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.     

Evaluation of the buffer capacity and permeability constant for protons in chromatophores from Rhodobacter capsulatus

1. The kinetics of decay in the dark of the transmembrane pH difference (delta pH) induced by light in nonphosphorylating chromatophores of Rhodobacter capsulatus were studied using the fluorescent probe 9-aminoacridine, in the presence of 50 mM KCl and 2 microM valinomycin. The transient fluorescence changes induced by acid to base transitions of chromatophore suspensions were used as an empirical calibration [Casadio, R. & Melandri, B. A. (1985) Arch. Biophys. Biochem. 238, 219-228]. The kinetic competence of the probe response was tested by accelerating the delta pH decay with the ionophore nigericin. 2. The time course in the dark of the increase in the internal pH in pre-illuminated chromatophores was analyzed on the basis of a model which assumes a certain number of internal buffers in equilibrium with the free protons and a diffusion-controlled H+ efflux [Whitmarsh, J. (1987) Photosynt. Res. 12, 43-62]. This model was extended to include the effects of the transmembrane electric potential difference on the H+ efflux. 3. The diffusion constant for proton efflux was measured at different values of the internal pH by evaluating the frequency of trains of single-turnover flashes capable of maintaining different delta pH in a steady state. The steady-state equation derived from the model does not include any parameter relative to the internal buffers and allows unequivocal determination of the diffusion constant on the basis of the known H+/e- ratio (equal to two) for the active proton translocation by the bacterial photosynthetic chain. A value for the first-order diffusion constant corresponding to a permeability coefficient, PH = 0.2 micron.s-1, was obtained at an external pH of 8.0; this value was constant for an internal pH ranging over 7.0-4.7. 4. Using the value of the diffusion constant determined experimentally, a satisfactory fitting of the kinetics of delta pH decay in the dark could be obtained when the presence of two internal buffers (with pK values of 3.6 and 6.7, respectively) was assumed. For these calculations, the time course of the transmembrane electric potential difference was evaluated from the electrochromic signal of carotenoids, calibrated with K(+)-induced diffusion potentials. The two internal buffers, suitable for modelling the behaviour of the system, were at concentrations of 250 mM (pK = 3.6) and 24 mM (pK = 6.7) respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of Steady-State Laurdan Fluorescence to Detect Changes in Liquid Ordered Phases in Human Erythrocyte Membranes

In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains.     

Electrochemical proton gradient and lactate concentration gradient in Streptococcus cremoris cells grown in batch culture.

The lactate concentration gradient and the components of the electrochemical proton gradient (delta micro H+) were determined in cells of Streptococcus cremoris growing in batch culture. The membrane potential (delta psi) and the pH gradient (delta pH) were determined from the accumulation of the lipophilic cation tetraphenylphosphonium and the weak acid benzoate, respectively. During growth the external pH decreased from 6.8 to 5.3 due to the production of lactate. Delta pH increased from 0 to -35 mV, inside alkaline (at an external pH of 5.7), and fell to zero directly after growth stopped. Delta psi was nearly constant at -90 mV during growth and also dissipated within 40 min after termination of growth. The internal lactate concentration decreased from 200 mM at the beginning of growth (at pH 6.8) to 30 mM at the end of growth (at pH 5.3); the external lactate concentration increased from 8 to 30 mM due to the fermentation of lactose. Thus, the lactate gradient decreased from 80 mV to zero as growth proceeded and the external pH decreased. From the data obtained on delta psi, delta pH, and the lactate concentration gradient, the H+/lactate stoichiometry (n) was calculated. The value of n varied with the external pH from 1.9 (at pH 6.8) to 0.9 (at pH values below 6). This implies that especially at high pH values the carrier-mediated efflux of lactate supplies a significant quantity of metabolic energy to S. cremoris cells. At pH 6.8 this energy gain was almost two ATP equivalents per molecule of lactose consumed if the H+/ATP stoichiometry equals 2. These results supply strong experimental evidence for the energy recycling model postulated by Michels et al.     

Simultaneous determination of membrane potential and pH gradient by photodiode array spectroscopy

Membrane potential (delta psi) and pH difference (delta pH) were simultaneously determined in liposomes using a photodiode array spectrophotometer. By the use of a cyanine dye (DiS-C3(5)) and 9-aminoacridine for delta psi and delta pH probes, respectively, both changes of delta psi and delta pH could be successfully determined by photodiode array spectrometry. Each dye did not disturb the fluorescence spectrum of the other probe when its concentration was lower than 5 microM. The K+-diffusion potential-driven, FCCP(protonophore)-mediated H+-influx process in the K+-loaded liposomes was analyzed by this method. Results indicate that the kinetic behavior of H+ influx changes at a FCCP concentration of approx. 30 nM. The rate of delta pH formation increased quantitatively with increasing concentrations of FCCP up to 30 nM, but was markedly enhanced at higher concentrations, although the maximal delta pH attained was about 3 pH units in any case when a K+-diffusion potential of -180 mV was applied.     

Transmembrane electrical potential in Rickettsia prowazekii and its relationship to lysine transport.

The transmembrane electrical potential (delta psi) generated by Rickettsia prowazekii metabolizing glutamic acid or ATP was determined by flow dialysis with the lipophilic cation tetraphenylphosphonium and with lysine. At pH 7.0, the rickettsiae generated a delta psi as measured by tetraphenylphosphonium distribution of 90 mV. Under similar conditions, cells of R.prowazekii concentrated lysine to a gradient indicating a delta psi of 90 mV. Energy-starved cells of R. prowazekii were able to utilize exogenously supplied ATP as well as glutamic acid to generate a delta psi of 110 mV at pH 8.0. Lysine transport was markedly affected by environmental pH, the optimum pH ranging from 8.0 to 8.5. delta psi as measured with tetraphenyl-phosphonium was similarly affected in this system, with values ranging from 70 mV at pH 6.0 to 100 mV at pH 8.0. Respiration rates were also affected by the external pH, with a maximum rate of 28 nmol of O2 consumed per min per mg of rickettsial protein occurring at pH 8.0. The pH effects were readily reversible and with a rapid onset.     

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.     

The membrane potential ofMethanobacterium thermoautotrophicum under different external conditions

The membrane potential (delta psi) of whole cells of Methanobacterium thermoautotrophicum strain delta H was estimated under different external conditions using a TPP(+)-sensitive electrode. The results show that the delta psi values of M. thermoautotrophicum at alkaline pHout (8.5) are comparable with delta psi values under slightly acidic conditions (pH 6.8; 230 and 205 mV, respectively). On the other hand, the size of colonies on Petri dishes was remarkably smaller at pH 8.5 than at 6.8. The delta psi was insensitive to relevant ATPase inhibitors. At pH 6.8, the protonophore 3,3',4',5-tetrachlorosalicylanilide (TCS) strongly inhibited delta psi formation and ATP synthesis driven by methanogenic electron transport. On the other hand, at pH 8.5 the CH4 formation and ATP synthesis were insensitive to TCS and a protonophore-resistant delta psi of approximately 150 mV was determined. The finding of a protonophore-resistant delta psi at pH 8.5 indicates that at alkaline pHout these cells can switch from H(+)-energetics to Na(+)-energetics, when the delta [symbol: see text] H+ becomes limited. The results strongly support the hypothesis that at alkaline pHout Na+ ions might fully substitute for H+ in these cells as the coupling ions.     

Conformational properties of streptokinase

The conformational properties of streptokinase (SK) have been assessed by the techniques of differential scanning calorimetry, circular dichroism (CD), and through a combinational approach employing several algorithms which are predictive of secondary structural characteristics. In low ionic strength buffers, SK undergoes a reversible two-state thermal transition with a temperature of maximum heat capacity (Tm) of 46.1 +/- 0.9, a delta Hcal of 98 +/- 11 kcal/mol and a delta Hcal/delta HvH of approximately 1. In high ionic strength buffers, similar calorimetric properties were obtained with the exception that the delta Hcal/delta HvH values were considerably less than 1, indicating the existence of an additional irreversible thermally induced alteration in the molecule, most likely resulting in its aggregation. The effect of pH on the thermal unfolding properties of SK was determined. The results demonstrated that single two-state thermal transitions were obtained, with progressively decreasing Tm values, as the pH was reduced from 6.4 to 3.4, indicating a destabilization of the entire molecule at reduced pH. In the alkaline region, between pH 8.4 and 9.4, stabilization of a separate region of the molecule was obtained, as evidenced by an increase in the delta Hcal/delta HvH to values approximating 2. CD analysis was performed in order to estimate secondary structural characteristics of SK. The best fit of secondary structural parameters to the experimental CD spectrum provided estimates of 17% helices, 28% beta-sheet, 21% beta-turns, and 34% disordered structures. Both the intensity of the spectral band at 208 nm and the level of antiparallel beta-sheet strongly suggest that SK is an alpha + beta protein.     

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.     

Proton motive force and the physiological basis of delta pH maintenance in thiobacillus acidophilus.

At optimal growth pH (3.0) Thiobacillus acidophilus maintained an internal pH of 5.6 (delta pH of 2.6 units) and a membrane potential (delta psi) of some +73 mV, corresponding to a proton motive force (delta p) of -83 mV. The internal pH remained poised at this value through external pH values of 1 to 5, so that the delta pH increased with decreasing external pH. The positive delta psi increased linearly with delta pH: above a delta pH of 0.6 units, some 60% of the increase in delta pH was compensated for by an opposing increase in delta psi. The highest magnitude of delta pH occurred at an external pH of 1.0, where the cells could not respire. Inhibiting respiration by CN- or azide in cells at optimal pH decreased delta pH by only 0.4 to 0.5 units and caused a corresponding opposite increase in delta psi. Thus, a sizable delta pH could be maintained in the complete absence of respiration. Treatment of cells with thiocyanate to abolish the delta psi resulted in a time-dependent collapse of delta pH, which was augmented by protonophores. We postulate that T. acidophilus possesses unusual resistance to ionic movements. In the presence of a large delta pH (greater than 0.6 pH units), limited diffusion of H+ into the cell is permitted, which generates a positive delta psi because of resistance to compensatory ionic movements. This delta psi, by undergoing fluctuations, regulates the further entry of H+ into the cell in accordance with the metabolic state of the organism. The effect of protonophores was anomalous: the delta p was only partially collapsed, and respiration was strongly inhibited. Possible reasons for this are discussed.     

The proton motive force generated in Leuconostoc oenos by L-malate fermentation.

In cells of Leuconostoc oenos, the fermentation of L-malic acid generates both a transmembrane pH gradient, inside alkaline, and an electrical potential gradient, inside negative. In resting cells, the proton motive force ranged from -170 mV to -88 mV between pH 3.1 and 5.6 in the presence Of L-malate. Membrane potentials were calculated by using a model for probe binding that accounted for the different binding constants at the different pH values at the two faces of the membrane. The delta psi generated by the transport of monovalent malate, H-malate-, controlled the rate of fermentation. The fermentation rate significantly increased under conditions of decreased delta psi, i.e., upon addition of the ionophore valinomycin in the presence of KCl, whereas in a buffer depleted of potassium, the addition of valinomycin resulted in a hyperpolarization of the cell membrane and a reduction of the rate of fermentation. At the steady state, the chemical gradient for H-malate- was of the same magnitude as delta psi. Synthesis of ATP was observed in cells performing malolactic fermentation.     

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.     

Potassium extrusion by the moderately halophilic and alkaliphilic methanogen methanolobus taylorii GS-16 and homeostasis of cytosolic pH.

Methanolobus taylorii GS-16, a moderately halophilic and alkaliphilic methanogen, grows over a wide pH range, from 6.8 to 9.0. Cells suspended in medium with a pH above 8.2 reversed their transmembrane pH gradient (delta pH), making their cytosol more acidic than the medium. The decreased energy in the proton motive force due to the reversed delta pH was partly compensated by an increased electric membrane potential (delta psi). The cytosolic acidification by M. taylorii at alkaline pH values was accompanied by K+ extrusion. The cytosolic K+ concentration was 110 mM in cells suspended at pH 8.7, but it was 320 mM in cells suspended at neutral pH values. High external K+ concentrations (210 mM or higher) inhibited the growth of M. taylorii at alkaline pH values, perhaps by preventing K+ extrusion. Cells suspended at pH 8.5 and 300 mM external K+ failed to acidify their cytosol. The key observation indicative of the involvement of K+ transport in cytosolic acidification was that valinomycin (0.8 microM), a K+ uniporter, inhibited the growth of M. taylorii only at alkaline pH values. Experiments with resting cells indicated that at alkaline pH values valinomycin uncoupled catabolic reactions from ATP synthesis. Thus, K+/H+ antiport activity was proposed to account for the K+ extrusion and the uncoupling effect of valinomycin at alkaline pH values. Such antiport activity was demonstrated by the sharp drop in pH of the bulk medium of the cell suspension upon the addition of 0.1 M KCl. The antiporter appeared to be active only at alkaline pH values, which was in accordance with a possible role in pH homeostasis by M. taylorii growing at alkaline pH values.     

Homonyms, synonyms and mutations of the sequence/structure vocabulary

The effect of pH and temperature on the association equilibrium constant (Ka) for bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor) binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B has been investigated. Ka values decrease with decreasing pH, reflecting the acid-midpoint and pK shifts, upon BPTI binding, of a three-proton co-operative transition, between pH 3 and 5, and of a single ionizable group, between pH 5 and 9. At pH 8, the values of delta H degree (between 7 degrees C and 42 degrees C) and delta S degree (at 21 degrees C) for BPTI binding to the glandular kallikreins considered were determined. In particular, the delta H degree values have been found to be independent of temperature and the following values have been obtained by van't Hoff plots: +1.8 kcal/mol, +2.3 kcal/mol and +2.4 kcal/mol (1 kcal = 4184 J) for the inhibitor binding to human urinary kallikrein and porcine pancreatic beta-kallikreins A and B, respectively. Considering the known molecular structures of free porcine pancreatic beta-kallikrein A and BPTI, and of their complex, the stereochemistry of the enzyme : inhibitor contact regions was analysed for the three serine proteinases, in relation to their respective types of behaviour.     

Stability mutants of staphylococcal nuclease: large compensating enthalpy-entropy changes for the reversible denaturation reaction

By use of intrinsic fluorescence to determine the apparent equilibrium constant Kapp as a function of temperature, the midpoint temperature Tm and apparent enthalpy change delta Happ on reversible thermal denaturation have been determined over a range of pH values for wild-type staphylococcal nuclease and six mutant forms. For wild-type nuclease at pH 7.0, a Tm of 53.3 +/- 0.2 degrees C and a delta Happ of 86.8 +/- 1.4 kcal/mol were obtained, in reasonable agreement with values determined calorimetrically, 52.8 degrees C and 96 +/- 2 kcal/mol. The heat capacity change on denaturation delta Cp was estimated at 1.8 kcal/(mol K) versus the calorimetric value of 2.2 kcal/(mol K). When values of delta Happ and delta Sapp for a series of mutant nucleases that exhibit markedly altered denaturation behavior with guanidine hydrochloride and urea were compared at the same temperature, compensating changes in enthalpy and entropy were observed that greatly reduce the overall effect of the mutations on the free energy of denaturation. In addition, a correlation was found between the estimated delta Cp for the mutant proteins and the d(delta Gapp)/dC for guanidine hydrochloride denaturation. It is proposed that both the enthalpy/entropy compensation and this correlation between two seemingly unrelated denaturation parameters are consequences of large changes in the solvation of the denatured state that result from the mutant amino acid substitutions.     

Immobilization of Clostridium thermocellum cells on bituminous coal particles

The immobilization isotherms of Clostridium thermocellum cells on bituminous coal particles of approximately 0.15- to 0.18-mm diameter were experimentally measured at 60, 45, and 30 degrees C with a pH value of 7.0 and with pH values of 6.0 and 5.0 at 60 degrees C. The immobilization data were correlated into Langmuir forms and their characteristic coefficients were obtained. A method to obtain thermodynamic quantities delta G, delta H, and delta S for the immobilization is also demonstrated.     

The electrochemical gradient of H+ in Candida albicans and its relevance to the uptake of nutrients

The electrochemical gradient of protons, delta microH+, in Candida albicans was estimated between pH 3.5 and 8.5. The electrical potential difference (delta psi) and the chemical proton gradient (delta pH) were measured by steady-state distribution of tetraphenylphosphonium ion and of propionic acid across the plasma-membrane, respectively. In the pH range tested, the intracellular pH was maintained fairly constant at values between 7.3 and 8.1. On the other hand, there was an up to three fold enhancement of delta psi under similar conditions. The uptake of a neutral (glycine), an acidic (L-glutamate) and a basic (L-arginine) amino-acids and of the aldopentose (D-xylose) was determined under different values of delta microH+, which was manipulated by varying the pH of the cell suspension. The rate of uptake of D-xylose and glycine appeared to follow delta microH+ while the uptake velocity of L-arginine could be correlated to changes in delta psi. The rate of uptake of L-glutamate, although at highest among the rates of tested nutrients, was, however, largely independent of delta microH+. This and other reasons (discussed below) indicate that delta microH+ may not be the sole driving force of nutrients uptake in C. albicans.     

SNARF-1 as an intracellular pH indicator in laser microspectrofluorometry: a critical assessment

The use of SNARF-1-AM (seminaphtorhodafluor-1-acetoxymethylester) to measure the internal pH of a single living cell by laser microspectrofluorometry has been analyzed with a lymphocyte murine B cell line A20. After incubation of the cells at 37 degrees C in the presence of 10 microM SNARF-1-AM, the internal concentration of SNARF-1 was approximately 200 microM. The enhancement of fluorescent intensity of the probe is concomitant with its leakage out of the cells. During the measurement period, this induces a continuous increase of the contribution of the external probe to the total fluorescence intensity. This prevented classical spectrofluorometry measurements, but did not preclude microspectrofluorometry measurements of internal pH. The ratio R was calculated from fluorescence intensities at 635 and 590 nm and used as an indicator of the intracellular pH. Calibration curves of the intracellular pH were obtained in the presence of nigericin and valinomycin. It appeared that both the fluorescence intensity and the ratio R were lower inside the cell than those values obtained in aqueous solutions. Possible interactions with the main biological macromolecules (i.e., DNA, proteins, membranes) were investigated as well as a possible compartmentation of the probe in cellular organelles. The modifications of probe characteristics inside the cells were attributed to the binding of the probe to cellular proteins. The intracellular pH of A20 cells, measured by SNARF-1 on 84 cells, was found to be 7.18 +/- 0.10 (with an external pH of 7.40 +/- 0.05), which corresponded with values obtained by conventional fluorometric methods.     

Characterization of the Na+/H+ antiporter of alkalophilic bacilli in vivo: delta psi-dependent 22Na+ efflux from whole cells.

The Na+/H+ antiporter of Bacillus alcalophilus was studied by measuring 22Na+ efflux from starved, cyanide-inhibited cells which were energized by means of a valinomycin-induced potassium diffusion potential, positive out (delta psi). In the absence of a delta psi, 22Na+ efflux at pH 9.0 was slow and appreciably inhibited by N-ethylmaleimide. Upon imposition of a delta psi, a very rapid rate of 22Na+ efflux occurred. This rapid rate of 22Na+ efflux was competitively inhibited by Li+ and varied directly with the magnitude of the delta psi. Kinetic experiments with B. alcalophilus and alkalophilic Bacillus firmus RAB indicated that the delta psi caused a pronounced increase in the Vmax for 22Na+ efflux. The Km values for Na+ were unaffected by the delta psi. Upon imposition of a delta psi at pH 7.0, a retardation of the slow 22Na+ efflux rate at pH 7.0 was caused by the delta psi. This showed that inactivity of the Na+/H+ antiporter at pH 7.0 was not secondary to a low delta psi generated by respiration at this pH. Indeed, 22Na+ efflux activity appeared to be inhibited by a relatively high internal proton concentration. By contrast, at a constant internal pH, there was little variation in the activity at external pH values from 7.0 to 9.0; at an external pH of 10.0, the rate of 22Na+ efflux declined. This decline at typical pH values for growth may be due to an insufficiency of protons when a diffusion potential rather than respiration is the driving force. Non-alkalophilic mutant strains of B. alcalophilus and B. firmus RAB exhibited a slow rate of 22Na+ efflux which was not enhanced by a delta psi at either pH 7.0 or 9.0.