Proton Electrochemical Potential Gradient Research Articles

Algal PETC-Pro171-Leu suppresses electron transfer in cytochrome b6f under acidic lumenal conditions.

Linear photosynthetic electron flow (LEF) produces NADPH and generates a proton electrochemical potential gradient across the thylakoid membrane to synthesize ATP, both of which are required for CO2 fixation. As cellular demand for ATP and NADPH varies, cyclic electron flow (CEF) between Photosystem I and the cytochrome b6f complex (b6f) produces extra ATP. b6f regulates LEF and CEF via photosynthetic control, which is a pH-dependent b6f slowdown of plastoquinol oxidation at the lumenal site. This protection mechanism is triggered at more alkaline lumen pH in the pgr1 (proton gradient regulation 1) mutant of the vascular plant Arabidopsis (Arabidopsis thaliana), which contains a Pro194Leu substitution in the b6f Rieske Iron-sulfur protein Photosynthetic Electron Transfer C (PETC) subunit. In this work, we introduced the equivalent pgr1 mutation in the green alga Chlamydomonas reinhardtii to generate PETC-P171L. Consistent with the pgr1 phenotype, PETC-P171L displayed impaired NPQ induction along with slower photoautotrophic growth under high light conditions. Our data provide evidence that the ΔpH component in PETC-P171L depends on oxygen availability. Only under low oxygen conditions was the ΔpH component sufficient to trigger a phenotype in algal PETC-P171L where the mutant b6f was more restricted to oxidize the plastoquinol pool and showed diminished electron flow through the b6f complex. These results demonstrate that photosynthetic control of different stringency are established in C. reinhardtii depending on the cellular metabolism, and the lumen pH-sensitive PETC-P171L was generated to read out various associated effects.

Nonylphenol and Octylphenol Differently Affect Cell Redox Balance by Modulating the Nitric Oxide Signaling.

Nonylphenol (NP) and octylphenol (OP) are pervasive environmental contaminants belonging to the broader class of compounds known as alkylphenols, with potential human toxic effects. Classified as “xenoestrogens,” NP and OP are able to interfere with the cell endocrine physiology via a direct interaction with the estrogen receptors. Here, using HepG2 cells in culture, the changes of the cell redox balance and mitochondrial activity induced by OP and NP have been investigated at μM concentrations, largely below those provoking acute toxicity, as those typical of environmental contaminants. Following 24 h cell exposure to both OP and NP, ROS production appeared significantly increased (p ≤ 0.01), together with the production of higher NO oxides (p = 0.003) and peroxynitrated protein-derivatives (NP versus CTR, p = 0.003). The mitochondrial proton electrochemical potential gradient instead was decreased (p ≤ 0.05), as the oxygen consumption by complex IV, particularly following incubation with NP (NP versus CTR, p = 0.017). Consistently, the RT-PCR and Western blot analyses proved that the OP and NP can modulate to a different extent the expression of the inducible NOS (NP versus CTR, p ≤ 0.01) and the endothelial NOS (OP versus CTR, p ≤ 0.05), with a significant variation of the coupling efficiency of the latter (NP versus CTR, p ≤ 0.05), a finding that may provide a novel clue to understand the specific xenoestrogenic properties of OP and NP.

Heterogeneity of the Hydrophobic Core of a Membrane Protein Complex

The cytochrome bc1 complex utilizes two pairs of b-hemes in a mostly symmetric dimer to accomplish trans-membrane electron transfer and generation of a trans-membrane proton electrochemical potential gradient. Analysis of the structure of the complex in the purple photosynthetic bacterium, Rhodobacter capsulatus, allowed determination of the identity of the strongly interacting heme pair. Determination of the preferred heme sites of electron residence utilized Soret band excitonic circular dichroism (CD) spectra to assay heme−heme interactions. Simultaneous kinetics of heme reduction and generation of the CD spectra imply that the thermodynamically favorable pair-wise heme occupancy utilizes the intra-monomer b-heme pair that exhibits different heme redox potentials on the electrochemically positive and negative sides of the cytochrome complex. This contradicts the expectation, based on the assumption of a dielectrically homogeneous protein environment, that the two higher potential hemes on the electronegative cytoplasmic side of the complex would be preferentially reduced. Relative to the protein medium in the structure that separates the two higher potential hemes on the electronegative side of the complex, a relatively large dielectric constant must exist between the intra-monomer b-hemes on the two sides of the complex, thus allowing increased polarizabilty and a smaller electrostatic repulsion between the reduced hemes. Thus, the distribution of dielectric constants in the core of the intra-membrane cytochrome complex, which mediate inter-heme electrostatic interactions, and which are a measure of local hydrophobicity, is heterogeneous.

Role of Met58 in the regulation of electron/proton transfer in trihaem cytochrome PpcA from Geobacter sulfurreducens

The bacterium Gs (Geobacter sulfurreducens) is capable of oxidizing a large variety of compounds relaying electrons out of the cytoplasm and across the membranes in a process designated as extracellular electron transfer. The trihaem cytochrome PpcA is highly abundant in Gs and is most probably the reservoir of electrons destined for the outer surface. In addition to its role in electron transfer pathways, we have previously shown that this protein could perform e−/H+ energy transduction. This mechanism is achieved by selecting the specific redox states that the protein can access during the redox cycle and might be related to the formation of proton electrochemical potential gradient across the periplasmic membrane. The regulatory role of haem III in the functional mechanism of PpcA was probed by replacing Met58, a residue that controls the solvent accessibility of haem III, with serine, aspartic acid, asparagine or lysine. The data obtained from the mutants showed that the preferred e−/H+ transfer pathway observed for PpcA is strongly dependent on the reduction potential of haem III. It is striking to note that one residue can fine tune the redox states that can be accessed by the trihaem cytochrome enough to alter the functional pathways.

Acr3p is a plasma membrane antiporter that catalyzes As(III)/H+ and Sb(III)/H+ exchange in Saccharomyces cerevisiae

Resistance to arsenical compounds in Saccharomyces cerevisiae as well as in a growing number of prokaryotes and eukaryotes is mediated by members of the Acr3 family of transporters. In yeast cells, it has been clearly shown that Acr3p is localized to the plasma membrane and facilitates efflux of trivalent arsenic and antimony. However, until now, the energy dependence and kinetic properties of Acr3 proteins remained uncharacterized. In this work, we show that arsenite and antimonite uptake into everted membrane vesicles via the yeast Acr3 transporter is coupled to the electrochemical potential gradient of protons generated by the plasma membrane H+-translocating P-type ATPase. These results strongly indicate that Acr3p acts as a metalloid/H+ antiporter. Two differential kinetic assays revealed that Acr3p-mediated arsenite/H+ and antimonite/H+ exchange demonstrates Michaelis–Menten-type saturation kinetics characterized by a maximum flux for permeating metalloids. The approximate Km values for arsenite and antimonite transport were the same, suggesting that Acr3p exhibits similar low affinity for both metalloids. Nevertheless, the maximal velocity of the transport at saturation concentrations of metalloids was approximately 3 times higher for arsenite than for antimonite. These findings may explain a predominant role of Acr3p in conferring arsenite tolerance in S. cerevisiae.

Protein Transport Through the Anthrax Toxin Channel: Molecular Mechanisms

Bacillus anthracis, the causative agent of anthrax, produced a toxin composed of a translocase heptameric channel, (PA63)7, which allows its two substrate proteins, lethal and edema factor (LF and EF), to translocate across a host cell's endosomal membrane, disrupting the cell's normal function. Protein translocation through the channel, reconstituted in lipid bilayers, is driven (N-terminal end first) by a proton electrochemical potential gradient. The (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins, and hence the acidic side chains on LFN (the N-terminal 263 residues of LF) enter protonated; these protons are released into the trans solution upon exiting the channel, thereby making this a proton-protein symporter. Consistent with this idea, a single SO3-, which is essentially not titrateable, introduced at most positions in LFN, drastically inhibited voltage-driven LFN translocation. The lumen of the (PA63)7 14-strand β barrel is ∼15 A wide and can barely accommodate an alpha-helix with its side chains. Translocation through the lumen thus requires the substrates to unfold. Here we present an approach using biotin-streptavidin chemistry to determine the length of the translocating polypeptide chain within the channel as it is traversing the (PA63)7 channel lumen, with the goal of shedding light on the structure of the polypeptide chain as it crosses the channel. We created a stopper at the LFN C terminus and attached a biotin at the N terminus. Translocation proceeds until the C terminus reaches the channel's cis entrance, and binding of the N-terminal biotin with streptavidin added to the trans side of the membrane, locks the polypeptide chain within the channel. By reducing the distance between the N-terminal biotin and the C-terminal stopper by deletion constructs, we can determine the minimum length that allows streptavidin to grab the N-terminal biotin.

Structure-Function, Stability, and Chemical Modification of the Cyanobacterial Cytochrome b6f Complex from Nostoc sp. PCC 7120

The crystal structure of the cyanobacterial cytochrome b(6)f complex has previously been solved to 3.0-A resolution using the thermophilic Mastigocladus laminosus whose genome has not been sequenced. Several unicellular cyanobacteria, whose genomes have been sequenced and are tractable for mutagenesis, do not yield b(6)f complex in an intact dimeric state with significant electron transport activity. The genome of Nostoc sp. PCC 7120 has been sequenced and is closer phylogenetically to M. laminosus than are unicellular cyanobacteria. The amino acid sequences of the large core subunits and four small peripheral subunits of Nostoc are 88 and 80% identical to those in the M. laminosus b(6)f complex. Purified b(6)f complex from Nostoc has a stable dimeric structure, eight subunits with masses similar to those of M. laminosus, and comparable electron transport activity. The crystal structure of the native b(6)f complex, determined to a resolution of 3.0A (PDB id: 2ZT9), is almost identical to that of M. laminosus. Two unique aspects of the Nostoc complex are: (i) a dominant conformation of heme b(p) that is rotated 180 degrees about the alpha- and gamma-meso carbon axis relative to the orientation in the M. laminosus complex and (ii) acetylation of the Rieske iron-sulfur protein (PetC) at the N terminus, a post-translational modification unprecedented in cyanobacterial membrane and electron transport proteins, and in polypeptides of cytochrome bc complexes from any source. The high spin electronic character of the unique heme c(n) is similar to that previously found in the b(6)f complex from other sources.

Evidence for a Proton–Protein Symport Mechanism in the Anthrax Toxin Channel

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LFN enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton–protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO3−, which is essentially not titratable, is introduced at most positions in LFN, through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LFN translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA63)7 channel resides at or near its Φ-clamp, the ring of seven phenylalanines near the channel's entrance.

Evidence for Proton-Coupled Protein Transport through the Anthrax Toxin Channel

The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of a translocase heptameric channel, (PA63)7, formed from protective antigen (PA), which allows its two substrate proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting the cellular homeostasis. It has been shown that (PA63)7 incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF, EF and other small proteins. Protein translocation through the channel is driven by a proton electrochemical potential gradient, on a time scale of seconds with S-shaped kinetics. A paradoxical aspect of this is that although LFN (the N-terminal 263 residues of LF), on which most of our experiment were performed, has a net negative charge, it is driven through the channel by a cis positive voltage. We have explained this by claiming that the (PA63)7 channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LFN enter protonated (i.e. neutralized), and therefore the translocated species is positively charged. Upon exiting the channel, these protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO3−, which is essentially not titrateable, is introduced at most positions in LFN, through the reaction of a cysteine-modified residue at those positions with (2-Sulfonatoethyl) Methanethiosulfonate (MTS-ES), voltage-driven LFN translocation is drastically inhibited.

Iron Imports. II. Iron uptake at the apical membrane in the intestine

How does iron enter enterocytes? Ablating SLC11A2, the gene for the divalent metal ion transporter DMT1, supports evidence from the Belgrade rat and mk mouse models establishing DMT1 as the primary mechanism serving apical uptake of nonheme iron. DMT1 harnesses the energy from the proton electrochemical potential gradient to drive active transport of Fe(2+) (and perhaps Mn(2+) and other metal ions) into enterocytes. Fe(III) must first be reduced by ascorbic acid and surface ferrireductases. Among these is duodenal cytochrome B (DcytB), but lack of an obvious phenotype in DcytB (Cybrd1) knockout mice suggests ferrireductase redundancy. Our understanding of heme absorption has lagged, but the time is ripe for gains.

Nitrogenase activity and membrane electrogenesis in the cyanobacterium Anabaena variabilis Kütz.

Relationships between nitrogenase activity and individual components of the proton electrochemical potential gradient (delta microH+) in Anabaena variabilis have been investigated. The ionophore nigericin was found to collapse delta pH in favour of the membrane potential (delta psi); hyperpolarization of delta psi was correlated with an increase in nitrogenase activity. A positive relationship between nitrogenase activity and membrane potential was also observed using the ionophore valinomycin and the uncoupler carbonylcyanide m-chlorophenylhydrazone. Furthermore, using the energy transfer inhibitor N,N'-dicyclohexylcarbodiimide, nitrogenase activity appeared to be limited by the supply of reductant rather than ATP. The data suggest, for intact filaments and isolated heterocysts, that delta psi may be important in regulating nitrogenase activity in vivo.

Uncoupling proteins 2 and 3: potential regulators of mitochondrial energy metabolism.

Mitochondria use energy derived from fuel combustion to create a proton electrochemical gradient across the mitochondrial inner membrane. This intermediate form of energy is then used by ATP synthase to synthesize ATP. Uncoupling protein-1 (UCP1) is a brown fat-specific mitochondrial inner membrane protein with proton transport activity. UCP1 catalyzes a highly regulated proton leak, converting energy stored within the mitochondrial proton electrochemical potential gradient to heat. This uncouples fuel oxidation from conversion of ADP to ATP. In rodents, UCP1 activity and brown fat contribute importantly to whole-body energy expenditure. Recently, two additional mitochondrial carriers with high similarity to UCP1 were molecularly cloned. In contrast to UCP1, UCP2 is expressed widely, and UCP3 is expressed preferentially in skeletal muscle. Biochemical studies indicate that UCP2 and UCP3, like UCP1, have uncoupling activity. While UCP1 is known to play an important role in regulating heat production during cold exposure, the biological functions of UCP2 and UCP3 are unknown. Possible functions include 1) control of adaptive thermogenesis in response to cold exposure and diet, 2) control of reactive oxygen species production by mitochondria, 3) regulation of ATP synthesis, and 4) regulation of fatty acid oxidation. This article will survey present knowledge regarding UCP1, UCP2, and UCP3, and review proposed functions for the two new uncoupling proteins.

Decreased ATP synthesis is phenotypically expressed during increased energy demand in fibroblasts containing mitochondrial tRNA mutations.

Mutations in the tRNA genes of mitochondrial DNA (mtDNA) cause the debilitating MELAS (mitochondrial, myopathy, encephalopathy, lactic acidosis and stroke-like episodes) and MERRF (myoclonic epilepsy and ragged-red fibres) syndromes. These mtDNA mutations affect respiratory chain function, apparently without decreasing cellular ATP concentration [Moudy et al. (1995) PNAS, 92, 729-733]. To address this issue, we investigated the role of mitochondrial ATP synthesis in fibroblasts from MELAS and MERRF patients. The maximum rate of mitochondrial ATP synthesis was decreased by 60-88%, as a consequence of the decrease in the proton electrochemical potential gradient of MELAS and MERRF mitochondria. However, in quiescent fibroblasts neither ATP concentration or the ATP/ADP ratio was affected by the lowered rate of ATP synthesis. We hypothesized that the low ATP demand of quiescent fibroblasts masked the mitochondrial ATP synthesis defect and that this defect might become apparent during higher ATP use. To test this we simulated high energy demand by titrating cells with gramicidin, an ionophore that stimulates ATP hydrolysis by the plasma membrane Na+/K+-ATPase. We found a threshold gramicidin concentration in control cells at which both the ATP/ADP ratio and the plasma membrane potential decreased dramatically, due to ATP demand by the Na+/K+-ATPase outstripping mitochondrial ATP synthesis. In MELAS and MERRF fibroblasts the corresponding threshold concentrations of gramicidin were 2-20-fold lower than those for control cells. This is the first demonstration that cells containing mtDNA mutations are particularly sensitive to increased ATP demand and this has several implications for how mitochondrial dysfunction contributes to disease pathophysiology. In particular, the increased susceptibility to plasma membrane depolarization will render neurons with dysfunctional mitochondria susceptible to excitotoxic cell death.

Deletion of amino acids 261-269 in the brown fat uncoupling protein converts the carrier into a pore.

The uncoupling protein (UCP) from brown adipose tissue mitochondria is a carrier that catalyzes proton re-entry into the matrix and thus dissipates the proton electrochemical potential gradient as heat. UCP activity is regulated: purine nucleotides inhibit while fatty acids activate transport. We have previously reported that sequence 261-269 of the UCP has a closely related counterpart in the adenine nucleotide translocator, as well as in the DNA binding domain of the estrogen receptor. Site-directed mutagenesis of the UCP showed that deletion of amino acids 267-269 in the UCP abolished nucleotide inhibition [Bouillaud, F., et al. (1994) EMBO J. 13, 1990-1997]. Complete deletion of the homologous domain (UCPDelta9) produced a highly deleterious mutant that collapsed the mitochondrial membrane potential and halted yeast growth. Since under our growth conditions revertants appeared rapidly, it was not possible to characterize this mutant. In this article, we have designed conditions to isolate mitochondria containing significant amounts of the UCPDelta9 mutant protein. These mitochondria show no respiratory control and are insensitive to nucleotides. Investigation of the permeability properties revealed that UCPDelta9 mitochondria swell rapidly in potassium salts in the absence of valinomycin, thus indicating a loss of specificity. The size exclusion properties of this mutant were determined with polyethylene glycols of various molecular masses (400-20000 Da), and it was found that UCPDelta9 can catalyze permeation of molecules of up to 1000 Da. We conclude that the deletion of amino acids 261-269 converts the UCP into an unspecific pore.

Thermodynamic significance of the lactate gradient.

The theory that some bacteria can save energy by an energy-recycling process, in which protons are excreted with metabolic end-products with variable stoichiometry, has been examined by 1H-NMR. A method has been developed that utilises observed differences in the Hahn T2 relaxation of metabolites in the intracellular and extracellular compartments to distinguish and quantify metabolite signals originating from both compartments. It was found that the lactate electrochemical-potential gradient calculated from the fraction of lactate that is sufficiently mobile to contribute to the NMR signal was in exact balance with the proton electrochemical-potential gradient over a wide range of pH values. The conclusion was reached that previous reports of variable stoichiometry were due to 'bound' lactate at high intracellular pH that could neither contribute neither to the NMR signal nor to the lactate electrochemical-potential gradient.

Proton flux measurements from tissues in buffered solution

ABSTRACTProton movement across plant cell membranes is part of many important physiological processes. The net proton flux to or from tissues can be determined non‐invasively by measuring the proton electrochemical potential gradient in the adjacent solution. In buffered solution, some of the protons crossing the tissue boundary diffuse as proto‐nated buffer whose flux is not included in the flux calculated from the proton (hydrogen ion) electrochemical gradient. In this theoretical paper, it is shown how experimenters can calculate the protonated buffer flux from the measured proton flux in solution. The ratio of these two components of total proton flux depends on the pH of the solution and on the concentration and pK of the buffer. For a given concentration of a buffer which has a single pK, the flux ratio rises with pH when the solution pH is lower than the buffer pK. The slope is about 2 on a log10 scale. As the pH increases above the pK, the flux ratio levels off to approach its maximum. With mixed buffers, or one having two or more pK values, the flux ratios are additive: each buffer acts independently based on its concentration and its pK value. Unbuffered solutions always have the buffering effects of water itself and also of carbonates due to carbon dioxide dissolved from the atmosphere. In unbuffered solutions at pH 6, the flux carried by water and carbonate is about 1 % of the measured proton flux. This validates measurements of proton flux from tissues, made by a number of workers, in unbuffered solutions below pH 6.

Lonidamine-mediated respiratory changes in rat heart myocytes: A re-examination of the functional response of mitochondrial cytochrome c oxidase

Respiratory activity of intact cardiac myocytes isolated from rats treated with lonidamine (LND) has been examined under conditions where cytochrome oxidase turns over at its maximal rate. Compared to myocytes isolated from control rat hearts, those treated with LND displayed a 60% increase in the cytochrome oxidase-dependent rate of respiration; electron microscopy revealed, in agreement with the literature, that the membrane structure of the mitochondrion had become disorganized. The increase in the rate of oxygen consumption was correlated with the (partial) impairment of the membrane ability to maintain the proton electrochemical potential gradient which normally inhibits oxidase activity. Results are discussed with reference to previous reports showing no effect of LND on cytochrome c oxidase activity. The evidence reported better clarifies the contribution of cytochrome oxidase to the demonstrated energetic failure displayed by cells treated with LND.

Proton-translocating transhydrogenase and NAD- and NADP-linked isocitrate dehydrogenases operate in a substrate cycle which contributes to fine regulation of the tricarboxylic acid cycle activity in mitochondria.

H(+)-transhydrogenase (H(+)-Thase) and NADP-linked isocitrate dehydrogenase (NADP-ICDH) are very active in animal mitochondria but their physiological function is only poorly understood. This is especially so in the case of the heart and muscle, where there are no major consumers of NADPH. We propose here that H(+)-Thase and NADP-ICDH have a combined function in the fine regulation of the activity of the tricarboxylic acid (TCA) cycle, providing enhanced sensitivity to changes in energy demand. This is achieved through cycling of substrates by NAD-linked ICDH, NADP-linked ICDH and H(+)-Thase. It is proposed that NAD-ICDH operates in the forward direction of the TCA cycle, but NADP-ICDH is driven in reverse by elevated levels of NADPH resulting from the action of the transmembrane proton electrochemical potential gradient (delta p) on H(+)-Thase. This has the effect of increasing the sensitivity to allosteric modifiers of NAD-ICDH (NADH, ADP, ATP, Ca2+ etc), potentially giving rise to large changes in the net flux from iso-citrate to alpha-ketoglutarate. Furthermore, changes in the level of delta p resulting from changes in the demand for ATP would, via H(+)-Thase, shift the redox state of the NADP pool and this, in turn, would lead to a change in the rate of the reaction catalysed by NADP-ICDH and hence to an additional and complementary effect on the net metabolic flux from isocitrate to alpha-ketoglutarate. Other consequences of this substrate cycle are, (i) the production of heat at the expense of delta p, which may contribute to thermoregulation in the animal, and (ii) an increased rate of dissipation of delta p (leak).

Ca 2+ transport in isolated mitochondrial vesicles from Leishmania braziliensis promastigotes

Leishmania braziliensis maintained very low (50±20 nM) intracellular concentrations of calcium ions under normal conditions, as shown by the fluorimetric indicator QUIN2. Digitonin-permeabilized cells liberated large amounts of calcium ions in the presence of the ionophore A23187, indicating the presence of a large intracellular reservoir for this ion. Given the extraordinary extension of the single giant mitochondrion of Kinetoplastida and the known capacity of mitochondria from other sources to accumulate calcium, we tested the capacity of this organelle to accumulate calcium ions in Leishmania. Coupled mitochondrial vesicles, five-fold enriched in succinate-cytochrome c oxidoreductase, were obtained from promastigotes by gentle grinding (45 s) with glass beads in hypertonic buffer solution, followed by differential centrifugation. These vesicles had a respiratory control ratio of 1.82±0.15, and two phosphorylation sites (sites II and III) using succinate as electron donor, and were capable of calcium uptake in the presence of several respiratory substrates; this uptake was enhanced in the presence of ADP and P i and was blocked by classical electron transport inhibitors. Uncouplers such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP) and the calcium ionophore A23187 released previously accumulated calcium ions, suggesting that the driving force for the calcium uptake by the vesicles is the respiratory generated electrochemical potential gradient of protons. A study of the affinity of this system for calcium showed that even at 90μM free calcium, succinate-induced calcium uptake is not saturated while approaching a level of 200 nmol min −1 (mg protein) −1, indicating a low-affinity, large-capacity system. These results indicate the the Leishmania mitochondrion is involved in calcium ion homeostasis in these cells; not in the maintenance of the basal cytoplasmic levels, but most probably in the recovery from large fluctuations.

Uncoupling of oxidative phosphorylation. 1. Protonophoric effects account only partially for uncoupling.

The mechanism of uncoupling of oxidative phosphorylation by carbonyl cyanide p-trifluoromethoxy)phenylhydrazone (FCCP), a typical weak acid protonophore, oleic acid, a fatty acid, and chloroform, a general anesthetic, has been investigated by measuring in mitochondria their effect on (i) the transmembrane proton electrochemical potential gradient (delta mu H) and the rates of electron transfer and adenosine 5'-triphosphate (ATP) hydrolysis in static head, (ii) delta mu H and the rates of electron transfer and ATP synthesis in state 3, and (iii) the membrane proton conductance. Both FCCP and oleic acid increase the membrane proton conductance, and accordingly, they cause a depression of delta mu H [generated by either the redox proton pumps or the adenosinetriphosphatase (ATPase) proton pumps]. Although their effects on ATP synthesis/hydrolysis, respiration, and delta mu H are qualitatively consistent with a pure protonophoric uncoupling mechanism and an additional inhibitory action of oleic acid on both the ATPases and the electron-transfer enzymes, a quantitative comparison between the dissipative proton influx and the rate of either electron transfer or ATP hydrolysis (multiplied by either the H+/e- or the H+/ATP stoichiometry, respectively) at the same delta mu H shows that the increase in membrane conductance induced by FCCP and oleic acid accounts for the stimulation of the rate of ATP hydrolysis but not for that of the rate of electron transfer. Chloroform (at concentrations that fully inhibit ATP synthesis) only very slightly increases the proton conductance of the mitochondrial membrane and causes only a little depression of delta mu H.(ABSTRACT TRUNCATED AT 250 WORDS)