An immediate and steep increase in ATP concentration in response to reduced turgor pressure in Escherichia coli B

Osmotic treatment with sodium chloride of Escherichia coli B growing in the logarithmic phase induced an immediate increase in ATP concentration in response to increased concentrations of added solute in its growth medium in the first 10 min of the addition. After that, ATP concentration decreased gradually. Sodium arsenate and potassium fluoride almost abolished the ATP increase. The time course of the increase was quite different from that of cells treated with inhibitors of protein synthesis. The osmotic treatment did not decrease the viability of cells. In addition, there was no degradation of RNA by 5 min after sodium chloride addition, and, further, the lag time of ATP increase was extended by an inhibitor of nucleotide synthesis. These results indicated that a major fraction of the stress-increased ATP resulted from de novo synthesis, and that it was mainly dependent upon the reaction of substrate-level phosphorylation, which is regulated by turgor pressure.     

Accumulation of guanosine tetraphosphate (ppGpp) under nitrogen starvation in Anacystis nidulans,a cyanobacterium

The effect of nitrate deprivation on cell growth and nucleotide level was studied in Anacystis nidulans. A 10-fold reduction in nitrate level resulted in a drastic slowdown of growth. Upon addition of nitrate to the starving cultures, after a lag period, the cells resumed growth.Nutritional shift-down induced a transitory expansion of the guanosine tetraphosphate (ppGpp) pool, preceeded by a transitory increase in GTP and ATP concentrations. After having reached peak values, the concentration of ppGpp, GTP and ATP dropped to the respective base levels. The expansion of the ppGpp pool was found to be due to an increase in ppGpp synthesis, rather than to a decrease in ppGpp breakdown. After nutritional shift-up, no decrease in the ppGpp level was found.In starving cells, a decrease in free amino acids was observed to occur concomitantly with the expansion of the ppGpp pool. The level of free amino acids started to increase simultaneously with the contraction of the ppGpp pool.     

Somatic Embryogenesis in Carrot Cell Suspension: I. PATTERN OF PROTEIN AND NUCLEIC ACID SYNTHESIS

The role of macromolecule synthesis during the early hours ofundifferentiated growth of carrot cell suspension in a mediumcontaining the synthetic auxin, 2,4-dichlorophenoxyacetic acidand during embryogenic growth in an auxin-free medium was investigated.For each set of cultures, total protein and RNA, rate of proteinsynthesis, changes in ATP pool, and rate of RNA synthesis weredetermined. Although no changes in protein and RNA contentsof cells were noted during the first 48 h of their growth infresh media, the rate of protein synthesis in the embryogeniccells markedly increased over that of the non-embryogenic cellsas early as 2 h. ATP pool size in both types of cells stabilizedwithin 1 h and it registered only a slight decrease over a 24h period. Rate of RNA synthesis increased in the embryogeniccells from 4 to 12 h following their transfer to fresh medium,after which it decreased. Transfer of carrot cells of zero embryogenicpotential to an auxin-free medium did not however elicit themacromolecule synthetic pattern characteristic of the highlyembryogenic cells.     

On the mechanism of xylitol-dependent inhibition of glycolysis in Streptococcus sobrinus OMZ 176.

1. The mechanism of xylitol-dependent inhibition of glycolysis in Streptococcus sobrinus OMZ 176 was investigated in aerobically and anaerobically grown cells. 2. Glucose-stimulated glycolysis was followed polarographically, by radio-HPLC-analyses of glycolytic intermediates, by measurement of ATP generated, and spectrophotometric monitoring of extent of NAD(P)+/NADPH-status. 3. Xylitol added to suspensions of S. sobrinus inhibited O2 uptake by approximately 20%, and led to a corresponding decrease in rate of lactate formation in aerobic and anaerobic cells. 4. Xylitol also delayed the onset of the glucose-dependent rapid reduction of NAD(P)+ by approximately 1 min, although the total extent of reduction was not significantly affected compared to control cells. 5. The inhibitory effect of xylitol on glucose dependent ATP synthesis, however, was decreased by 70-80%. 6. Hence the dramatic decrease in glucose-dependent synthesis of ATP may be the direct cause of decreased bacterial growth in the presence of xylitol. 7. A mechanism explaining the observed phenomena is proposed.     

The dependence of intracellular ATP level on the nutrition mode of the acidophilic bacteria Sulfobacillus thermotolerans and Alicyclobacillus tolerans

The dynamics of the ATP pool in the aerobic spore-forming acidothermophilic mixotrophic bacteria Sulfobacillus thermotolerans Kr1T and Alicyclobacillus tolerans K1T were studied in the course of their chemolithoheterotrophic, chemoorganoheterotrophic, and chemolithoautotrophic growth. It was established that, during mixotrophic growth, the maximum ATP concentrations in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 3.8 and 0.6 nmol/mg protein, respectively. The ATP concentrations in sulfobacilli and alicyclobacilli during organotrophic growth were 2.2 and 3.1 nmol/mg protein, respectively. In the cells of the obligately heterotrophic bacterium Alicyclobacillus cycloheptanicus 4006T, the maximum ATP concentration was several times higher and reached 12.3 nmol/mg protein. During lithotrophic growth, the maximum values of the ATP concentration in the cells of S. thermotolerans Kr1 and A. tolerans K1 were 0.3 and <0.1 nmol/mg protein, respectively; in the cells of the autotrophic bacterium Acidithiobacillus ferrooxidans TFBk, the ATP content was about 60-300 times higher (17.0 nmol/mg protein). It is concluded that low ATP content is among the possible causes of growth cessation of S. thermotolerans Kr1 and A. tolerans K1 under auto- and heterotrophic conditions after several culture transfers.     

Alteration of the Intracellular Energetic and Ionic Conditions by Mengovirus Infection of Ehrlich Ascites Tumor Cells and Its Influence on Protein Synthesis in the Midphase of Infection

Mengovirus infection of Ehrlich ascites tumor cells caused a change of the intracellular ATP concentration. It increased by 35% within the first 3 h postinfection and then declined to zero within the next 5 h. The decrease in the ATP concentration was due, at least in part, to leakage of ATP into the medium, where it could be demonstrated by the luciferin-luciferase assay. Gross leakage of ATP was observed at 4.5 h postinfection, concomitant with the production of the first intracellular, infectious virus particles. A similar concentration decrease was detected for Mg(2+), the polyamines, and K(+), whereas an increase in the Na(+) concentration was observed. The intracellular Mg(2+) concentration varied synchronously with the ATP level, rising by 16% during the first 3 h postinfection and then progressively falling to lower values in the late period of the infectious cycle. After an initial slight enhancement, the putrescine, spermidine, and spermine concentrations declined at about 1.5 h postinfection. Wherease the intracellular K(+) concentration increased by 17% during the first hour postinfection, the Na(+) concentration diminished by the same value within the same time period, leaving the internal ionic strength unchanged early in infection. Three hours after the beginning of virus infection, there was a rapid decline of K(+) and enhancement of Na(+) within the cell. These alterations of the intracellular energetic and ionic conditions seem to be, at least in part, responsible for the cessation of virus-specific protein synthesis in mengovirus-infected Ehrlich ascites tumor cells commencing 3 to 3.5 h postinfection.     

Nicotinamide methylation and its relation to NAD synthesis in rat liver tissue culture. Biochemical basis for the physiological activities of 1-methylnicotinamide

The mode of [14C]nicotinamide conversion to NAD and 1-methylnicotinamide and the effects of exogenous 1-methylnicotinamide on this metabolic conversion were studied using rat liver slices incubated in a chemically defined culture medium. It was shown that at the physiological nicotinamide concentrations tested (11-500 microM), 1-methylnicotinamide is preferentially produced, rather than NAD. Upon increasing nicotinamide concentration to the levels that cause cytotoxicity (1-10 mM and higher), the rate of NAD synthesis dramatically increased and reached a level 6-fold higher than that of 1-methylnicotinamide. A dose-dependent inhibition (up to 60%) of NAD synthesis was seen by the exogenous addition of 1-methylnicotinamide; the degree of inhibition is affected also by the concentration of nicotinamide present as a precursor. A large depletion of intracellular ATP, associated with a marked accumulation of NAD, occurred in slices in response to the addition of high amounts of nicotinamide. However, the loss of ATP was overcome, when nicotinamide was given together with 1-methylnicotinamide. Finally, 1-methylnicotinamide per se was proven active in regulating cell growth by comparing the cytosolic activity of 1-methylnicotinamide oxidation of cultured RLC cells with that of rat liver. Thus, the previously observed growth stimulation of hepatic cells by 1-methylnicotinamide can reasonably been explained by its ATP-sparing effect due to the inhibition of NAD synthesis, a reaction which requires ATP.     

Relationships between the rate of synthesis of ATP and the concentrations of reactants and products of ATP hydrolysis in maize root tips, determined by 31P nuclear magnetic resonance

Using 31P NMR spectroscopy, we have measured the rate of ATP synthesis, and the free concentrations of ATP, ADP, cytoplasmic Pi, and H+ in maize root tips under a wide range of conditions. We show that the ratio [ATP]/[ADP] in normoxic root tips is greater than 25. We found no simple relationship between the concentration of ATP and the rate of ATP synthesis: when the rate of ATP synthesis decreases in response to different treatments, the concentration of ATP can increase, decrease, or remain unchanged. Clear relationships were obtained, however, when the rate of synthesis of ATP was plotted against the logarithm of the ratio psi, defined as [ATP]/[ADP][Pi][H+]. Two curves were obtained, depending on which of two situations pertained. First, if mitochondrial ATP synthesis was inhibited, e.g., by KCN or hypoxia, ln psi decreased monotonically as rates of ATP synthesis decreased. The decrease in ln psi may account for decreases in the rates of biosynthetic reactions dependent on ATP, such as protein synthesis, as they approach equilibrium. Second, if consumption of ATP for biosynthetic reactions was inhibited, by treatment with succinate, ln psi increased as rates of ATP synthesis decreased. The increase in ln psi may account for decreases in the rate of ATP synthesis, as oxidative phosphorylation approaches equilibrium.     

Influence of ATP on Ehrlich ascites carcinoma cell free cytoplasmic calcium concentration in the course of tumor growth

The changes in free cytoplasmic calcium concentration ([Ca2+](in)) and the effects of extracellular ATP on [Ca2+](in) have been studied in Ehrlich ascites carcinoma cells in the dynamics of their growth. The basal level of [Ca2+](in) and the effects of ATP on the ascites cells were determined by the stage of tumor growth and depended on the content of reactive oxygen species (ROS). The sharp increase in basal and ATP-induced elevation of [Ca2+](in) levels were observed at the 12th day of ascites cell growth. Inhibition of ROS formation by N-acetyl-L-cysteine decreased [Ca2+](in) and suppressed the cell reaction to ATP. We suggest that the increased sensitivity of the ascites cells to ATP observed on the 12th day may be also attributed to a decrease in ecto-ATPase activity.     

Effects of glycerol and fructose on purine synthesis de novo and on PP-ribose-P availability in rat liver cells

Incubation of freshly isolated rat liver cells with glycerol resulted in an initial decrease, followed by an increase in purine synthesis de novo and in PP-ribose-P availability. The magnitude of these effects was dependent on the concentration of glycerol; as it increased, the initial period of latency or inhibition was prolonged, and the extent of the subsequent stimulation was greater. The intracellular Pi concentration and the [14C]ATP/[14C]ADP ratio were also initially decreased in these cells, and they too returned subsequently to normal values. All these changes were similar to those induced by fructose under the same conditions. The increase in PP-ribose-P availability always preceded that in purine synthesis de novo, indicating that, under most circumstances, PP-ribose-P availability is limiting for purine synthesis de novo. Finally, PP-ribose-P synthesis in these cells varied in parallel with the intracellular Pi concentration and with the ATP/ADP and ATP/AMP ratios.     

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.     

Difference in ionic specificity of ATP synthesis in extremely alkalophilic sulfate-reducing and acetogenic bacteria

Ionic specificity of oxidative phosphorylation was studied in Natroniella acetigena and Desulfonatronum lacustre, which are new alkaliphilic anaerobes that were isolated from soda lakes and have a pH growth optimum of 9.5-9.7. The ability of their cells to synthesize ATP in response to the imposition of artificial delta pH+ and delta pNa+ gradients was studied. As distinct from other marine and freshwater sulfate reducers and extremely alkaliphilic anaerobes, D. lacustre uses a Na(+)-translocating ATPase for ATP synthesis. The alkaliphilic acetogen N. acetigena, which develops at a much higher Na+ concentration in the medium, generated primary delta pH+ for ATP synthesis. Thus, the high Na+ concentrations and alkaline pH values typical of soda lakes do not predetermine the type of bioenergetics of their inhabitants.     

Cellular ATP levels and nitrogenase switchoff upon oxygen stress in chemostat cultures of Azotobacter vinelandii.

When Azotobacter vinelandii, growing diazotrophically in chemostat culture, was subjected to sudden increases in the ambient oxygen concentration (oxygen stress), nitrogenase activity was switched off and cellular ATP pools decreased at rates depending on the stress level. Following a fast decrease, the ATP pool approached a lower level. When the stress was released, these effects were reversed. The reversible decrease of the ATP pool upon oxygen stress could also be observed with cultures assimilating ammonium and, at the same time, fixing dinitrogen because of growth at a high C/N ratio but not with cultures growing only at the expense of ammonium. When strains OP and UW136 of A. vinelandii were subjected to long-term increases in ambient oxygen, the sizes of cellular ATP pools eventually started to increase to the level before stress and diazotrophic growth resumed. The cytochrome d-deficient mutant MK5 of A. vinelandii, however, impaired in aerotolerant diazotrophic growth, was unable to recover from stress on the basis of its ATP pool. The results suggest that adaptation to higher ambient oxygen depends on increased ATP synthesis requiring increased electron flow through the entire respiratory chain, which is possible only in combination with the more active, yet possibly uncoupled, branch terminated by cytochrome d. It is proposed that the decrease of the cellular ATP level under oxygen stress resulted from the increased energy and electron donor requirement of nitrogenase in reacting with oxygen.     

Features of apparent nonchemiosmotic energization of oxidative phosphorylation by alkaliphilic Bacillus firmus OF4.

Oxidative phosphorylation by extremely alkaliphilic Bacillus species violates two major predictions of the chemiosmotic hypothesis: the magnitude of the chemiosmotic driving force, the delta p (electrochemical proton gradient), is too low to account for the phosphorylation potentials observed during growth at pH 10.5 without using a much higher H+/ATP stoichiometry than used during growth at pH 7.5, and artificially imposed diffusion potentials fail to energize ATP synthesis above about pH 9.5 (Guffanti, A. A., and Krulwich, T. A. (1989) Annu. Rev. Microbiol. 43, 435-463). To further examine the latter observation, large valinomycin-mediated potassium diffusion potentials were imposed across starved cells of Bacillus firmus OF4 at various pH values from pH 7.5 to 10.5. As the external pH increased above pH 8, there was a sharp decrease in the rate of ATP synthesis in response to an imposed diffusion potential. The rate of ATP synthesis fell to zero by pH 9.2 and 9.4, respectively, in the presence and absence of a small inwardly directed Na+ gradient. Electrogenic Na+/H+ antiport and Na+/alpha-aminoisobutyric acid symport proceeded at substantial rates throughout. When synthesis was energized by an electron donor, cells under comparable conditions synthesized ATP at rapid rates up to pH 10.5. The proton transfers that occur during respiration-dependent oxidative phosphorylation at pH 10.5 may depend upon specific complexes. Cells grown at pH 7.5, which have one-third the levels of the caa3-type terminal oxidase, and slightly lower levels of certain other respiratory chain complexes than pH 10.5-grown cells, support only low rates of ATP synthesis at pH 10.5, although energy-dependent symport and antiport rates are comparable with those in pH 10.5-grown cells. A model is presented for oxidative phosphorylation by the alkaliphilic Bacillus that involves a nonchemiosmotic direct intramembrane transfer of protons from specific respiratory chain complexes to the F0 sector of the ATPase, whereas remaining respiratory chain complexes extrude protons into the bulk to generate the bulk potential required both for ATP synthesis and other bioenergetic work. A pK-regulated gate or a delocalized proton pathway that fails to work above pH 9.5 are suggested as possible features that account for the loss of efficacy of a bulk-imposed diffusion potential in energizing ATP synthesis above pH 9.4.     

Dramatic effect of glycolysis inhibition on the cerebellar granule cells bioenergetics

Cultured cerebellar granule cells were co-loaded with Ca²⁺-sensitive dye fura-2FF and rhodamine-123 sensitive to changes in the mitochondrial potential (????_m). A 60-min incubation of cells in glucose-free solution containing 2-deoxy-D-glucose (DG) induced a slow developing mitochondrial depolarization (sMD) without appreciable changes in basal [Ca²⁺]ⁱ. This sMD was insensitive to a removal of external Ca²⁺ or to the NMDA channels blocker memantine but could be readily suppressed by oligomycin due to inhibition of the inward proton current through the Fo channel of mitochondrial ATP synthase. In resting cells glucose deprivation caused a progressive decrease in mitochondrial NADH content ([NADH]), which strikingly enhanced the ability of glutamate to induce a delayed Ca²⁺ deregulation (DCD) associated with a profound mitochondrial depolarization. In glucose-containing medium this DCD appeared in young cells (usually 6?C8 days in vitro) after a prolonged latent period (lag phase). Substitution of glucose by DG led to a dramatic shortening of this lag phase, associated with a critical decrease in [NADH] in most neurons. Addition of pyruvate or lactate to DG-containing solution prevented the sMD and [NADH] decrease in resting cells and greatly diminished the number of cells exhibiting glutamate-induced DCD in glucose-free medium. Measurement of intracellular ATP level ([ATP]) in experiments on sister cells showed that glucose deprivation decreased [ATP] in resting cells and considerably deepened the fall of [ATP] caused by glutamate. This decrease in [ATP] was only slightly attenuated by pyruvate and lactate, despite their ability to prevent the shortening of lag phase preceding the DCD appearance under these conditions. Simultaneous monitoring of cytosolic ATP concentration ([ATP]_c) and ????_m changes in individual CGC expressing fluorescent ATP sensor (AT1.03) revealed that inhibition of either mitochondrial respiration or glycolysis caused a relatively small decrease in [ATP]_c and ????_m. Complete blockade of ATP synthesis in resting CGC with oligomycin in glucose-free DG-containing buffer caused fast ATP depletion and mitochondrial repolarization, indicating that mitochondrial respiratory chain still possess a reserve fuel to support ????_m despite inhibition of glycolysis. The data obtained suggest that the extraordinary enhancement of glutamate-induced deterioration in Ca²⁺ homeostasis caused by glucose deprivation in brain neurons is mainly determined by NADH depletion.     

Regulation of the synthesis of total cellular proteins and monoclonal antibodies in a hybridoma cell culture

A study was made of the regulation of total protein synthesis in cells of the mouse hybridoma producing monoclonal antibodies (McAb) against lambda phage, and in the course of hybridoma growth and at the change of fetal bovine serum (FBS) concentration. FBS strictly affected proliferation of hybridoma cells, the specific production of McAb per cell being unchanged. The rate of total cellular protein synthesis does depend on FBS concentration in the medium, whereas the rates of protein degradation and secretion do not. Evidence is presented that the reduction in the protein-synthesis rate, after the removal of FBS from the medium, is caused by a coordinated decrease in both the rate of protein synthesis initiation and the rates of polypeptide chain elongation and translation termination. The decrease in the protein synthesis rate at the stationary phase of cell growth was shown to be related to the three main factors: 1) a 15-25% decrease in ribosome content per cell; 2) a two-fold decrease of the ribosome portion involved in mRNA translation; 3) a 5 to 15% decrease in the rate of mRNA translation. Evidence is presented that the decrease in the portion of mRNA translating ribosomes is due to the decrease in the rate of protein synthesis initiation.     

Screening of protein kinase inhibitors and knockdown experiments identified four kinases that affect mitochondrial ATP synthesis activity

Mitochondrial ATP synthase, a major ATP supplier in respiring cells, should be regulated in amount and in activity to respond to the varying demands of cells for ATP. We screened 80 protein kinase inhibitors and found that HeLa cells treated with four inhibitors exhibited reduced mitochondrial ATP synthesis activity. Consistently, knockdown of their target kinases (PKA, PKCδ, CaMKII and smMLCK) resulted in a decrease in mitochondrial ATP synthesis activity. Among them, mitochondria of smMLCK-knockdown cells contained only a small amount of ATP synthase, while the α- and β-subunits of ATP synthase were produced normally, suggesting that smMLCK affects assembly (or decay) of ATP synthase.     

Phosphorus-31 nuclear magnetic resonance spectroscopy of human retinoblastoma cells: correlation with metabolic indices

The 31P nuclear magnetic resonance spectrum of cultured human Y-79 retinoblastoma cells was obtained at 121 MHz on intact cells trapped in agarose threads. The spectrum was dominated by monoester peaks, which varied in relative concentration from preparation to preparation. Resonances from phosphocreatine, phosphodiesters and diphosphodiesters also exhibited variability relative to ATP. The main monoester was identified as phosphorylcholine by 31P-NMR of perchloric acid extracts. It was determined that the changes in monoester concentration correlated with feeding pattern. Phosphorus spectra of cells 1, 2 and 3 days post feeding showed a 40% decrease in the relative concentration of phosphorylcholine concentration over the 3 day period. Phosphocreatine, phosphodiesters and diphosphodiesters increased relative to ATP during the same period. Growth curve experiments and oxygen consumption measurements indicated that the decrease in phosphorylcholine correlated with a decrease in cellular growth and oxygen consumption. We conclude that monoester concentration may be a useful indicator of nutritional status in these cells and possibly in intact tumors.     

Energetic and Morphological Plasticity of C6 Glioma Cells Grown on 3-D Support; Effect of Transient Glutamine Deprivation

The energetic metabolism of rat C6 glioma cells has been investigated as a function of the proliferative and differentiation states under three-dimensional (3-D) growing conditions on microcarrier beads. First, the transient deprivation of glutamine from the culture medium induced a marked decrease in the growth rate and a differentiation of C6 cells through the oligodendrocytic phenotype. Second, the respiratory capacity of the C6 cells during short-term subcultures with or without glutamine continuously declined as a function of the cell density, in part due to the mitochondrial content decrease. During the transition from the early exponential to the plateau growth phase in glutamine-containing medium, the oxygen consumption rate per single cell decreased concomitantly with a decrease in the glucose consumption and lactate production rates. This phenomenon led to a sixfold decrease in the total ATP production flux, without significantly affecting the cellular ATP/ADP ratio, thus indicating that some ATP-consuming processes were simultaneously suppressed during C6 proliferation. In glutamine-free medium, the cellular ATP/ADP ratio transiently increased due to growth arrest and to a reduced ATP turnover. Moreover, the results indicated that glutamine is not an essential respiratory substrate for rat C6 glioma under short-term glutamine deprivation. Worth noting was the high contribution of the mitochondrial oxidative phosphorylation toward the total ATP synthesis (about 80%), regardless of the proliferation or the differentiation status of the C6 cells.