Regulation of Glycolytic Oscillations by Mitochondrial and Plasma Membrane H-ATPases

We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3′-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H⁺-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP/ATP antiporter and the mitochondrial F₀F₁-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F₀F₁-ATPase and plasma membrane H⁺-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations in mitochondrial membrane potential.     

31P nuclear magnetic resonance studies on relaxation parameters and line broadening of intracellular metabolites of HeLa cells.

The 40-MHz ³¹P nuclear magnetic resonance (nmr) spectrum of intact HeLa cells contains seven broad peaks with some detectable splittings. The linewidths were significantly broader than for those of cell-free systems such as cell extracts, indicating that the cellular environment is responsible for the unusual line broadening. Resolution of these peaks at 40 MHz is sufficient to make certain assignments and the relaxation parameters of some of the intracellular metabolites have been measured. The spin-lattice relaxation times (T₁) ranged from 0.3 s for adenosine triphosphate (ATP) to about 3 s for inorganic phosphate (P_i) and monophosphate compounds. Nuclear Overhauser enhancements (NOE) were induced by proton irradiation with the possible exception of ATP. The relaxation parameters were compared to those of cell-free compounds and in all cases T₁ and NOE were smaller for the intracellular metabolites. The relaxation parameters for ATP were affected the most. This behavior was mimicked with mixtures of cell-free metabolites containing paramagnetic ions. The larger change in both T₁ and NOE of intracellular ATP could be accounted for by selective binding of paramagnetic ions. This phenomenon also explains some of the line broadening in the cell spectrum especially that of ATP. The spin-spin relaxation times (T₂) of P₁ and monophosphate compounds as measured by a pulse technique did not account for the observed linewidths. This is due to the presence of chemical shift envelopes arising from pH heterogeneity. All resonances were broader at 146 MHz because of the line broadening by paramagnetic ions and the presence of chemical shift envelopes. Other mechanisms of line broadening may also be significant. There was little difference in resolution of spectra at 40 and 146 MHz. Water proton linewidths and T₂ values were measured for HeLa cells and for some minced tissue preparations. The water linewidth in tissue samples was broader than that in the cell suspension. The large linewidths in tissues arise mainly from chemical shift envelopes caused by magnetic field nonuniformity in the tissue samples. There appears to be a small chemical shift envelope from magnetic nonuniformity in HeLa cells as well. The ¹H results on envelopes were extrapolated to ³¹P studies on cells and tissues. Possible methods for reducing linewidths arising from the various proposed broadening mechanisms were discussed.     

Pools of water in anhydrobiotic organisms: A thermally stimulated depolarization current study

The ability to survive the removal of water in anhydrous biosystems is especially remarkable as a departure from the manifold structural and functional dependences on the presence of H₂O molecules. Identifiable pools of water present in dry soybean axes were investigated by means of the thermally stimulated depolarization current method. Samples were examined in the temperature range 100-340 K and over water contents (h, in gram H₂O per gram sample dry weight) ranging from h = 0.05 to 0.30 g/g. Three water-dependent relaxation mechanisms were detected; one attributed to dipolar reorientation of H₂O molecules hydrogen-bonded to other water molecules, one to reorientation of CH₂OH groups, and one to a glass transition in sugar-water domains. These glassy domains can protect intracellular components against destruction in the dehydrated state. Interestingly, protecting glassy domains were not found in dehydration intolerant seeds, supporting the hypothesis that the ability to withstand dehydration is associated with intracellular glass formation. A model for the state of cell water at interfaces is proposed.     

Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor, while mitochondrial membrane potential was measured using the fluorescent dye DiOC 2(3). The results show that, as opposed to NADH and other intermediates in glycolysis, intracellular glucose is not oscillating. Furthermore, oscillations in NADH and membrane potential are inhibited by the ATP/ADP antiporter inhibitor atractyloside and high concentrations of the ATPase inhibitor N, N'-dicyclohexylcarbodiimide, suggesting that there is a strong coupling between oscillations in mitochondrial membrane potential and oscillations in NADH mediated by the ATP/ADP antiporter and possibly also other respiratory components.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

Time-resolved measurements of intracellular ATP in the yeast Saccharomyces cerevisiae using a new type of nanobiosensor

Adenosine 5'-triphosphate is a universal molecule in all living cells, where it functions in bioenergetics and cell signaling. To understand how the concentration of ATP is regulated by cell metabolism and in turn how it regulates the activities of enzymes in the cell it would be beneficial if we could measure ATP concentration in the intact cell in real time. Using a novel aptamer-based ATP nanosensor, which can readily monitor intracellular ATP in eukaryotic cells with a time resolution of seconds, we have performed the first on-line measurements of the intracellular concentration of ATP in the yeast Saccharomyces cerevisiae. These ATP measurements show that the ATP concentration in the yeast cell is not stationary. In addition to an oscillating ATP concentration, we also observe that the concentration is high in the starved cells and starts to decrease when glycolysis is induced. The decrease in ATP concentration is shown to be caused by the activity of membrane-bound ATPases such as the mitochondrial F(0)F(1) ATPase-hydrolyzing ATP and the plasma membrane ATPase (PMA1). The activity of these two ATPases are under strict control by the glucose concentration in the cell. Finally, the measurements of intracellular ATP suggest that 2-deoxyglucose (2-DG) may have more complex function than just a catabolic block. Surprisingly, addition of 2-DG induces only a moderate decline in ATP. Furthermore, our results suggest that 2-DG may inhibit the activation of PMA1 after addition of glucose.     

Activation of an ATP-dependent K+ conductance in Xenopus oocytes by expression of adenylate kinase cloned from renal proximal tubules

In rabbit proximal convoluted tubules, an ATP-sensitive K⁺ (K_ATP) channel has been shown to be involved in membrane cross-talk, i.e. the coupling (most likely mediated through intracellular ATP) between transepithelial Na⁺ transport and basolateral K⁺ conductance. This K⁺ conductance is inhibited by taurine. We sought to isolate this K⁺ channel by expression cloning in Xenopus oocytes. Injection of renal cortex mRNA into oocytes induced a K⁺ conductance, largely inhibited by extracellular Ba²⁺ and intracellular taurine. Using this functional test, we isolated from our proximal tubule cDNA library a unique clone, which induced a large K⁺ current which was Ba²⁺-, taurine- and glibenclamide-sensitive. Surprisingly, this clone is not a K⁺ channel but an adenylate kinase protein (AK3), known to convert NTP+AMP into NDP+ADP (N could be G, I or A). AK3 expression resulted in a large ATP decrease and activation of the whole-cell currents including a previously unknown, endogenous K⁺ current. To verify whether ATP decrease was responsible for the current activation, we demonstrated that inhibition of glycolysis greatly reduces oocyte ATP levels and increases an inwardly rectifying K⁺ current. The possible involvement of AK in the K_ATP channel’s regulation provides a means of explaining their observed activity in cytosolic environments characterized by high ATP concentrations.     

The State of Water in Muscle Tissue as Determined by Proton Nuclear Magnetic Resonance

The nuclear magnetic resonance (NMR) of water protons in live and glycerinated muscle, suspensions of glycerinated myofibrils, and solutions of several muscle proteins has been studied. T₁ and T₂, measured on partially hydrated proteins by pulsed spin-echo techniques, decreased as the ratio of water to protein decreased, showing that the water which is tightly bound by the protein has short relaxation times. In live muscle fibers the pulse techniques showed that, after either a 180 or a 90° pulse, the relaxation of the magnetization is described by a single exponential. This is direct evidence that a fast exchange of protons occurs among the phases of the intracellular water. The data can be fitted with a model in which the bulk of the muscle water is in a phase which has properties similar to those of a dilute salt solution, while less than 4-5% of the total water is bound to the protein surface and has short relaxation times. Measurements of T₁ and T₂ in protein solutions showed that no change in the proton relaxation times occurred when heavy meromyosin was bound to actin, when myofibrils were contracted with adenosine triphosphate (ATP), or when globular actin was polymerized.     

Studies on the relationship between glycolysis and (Na++K+)-ATPase in cultured cells

In several tissues a coupling between glycolysis and (Na⁺+K⁺)-ATPase has been observed. We report here studies on the coupling of glycolysis and (Na⁺+K⁺)-ATPase in Rous-transformed hamster cells and Ehrlich ascites tumor cells. The rate of (Na⁺+K⁺)-ATPase was estimated by the initial rate of ouabain-sensitive K⁺ influx after K⁺ reintroduction to K⁺-depleted cells. Experiments were performed with cells producing ATP via oxidative phosphorylation alone (i.e., lactate sole substrate), glycolysis alone (i.e., glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e., glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K⁺-influx was approx. 2-fold higher when AtP was produced from glycolysis. Changes in cell Na⁺ due to other transport processes related to glycolysis, such as Na⁺-H⁺ exchange, Na⁺-glucose cotransport, and K⁺-H⁺ exchange were ruled out as mediators of this effect on (Na⁺+K⁺)-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to (Na⁺+K⁺)-ATPase to these cultured cells.     

Control of aerobic glycolysis in the brain in vitro

Protoveratrine-(5 M) stimulated aerobic glycolysis of incubated rat brain cortex slices that accompanies the enhanced neuronal influx of Na⁺ is blocked by tetrodotoxin (3 M) and the local anesthetics, cocaine (0.1 mM) and lidocaine (0.5 mM). On the other hand, high [K⁺]-stimulated aerobic glycolysis that accompanies the acetylcholine-sensitive enhanced glial uptakes of Na⁺ and water is unaffected by acetylcholine (2 mM). Experiments done under a variety of metabolic conditions show that there exists a better correlation between diminished ATP content of the tissue and enhanced aerobic glycolysis than between tissue ATP and the ATP-dependent synthesis of glutamine. Whereas malonate (2 mM) and amino oxyacetate (5 mM) suppress ATP content and O₂ uptake, stimulate lactate formation, but have little effect on glutamine levels, fluoroacetate (3 mM) suppresses glutamine synthesis in glia, presumably by suppressing the operation of the citric acid cycle, with little effect on ATP content, O₂ uptake, and lactate formation. Exogenous citrate (5 mM), which may be transported and metabolized in glia but not in neurons, inhibits lactate formation by cell free acetone-dried powder extracts of brain cortex but not by brain cortex slices. These results suggest that the neuron is the major site of stimulated aerobic glycolysis in the brain, and that under our experimental conditions glycolysis in glia is under lesser stringent metabolic control than that in the neuron. Stimulation of aerobic glycolysis by protoveratrine occurs due to diminution of the energy charge of the neuron as a result of stimulation of the sodium pump following tetrodotoxin-sensitive influx of Na⁺; stimulation by high [K⁺, NH₄ ⁺, or Ca²⁺ deprivation occurs partly by direct stimulation of key enzymes of glycolysis and partly by a fall in the tissue ATP concentration.     

The effect of changing extracellular osmolality on water transport in the human red blood cell as measured by the cell water residence time and the activation energy of water transport

A pulse NMR technique employing low extracellular Mn²⁺ concentrations has been used in following the effect of variations in extracellular osmolality on water transport through the human red blood cell membrane. We report results including the effect of osmolality on the cell water lifetime (τ_a) and, for the first time, the effect on the proton spin-spin relaxation of the intracellular water (T_2a) and the activation energy for the water transport process. Current results are encouraging in correlating the effects seen in this study with suspected membrane functional changes occurring in both in vivo and in vitro aging and during in vitro preservation attempts.     

Manganese substrate complexes of phosphofructokinase studies by pulsed magnetic resonance

The formation of binary, ternary, and quaternary complexes between phosphofructokinase, manganese, and substrates has been demonstrated by use of pulsed nuclear magnetic resonance techniques. A Scatchard plot of the interaction of manganese with phosphofructokinase as determined by electron paramagnetic resonance shows two types of manganese binding sites. Phosphofructokinase seems to contain one or two of the metal binding sites with K_d = 20 μm and ?_b ≦ 4, and perhaps, as many as 14 binding sites with K_d ~ 0.8 mm and ?_b ≦ 12 ± 2 per enzyme. Addition of ATP or ADP results in a further enhancement of the relaxation rate indicating ternary complex formation. The concentration of ATP and ADP which results in half maximal change of enhancement is 30–100 μm and 80 μm, respectively. No change in the water proton relaxation rate was detected upon addition of fructose-6-P or fructose-1,6-bisphosphate. A quaternary complex was detected by proton relaxation measurements upon addition of fructose-6-P to a reaction mixture containing β, γ-methylene ATP, manganese, and enzyme with 50 μm fructose-6-P required to obtain the half maximal observed effect. This evidence for a quaternary complex is consistent with a sequential reaction mechanism for phosphofructokinase.     

EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: An application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates

Electron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T₁ of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T₁ is longer, no modulation of the coupling between metal centers can be detected.     

The effect of the cryoprotectants,dimethylsulfoxide and glycerol on water transport in the human red blood cell

The response of human red blood cells to the cryoprotective agents, DMSO and glycerol, has been investigated using a pulsed NMR method. The experimentally determined parameters are: (1) the intracellular transverse relaxation time, T_2a; (2) the mean residence time of intracellular water, τ_a, which is effectively a reciprocal measure of the rate of water transport across the red blood cell membrane; and (3) the activation energy for this process. The quantitative data indicate that the observed effects are colligative rather than species-specific in origin.     

Changes in Membrane Permeability of Winter Wheat Cells following Freeze-Thaw Injury as Determined by Nuclear Magnetic Resonance

Nuclear magnetic resonance (NMR) relaxation times were studied in acclimated and nonacclimated Kharkov winter wheat (Triticum aestivum L.) crowns and acclimated cell aggregates to determine if membrane permeability was altered by freezing. The NMR water signal decay consisted of two exponential components: a short one arising from extracellular water, and a long one arising from intracellular water. A slow freezethaw treatment of nonacclimated and 1-week acclimated crowns decreased the long relaxation time, suggesting membrane injury. Similar results were obtained for nonacclimated and acclimated crowns killed directly in liquid N₂.

A significant increase in plasma membrane permeability to Mn²⁺ was observed in acclimated freeze-killed crowns and cell aggregates. Freezing injury to plant tissue appears to be a membrane-related phenomenon, but more extensive injury occurs to nonacclimated and acclimated tissue with a high water content (cell aggregates) compared to acclimated tissue with a low water content (crowns).

     

Methanogenesis from trimethylamine + H2 by Methanosarcina barkeri is coupled to ATP formation by a chemiosmotic mechanism

Cell suspensions of Methanosarcina barkeri catalyzed the conversion of trimethylamine and molecular hydrogen to methane according to the equation (CH₃)₃NH⁺ + 3 H₂ → 3 CH₄ + NH⁺₄. The onset of methane formation resulted in an increase of the intracellular ATP content from 2 to 4.6 nmol/mg protein and in the generation of a protonmotive force (Δp) of −130 mV, of which the Δψ contributed 90%. The addition of the uncoupler led to a drastic decrease of the intracellular ATP content and the Δψ, but stimulated methanogenesis. The ATPase inhibitor DCCD caused a rapid exhaustion of the ATP pool and inhibited methane formation, whereas Δψ was not affected. The inhibition of methane formation by DCCD could be relieved by addition of TCS, indicating a chemiosmotic coupling between methane formation according to the above equation and ATP synthesis.     

Regulation of glucose metabolism in rat lung: Subcellular distribution,isozyme pattern,and kinetic properties of hexokinase

The subcellular distribution and isozyme pattern of hexokinase in rat lung were studied. Of the total hexokinase activity of lung, one-third was bound to mitochondria and one-third of the mitochondrial activity was in a latent form. The overt-bound mitochondrial hexokinase was specifically solubilized by physiological concentrations of glucose 6-phosphate and ATP. Inorganic phosphate partially prevented the solubilization by glucose 6-phosphate (Glc 6-P), whereas Mg²⁺ ions promoted rebinding of the solubilized enzyme to mitochondria. Thus, the distribution of hexokinase between soluble and particulate forms in vivo is expected to be controlled by the relative concentrations of Glc 6-P, ATP, P_i, and Mg²⁺. Study of the isozyme pattern showed that hexokinase types I, II, and III constitute the cell-sap enzyme of lung. The overt and latent hexokinase activities could be separately isolated by successive treatments of mitochondria with Glc 6-P and Triton X-100. The overt-bound activity consisted primarily of hexokinase type I, with a small proportion of type II isozyme. The latent activity, on the other hand, exclusively consisted of type I isozyme. Type I hexokinase, the predominant isozyme in lung, was strongly inhibited by intracellular concentration of Glc 6-P and this inhibition was counteracted by P_i. The bound form of hexokinase exhibited a significantly higher apparent K_i for Glc 6-P inhibition and a lower apparent K_m for ATP as compared to the soluble form. Thus, the particulate form of hexokinase is expected to promote glycolysis and may provide a mechanism for the high rate of aerobic glycolysis in lung.     

Analysis of Sub-τc and Supra-τc Motions in Protein Gβ1 Using Molecular Dynamics Simulations

The functions of proteins depend on the dynamical behavior of their native states on a wide range of timescales. To investigate these dynamics in the case of the small protein Gβ1, we analyzed molecular dynamics simulations with the model-free approach of nuclear magnetic relaxation. We found amplitudes of fast timescale motions (sub-τ_c, where τ_c is the rotational correlation time) consistent with S² obtained from spin relaxation measurements as well as amplitudes of slow timescale motions (supra-τ_c) in quantitative agreement with S² order parameters derived from residual dipolar coupling measurements. The slow timescale motions are associated with the large variations of the ³J couplings that follow transitions between different conformational substates. These results provide further characterization of the large structural fluctuations in the native states of proteins that occur on timescales longer than the rotational correlation time.     

Long-lived spin state of a tripeptide in stretched hydrogel

The longitudinal (T ₁), transverse (T ₂), and singlet state (T _s) relaxation times of the geminal backbone protons (CH₂) of l-Leu-Gly-Gly were studied by NMR spectroscopy at 9.4 T in a bovine hide gelatin gel composed in D₂O at 25 °C. Gelatin granules were dissolved in a hot solution of the tripeptide and then the solution was allowed to gel inside a flexible silicone tubing. With increases in gelatin content, the T ₂ and T _s of the CH₂ protons correspondingly decreased (T _s/T ₂ ~ constant), while the change in T ₁ was relatively small. The largest observed T _s/T ₁ value was 3.3 at 46 % w/v gelatin that was the lowest gelatin content examined. Stretching the tubing, and hence the gel, brought about anisotropic alignment of the constituents resulting in residual quadrupolar splitting of the resonance from D₂O in ²H NMR spectra, and residual dipolar splitting of the CH₂ resonance in ¹H NMR spectra. WALTZ-16 decoupling during the relaxation intervals extended the singlet state relaxation time, but the efficacy diminished as the gels were stretched. Theoretically predicted T ₁, T ₂, and T _s values, assuming intramolecular dipolar coupling as the only source of relaxation, were within the same order of magnitude as the experimentally observed values. Overall we showed that it is possible to observe a long-lived spin state in an anisotropic medium when T ₂ is shorter than T ₁ in the presence of non-zero residual dipolar couplings.     

Control of glycolysis by orthophosphate and adenosine triphosphate in soluble extracts of germinating pea seeds

Summary Control of aerobic glycolysis by adenosine triphosphate and orthophosphate has been studied in cell-free extracts of germinating pea seeds. Orthophosphate accelerates glycolysis under all conditions studied. At high concentrations of magnesium ion ATP accelerates glycolysis, whereas at lower magnesium concentrations ATP severely inhibits glycolysis. The inhibitory effect of ATP is markedly relieved by orthophosphate. Metabolite analyses suggest an important regulatory role of phosphofructokinase and show that low ratios of F-6-P: FDP accompany the appearance of a high rate of glycolysis, and vice versa. Thus, ATP raises the F-6-P: FDP ratio at low magnesium levels, while P_i lowers this ratio. At high Mg²⁺ (where ATP accelerates glycolysis), ATP causes a low F-6-P: FDP ratio to appear. At low Mg²⁺ concentration, orthophosphate accelerates glycolysis by activation of phosphofructokinase; at high magnesium concentration, the chief effect of orthophosphate is its long-known role in facilitating the oxidation of triose phosphate.