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1.
This work reports the intracellular pH (pHi) dynamics of Saccharomyces cerevisiae cells in sporulation medium. Cells loaded with the pH-sensitive dye carboxy-seminaphthorhodafluor-1 (C.SNARF-1) exhibited an alkalization of the pHi following the extracellular pH during sporulation in the absence of buffer and almost no change in pHi or ΔpH when sporulation was carried out in buffered medium. The results indicate that the pH gradient does not appear to be directly involved in the regulation of acetate uptake during sporulation. However, the alkalization of pHi by eliciting a decrease in metabolic fluxes could account for a lower demand for acetate. 相似文献
2.
Measurement of the Effects of Acetic Acid and Extracellular pH on Intracellular pH of Nonfermenting, Individual Saccharomyces cerevisiae Cells by Fluorescence Microscopy
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The effects of acetic acid and extracellular pH (pHex) on the intracellular pH (pHi) of nonfermenting, individual Saccharomyces cerevisiae cells were studied by using a new experimental setup comprising a fluorescence microscope and a perfusion system. S. cerevisiae cells grown in brewer’s wort to the stationary phase were stained with fluorescein diacetate and transferred to a perfusion chamber. The extracellular concentration of undissociated acetic acid at various pHex values was controlled by perfusion with 2 g of total acetic acid per liter at pHex 3.5, 4.5, 5.6, and 6.5 through the chamber by using a high-precision pump. The pHi of individual S. cerevisiae cells during perfusion was measured by fluorescence microscopy and ratio imaging. Potential artifacts, such as fading and efflux of fluorescein, could be neglected within the experimental time used. At pHex 6.5, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.035 g/liter, whereas at pHex 3.5, 4.5, and 5.6, the pHi of individual S. cerevisiae cells decreased as the extracellular concentration of undissociated acetic acid increased from 0 to 0.10 g/liter. At concentrations of undissociated acetic acid of more than 0.10 g/liter, the pHi remained constant. The decreases in pHi were dependent on the pHex; i.e., the decreases in pHi at pHex 5.6 and 6.5 were significantly smaller than the decreases in pHi at pHex 3.5 and 4.5. 相似文献
3.
T. S. Harter R. B. Shartau D. W. Baker D. C. Jackson A. L. Val C. J. Brauner 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2014,184(6):709-718
Preferential intracellular pH (pHi) regulation, where pHi is tightly regulated in the face of a blood acidosis, has been observed in a few species of fish, but only during elevated blood PCO2. To determine whether preferential pHi regulation may represent a general pattern for acid–base regulation during other pH disturbances we challenged the armoured catfish, Pterygoplichthys pardalis, with anoxia and exhaustive exercise, to induce a metabolic acidosis, and bicarbonate injections to induce a metabolic alkalosis. Fish were terminally sampled 2–3 h following the respective treatments and extracellular blood pH, pHi of red blood cells (RBC), brain, heart, liver and white muscle, and plasma lactate and total CO2 were measured. All treatments resulted in significant changes in extracellular pH and RBC pHi that likely cover a large portion of the pH tolerance limits of this species (pH 7.15–7.86). In all tissues other than RBC, pHi remained tightly regulated and did not differ significantly from control values, with the exception of a decrease in white muscle pHi after anoxia and an increase in liver pHi following a metabolic alkalosis. Thus preferential pHi regulation appears to be a general pattern for acid–base homeostasis in the armoured catfish and may be a common response in Amazonian fishes. 相似文献
4.
Hanns Frohnmeyer Alexander Grabov Michael R. Blatt 《The Plant journal : for cell and molecular biology》1998,13(1):109-116
A role for cytosolic pH (pHi) in hormonal signalling and transport control in plants has long been mooted. Yet, while changes in pHi are a common consequence of hormonal stimuli in plant cells and contribute to hormonally evoked ion channel control, the origins of these changes remain unknown. To examine a possible role for the tonoplast and vacuolar compartment in these events, pHi was measured in the presence of auxins and during cytosolic H+ loading with weak acid in vacuolate and evacuolate protoplasts, both from mesophyll and guard cells of Vicia faba L. Evacuolate protoplasts were obtained following ultracentrifugation on Percoll gradients, and pHi of single protoplasts was recorded in both vacuolate and evacuolate preparations using fluorescence ratio microphotometry and the pH-sensitive dye BCECF. External pH measurements indicated a roughly twofold increase in the rate of net H+ secretion in evacuolate compared with vacuolate protoplasts, and showed that evacuolate protoplasts retained the characteristic stimulation of H+ secretion in the presence of auxin. BCECF fluorescence recording gave resting pHi values near 7.5, and evacuolation had no significant effect on this parameter. Reversible decreases of 0.1–0.2 units in pHi were evoked in vacuolate protoplasts by 10 μM concentrations of the auxins 1-naphthalene acetic acid and 3-indoyl-acetic acid, and not by the inactive (anti-auxin) analogue 2-naphthalene-acetic acid. However, auxin treatments failed to evoke a change in pHi in all but one of 12 experiments with evacuolate protoplasts. Evacuolation also appeared to reduce the transient, dynamic H+ buffering capacity of the protoplasts in the face of acid pHi loads imposed by adding Na+-butyrate to the bath. These results implicate the tonoplast or vacuolar compartment in short-term pHi homeostasis and generation of hormonally evoked H+ signalling in plant cells; they also conform with the view that the decrease in pHiper se is not a primary determinant in the stimulation of H+ secretion by auxin. 相似文献
5.
Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis
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The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pHo). A decrease in either the dilution rate or the pHo led to a decrease in the cytoplasmic pH (pHi) of the cells, and similar levels of acid tolerance were observed at any specific pHi irrespective of whether the pHi resulted from manipulation of the growth rate, manipulation of the pHo, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pHo of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pHo of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pHi. The rate of ATP formation was not correlated with pHi, but a good correlation between the cellular level of H+-ATPase and pHi was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pHi was established. Each pHi below 6.6 was characterized by unique levels of ATR, H+-ATPase, and ATP. High levels of H+-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H+-ATPase is one of the ATR proteins induced by acid pHi through growth at an acid pHo or a slow growth rate. 相似文献
6.
The food pathogen Bacillus cereus is likely to encounter acidic environments (i) in food when organic acids are added for preservation purposes, and (ii) during the stomachal transit of aliments. In order to characterise the acid stress response of B. cereus ATCC14579, cells were grown in chemostat at different pH values (pHo from 9.0 to 5.5) and different growth rates (μ from 0.1 to 0.8 h−1), and were submitted to acid shock at pH 4.0. Cells grown at low pHo were adapted to acid media and induced a significant acid tolerance response (ATR). The ATR induced was modulated by both pHo and μ, and the μ effect was more marked at pHo 5.5. Intracellular pH (pHi) was affected by both pHo and μ. At a pHo above 6, the pHi decreased with the decrease of pHo and the increase of μ. At pHo 5.5, pHi was higher compared to pHo 6.0, suggesting that mechanisms of pHi homeostasis were induced. The acid survival of B. cereus required protein neo-synthesis and the capacity of cells to maintain their pHi and ΔpH (pHi - pHo). Haemolysin BL and non-haemolytic enterotoxin production were both influenced by pHo and μ. 相似文献
7.
Anand Rotte Melanie Eichenmüller Michael Föller 《Biochemical and biophysical research communications》2010,398(4):677-510
AMP-activated protein kinase (AMPK) is activated upon energy depletion and serves to restore energy balance by stimulating energy production and limiting energy utilization. Specifically, it enhances cellular glucose uptake by stimulating GLUT and SGLT1 and glucose utilization by stimulating glycolysis. During O2 deficiency glycolytic degradation of glucose leads to formation of lactate and H+, thus imposing an acid load to the energy-deficient cell. Cellular acidification inhibits glycolysis and thus impedes glucose utilization. Maintenance of glycolysis thus requires cellular H+ export. The present study explored whether AMPK influences Na+/H+ exchanger (NHE) activity and/or Na+-independent acid extrusion. NHE1 expression was determined by RT-PCR and Western blotting. Cytosolic pH (pHi) was estimated utilizing BCECF fluorescence and Na+/H+ exchanger activity from the Na+-dependent re-alkalinization (ΔpHi) after an ammonium pulse. As a result, human embryonic kidney (HEK) cells express NHE1. The pHi and ΔpHi in those cells were significantly increased by treatment with AMPK stimulator AICAR (1 mM) and significantly decreased by AMPK inhibitor compound C (10 μM). The effect of AICAR on pHi and ΔpHi was blunted in the presence of the Na+/H+ exchanger inhibitor cariporide (10 μM), but not by the H+ ATPase inhibitor bafilomycin (10 nM). AICAR significantly enhanced lactate formation, an effect significantly blunted in the presence of cariporide. These observations disclose a novel function of AMPK, i.e. regulation of cytosolic pH. 相似文献
8.
Internal pH (pHi) was determined inEmiliania huxleyi (Lohmann) using the probe 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pHi and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pHi was 7.29 (±0.11) for cells in the presence of 2 mM HCO
3
–
. In the absence of HCO
3
–
the pHi was decreased by 0.8 pH unit. The importance of these changes in pHi is considered in relation to inorganic-carbon uptake.Abbreviations AM
acetoxymethylester
- BCECF
2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein
- Hepes
4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid
- pHi
intracellular pH 相似文献
9.
The activation by abscisic acid (ABA) of current through outward-rectifying K+ channels and its dependence on cytoplasmic pH (pHi) was examined in stomatal guard cells of Vicia faba L. Intact guard cells were impaled with multibarrelled and H+-selective microelectrodes to record membrane potentials and pHi during exposures to ABA and the weak acid butyrate. Potassium channel currents were monitored under voltage clamp and, in some experiments, guard cells were loaded with pH buffers by iontophoresis to suppress changes in pHi. Following impalements, stable pHi values ranged between 7.53 and 7.81 (7.67±0.04, n = 17). On adding 20 M ABA, pHi rose over periods of 5–8 min to values 0.27±0.03 pH units above the pHi before ABA addition, and declined slowly thereafter. Concurrent voltage-clamp measurements showed a parallel rise in the outward-rectifying K+ channel current (IK, out) and, once evoked, both pHi and IK, out responses were unaffected by ABA washout. Acid loads, imposed with external butyrate, abolished the ABA-evoked rise in IK, out. Butyrate concentrations of 10 and 30 mM (pH0 6.1) caused pHi to fall to values near 7.0 and below, both before and after adding ABA, consistent with a cytoplasmic buffer capacity of 128±12 mM per pH unit (n = 10) near neutrality. Butyrate washout was characterised by an appreciable alkaline overshoot in pHi and concomitant swell in the steady-state conductance of IK, out. The rise in pHi and iK, out in ABA were also virtually eliminated when guard cells were first loaded with pH buffers to raise the cytoplasmic buffer capacity four- to sixfold; however, buffer loading was without appreciable effect on the ABA-evoked inactivation of a second, inward-rectifying class of K+ channels (IK, in). The pHi dependence of IK, out was consistent with a cooperative binding of at least 2H+ (apparent pKa = 8.3) to achieve a voltage-independent block of the channel. These results establish a causal link previously implicated between cytoplasmic alkalinisation and the activation of IK, out in ABA and, thus, affirm a role for H+ in signalling and transport control in plants distinct from its function as a substrate in H+-coupled transport. Additional evidence implicates a coordinate control of IK, in by cytoplasmic-free [Ca2+] and pHi.Abbreviations ABA
abscisic acid
- [Ca2+]i
cytoplasmic free [Ca2+]i
- EK
K+ equilibrium potential
- IK, out, IK, in
outward-, inward-rectifying K+ channel (current)
- I-V
current-voltage (relation)
- Mes
2-(N-morpholino)ethanesulfonic acid
- pHi
cytoplasmic pH
- Tes
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-amino}ethanesulfonic acid
- Vm
membrane potential
We are grateful to G. Thiel (Pflanzenphysiologisches Institut, Universität Göttingen, Germany) for helpful discussions. This work was possible with equipment grants-in-aid from the Gatsby Charitable Foundation, the Royal Society and the University of London Central Research Fund. F.A. holds a Sainsbury Studentship. 相似文献
10.
(1) In isolated chloroplasts (class B) electron flow is controlled mainly by the intrathylakoid pH (pHin). A decrease in pHin due to the light-driven injection of protons inside the thylakoid leads to the retardation of electron flow between two photosystems. This effect can be abolished by uncouplers or under photophosphorylation conditions (addition of Mg2+-ADP with Pi); Mg2+-ATP does not influence the steady-state rate of electron flow, (2) The steady-state pH difference, ΔpH, across the thylakoid membrane was estimated from quantitative analysis of the rate of P-700+ reduction. In chloroplasts, without adding Mg2+-ADP, ΔpH increases from 1.6 to 3.2 as the external pH rises from 6 to 9.5. Under the photophosphorylation conditions, ΔpH decreases showing a minimum at the external pH 7.5 (). (3) The value of photosynthetic control, K, measured as the ratio of the steady-state rates of P-700+ reduction in the presence of Mg2+-ADP (with Pi) and without adding Mg2+-ADP is dependent on external pH variations, showing a maximum value of at pHout 7.5. This pH dependence coincides with that of the ADP-stimulated ΔpH decrease. (4) Experiments with spin labels provide evidence that the light-induced changes in the thylakoid membrane are sensitive to the addition of uncouplers and are affected only slightly by the addition of Mg2+-ADP and Pi. 相似文献
11.
Intracellular pH (pHi) was assayed during the hormonally induced maturation of oocytes of the starfish Pisaster ochraceus. Cytoplasmic pH was measured by the DMO method, and concurrently, the initiation of maturation was determined by germinal vesicle breakdown (GVBD) and the increase in protein synthesis (percentage incorporation of amino acids). Our results indicate that (1) oocyte pHi rises slightly after initiation of maturation; (2) GVBD is not inhibited by acidifying pHi; and (3) amino acid incorporation can be affected by large changes in pHi, but not by the small pHi change promoted by maturation. Therefore, activation of GVBD and amino acid incorporation must proceed by mechanisms which do not include changing pHi. These conclusions appear to be true of oocyte activation by fertilization as well, for the pHi change following insemination is even smaller than during maturation. These results are discussed in terms of mechanisms by which dormancy is controlled. 相似文献
12.
Charles S. Owen Patricia M. Pooler Miriam L. Wahl Ronald A. Coss Dennis B. Leeper 《Journal of cellular physiology》1997,173(3):397-405
Intracellular pH (pHi) homeostasis is crucial to cell survival. Cells that are chronically exposed to a low pH environment must adapt their hydrogen ion extrusion mechanisms to maintain their pHi in the physiologic range. An important component of the adaptation to growth at low pH is the upregulation of pHi relative to the extracellular pH (pHe). To test the ability of low pHe adapted cells to respond to a pHi lowering challenge, a fluorescence assay was used that directly monitors proton removal as the rate of change of pHi during recovery from cytosolic acidification. Two cell lines of Chinese hamster origin (ovarian carcinoma and ovary fibroblastoid cells) were compared, both of which showed altered proton extrusion after adaptation to growth at low pHe = 6.70. In the ovarian carcinoma (OvCa) cell line, the pattern was consistent with an upregulation by means of an increase in the number of functional proton transporters in the plasma membrane. In the ovary fibroblastoid (CHO-10B) cell line, pHi was consistently elevated in adapted cells as compared with cells grown at normal pHe = 7.30 without an increase in maximum extrusion rate. This upregulation was consistent with a shift in the activating pHi of proton transporters without an increase in the number of transporters, i.e., a change in substrate affinity of the transporter. In OvCa cells, recovery from acidification could be blocked by amiloride, an inhibitor of Na+/H+ exchange. In contrast, a more modest effect of amiloride on CHO cells was observed but a complete inhibition was seen with the Cl−/HCO−3 exchange inhibitor 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS). These data indicate that the two cell lines rely to different degrees on the two major pathways for pH regulation during recovery from cytosolic acidification. J. Cell. Physiol. 173:397–405, 1997. © 1997 Wiley-Liss, Inc. 相似文献
13.
This study presents the effects of citric acid and extracellular pH (pHe) on the intracellular pH (pHi) of wild-type and citrate negative variants (cit−) Leuconostoc mesenteroides subsp. mesenteroides (Ln. mesenteroides M) and Lactococcus lactis subsp. lactis bv. diacetylactis (L. lactis LD). A recent method using a pH-sensitive fluorescent indicator carboxyfluorescein succinimidyl ester (cFSE) was adapted
to measure the pHi of these two lactic acid bacteria in resting cells. Energized cells with 10 mM lactose of Ln. mesenteroides M and L. lactis LD modified their pH gradient (ΔpH) in the same manner; when the pHe was decreased from 7 to 4, the pHi decreased from 7 to about 5. The adjunction of 10 mM citric acid had no effect on the pHi of wild-type and cit(−) variant of L. lactis LD, nor on the pHi of Ln. mesenteroides cit(−) variant. Nevertheless, in Ln. mesenteroides M wild-type, citric acid utilization increased the pHi, which was maintained at about 6.5–7.0 when the pHe was decreased from 7 to 4. It could be concluded that citric acid allows the maintenance of pH homeostasis in Leuconostoc mesenteroides.
Received: 7 March 1997 / Accepted: 14 April 1997 相似文献
14.
Intracellular pH (pHi) affects smooth muscle function, yet little is known concerning its regulation. I have therefore investigated pH regulation in rat uterus, using 31P-NMR spectroscopy. A change in extracellular pH(pHe) of 1 pH unit (7.4 to 6.4) elicited a 0.29 change in pHi; smaller changes in pHo were accompanied by proportionately smaller changes in pHi. The pH changes were reversible. There was no fall of uterine ATP or phosphocreatine during the pH changes. 相似文献
15.
Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques
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Lana Shabala Birgitte Budde Tom Ross Henrik Siegumfeldt Mogens Jakobsen Tom McMeekin 《Applied microbiology》2002,68(4):1794-1802
Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H+ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H+ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pHo) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pHi) and net H+ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pHi remained above 7.0 within a pHo range of 4 to 6 and decreased below pHo 4. Above pHo 4, H+ extrusion increased correspondingly, with the maximum value at pHo 5.5, and below pHo 4, a net H+ influx was observed. Without glucose in the medium, the pHi decreased, and a net H+ influx was observed below pHo 5.5. A high correlation (R = 0.75 to 0.92) between the pHi and net H+ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pHi homeostasis in L. monocytogenes subjected to acid stress. 相似文献
16.
《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1988,972(3):299-301
Intracellular pH (pHi) affects smooth muscle function, yet little is known concerning its regulation. I have therefore investigated pH regulation in rat uterus, using 31P-NMR spectroscopy. A change in extracellular pH(pHe) of 1 pH unit (7.4 to 6.4) elicited a 0.29 change in pHi; smaller changes in pHo were accompanied by proportionately smaller changes in pHi. The pH changes were reversible. There was no fall of uterine ATP or phosphocreatine during the pH changes. 相似文献
17.
Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH 总被引:8,自引:3,他引:5
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We describe the dynamics of changes in the intracellular pH (pHi) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pHex). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pHi within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pHex from 7.0 to 5.0. Under these conditions, the pHi of L. innocua remained neutral (between 7 and 8). In contrast, the pHi values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pHex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pHi at pHex 7.0, while different kinetics of pHi regulation were observed in different species and also in different strains of S. thermophilus. 相似文献
18.
Miriam L. Wahl Ronald A. Coss Suzanne B. Bobyock Dennis B. Leeper Charles S. Owen 《Journal of cellular physiology》1996,166(2):438-445
As an in vitro model for the low extracellular pH (pHe) which has frequently been observed in tumors, cell lines have been grown in a low-pH medium in order to allow cell adaptation to that milieu. Two Chinese hamster cell lines [Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa)] were compared, both of which acquired thermotolerance during 42°C heating in pHe = 7.3 buffer, but not in pHe = 6.7 medium unless grown at that pH long enough to become adapted. CHO cells, even when acutely acidified, showed higher intracellular pH (pHi) values in a suspension assay than OvCa cells, which confirmed the danger of comparing absolute values of pHi between cell lines. Despite this fundamental difference, relative changes in pHi were similar in that both lines showed a higher pHi in adapted than in unadapted cells, over the range of pHe values tested. The upregulation of pHi was statistically significant, but the two lines differed in the time frame over which adaptation occurred. OvCa cells acquired an enhanced ability to develop tolerance to 42° heat at pHe = 6.7 in 4 days, but the CHO cells acquired this ability more progressively, achieving a maximum ability at approximately 100 days. In contrast, both lines were able to upregulate their pHi within 4 hours of being exposed to pH 6.7 medium. A further indication of different biochemical mechanisms at work was the opposite effects seen on pHi in the two cell lines upon the removal of extracellular CO2/HCO−3. The differential between adapted and unadapted OvCa cells was enhanced by removal of bicarbonate, whereas CHO cells seemed less stable and the data with greater scatter failed to show any difference between adapted and unadapted cells. © 1996 Wiley-Liss, Inc. 相似文献
19.
Karen Trchounian Syuzanna Blbulyan Armen Trchounian 《Journal of bioenergetics and biomembranes》2013,45(3):253-260
Proton motive force (Δp) generation by Escherichia coli wild type cells during glycerol fermentation was first studied. Its two components, electrical—the membrane potential (?φ) and chemical—the pH transmembrane gradient (ΔpH), were established and the effects of external pH (pHex) were determined. Intracellular pH was 7.0 and 6.0 and lower than pHex at pH 7.5 and 6.5, respectively; and it was higher than pHex at pH 5.5. At high pHex, the increase of ?φ (?130 mV) was only partially compensated by a reversed ΔpH, resulting in a low Δp. At low pHex ?φ and consequently Δp were decreased. The generation of Δp during glycerol fermentation was compared with glucose fermentation, and the difference in Δp might be due to distinguished mechanisms for H+ transport through the membrane, especially to hydrogenase (Hyd) enzymes besides the F0F1-ATPase. H+ efflux was determined to depend on pHex; overall and N,N’-dicyclohexylcarbodiimide (DCCD)-inhibitory H+ efflux was maximal at pH 6.5. Moreover, ΔpH was changed at pH 6.5 and Δp was different at pH 6.5 and 5.5 with the hypF mutant lacking all Hyd enzymes. DCCD-inhibited ATPase activity of membrane vesicles was maximal at pH 7.5 and decreased with the hypF mutant. Thus, Δp generation by E. coli during glycerol fermentation is different than that during glucose fermentation. Δp is dependent on pHex, and a role of Hyd enzymes in its generation is suggested. 相似文献
20.
Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes.
In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere
with intracellular pH (pH
i
) changes in the next. Using the pH-sensitive fluorescent dye 2′,7′-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured
pH
i
in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pH
i
decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pH
i
increase caused by elevating extracellular [K+], and (iii) an acid shift in the pH
i
dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride
(EIPA). However, contamination had no effect on the pH
i
dependence of Na-H exchange, computed by adding the pH
i
dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by
using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each
experiment.
Received: 14 October 1998/Revised: 2 March 1999 相似文献