Transmembrane electrical potential and transmembrane pH gradient in the acidophile Thiobacillus ferro-oxidans.
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ABSTRACT:
SUBMITTER: Cox JC
PROVIDER: S-EPMC1186496 | biostudies-other | 1979 Jan
REPOSITORIES: biostudies-other
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Project description:1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.
| S-EPMC1162762 | biostudies-other
Project description:ATP synthase is the key player of Mitchell's chemiosmotic theory, converting the energy of transmembrane proton flow into the high energy bond between ADP and phosphate. The proton motive force that drives this reaction consists of two components, the pH difference (?pH) across the membrane and transmembrane electrical potential (??). The two are considered thermodynamically equivalent, but kinetic equivalence in the actual ATP synthesis is not warranted, and previous experimental results vary. Here, we show that with the thermophilic Bacillus PS3 ATP synthase that lacks an inhibitory domain of the ? subunit, ?pH imposed by acid-base transition and ?? produced by valinomycin-mediated K(+) diffusion potential contribute equally to the rate of ATP synthesis within the experimental range examined (?pH -0.3 to 2.2, ?? -30 to 140 mV, pH around the catalytic domain 8.0). Either ?pH or ?? alone can drive synthesis, even when the other slightly opposes. ?? was estimated from the Nernst equation, which appeared valid down to 1 mm K(+) inside the proteoliposomes, due to careful removal of K(+) from the lipid.
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Project description:Reliable ammonia quantification assays are essential for monitoring ammonemia in patients with liver diseases. In this study, we describe the development process of a microplate-based assay for accurate, precise, and robust ammonia quantification in biological fluids, following regulatory guidelines on bioanalytical method validation. The assay is based on transmembrane pH-gradient polymersomes that encapsulate a pH-sensitive ratiometric fluorophore, the fluorescence signal of which correlates with the ammonia concentration in the sample. Using a four-parameter logistic regression, the assay had a large quantification range (30-800 μM ammonia). As for selectivity, the presence of amino acids or pyruvate (up to clinically relevant concentrations) showed no assay interference. In samples with low bilirubin levels, polymersomes containing the fluorophore pyranine provided accurate ammonia quantification. In samples with high bilirubin concentrations, billirubin's optical interference was alleviated when replacing pyranine with a close to near-infrared hemicyanine fluorophore. Finally, the assay could correctly retrieve the ammonia concentration in ammonia-spiked human plasma samples, which was confirmed by comparing our measurements with the data obtained using a commercially available point-of-care device for ammonia.
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Project description:1. The effects of various inhibitors of electron transport and of oxidative phosphorylation and the effects of ionophores on the uptake of native aspartate aminotransferase into mitochondria were investigated. 2. Both antimycin and cyanide completely inhibited the uptake of the enzyme. On the other hand, uptake was stimulated to ATP and by oligomycin; however, the stimulation by ATP is inhibited by oligomycin. 3. The effects of ionophores of the valinomycin type in media containing K+ ions depended on the conditions used. Valinomycin alone stimulated the uptake of the enzyme, but in the presence of phosphate ions uptake was abolished. Nonactin was without effect at a low K+ concentration, but was stimulatory at 100 mM-KCl. Gramicidin also stimulated the uptake process. 4. Nigericin completely abolished uptake of aspartate aminotransferase into mitochondria. 5. The uptake of te enzyme was decreased by 18% in the absence of inhibitors or ionophores when the external pH was increased from 6.9 to 7.6. 6. These results indicate that ATP is not directly involved in the uptake of aspartate aminotransferase into mitochondria, neither is there a requirement for a cation gradient. Rather the uptake depends on the maintenance of a pH gradient across the mitochondrial inner membrane.
| S-EPMC1158118 | biostudies-other
Project description:The 'blue' copper-containing protein rusticyanin was purified to homogeneity from cells of the chemolithotrophic bacterium Thiobacillus ferro-oxidans by (NH4)SO4 fractionation and ion-exchange chromatography. The protein, which is stable at low pH, consists of a single polypeptide chain of mol. wt. 16500 and possesses 0.79 (+/- 0.28)g-atom of Cu/mol. The protein, which does not contain arginine residues, has optical absorbance maxima at 287, 450, 597 and 750 nm and is generally similar to azurin. The isolated protein is reduced directly by Fe2+ with a 1:1 stoicheiometry to Cu. On reduction by Fe2+ the absorption peaks at 450, 597 and 750 nm are abolished, with the appearance of a new absorption band at 320 nm. The results obtained are consistent with rusticyanin being the initial acceptor of electrons from Fe2+ during respiratory iron oxidation.
| S-EPMC1185940 | biostudies-other
Project description:Suspended aqueous aerosol droplets (<50 µm) are microreactors for many important atmospheric reactions. In droplets and other aquatic environments, pH is arguably the key parameter dictating chemical and biological processes. The nature of the droplet air/water interface has the potential to significantly alter droplet pH relative to bulk water. Historically, it has been challenging to measure the pH of individual droplets because of their inaccessibility to conventional pH probes. In this study, we scanned droplets containing 4-mercaptobenzoic acid-functionalized gold nanoparticle pH nanoprobes by 2D and 3D laser confocal Raman microscopy. Using surface-enhanced Raman scattering, we acquired the pH distribution inside approximately 20-µm-diameter phosphate-buffered aerosol droplets and found that the pH in the core of a droplet is higher than that of bulk solution by up to 3.6 pH units. This finding suggests the accumulation of protons at the air/water interface and is consistent with recent thermodynamic model results. The existence of this pH shift was corroborated by the observation that a catalytic reaction that occurs only under basic conditions (i.e., dimerization of 4-aminothiophenol to produce dimercaptoazobenzene) occurs within the high pH core of a droplet, but not in bulk solution. Our nanoparticle probe enables pH quantification through the cross-section of an aerosol droplet, revealing a spatial gradient that has implications for acid-base-catalyzed atmospheric chemistry.
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Project description:Glucagon treatment of rats allowed the isolation of liver mitochondria with enhanced rates of pyruvate metabolism measured in either sucrose or KCl media. No change in the activity of the pyruvate carrier itself was apparent, but under metabolizing conditions, use of the inhibitor of pyruvate transport, alpha-cyano-4-hydroxycinnamate, demonstrated that pyruvate transport limited the rate of pyruvate metabolism. The maximum rate of transport under metabolizing conditions was enhanced by glucagon treatment. Problems involved in measuring the transmembrane pH gradient under metabolizing conditions are discussed and a variety of techniques are used to estimate the matrix pH. From the distribution of methylamine, ammonia and D-lactate and the Ki for inhibition by alpha-cyano-4-hydroxycinnamate it is concluded that the matrix is more acid than the medium and that the pH of the matrix rises after glucagon treatment. The increase in matrix pH stimulates pyruvate transport. The membrane potential, ATP concentration and O2 uptake were also increased under metabolizing conditions in glucagon-treated mitochondria. These changes were correlated with a stimulation of the respiratory chain which can be observed in uncoupled mitochondria [Yamazaki (1975) J. Biol. Chem. 250, 7924--7930]. The mitochondrial Mg2+ content (mean +/- S.E.M.) was increased from 38.8 +/- 1.2 (n = 26) to 47.5 +/- 2.0 (n = 26) ng-atoms/mg by glucagon and the K+ content from 126.7 +/- 10.3 (n = 19) ng-atoms/mg. This may represent a change in membrane potential induced by glucagon in vivo. The physiological significance of these results in the control of gluconeogenesis is discussed.
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Project description:The mitochondrial electrochemical gradient (?p), which comprises the pH gradient (?pH) and the membrane potential (??), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (·O2-) and H2O2 overproduction leading to oxidative injury. However, the role of ?pH and ?? in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced ·O2- and H2O2 production when compared to state 2 conditions. Oligomycin (increases ?pH by inhibiting ATP synthase) increased ·O2- and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial ·O2- and H2O2 generation is dependent on ?pH and that IR impaired the ?pH of normal mitochondria. Conversely, nigericin (dissipates ?pH) dramatically reduced ·O2- and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ?pH impairment in normal mitochondria. Valinomycin, a K+ ionophore used to dissipate ??, drastically diminished ·O2- and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ?? also contributed to ·O2- generation by normal mitochondria and that IR mediated ?? impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ?pH restricts electron flow, controls a limited amount of ·O2- generation, and results in a more reduced myocardium; however, IR causes ?pH impairment and prompts a more oxidized myocardium.
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Project description:The ventricular gradient, an electrocardiographic concept calculated by integrating the area under the QRS complex and T-wave, represents the degree and direction of myocardial electrical heterogeneity. Although the concept of the ventricular gradient was first introduced in the 1930s, it has not yet found a place in routine electrocardiography. In the modern era, it is relatively simple to calculate the ventricular gradient in three dimensions (the spatial ventricular gradient (SVG)), and there is now renewed interest in using the SVG as a tool for risk stratification of ventricular arrhythmias and sudden cardiac death. This manuscript will review the history of the ventricular gradient, describe its electrophysiological meaning and significance, and discuss its clinical utility.
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Project description:Solid tumors have an acidic extracellular pH (pHe ) but near neutral intracellular pH (pHi ). Because acidic pHe milieu is conducive to tumor growth and builds resistance to therapy, simultaneous mapping of pHe inside and outside the tumor (i.e., intratumoral-peritumoral pHe gradient) fulfills an important need in cancer imaging. We used Biosensor Imaging of Redundant Deviation in Shifts (BIRDS), which utilizes shifts of non-exchangeable protons from macrocyclic chelates (e.g., 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylene phosphonate) or DOTP(8-) ) complexed with paramagnetic thulium (Tm(3) (+) ) ion, to generate in vivo pHe maps in rat brains bearing 9L and RG2 tumors. Upon TmDOTP(5-) infusion, MRI identified the tumor boundary by enhanced water transverse relaxation and BIRDS allowed imaging of intratumoral-peritumoral pHe gradients. The pHe measured by BIRDS was compared with pHi measured with (31) P-MRS. In normal tissue, pHe was similar to pHi , but inside the tumor pHe was lower than pHi . While the intratumoral pHe was acidic for both tumor types, peritumoral pHe varied with tumor type. The intratumoral-peritumoral pHe gradient was much larger for 9L than RG2 tumors because in RG2 tumors acidic pHe was found in distal peritumoral regions. The increased presence of Ki-67 positive cells beyond the RG2 tumor border suggested that RG2 was more invasive than the 9L tumor. These results indicate that extensive acidic pHe beyond the tumor boundary correlates with tumor cell invasion. In summary, BIRDS has sensitivity to map the in vivo intratumoral-peritumoral pHe gradient, thereby creating preclinical applications in monitoring cancer therapeutic responses (e.g., with pHe -altering drugs). Copyright © 2016 John Wiley & Sons, Ltd.
| S-EPMC4769673 | biostudies-literature