Transmembrane Proton Flux Research Articles

A method for separating tonoplast from wheat

Vacuoles account for 90% of plant cell volume and play important roles in maintaining osmotic pressure, storing metabolites and lysosomes, compartmentalizing harmful ions, and storing and reusing minerals. These functions closely relay on the ion channels and transporters located on the tonoplast. The separation of intact vacuoles from plant cells is the key technology utilized in the study of tonoplast-located ion channels and transporters. However, the current vacuole separation methods are available for Arabidopsis and some other dicotyledons but are lacking for monocot crops. In this study, we established a new method for the vacuole separation from wheat mesophyll cells and investigated the transmembrane proton flux of tonoplasts with non-invasive micro-test technology (NMT). Moreover, our study provides a technology for the study of vacuole functions in monocot crops.

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering.

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (>10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 µL), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

Microfluidic formation and manipulation of OLA-based liposomes for synthetic biology research.

Bottom-up synthetic biology aims to create biomimetic systems from the basic elements of cellular life. Such synthetic cell analogs may support scientists in understanding the fundamental biological behaviors at the cellular level. As microscopic aqueous compartments confined within a lipid bilayer, liposomes have been widely used as simplistic cellular mimics for synthetic cell studies. Here, we describe the manufacturing protocol for octanol-assisted liposome assembly (OLA) to produce unilamellar, monodispersed, cell-sized, and biocompatible liposomes as well as in situ liposome experiments. We use maskless, direct-write lithography, which allows for rapid prototyping, to fabricate PDMS-based lab-on-a-chip devices. OLA makes use of the double emulsion generation and dewetting purification approach, with the membrane composition of the formed liposomes and the nature of the encapsulated solution widely adjustable. Using highly responsive pressure-driven flow, the liposome size is tuneable with a steady liposome generation, and the experiment requires very low sample volumes (∼50 µl). We enrich the liposomes from the waste products using density-based on-chip separation techniques, which allows us to conduct experiments on the same device with real-time observation, right after production. As a proof-of-principle synthetic biology application, we demonstrate the capability of adjusting the microenvironment by inducing pH-controlled coacervation between ATP and poly-Llysine molecules inside the liposomes via transmembrane proton flux. Such liquid-liquid phase separation, creating multiple distinct phases from a single homogeneous mixture, is known to play a key role in a variety of cellular processes, including the formation of membraneless organelles as well as numerous other supramolecular assemblies. Our technique shows promising potential for synthetic cell research, particularly in terms of step-by-step construction and experimental control.

Proton coupling and the multiscale kinetic mechanism of a peptide transporter

Proton-coupled peptide transporters (POTs) are crucial for the uptake of di- and tripeptides as well as drug and prodrug molecules in prokaryotes and eukaryotic cells. We illustrate from multiscale modeling how transmembrane proton flux couples within a POT protein to drive essential steps of the full functional cycle: 1) protonation of a glutamate on transmembrane helix 7 (TM7) opens the extracellular gate, allowing ligand entry; 2) inward proton flow induces the cytosolic release of ligand by varying the protonation state of a second conserved glutamate on TM10; 3) proton movement between TM7 and TM10 is thermodynamically driven and kinetically permissible via water proton shuttling without the participation of ligand. Our results, for the first time, give direct computational confirmation for the alternating access model of POTs, and point to a quantitative multiscale kinetic picture of the functioning protein mechanism.

PH-Controlled Coacervate-Membrane Interactions within Liposomes.

Membraneless organelles formed by liquid–liquid phase separation are dynamic structures that are employed by cells to spatiotemporally regulate their interior. Indeed, complex coacervation-based phase separation is involved in a multitude of biological tasks ranging from photosynthesis to cell division to chromatin organization, and more. Here, we use an on-chip microfluidic method to control and study the formation of membraneless organelles within liposomes, using pH as the main control parameter. We show that a transmembrane proton flux that is created by a stepwise change in the external pH can readily bring about the coacervation of encapsulated components in a controlled manner. We employ this strategy to induce and study electrostatic as well as hydrophobic interactions between the coacervate and the lipid membrane. Electrostatic interactions using charged lipids efficiently recruit coacervates to the membrane and restrict their movement along the inner leaflet. Hydrophobic interactions via cholesterol-tagged RNA molecules provide even stronger interactions, causing coacervates to wet the membrane and affect the local lipid-membrane structure, reminiscent of coacervate–membrane interactions in cells. The presented technique of pH-triggered coacervation within cell-sized liposomes may find applications in synthetic cells and in studying biologically relevant phase separation reactions in a bottom-up manner.

Tuning biomimetic membrane barrier properties by hydrocarbon, cholesterol and polymeric additives

The barrier properties of cellular membranes are increasingly attracting attention as a source of inspiration for designing biomimetic membranes. The broad range of potential technological applications makes the use of lipid and lately also polymeric materials a popular choice for constructing biomimetic membranes, where the barrier properties can be controlled by the composition of the membrane constituent elements. Here we investigate the membrane properties reported by the light-induced proton pumping activity of bacteriorhodopsin (bR) reconstituted in three vesicle systems of different membrane composition. Specifically we quantify how the resulting proton influx and efflux rates are influenced by the membrane composition using a variety of membrane modulators. We demonstrate that by adding hydrocarbons to vesicles with reconstituted bR formed from asolectin lipids the resulting transmembrane proton fluxes changes proportional to the carbon chain length when compared against control. We observe a similar proportionality in single-component 1,2-Dioleoyl-sn-glycero-3-phosphocholine model membranes when using cholesterol. Lastly we investigate the effects of adding the amphiphilic di-block co-polymer polybutadiene–polyethyleneoxide (PB12–PEO10) to phospholipid membranes formed from 1,2-Dioleoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphatidylethanolamine, and 1,2-Dioleoyl-sn-glycero-3-phosphatidylserine. The proton pumping activity of bR (measured as a change in extra-vesicular pH) in mixed lipid/PB12–PEO10 lipid systems is up to six-fold higher compared to that observed for bR containing vesicles made from PB12–PEO10 alone. Interestingly, bR inserts with apparent opposite orientation in pure PB12–PEO10 vesicles as compared to pure lipid vesicles. Addition of equimolar amounts of lipids to PB12–PEO10 results in bR orientation similar to that observed for pure lipids. In conclusion our results show how the barrier properties of the membranes can be controlled by the composition of the membrane. In particular the use of mixed lipid-polymer systems may pave the way for constructing biomimetic membranes tailored for optimal properties in various applications including drug delivery systems, biosensors and energy conservation technology.

Novel Drugs Targeting the c-Ring of the F1FO-ATP Synthase.

Increasing evidence highlights the role of the ATP synthase/hydrolase, also known as F1FO-complex, as key molecular and enzymatic switch between cell life and death, thus increasing the enzyme attractiveness as drug target in pharmacology. Being inhibition of ATP production usually linked to antiproliferative properties, drugs targeting the enzyme complex have been mainly considered to fight pathogen parasites and cancer. In recent years, a number of natural macrolides, produced by bacterial fermentation and structurally related to the classical enzyme inhibitor oligomycin, have been shown to bind to the membrane-embedded FO sector and to inhibit the enzyme complex by an oligomycin-like mechanism, namely by interacting with the c-ring. Other than natural macrolide antibiotics, which display variegated inhibition power on different F1FO-complexes, synthetic compounds from the diarylquinoline and organotin families also target the c-ring and strongly inhibit the enzyme. Bioinformatic insights address drug design to target FO subunits. Additionally, the possible modulation of the drug inhibition power, by amino acid substitutions or post-translational modifications of c-subunits, adds further interest to the target. The present survey on compounds targeting the c-ring and bi-directionally blocking the transmembrane proton flux which drives ATP synthesis/hydrolysis, discloses new therapeutic options to fight cancer and infections sustained by therapeutically recalcitrant microorganisms. Additionally, c-ring targeting compounds may constitute new tools to eradicate undesired biofilms and to address at the molecular level the therapy of mammalian diseases linked to mitochondrial dysfunctions. In summary, studies on the only partially known molecular interactions within the c-ring of the F1FO-complex may renew hope to counteract mammalian diseases.

A biophysical study on molecular physiology of the uncoupling proteins of the central nervous system.

Mitochondrial inner membrane uncoupling proteins (UCPs) facilitate transmembrane (TM) proton flux and consequently reduce the membrane potential and ATP production. It has been proposed that the three neuronal human UCPs (UCP2, UCP4 and UCP5) in the central nervous system (CNS) play significant roles in reducing cellular oxidative stress. However, the structure and ion transport mechanism of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1in bacterial membranes and applied this system to obtain highly pure neuronal UCPs in high yields. In the present study, we report on the structure and function of the three neuronal UCP homologues. Reconstituted neuronal UCPs were dominantly helical in lipid membranes and transported protons in the presence of physiologically-relevant fatty acid (FA) activators. Under similar conditions, all neuronal UCPs also exhibited chloride transport activities that were partially inhibited by FAs. CD, fluorescence and MS measurements and semi-native gel electrophoresis collectively suggest that the reconstituted proteins self-associate in the lipid membranes. Based on SDS titration experiments and other evidence, a general molecular model for the monomeric, dimeric and tetrameric functional forms of UCPs in lipid membranes is proposed. In addition to their shared structural and ion transport features, neuronal UCPs differ in their conformations and proton transport activities (and possibly mechanism) in the presence of different FA activators. The differences in FA-activated UCP-mediated proton transport could serve as an essential factor in understanding and differentiating the physiological roles of UCP homologues in the CNS.

Molecular Physiology of Uncoupling Proteins in the Central Nervous System: Self-Association and Proton Transport

Uncoupling proteins (UCPs), located in the mitochondrial inner membrane, facilitate the transmembrane proton flux and, consequently, reduce the membrane potential and ATP production. Found in the central nervous system (CNS), based on several studies on animal models and clinical investigations, three human UCP homologs (UCP2, UCP4, and UCP5) could have crucial physiological functions. Despite these studies, the detailed structural features and molecular physiology landscape of these proteins remain relatively unexplored. Recently, we reported a novel expression system for obtaining functionally folded UCP1 in bacterial membranes (Hoang et al., 2013). In the current study, employing similar expression and reconstitution methods, we will report our new findings for the three human neuronal UCP homologs. The reconstituted CNS proteins display high helical contents and transport protons in the presence of several physiologically-relevant fatty acid activators. In addition, experimental results from CD, fluorescence spectroscopy, mass spectrometry and semi-native electrophoresis suggest self-association of these proteins in the membranes. While sharing comparable secondary structures in the liposomes, neuronal UCPs differ in their proton transport rates (and possibly mechanism) in the presence of different fatty acid activators. The protein-fatty acids interaction is further investigated using near-UV CD spectroscopy. The differences in fatty-acid activated UCP-mediated proton transport could serve as an essential clue in understanding and differentiating the physiological roles of UCP homologs in the CNS.Hoang T, Smith MD, Jelokhani-Niaraki M (2013) Expression, folding, and proton transport activity of human uncoupling protein-1 (UCP1) in lipid membranes: evidence for associated functional forms. J. Biol. Chem. 288, 36244-36258

SkQ3: The new member of the class of membranotropic uncouplers

In our previous studies we have described the non-equilibrium binding of hydrogen ions to the membranes upon induction of transmembrane proton flux in the model system (BLM) and in mitochondria. In 2009 the investigation program aimed to find uncouplers selectively interacting with the non-equilibrium bound protons was started. This study is devoted to a new representative of this class of uncouplers. The effect of respiratory stimulation evoked by this compound can be suppressed by 50–90% through the elimination of non-equilibrium fraction of protons associated with the outer side of the inner mitochondrial membrane. The peculiarity of this compound results from its belonging to the class of quinones with a high affinity to lipid membranes. It becomes a weak acid (hydroquinone) only after reduction. The formation of hydroquinone in the mitochondria determines the observed effect of respiratory stimulation. Thus, the artificial induction of interaction between redox-reactions and reactions of proton association and dissociation occurring on the surface of the inner mitochondrial membrane was demonstrated for the first time.

Resurrection of the flagellar rotary motor near zero load

Flagellated bacteria, such as Escherichia coli, are propelled by helical flagellar filaments, each driven at its base by a reversible rotary motor, powered by a transmembrane proton flux. Torque is generated by the interaction of stator proteins, MotA and MotB, with a rotor protein FliG. The physiology of the motor has been studied extensively in the regime of relatively high load and low speed, where it appears to operate close to thermodynamic equilibrium. Here, we describe an assay that allows systematic study of the motor near zero load, where proton translocation and movement of mechanical components are rate limiting. Sixty-nanometer-diameter gold spheres were attached to hooks of cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image plane of a microscope through a small pinhole. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23 degrees C by expression of wild-type MotA, and speeds jumped from zero to a maximum value ( approximately 300 Hz) in one step. Thus, near zero load, the speed of the motor is independent of the number of torque-generating units. Evidently, the units act independently (they do not interfere with one another), and there are no intervals during which a second unit can add to the speed generated by the first (the duty ratio is close to 1).

Polarity of alveolar epithelial cell acid-base permeability.

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO-free Na(+) buffer (140 mM Na(+), 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked > or =80% by 100 microM dimethylamiloride, an inhibitor of Na(+)/H(+) exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na(+)/H(+) exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.

Effect of exogenous calmodulin and ATP on the activity of ethylene forming enzyme obtained from tomatoes and green pea pods

Ethylene forming enzymes (EFE) from both peas and tomatoes were prepared. EFE from both sources were inhibited by adenosine triphosphate (ATP). Exogeneous calmodulin had no effect on EFE activity either in the presence or absence of ATP. The presence of gramicidin in pea microsomal membrane EFE did not affect EFE activity in the presence of ATP. This suggests that ATP is inhibiting EFE activity through phosphorylation and not through transmembrane proton flux. However, inhibition of EFE activity by ATP was partially reversed by 60 μMN-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) and 15 μM trifluoperazine (TFP) but not by 60 μMN-6(aminohexyl)-1-naphthalenesulphonamide (W5), which suggests that the ATP inhibitory affect on EFE is probably controlled by calmodulin. © 1998 SCI.

Effect of exogenous calmodulin and ATP on the activity of ethylene forming enzyme obtained from tomatoes and green pea pods

Ethylene forming enzymes (EFE) from both peas and tomatoes were prepared. EFE from both sources were inhibited by adenosine triphosphate (ATP). Exogeneous calmodulin had no effect on EFE activity either in the presence or absence of ATP. The presence of gramicidin in pea microsomal membrane EFE did not affect EFE activity in the presence of ATP. This suggests that ATP is inhibiting EFE activity through phosphorylation and not through transmembrane proton flux. However, inhibition of EFE activity by ATP was partially reversed by 60 μM N-6(aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) and 15 μM trifluoperazine (TFP) but not by 60 μM N-6(aminohexyl)-1-naphthalenesulphonamide (W5), which suggests that the ATP inhibitory affect on EFE is probably controlled by calmodulin. © 1998 SCI.

The relationship between membrane fluidity and permeabilities to water, solutes, ammonia, and protons.

Several barrier epithelia such as renal collecting duct, urinary bladder, and gastric mucosa maintain high osmotic pH and solute gradients between body compartments and the blood by means of apical membranes of exceptionally low permeabilities. Although the mechanisms underlying these low permeabilities have been only poorly defined, low fluidity of the apical membrane has been postulated. The solubility diffusion model predicts that lower membrane fluidity will reduce permeability by reducing the ability of permeant molecules to diffuse through the lipid bilayer. However, little data compare membrane fluidity with permeability properties, and it is unclear whether fluidity determines permeability to all, or only some substances. We therefore studied the permeabilities of a series of artificial large unilamellar vesicles (LUV) of eight different compositions, exhibiting a range of fluidities encountered in biological membranes. Cholesterol and sphingomyelin content and acyl chain saturation were varied to create a range of fluidities. LUV anisotropy was measured as steady state fluorescence polarization of the lipophilic probe DPH. LUV permeabilities were determined by monitoring concentration-dependent or pH-sensitive quenching of entrapped carboxyfluorescein on a stopped-flow fluorimeter. The relation between DPH anisotropy and permeability to water, urea, acetamide, and NH3 was well fit in each instance by single exponential functions (r > 0.96), with lower fluidity corresponding to lower permeability. By contrast, proton permeability correlated only weakly with fluidity. We conclude that membrane fluidity determines permeability to most nonionic substances and that transmembrane proton flux occurs in a manner distinct from flux of other substances.

Ion-translocating ATPases in tendrils of Bryonia dioica Jacq.

Procedures have been developed which allow the preparation of highly pure endoplasmic reticulum and plasma membrane from tendrils of Bryonia dioica. These and further membrane fractions were used to study vanadate-sensitive ATPase activity as well as Mg2+ATP-driven transport of 45Ca2+. Calcium-translocating ATPases were detected in the endoplasmic reticulum, the plasma membrane and the mitochondrial fraction and characterized kinetically and with respect to the effects of various inhibitors. The endoplasmic-reticulum Ca2+-translocating ATPase was stimulated by KCl and was calmodulin-dependent. The plasma-membrane enzyme was not affected by these agents. These, as well as the inhibitor data, show that the Ca2+-translocating ATPases of the endoplasmic reticulum and the plasma membrane are distinctly different enzymes. Upon mechanical stimulation, the activities of the vanadate-sensitive K+, Mg2+-ATPase and the Ca2+-translocating ATPase(s) increased rapidly and transiently, indicating that increasing transmembrane proton and calcium fluxes are involved in the early stages of tendril coiling.

Diclofop and Fenoxaprop Resistance in Wild Oat Is Associated with an Altered Effect on the Plasma Membrane Electrogenic Potential

We have examined the mechanism of herbicide resistance in a biotype of wild oat ( Avena fatua L.) that is resistant to diclofop-methyl and many other acetyl-coenzyme A carboxylase (ACCase) inhibitors. Resistance to diclofop-methyl and fenoxaprop-ethyl was not based on reduced uptake nor on enhanced metabolism of the herbicides to inactive products. In in vitro assays of crude or partially purified preparations, ACCase from the resistant (UM-1) and susceptible (UM-5) biotypes was equally sensitive to diclofop, with I 50 values of 6.1 and 7.3 μ M for UM-1 and UM-5, respectively. Corresponding values for fenoxaprop were 2.5 and 1.0 μ M. These results suggest that the high level of resistance observed toward these herbicides is not based on an altered target enzyme. Root tissue from both UM-1 and UM-5 acidified an unbuffered bathing solution. Addition of 100 μ M diclofop or fenoxaprop prevented acidification of the bathing medium by UM-1, but alkalinization occurred rapidly with UM-5. When diclofop was removed from the treatment solution, UM-1 resumed acidification of the solution, whereas the pH of the UM-5 bathing solution continued to increase. Diclofop (50 μ M) rapidly depolarized the cell membrane in peeled coleoptile sections, with no difference between UM-1 and UM-5. However, when diclofop was removed from the treatment solution, the electrogenic membrane potential was quickly reestablished in UM-1, but remained collapsed in UM-5. These results provide support for the hypothesis that the effect of diclofop on the plasma membrane potential is an important component of its herbicidal activity. The reversibility of the effect of diclofop and fenoxaprop on transmembrane proton flux in UM-1 appears to be associated with resistance to these herbicides.

Electrochemical aspects of biological energy conversion

The phosphorylating membrane is treated as an electrochemical energy converter with protons instead of electrons as charge-separating entities. Scalar chemical reactions are taken to be coupled to proton transfer across the membrane—solution interface. A Butler—Volmer type of equation set-up in terms of the electrochemical potential difference of the proton is taken to govern interfacial as well as transmembrane proton flux. This is formally similar to the Butler—Volmer equation used by Gurevich and Kharkats (Yu. Y. Gurevich and Yu. I. Kharkats, J. electroanal. Chem. 200, 3, 1986) for ion transfer across a phase boundary, taking into account surface concentrations of ions not at equilibrium with the bulk. On the basis of the theory of metastable states and hysteresis of polyelectrolytes brought forward by Katchalsky and Spangler (A. Katchalsky and R. Spangler, Q. Rev. Biophys. 1, 127, 1986) the μ ̃ operating at the membrane/solution interface is also shown to act as modulator of self-induced oscillations of proton flux coupled to cooperative phase transitions. This provides an explanation of conformational fluctuations of membrane proteins observed at biological membranes in connection with energy conversion.

On the Inhibition of the Mitochondrial Inner Membrane Anion Uniporter by Cationic Amphiphiles and Other Drugs

Depleting the mitochondrial matrix of divalent cations with the ionophore A23187 activates a pH-sensitive, anion uniport pathway which can transport many anions normally regarded as impermeant (Beavis, A. D., and Garlid, K. D. (1987) J. Biol. Chem. 262, 15085-15093). Addition of valinomycin to respiring mitochondria can also induce the uptake of a wide variety of anions; however, the mechanism of anion transport during this "respiration-induced" swelling is less certain. In this paper, I demonstrate that both of these processes are inhibited by a variety of cationic amphiphiles including propranolol, quinine, amiodarone, imipramine and amitriptyline, and the benzodiazepine R05-4864. Although the IC50 values for the two processes are not equal, the ratio of IC50 values for the two processes appears to be the same for all drugs. Measurements of net transmembrane proton fluxes that occur during the assays reveal that respiration-induced swelling is associated with extensive proton ejection, the peak of which coincides with the maximum rate of anion transport. Moreover, from measurements of matrix buffering power, it is estimated that the matrix pH is 3 units more alkaline during respiration-induced swelling than during A23187-induced swelling. It is also shown that the IC50 for A23187-induced transport is pH-dependent in a manner consistent with modulation of drug binding by protonation of two sites. These findings allow the difference in IC50 values for the two types of assay to be explained by the pH dependence of the binding constant for the drug. Furthermore, the pH gradient generated during respiration-induced swelling is so large that the electrical component of the proton-motive force will be negligible. Thus, despite the fact that the mitochondria are "energized," rapid electrophoretic anion influx is possible. These data provide evidence that the transport of anions in these two types of assay occurs via the same pathway.

Coupling of Proton Flux to the Hydrolysis and Synthesis of ATP

The extensive experimental data reported since thc introduction of the chemiosmotic hypothesis (54, 55) and the isolation of proton-A TPases from mitochondria (43, 46, 67, 73, 83), chloroplasts (3, 51, 57, 87, 98), and various bacteria (5, 39,45, 85, 105) have led to the general acceptance of the notion that catalytic hydrolysis and synthesis of adenosine 5' -triphosphate(A TP) by energy-transducing membranes are coupled to transmembrane proton flux. All proton-A TPases that have been studied consist of two parts: a hydrophobic moiety (F 0)' which contains a proton channel and is located in the energy-transducing mcmbrane, and a hydrophilic moiety (F 1), which catalyzes the hydrolysis and synthesis of ATP and is exposed to the aqueous phase but is attached to F o' The subunit structures of F 0 and F 1 and their interaction with each other have been discussed in a number of excellent reviews (6, 10, 11, 17, 24, 27, 40, 50, 58, 82). Recently, the amino acid sequences of all the subunits of the F of 1 complex from Escherichia coli K12 have been deduced from its coding sequence (30, 31, 41, 77). The F of 1 complexes are called proton-A TPases because they catalyze the synthesis of A TP by transmembrane proton flux in the direction from F 0 to F 1 as well as the generation of transmembrane proton flux in the opposite direction by the hydrolysis of A TP. Phenomenologically, these coupled catalytic processes may be represented as follows: