Citrate transport in Klebsiella pneumoniae

Sodium ions were specifically required for citrate degradation by suspensions of K. pneumoniae cells which had been grown anaerobically on citrate. The rate of citrate degradation was considerably lower than the activities of the citrate fermentation enzymes citrate lyase and oxaloacetate decarboxylase, indicating that citrate transport is rate limiting. Uptake of citrate into cells was also Na+ -dependent and was accompanied by its rapid metabolism so that the tricarboxylic acid was not accumulated in the cells to significant levels. The transport could be stimulated less efficiently by LiCl. Li+ ions were cotransported with citrate into the cells. Transport and degradation of citrate were abolished with the uncoupler [4-(trifluoromethoxy)phenylhydrazono]propanedinitrile (CCFP). After releasing outer membrane components and periplasmic binding proteins by cold osmotic shock treatment, citrate degradation became also sensitive towards monensin and valinomycin. The shock procedure had no effect on the rate of citrate degradation indicating that the transport is not dependent on a binding protein. Citrate degradation and transport were independent of Na+ ions in K. pneumoniae grown aerobically on citrate and in E. coli grown anaerobically on citrate plus glucose. An E. coli cit+ clone obtained by transformation of K. pneumoniae genes coding for citrate transport required Na specifically for aerobic growth on citrate indicating that the Na-dependent citrate transport system is operating. Na+ and Li+ were equally effective in stimulating citrate degradation by cell suspensions of E. coli cit+. Citrate transport in membrane vesicles of E. coli cit+ was also Na+ dependent and was energized by the proton motive force (delta micro H+). Dissipation of delta micro H+ or its components delta pH or delta psi by ionophores either totally abolished or greatly inhibited citrate uptake. It is suggested that the systems energizing citrate transport under anaerobic conditions are provided by the outwardly directed cotransport of metabolic endproducts with protons yielding delta pH and by the decarboxylation of oxaloacetate yielding delta pNa+ and delta psi. In citrate-fermenting K. pneumoniae an ATPase which is activated by Na+ was not found. The cells contain however a proton translocating ATPase and a Na+/H+ antiporter in their membrane.     

Coupling between the sodium and proton gradients in respiring Escherichia coli cells measured by 23Na and 31P nuclear magnetic resonance

The relationship between the steady-state sodium gradient (delta pNa) and the protonmotive force developed by endogenously respiring Escherichia coli cells has been studied quantitatively, using 23Na NMR for measurement of intracellular and extracellular sodium concentrations, 31P NMR for measurement of intracellular and extracellular pH, and tetraphenylphosphonium distribution for measurement of membrane potential. At constant protonmotive force, the sodium concentration gradient was independent of extracellular concentrations over the measured range of 4-285 mM, indicating that intracellular sodium concentration is not regulated. The magnitude of delta pNa was measured as a function of the composition and magnitude of the protonmotive force. At external pH values below 7.2, delta pNa was parallel to delta pH but showed no simple relationship to the membrane potential; above pH 7.2 the parallel relationship began to diverge, with delta pH continuing to decrease but delta pNa starting to level off or increase. Although plots of delta pNa versus delta pH had slopes of close to 1, the value of delta pNa consistently exceeded that of delta pH by approximately 0.4 units, indicating a partially electrogenic character to the putative H+/Na+ antiport. The apparent stoichiometry was 1.13 +/- 0.01 at external pH below 7.2. The possible significance of this nonintegral stoichiometry is discussed according to a model in which two distinct integral stoichiometries (possibly 1H+/1Na+ and 2H+/1Na+) are available with some relative probability; the model predicts futile cycling of sodium ions and a dissipative proton current. In the course of this study, we discovered that the magnitude of the pH gradient developed by the cells was osmolarity-dependent, yielding steady-state intracellular pH values that varied from 7.1 at 100 mosm to 7.7 at 800 mosm.     

Interaction of membrane proton conductivity,membrane and oxidation-reduction potential in Escherichia coli

It was shown that the proton conductivity of Escherichia coli membranes depends on pH and other conditions of bacterial growth. It is considerably lower in cells fermenting glucose and accomplishing the nitrate-nitrite respiration compared with cells accomplishing the oxygen respiration. Proton conductivity increases substantially with decreasing pH of medium. It was found that proton conductivity is related to the redox and membrane potentials of cells. The energy-dependent flux of protons from cells and the ATPase activity of membrane vesicles considerably vary depending on whether bacteria are grown under aerobic or anaerobic conditions. The H+ flux from cells fermenting glucose (pH 7.5) was 1.7 times greater than the H+ flux from cells that accomplish the nitrate-nitrite and oxygen respiration. The N,N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity increased 2.5 times as K+ concentration increased to 100 mM (including residual K+ in potassium-free medium). The DCCD-sensitive ATPase activity considerably decreased with decreasing pH of medium, whereas the ATPase activity that was not suppressed by DCCD was stimulated. These results can be used for establishing the relationship between membrane proton conductivity and the energy-dependent H+ flux and ATPase activity.     

31 P nuclear magnetic resonance measurements of phosphate metabolites and intracellular pH in turbot Psetta maxima red blood cells using a novel flow method

A method for oxygenating and mixing suspensions of turbot Psetta maxima red blood cells (RBC) was tested in ³¹P nuclear magnetic resonance (NMR) spectroscopy. In normoxia, the levels of inorganic phosphate (Pi) and nucleoside triphosphates (NTP) were stable up to 140 min and intracellular pH (pHi) was maintained and decreased oxygen partial pressure ( P _{O 2}) from 30 to 15 and 600 Pa produced a significant fall in the intensity of NTP resonance, balanced by an increase in the Pi signal. Treatment of RBC with 0· 5 M isoproterenol during hypoxia exposure did not affect the pattern of changes in NTP or pHi induced by hypoxia and the effect was manifest only on Pi levels.     

The proton-translocating ATPase of Candida tropicalis plasma membrane

Proton translocation activity of Candida tropicalis plasma membrane ATPase has been demonstrated using a fluorescent delta pH probe (ACMA) and by direct pH measurements. Modifications in fluorescence intensity and H+ transport are highly specific for Mg2+ and ATP, and are sensitive to the well-known inhibitors of the plasma membrane ATPase, vanadate and DCCD. A H+/ATP ratio of 0.54 is found.     

Observations of aerobic, growing escherichia coli metabolism using an on-line nuclear magnetic resonance spectroscopy system

An experimental system has been constructed which enables on-line measurements of phosphorus-31 ((31)P) nuclear magnetic resonance (NMR) spectra for growing bacterial suspensions under anaerobic or aerobic conditions. A sample stream from a laboratory bioreactor is circulated to the NMR sample chamber in a gas exchange system which permits maintenance of aerobic conditions for high-cell-density cultures. (31)P NMR spectra with resolution comparable with those obtained traditionally using dense, concentrated, nongrowing cell suspensions can be obtained at cell densities above 25 g/L with acquisition times ranging from 14 to 3 minutes which decline as cell density increases. This system has been employed to characterize the changes in intracellular state of a stationary phase culture which is subjected to a transition from aerobic to anaerobic conditions. Both intracellular NTP level and cytoplasmic pH are substantially lower under anaerobic conditions. Also, the system has been employed to observe the response of a growing culture to external addition of acetate. Cells are able to maintain pH difference across the cytoplasmic membrane at extracellular acetate concentrations of 5 and 10 g/L. However, acetate concentrations of 20 g/L cause collapse of the transmembrane DeltapH and sharp reduction of the growth rate of the culture. The experimental configuration described should also permit NMR observations of many other types of microbial cultures and of other nuclei. (c) 1993 John Wiley & Sons, Inc.     

Active electrogenic transport H+ in plasma membrane vesicles of cow parsnip phloem cells

Generation of electric (delta psi) and chemical (delta pH) components of electrochemical proton gradient delta muH+, in plasma membrane vesicles of Heracleum sosnovskyi phloem cells was investigated. ATP-dependent generation of delta psi at pH 6.0 in the presence of Mg2+ and K+ was established with the help of fluorescent probes AU+ and ANS-. Protonophore CCCP and proton ATPase inhibitor DCCD suppressed generation, whereas oligomycin, the inhibitor of mitochondrial ATPases did not affect it. Measurings of delta psi value indicated its oscillations within the limits from 10 to 60 mV. ATP-dependent generation of delta pH was established by means of fluorescent probe 9-AA. The effect was eliminated by CCCP and stimulated by K+, that may testify to the transformation of a part of delta psi into delta pH at antiport H+/K+. Existence of H+-ATPase in the plasma membranes of higher plant cells insuring generation of delta muH+ is supposed.     

Effects of aerobiosis and nitrogen source on the proton motive force in growing Escherichia coli and Klebsiella pneumoniae cells.

The electrochemical gradient of hydrogen ions, or proton motive force (PMF), was measured in growing Escherichia coli and Klebsiella pneumoniae in batch culture. The electrical component of the PMF (delta psi) and the chemical component (delta pH) were calculated from the cellular accumulation of radiolabeled tetraphenylphosphonium, thiocyanate, and benzoate ions. In both species, the PMF was constant during exponential phase and decreased as the cells entered stationary phase. Altering the growth rate with different energy substrates had no effect on the PMF. The delta pH (alkaline inside) varied with the pH of the culture medium, resulting in a constant internal pH. During aerobic growth in media at pH 6 to 7, the delta psi was constant at 160 mV (negative inside). The PMF, therefore, was 255 mV in cells growing at pH 6.3, and decreased progressively to 210 mV in pH 7.1 cultures. K. pneumoniae cells and two E. coli strains (K-12 and ML), including a mutant deficient in the H+-translocating ATPase and a pleiotropically energy-uncoupled mutant with a normal ATPase, had the same PMF during aerobic exponential phase. During anaerobic growth, however, both species had delta psi values equal to 0. Therefore, the PMF in anaerobic cells consisted only of the delta pH component, which was 75 mV or less in cells growing at pH 6.2 or greater. These data thus met the expectation that cells deriving metabolic energy from respiration have a PMF above a threshold value of about 200 mV when the ATPase functions in the direction of H+ influx and ATP synthesis; in fermenting cells, a PMF below a threshold value was expected since the enzyme functions in the direction of H+ extrusion and ATP hydrolysis. K. pneumoniae cells growing anaerobically had no delta psi whether the N source added was N2, NH+4 or one of several amino acids; the delta pH was unaffected. Therefore, any energy cost incurred by the process of nitrogen fixation could not be detected as an alteration of the proton gradient.     

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.     

Nisin dissipates the proton motive force of the obligate anaerobe Clostridium sporogenes PA 3679.

The influence of nisin on the proton motive force (delta p) generated by glucose-energized cells of the obligate putrefactive anaerobe Clostridium sporogenes PA 3679 was determined. The components of delta p, the transmembrane potential (delta psi) and the pH gradient (delta pH), were determined from the distributions of the lipophilic cation [3H]TPP+ ([3H]tetraphenylphosphonium bromide) and [14C]salicylic acid, respectively. The cells maintained a constant delta p of -111 mV, consisting of a delta pH of 0.4 to 1.0 pH units at an external pH of 5 to 7 and a delta psi of -60 to -88 mV. Nisin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and N,N'-dicyclohexylcarbodiimide (DCCD) at pH 6.0 elicited the complete release of preaccumulated [3H]tetraphenylphosphonium bromide and [14C]salicylic acid, with a concomitant depletion of delta psi and delta pH. Nisin and DCCD caused rapid drops in intracellular ATP levels from 1.2 to 0.01 and 0.06 nmol/mg of cells (dry weight), respectively. Cells exposed to nisin and DCCD lost the ability to form colonies, thus suggesting that delta psi and delta pH are necessary for cell viability. The data suggest that depletion of delta p and exhaustion of cellular ATP reserves are the basis for nisin inhibition of C. sporogenes PA 3679.     

Stoichiometry of the H+-ATPase of growing and resting, aerobic Escherichia coli

The H+/ATP stoichiometry of the proton-translocating ATPase was investigated in growing and nongrowing, respiring cells of Escherichia coli. The protonmotive force, delta p, was determined by measuring the transmembrane chemical gradient of protons, delta pH, from the cellular accumulation of benzoate anions, and the electrical gradient, delta psi, from the accumulation of the lipophilic cation tetraphenylphosphonium (TPP+). The accumulation of lactose was also used to calculate the delta p in this lactose operon constitutive beta-galactosidase negative mutant. The phosphorylation potential, delta GP', was determined by measuring the cellular concentration of ATP, ADP, and inorganic phosphate. According to chemiosmotic principles, at steady state the phosphorylation potential is in thermodynamic equilibrium with the protonmotive force, and thus the ratio delta p/delta GP' can be used to determine the H+/ATP ratio. Respiring E. coli cells, in mid-exponential phase of growth or incubated in buffer, at external pHs from 6.25 to 8.25 had a constant delta GP' of about 500 mV. The H+/ATP ratio was found to be 3 when the delta p value derived from lactose accumulation levels was used. However, when the delta p values derived from delta pH and delta psi were used in the calculations, the H+/ATP ratio varied from about 2.5 at external pH 6.25 to about 4 at pH 8.25. Arguments are presented for the hypothesis that the delta psi values obtained from the TPP+ measurements are likely to be inaccurate and that a value of 3 H+/ATP, independent of the external pH, is likely to be the valid stoichiometry.     

Quantitative association between electrical potential across the cytoplasmic membrane and early gentamicin uptake and killing in Staphylococcus aureus

The relationship between the magnitude of the transmembrane electrical potential and the uptake of [14C]gentamicin was examined in wild-type Staphylococcus aureus in the logarithmic phase of growth. The electrical potential (delta psi) and the pH gradient across the cell membrane were determined by measuring the equilibrium distribution of [3H]tetraphenyl-phosphonium and [14C]acetylsalicylic acid, respectively. Incubation in the presence of the H+-ATPase inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) led to an increase in delta psi with no measurable effect on the pH gradient at external pHs ranging from 5.0 to 6.5, and the effect on delta psi was DCCD concentration dependent. In separate experiments, gentamicin uptake and killing were studied in the same cells under identical conditions. At pH 5.0 (delta psi = -140 mV), no gentamicin uptake occurred. In the presence of 40 and 100 microM DCCD, delta psi was increased to -162 and -184 mV, respectively, and gentamicin uptake was observed in a manner that was also dependent on the DCCD concentration. At pH 6.0 (delta psi = -164 mV), gentamicin uptake occurred in the absence of the carbodiimide but was enhanced in a concentration-dependent fashion by 40 and 100 microM DCCD (delta psi = -174 and -216 mV, respectively). In all cases increased gentamicin uptake was associated with an enhanced bactericidal effect. The results indicate that initiation of gentamicin uptake requires a threshold level of delta psi (-155 mV) and that above this level drug uptake is directly dependent on the magnitude of delta psi.     

Phosphorus-31 nuclear magnetic resonance analysis of internal pH during photosynthesis in the cyanobacterium Synechococcus

Phosphorus-31 nuclear magnetic resonance (31P NMR) spectra were obtained from actively photosynthesizing and darkened suspensions of the unicellular cyanobacterium Synechococcus. These spectra show intracellular resonances belonging to inorganic phosphate (Pi), a sugar phosphate (sugar-P), nucleotide di- and triphosphates, and poly-phosphates. The pH-dependent chemical shifts of Pi and sugar-P allowed the estimation of intracellular pH. When irradiated with high-intensity tungsten-halogen light (100 x 10(4) ergs . cm-2 . s-1, measured in the visible range), concentrated cell suspensions in the NMR spectrometer incorporated NaH14CO3 at approximately two-thirds the rate shown by a dilute suspension of cells at saturating light intensity. On the basis of NaH14CO3 incorporation, the effective light intensity obtained under NMR conditions would support growth at approximately one-fourth the maximum rate in dilute suspensions of cells. Irradiated cells maintained a cytoplasmic pH of 7.1--7.3 when exposed to an external pH from 6.4 to 8.3. At an external pH of 6.7, a darkness to light shift caused a 0.4 pH unit alkalinization of the cytoplasm. Treatment of cell suspensions with the uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), in light or darkness, collapsed the internal pH to the level of the external pH. The results suggest a strong light- or energy-dependent buffering of the cytoplasm over a range of external pH. The study demonstrates that 31P NMR can be used to investigate intracellular events in an actively photosynthesizing microorganism.     

Relationship of K+-Uptaking System with H+-Translocating ATPase in Enterococcus hirae, Grown at a High or Low Alkaline pH

Potassium ion pool was studied in glycolyzing Enterococcus hirae, grown at high or low alkaline pH (pH 9.5 and 8.0, respectively). Energy-dependent increase of K⁺ pool was lower for the wild-type cells, grown at pH 9.5, than that for the cells grown at pH 8.0. It was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD). The stoichiometry of DCCD-inhibited K⁺ influx to DCCD-inhibited H⁺ efflux for the wild-type cells, grown at pH 9.5 or 8.0, was fixed for different K⁺ external activity. DCCD-inhibited ATPase activity of membrane vesicles was significantly stimulated by K⁺ for the wild-type cells grown at pH 9.5, and required K⁺ for the wild-type cells grown at pH 8.0, while the levels of α and β subunits of the F₁ and b subunit of the F₀ were lower for the cells grown at pH 9.5 than that for the cells grown at pH 8.0. Such an ATPase activity was residual in membrane vesicles from the atpD mutant with a nonfunctional F₀F₁. ATPase activity of membrane vesicles from the mutant with defect in Na⁺-ATPase was higher for the cells grown at pH 9.5 than that for the cells grown at pH 8.0, and was inhibited by DCCD. An energy-dependent increase of K⁺ pool in this bacterium, grown at a high or low alkaline pH, is assumed to occur through a K⁺ uptaking system, most probably the Trk. The latter functions in a closed relationship with the H⁺-translocating ATPase F₀F₁. Received: 30 June 1997 / Accepted: 4 August 1997     

Functional comparison of DCCD-sensitive Na+/H+ antiporter in Halobacterium halobium with monensin

Membrane vesicles from Halobacterium halobium create a large, inside negative membrane potential (delta psi) and small, inside alkaline pH gradient (delta pH) by illumination in 3 M NaCl. delta psi was the major component of a proton electrochemical potential (delta microH+) over a pH range from 5 to 8. After DCCD treatment of the vesicles, delta psi was replaced by delta pH due to the inhibition of the intrinsic delta pH----delta psi transformation process: delta psi formation in light is markedly retarded and an inversely large delta pH is established at these pHs. DCCD-caused changes in delta psi and delta pH were completely restored to the control level by the addition of monensin, an electroneutral Na+/H+ exchanger. The ratio of DCCD-caused change in delta pH and delta psi was identical to that of monensin-recovered delta psi and delta pH. The delta psi/delta pH ratio was approximately 0.8, that is, 100 mV of delta pH was transformed into 78 mV of delta psi. The present results indicate that the intrinsic activity of the DCCD-sensitive delta pH----delta psi transformation is mediated by an electroneutral Na+/H+ exchange.     

Origin of the cytoplasmic pH changes during anaerobic stress in higher plant cells. Carbon-13 and phosphorous-31 nuclear magnetic resonance studies

We tested the contribution of nucleoside triphosphate (NTP) hydrolysis, ethanol, and organic acid syntheses, and H(+)-pump ATPases activity in the acidosis of anoxic sycamore (Acer pseudoplatanus) plant cells. Culture cells were chosen to alter NTP pools and fermentation with specific nutrient media (phosphate [Pi]-deprived and adenine- or glycerol-supplied). In vivo (31)P- and (13)C-nuclear magnetic resonance (NMR) spectroscopy was utilized to noninvasively measure intracellular pHs, Pi, phosphomonoesters, nucleotides, lactate, and ethanol. Following the onset of anoxia, cytoplasmic (cyt) pH (7.5) decreased to 6.8 within 4 to 5 min, whereas vacuolar pH (5.7) and external pH (6.5) remained stable. The NTP pool simultaneously decreased from 210 to <20 nmol g(-1) cell wet weight, whereas nuceloside diphosphate, nucleoside monophosphate, and cyt pH increased correspondingly. The initial cytoplasmic acidification was at a minimum in Pi-deprived cells containing little NTP, and at a maximum in adenine-incubated cells showing the highest NTP concentration. Our data show that the release of H(+) ions accompanying the Pi-liberating hydrolysis of NTP was the principal cause of the initial cyt pH drop and that this cytoplasmic acidosis was not overcome by H(+) extrusion. After 15 min of anoxia, a partial cyt-pH recovery observed in cells supplied with Glc, but not with glycerol, was attributed to the H(+)-consuming ATP synthesis accompanying ethanolic fermentation. Following re-oxygenation, the cyt pH recovered its initial value (7.5) within 2 to 3 min, whereas external pH decreased abruptly. We suggest that the H(+)-pumping ATPase located in the plasma membrane was blocked in anoxia and quickly reactivated after re-oxygenation.     

Differential sensitivity of the cellular compartments of Saccharomyces cerevisiae to protonophoric uncoupler under fermentative and respiratory energy supply.

The effect of a protonophoric uncoupler (CCCP) on the different cellular compartments was investigated in yeast grown aerobically on lactate. These cells were incubated in a resting cell medium under three conditions; in aerobiosis with lactate or glucose or in anaerobiosis with glucose as energetic substrate. For each condition, in vivo 31P NMR was used to measure pH gradients across vacuolar and plasma membrane and phosphorylated compound levels. Respiratory rate (aerobic conditions) and TPP+ uptake were measured independently. Concerning the polyphosphate metabolism, spontaneous NMR-detected polyphosphate breakdown occurred, in anaerobiosis and in the absence of CCCP. In contrast, in aerobiosis, polyphosphate hydrolysis was induced by addition of either CCCP or a vacuolar membrane ATPase-specific inhibitor, bafilomycin A1. Moreover, polyphosphates were totally absent in a null vacuolar ATPase activity mutant. The vacuolar polyphosphate content depended on two factors: vacuolar pH value, strictly linked to the vacuolar H(+)-ATPase activity, and inorganic phosphate concentration. CCCP was more efficient in dissipating the proton electrochemical gradient across vacuolar and mitochondrial membranes than across the plasma membrane. This discrepancy can be essentially explained by a difference of stimulability of each proton pump involved. As long as the energetic state (measured by NDP + NTP content) remains high, the plasma membrane proton ATPase is able to compensate the proton leak. Moreover, this ATPase contributes only partially to the generation of delta pH. The maintenance of the delta pH across the plasma membrane, that of the energetic state, and the cellular TPP+ uptake depend on the nature of the ATP-producing process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Diffusional limitations of immobilized Escherichia coli in hollow-fiber reactors: influence on 31P NMR spectroscopy

Escherichia coli cells were immobilized and grown in hollow-fiber reactors allowing simultaneous NMR spectroscopy and perfusion with nutrient medium. The extent to which the cells were starved due to inadequate mass transfer was predicted using a mathematical model of reaction and diffusion. Reactors were experimentally characterized using (35)S autoradiography to visualize spatial variations in protein synthesis rates and transmission electron microscopy to indicate spatial variations in cell morphology. Mass transfer limitations in reactors operated at 37 degrees C were shown to be severe, with regions of starved cells occupying up to 80% of the cell-containing region. Phosphorus-31 nuclear magnetic resonance (NMR) spectra of the immobilized, perfused cells revealed abnormally low volume-averaged concentrations of sugar phosphates, NTP, and ratios of NTP/NDP in these reactors. Intracellular pH was also depressed in the cells. In order to overcome mass transfer limitations in the cell layer, the reactor growth temperature was decreased. Sulfur-35 autoradiographs of a reactor operated at 16 degrees C did not indicate the presence of starved cells. The NMR spectra obtained from this reactor showed near-normal intracellular pH, metabolite concentrations, and NTP/NDP ratios. The presence of significant mass transfer limitations in a perfused cell sample during NMR spectroscopy is generally undesirable since the resulting spectra can be ambiguous and difficult to interpret. The strategy adopted in this work, namely estimation of the relative rates of reaction and diffusion in the cell mass and appropriate changes in reactor design and operating parameters, should prove generally applicable for the design of perfused cell samples for NMR spectroscopic experiments.     

19F-nuclear magnetic resonance: measurements of [O2] and pH in biological systems.

19F-nuclear magnetic resonance (NMR) has been used to determine both intracellular pH and oxygen concentrations in cell suspensions. Oxygen concentrations in Paracoccus denitrificans and insulinoma cells, RINm5F, in the NMR probe can be monitored directly by 1/T1 measurements of perfluorotripropylamine (FTPA)/lecithin emulsion added to the suspensions. With FTPA oxygen monitoring, we investigated the relative aeration capabilities of two types of NMR chambers. Both normal and transformed eucaryotic cells can be maintained in either chamber for at least 1-2 h at cytocrits of up to 20-25%, with 30% oxygen saturation and cell viabilities of 90-95%. Similar concentrations of procaryotes were maintained aerobic with high FTPA concentrations in the more efficient of the two NMR chambers. A new precursor molecule for the 19F-NMR pH indicator difluoromethylalanine, the para-chlorophenyl ester, has been tested and used in RINm5F cells and P. denitrificans, neither of which hydrolyzes methyl esters.     

DCCD sensitivity of electron and proton transfer by NADH: ubiquinone oxidoreductase in bovine heart submitochondrial particles--a thermodynamic approach

The sensitivity of the H+/2e- ratio of the redox-driven proton pumping by the NADH: ubiquinone reductase (complex I) of the submitochondrial particles to dicyclohexylcarbodiimide (DCCD) was studied by a thermodynamic approach, measuring the membrane potential and delta pH across the membrane and the redox potential difference across the complex I span of the respiratory chain. The delta Gr/delta muH+ ratio did not decrease upon additions of 50 or 100 nmol of DCCD per mg protein in the presence of oligomycin although the H+/2e- ratio has been demonstrated to decrease upon DCCD addition in kinetic experiments with mitochondria. Complex I then becomes reminiscent of the cytochrome bc1 complex, which shows DCCD sensitivity of the kinetically but not thermodynamically determined H+/2e- ratio.