the endogenous [12C]arginine pool. Both metabolic processes appear to use the same cytosolic arginine pool. It is calculated that the nonorganellar cytoplasm contains approximately 20% of the intracellular arginine pool when the cells are growing in arginine-supplemented medium. The results suggest that compartmentation of arginine is a significant factor in controlling arginine metabolism in Neurospora. The significance of these results for studies of amino acid metabolism in other eukaryotic systems is discussed.     

Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.     

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.     

A critical appraisal of the association between energy charge and cell damage

The association between the energy charge and cellular damage caused by metabolic inhibitors was investigated in a cellular system of quiescent fibroblasts. The cell damage was assessed by the release of lactate dehydrogenase (LDH) which indicates a severe change of membrane integrity. Inhibition of glycolysis resulted in release of LDH when the energy charge decreased below 0.5 at an ATP level of 10% of the original level. If oxidative phosphorylation was inhibited, the energy charge decreased to 0.1-0.35 (dependent on the type of inhibitor) a long time before release of LDH, and no change occurred in the energy charge when release of LDH started. The ATP level was 0.5-2% of the original at this time. Even a decrease of the energy charge to 0.1 could be reversed to a normal level, and at the same time the morphological cellular changes were fully reversed and no release of LDH occurred. The conclusion is that no simple correlation between energy charge and cell survival exists. The different levels of ATP at which release of LDH started after inhibition of glycolysis and oxidative phosphorylation indicate a special role of glycolysis in maintaining the membrane function and integrity. This was emphasized by measuring the potassium loss of the cells which was much more marked after inhibition of glycolysis.     

Variations in the Adenylate Energy Charge During Phased Growth (Cell Cycle) of Candida utilis Under Energy Excess and Energy-Limiting Growth Conditions

The variations in the levels of adenine nucleotides during the phased growth (cell cycle) of the yeast Candida utilis growing under nitrogen, sulfate, or iron limitation with glycerol as carbon source have been determined. Synchronous cultures were obtained by the continuous phasing technique, and the results were compared with those of chemostat cultures growing at similar growth rates and under the same types of nutrient limitation. Whereas the chemostat experiments indicated only the average energy status of cultures growing at random, results from phased cultures showed that the adenylate energy charge, defined as (ATP + (1/2)ADP)/(ATP + ADP + AMP) (where ATP, ADP, and AMP signify adenosine 5'-triphosphate, -diphosphate, and -monophosphate, respectively), varied during the phased growth of the yeast. These variations were related to the stage of development of the cells and to the type of nutrient limitation. In every case the energy charge dropped to a low value during the first half of the phasing cycle (cell cycle). Whereas the energy charge was maintained at relatively high levels (ranging from 0.78 to 0.94), for sulfate- or nitrogen-limited cultures, it was very low when iron was the growth-limiting nutrient (0.44 to 0.78). In spite of the low energy charge, the yeast continued to grow under iron limitation. The main component of the adenylate pool of the iron-limited culture was ADP and not ATP as observed with other types of nutrient limitation. It is concluded that under iron limitation the growth of the organism is limited by energy and that under energy-limited growth the energy charge of a growing organism is maintained at low levels. The reason for maintaining a low energy charge in an energy-limited culture is discussed.     

Charges of nicotinamide adenine nucleotides and adenylate energy charge as regulatory parameters of the metabolism in Escherichia coli.

Methods for measurements of catabolic reduction charge (defined as NADH/(NADH+NAD+)) and anabolic reduction charge (defined as NADPH/(NADPH + NADP+)) are described using [14C]nicotinamide labeling of Escherichia coli cultures. Together with these parameters the adenylate energy charge (ATP + 1/2ADP)/(ATP + ADP + AMP) was measured using labeling with [2-3H]adenine. These three charges were found under different exponential growth conditions to have values independent of the growth conditions: catabolic reduction charge, 0.05; anabolic reduction charge, 0.45; and adenylate energy charge, 0.9. The charges were examined during interruption of growth primarily affecting catabolism, respiration, or anabolism, leading to changes of the charges. The changes of charges are evaluated as a possible regulation of the metabolic rates utilizing or producing the nucleotides by their respective charges.     

Adenine nucleotide degradation during energy depletion in human lymphoblasts. Adenosine accumulation and adenylate energy charge correlation.

Adenine nucleotide breakdown to nucleosides and purine bases was measured in cultures of human lymphoblastoid cells following: 1) the inhibition of oxidative phosphorylation in the absence of glucose or 2) the addition of 2-deoxyglucose. A mutant cell line, deficient in adenosine kinase, in the presence of an adenosine deaminase inhibitor was used to measure utilization of the two pathways of AMP catabolism involving initial action of either purine 5'-nucleotidase or AMP deaminase. In such a system the appearance of adenosine induced by the oxidative phosphorylation inhibitor, rotenone, implies that approximately 70% of AMP breakdown occurs via dephosphorylation. By the same method, deamination accounts for 82% of AMP breakdown when 2-deoxyglucose is added. The occurrence of AMP dephosphorylation is not correlated with elevated concentrations of substrate or with decreased concentrations of the inhibitors of 5'-nucleotidase, ATP and ADP. Dephosphorylation occurs if, and only if, the adenylate energy charge decreases to about 0.6 in these experiments. In cultures deprived of glucose and oxygen, adenine nucleotide degradation via dephosphorylation results in recovery of normal energy charge values.     

Substrate-dependent modulation of ATPase activity by Na+ and K+ in roots of Plantago species

The equal rates of water vapour absorption by both bi- and trinucleate pollen indicate that their widely-differing rates of respiration have an intrinsic, biochemical basis. This was investigated with various metabolic inhibitors that were previously introduced into dry pollen via anhydrous acetone. The uncoupler, carbonyl cyanide m-chlorophenyl hydrazone, inhibited the O₂ uptake of rapidly respiring pollen and stimulated that of slowly respiring types to similar absolute values, that probably reflect the rates of substrate transport across the mitochondrial membranes. The extent of inhibition of the O₂ uptake by oligomycin, dicyclohexyl carbodiimide, antimycin A, and salicyl hydroxamic acid, alone and in combinations, indicates that hardly any oxidative phosphorylation and anabolic activities occur in slowly respiring, binucleate pollen species, having low-developed mitochondria and high energy charge values. The presence of the alternative pathway was insignificant. In other binucleate pollen species, characterized by recognizable mitochondria and low energy charge values, a limited ATP synthesis was established. The low energy charge values point to imbalance between phosphorylative and anabolic activities. In rapidly respiring, trinucleate pollen, containing well-developed mitochondria, a significant activity of the alternative oxidase was found. The energy charge values were high notwithstanding the large demand for ATP, mounting to 1.7 μmol h^?1 (mg pollen)^?1. In some pollen species, oligomycin highly stimulated the flow of electrons through the cytochrome pathway, which made an estimation of the ATP synthesis impossible.     

Tumor cell killing by macrophages activated in vitro with lymphocyte mediators. IV. Role of energy metabolism

The role of energy metabolism on tumor cell killing by in vitro activated macrophages was studied. Depletion of extracellular glucose had little effect on the cytotoxic capacity of mediator-activated macrophages. Respiratory antagonists did not inhibit cytotoxicity regardless of whether or not the assays were carried out in low-glucose-containing medium. Sodium fluoride, a known inhibitor of glycolysis, inhibited the killing of tumor cells by activated macrophages. 2-Deoxyglucose, an analog of glucose, was found to be an effective inhibitor of cytotoxicity. Three other analogs, 5-thio-d-glucose, 3-O-methylglucose, and 2-deoxy-d-galactose, were without effect. The concentrations of 2-DG that inhibited cytotoxicity did not lower cellular ATP levels to an appreciable extent. The combined addition of inhibitors of glycolysis and respiration resulted in a marked reduction in ATP levels. Under these experimental conditions, macrophage-mediated cytotoxicity was also significantly inhibited.