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PubMed1997

The proton permeability of liposomes made from mitochondrial inner membrane phospholipids: comparison with isolated mitochondria.

Unilamellar liposomes with native phospholipid fatty acid composition were prepared from rat liver mitochondrial inner membrane phospholipids by extrusion in medium containing 50 mm potassium. They were diluted into low potassium medium to establish a transmembrane potassium gradient. A known membrane potential was imposed by addition of valinomycin, and proton flux into liposomes was measured. Valinomycin in the range 10 pm-1nm was sufficient to fully establish membrane potential. Valinomycin concentrations above 3 nm catalyzed additional proton flux and were avoided. At 300 pm valinomycin, proton flux depended nonlinearly on membrane potential. At 160 mV membrane potential the flux was 30 nmol H+/min/mg phospholipid-approximately 5% of the proton leak flux under comparable conditions in isolated mitochondria, indicating that leak pathways through bulk phospholipid bilayer account for only a small proportion of total mitochondrial proton leak.

Animals↗
PubMed1979

The effect of antibiotics on the photocycle and protoncycle of purple membrane suspensions.

The interrelation was studied between the phototransient absorbing maximally at 412 nm (M412) and light-induced proton release under steady-state conditions in aqueous suspensions of 'purple membrane' derived from Halobacterium halobium. The decay of M412 was slowed down by the simultaneous application of the ionophoric antibiotics valinomycin and beauvericin. The former had only slight activity alone and the latter was effective only in conjunction with valinomycin. The steady-state concentration of M412 which was formed on illumination was a direct function of the concentration of valinomycin. Maximum stabilization of M412 was obtained when the valinomycin was approximately equimolar with the bacteriorhodopsin. Addition of salts to the medium increased the number of protons released per molecule of M412 without affecting the level of M412 which was produced by continuous illumination. The effectiveness of the salts in this respect depended on the nature of the cation. Ca2+ and their antagonists La3+ and ruthenium red were found to have especially high affinity for the system. The extent of light-induced acidification could not be enhanced by increasing the pH of the medium from 6.5 to 7.8. The possible mechanism of action of the ionophores and of the cations on the photocycle and on the proton cycle is discussed.

Anti-Bacterial Agents↗
PubMed1980

Bimodal effects of cellular amino acids on Na+-dependent amino acid transport in Ehrlich cells.

Cells depleted of amino acids show lower rates of glycine or aminoisobutyric acid uptake than do freshly isolated cells. In the amino acid-depleted cells, addition of valinomycin stimulates amino acid influx at least to the level observed in freshly isolated cells. In cells containing high levels of cellular amino acids, valinomycin has little effect on influx of amino acids. It is concluded that the transport of amino acids in freshly isolated cells is elevated compared to depleted cells because the cells are hyperpolarized by the continuous loss of cellular amino acids during the transport assay. During this hyperpolarization by amino acid loss, transport of amino acids is not further stimulated by valinomycin at low external [K+] (10 mM +/- 5 mM). With the exception of preloading with glycine, cells preloaded with a single amino acid to a concentration greater than 20 mM show reduced rates of glycine and aminoisobutyric acid influx at early times (less than 15 min) compared to amino acid-depleted cells. The reduction of influx is transient and by 30 min, influx is greater in preloaded than in amino acid-depleted cells. Knowing that increases and decreases in the membrane potential are achieved by using varying external [K+] in the presence of valinomycin and propranolol, and using amino acid-depleted cells, it can be shown that an increased membrane potential increases the V for glycine and aminoisobutyric acid influx. A decrease in the potential difference results in a decreased V. Changes in Km also occur when the membrane potential is varied.

Amino Acids↗
PubMed1988

The membrane potential modulates the ATP-dependent Ca2+ pump of cardiac sarcolemma.

The effect of membrane potential on the activity of the ATP-dependent Ca2+ pump of isolated canine ventricular sarcolemmal vesicles was investigated. The membrane potential was controlled by the intravesicular and extravesicular concentration of K+, and the initial rates of Ca2+ uptake both in the presence and the absence of valinomycin were determined. The rate of Ca2+ uptake was stimulated by a inside-negative potential induced in the presence of valinomycin. The valinomycin-dependent stimulation was enhanced by the addition of K+ channel blocker, tetraethylammonium ion or Ba2+. The electrogenicity of cardiac sarcolemmal ATP-dependent Ca2+ pump is suggested from the increase of Ca2+ uptake by negative potential induced by valinomycin.

Adenosine Triphosphate↗
PubMed1995

Reconstitution of bacteriorhodopsin and ATP synthase from Micrococcus luteus into liposomes of the purified main tetraether lipid from Thermoplasma acidophilum: proton conductance and light-driven ATP synthesis.

The archaebacterium Thermoplasma acidophilum is cultivated at 59 degrees C in a medium containing sulfuric acid of pH 2. The purified bipolar membrane spanning main phospholipid (MPL) of this organism can be used to produce stable liposomes of 100-500 nm in diameter either using a French pressure cell detergent dialysis or sonication. Despite a potassium diffusion potential of 186 mV very low ionic permeability of sonicated MPL liposomes was measured using the potassium binding fluorescent indicator benzofuran isophthalate PBF1, which measures net K+ uptake. The latter also remained very low, in the presence of the K(+) ionophore valinomycin and palmitic acid. Addition of valinomycin and the potent uncoupler carbonylcyanid-p-trifluormehoxyphenyl-hydrazone (FCCP), led to a stimulation in potassium uptake. The rate of proton flux can be calculated from the net K(+) uptake. Under these conditions MPL liposomes are 1-2 orders of magnitude less permeable than egg yolk lecithin vesicles. The difference in proton permeability becomes even more pronounced with increasing temperature, examined using the fluorescent pH indicator pyranine. Purified bacteriorhodopsin from Halobacterium halobium was reconstituted into MPL liposomes in order to study the light-driven proton uptake in 150 mM KCl following addition of valinomycin, gramicidin, FCCP and Triton X-100. The light-driven proton transport into the liposomes was increased 30-fold by addition of valinomycin decreased by gramicidin and FCCP, and abolished by Triton X-100. Co-reconstituted MPL proteoliposomes containing bacteriorhodopsin and ATP synthase from Micrococcus luteus were capable of light-driven ATP synthesis demonstrating the functional coupling of proton transport and nucleotide generation in liposomal MPL membranes.

Adenosine Triphosphate↗
PubMed1989

Cation-anion cotransport.

Two methods have been described for the study of cation-chloride cotransport systems. The zero-trans efflux method is designed to determine stoichiometric relationships between cotransported ions under conditions where ion exchanges cannot occur. These exchanges (e.g., Na+/Na+, K+/K+) may occur as partial or incomplete reactions of a cotransport process and can lead to erroneous determinations of the stoichiometry of the cotransport process. The zero-trans efflux method can also be used to study the effects of cell volume, pH, and intracellular ion concentrations on cotransport processes. The valinomycin method is used to determine the electrogenicity or electroneutrality of transport, and in this regard can be used in conjunction with other methods such as those employing potential-sensitive dyes or microelectrodes. Other, more recently developed ionophores with specificity for lithium rather than potassium have now been used to study the effect of Em on the ATP-dependent Na+/K+ pump. It may be possible to use such ionophores to confirm the suspected electroneutrality of (K+ + Cl-) cotransport systems, as well as for other studies of specific potassium transport processes in which valinomycin obviously cannot be used. Both methods discussed in detail in this chapter, and particularly the valinomycin method, were originally devised for use in red blood cells in order to take advantage of (or circumvent) properties of the red cell membrane, such as its low permeability to sodium and potassium and relatively high permeability to chloride. However, valinomycin has been used successfully to demonstrate the electroneutrality of (Na+ + K+ + 2Cl-) cotransport in MDCK cells, and the zero-trans efflux method should be applicable to the study of transport processes in other types of cells in suspension, so long as the transport system being studied can be accurately defined (e.g., as an inhibitor-sensitive or chloride-dependent cation flux) and comprises a significant fraction of the total salt efflux.

Animals↗
PubMed1991

Functional reconstitution of the gamma-aminobutyric acid transporter from synaptic vesicles using artificial ion gradients.

The gamma-aminobutyric acid transporter of rat brain synaptic vesicles was reconstituted in proteoliposomes, and its activity was studied in response to artificially created membrane potentials or proton gradients. Changes of the membrane potential were monitored using the dyes oxonol VI and 3,3'-diisopropylthiodicarbocyanine iodide, and changes of the H+ gradient were followed using acridine orange. An inside positive membrane potential was generated by the creation of an inwardly directed K+ gradient and the subsequent addition of valinomycin. Under these conditions, valinomycin evoked uptake of [3H]GABA which was saturable. Similarly, [3H]glutamate uptake was stimulated by valinomycin, indicating that both transporters can be driven by the membrane potential. Proton gradients were generated by the incubation of K(+)-loaded proteoliposomes in a buffer free of K+ or Na+ ions and the subsequent addition of nigericin. Proton gradients were also generated via the endogenous H+ ATPase by incubation of K(+)-loaded proteoliposomes in equimolar K+ buffer in the presence of valinomycin. These proton gradients evoked nonspecific, nonsaturable uptake of GABA and beta-alanine but not of glycine in proteoliposomes as well as protein-free liposomes. Therefore, transporter activity was monitored using glycine as an alternative substrate. Proton gradients generated by both methods elicited saturable glycine uptake in proteoliposomes. Together, our data confirm that the vesicular GABA transporter can be energized by both the membrane potential and the pH gradient and show that transport can be achieved by artificial gradients independently of the endogenous proton ATPase.

Animals↗
PubMed1982

Mitochondrial transmembrane pH and electrical gradients: evaluation of their energy relationships with respiratory rate and adenosine 5'-triphosphate synthesis.

Mitochondria from rat liver were suspended in ionic media, and parallel measurements were made of the respiratory rate, intramitochondrial volumes, and the phosphorylation state [[ATP]/([ADP][Pi])], as well as transmembrane pH gradients (from the distribution of labeled weak acids and bases) and electrical gradients (from the distribution of labeled lipid-soluble ions and of K+ in the presence of valinomycin). A decrease in the pH gradient was observed with additions of sodium propionate, acetate, or lactate. In the case of propionate, the pH gradient decreased from -0.94 +/- 0.10 (control) to -0.09 +/- 0.08 unit (with 3 mM propionate) without change in any of the other measured parameters. Variation of the intramitochondrial volumes was achieved by changing [K+] (+valinomycin) and by diluting the choline chloride media. The transmembrane electrical potential decreased from -150 mV under control conditions to approximately -75 mV in a 70 mosM medium or when 8 mM K+ (+valinomycin) was present. The changes in [ATP]/([ADP][Pi]) were as follows: from 1.8 X 10(4) (control) to 3.9 X 10(2) M-1 with 8 mM K+ (+valinomycin) or 1.0 X 10(4) M-1 when the osmolarity was decreased from 300 to 70 mosM. This corresponds to a decrease in the energy utilized for ATP synthesis from 14.1 to 11.9 kcal/mol (from 58.9 to 49.7 kJ/mol) with added K+ and to 13.8 kcal/mol (57.7 kJ/mol) with decreased osmolarity. No direct correlation was found between the changes in transmembrane electrical potential, pH gradient, or total proton electrochemical gradient and either the respiratory rate or [ATP]/([ADP][Pi]).

Adenosine Triphosphate↗
PubMed1978

Platelet 5-hydroxytryptamine transport, an electroneutral mechanism coupled to potassium.

Transport of 5-hydroxytryptamine into plasma membrane vesicles isolated from porcine blood platelets is stimulated when a potassium gradient (in greater than out) is imposed across the vesicle membrane. This stimulation occurs in the absence of measurable electrical potential across the membrane. Addition of valinomycin induces a membrane potential of approximately 50 mV (interior negative) as estimated by uptake of the lipophilic cation triphenylmethylphosphonium, but has surprisingly little effect on 5-hydroxytryptamine transport. Addition of 2,4-dinitrophenol dissipates the valinomycin-induced membrane potential. In the absence of valinomycin, 2,4-dinitrophenol has no effect on 5-hydroxytryptamine transport but valinomycin and 2,4-dinitrophenol together inhibit transport, probably by dissipation of the K+ gradient. These results are consistent with an electroneutral mechanism in which 5-hydroxytryptamine influx is directly coupled to potassium ion efflux and argue against an electrogenic mechanism in which there is a net influx of positive charge with 5-hydroxytryptamine.

Animals↗
PubMed2005

Antibiotic assisted molecular ion transport across a membrane in real time.

The transport of an organic cation across a 4-5 nm liposome bilayer is observed in real time using second harmonic generation. It is proposed that an electrostatic barrier between the inside and outside of the liposome develops as the cation crosses the bilayer. This would explain why the SHG signal does not approach zero at long times. To test this mechanism, the antibiotic valinomycin, which can transport alkali ions across a phospholipid bilayer, is introduced into the system. It is found that the transport time is reduced by a factor of three from 90+/-2 s to 30+/-1 s with 1.25 x 10(-8) M valinomycin concentration, and a factor of fifteen to 6.2+/-0.2 s with 1.25 x 10(-8) M valinomycin concentration. In addition, the SHG signal approaches zero, which further supports the presence of an electrostatic barrier that can be eliminated by the alkali ion transporter valinomycin.

Ion Transport↗
PubMed1990

Energy coupling of L-glutamate transport and vacuolar H(+)-ATPase in brain synaptic vesicles.

Energy coupling of L-glutamate transport in brain synaptic vesicles has been studied. ATP-dependent acidification of the bovine brain synaptic vesicles was shown to require CI-, to be accelerated by valinomycin and to be abolished by ammonium sulfate, nigericin or CCCP plus valinomycin, and K+. On the other hand, ATP-driven formation of a membrane potential (positive inside) was found to be stimulated by ammonium sulfate, not to be affected by nigericin and to be abolished by CCCP plus valinomycin and K+. Like formation of a membrane potential, ATP-dependent L-[3H]glutamate uptake into vesicles was stimulated by ammonium sulfate, not affected by nigericin and abolished by CCCP plus valinomycin and K+. The L-[3H]glutamate uptake differed in specificity from the transport system in synaptic plasma membranes. Both ATP-dependent H+ pump activity and L-glutamate uptake were inhibited by bafilomycin and cold treatment (common properties of vacuolar H(+)-ATPase). ATP-dependent acidification in the presence of L-glutamate was also observed, suggesting that L-glutamate uptake lowered the membrane potential to drive further entry of H+. These results were consistent with the notion that the vacuolar H(+)-ATPase of synpatic vesicles formed a membrane potential to drive L-glutamate uptake. ATPase activity of the vesicles was not affected by the addition of Cl-, glutamate or nigericin, indicating that an electrochemical H+ gradient had no effect on the ATPase activity.

Adenosine Triphosphate↗
PubMed1990

Ionophores for monovalent cations inhibit angiotensin-stimulated aldosteronogenesis.

We examined the effects of monensin (a sodium ionophore), valinomycin (a potassium ionophore), and nigericin (a nonspecific ionophore) on steroid production and its stimulation in bovine adrenal glomerulosa and fasciculata cells. All three ionophores at nanomolar concentrations inhibited angiotensin (AII)-stimulated aldosterone production; potassium-stimulated aldosteronogenesis was more sensitive, and cortisol synthesis by fasciculata cells was much less sensitive. Ionophores completely inhibited the early pathway of aldosteronogenesis and partially inhibited conversion of progesterone to aldosterone. Ionophores had no effect on pregnenolone production by isolated glomerulosa mitochondria. Monensin had no effect on AII binding, calcium flux, calcium transient, protein phosphorylation, or protein synthesis; valinomycin slightly inhibited these processes. Valinomycin lowered cell potassium and raised cell sodium, but its inhibition of aldosteronogenesis was not overcome by increasing extracellular potassium. Monensin and nigericin had no effect on cell potassium or sodium. Cellular ATP was decreased by valinomycin, but not by monensin or nigericin. Our results show that stimulation of aldosteronogenesis by AII and potassium is highly sensitive to ionophores of monovalent cations. Monensin and nigericin inhibit steroidogenesis at concentrations that have no other observed deleterious effects on glomeulosa cells. These results identify a distinguishing characteristic of adrenal glomerulosa cells and suggest a new pharmacologic approach to inhibition of aldosteronogenesis.

Adenosine Triphosphate↗
PubMed1974

Determination of membrane potentials in human and Amphiuma red blood cells by means of fluorescent probe.

1. Changes in the fluorescent intensity of the dye, 3,3'-dihexyl-2,2'-oxacarbocyanine, added to suspensions of human and Amphiuma red blood cells were measured in parallel with changes in the membrane potentials of these cells. In these studies the membrane potential was altered in three different ways: by the addition of valinomycin to alter the ratio, P(K)/P(Cl), by a change in the pH of the medium to alter the ratio, Cl(c)/Cl(0), and by the substitution of impermeant anions for Cl(0) again to alter the ratio, Cl(c)/Cl(0). In each case hyperpolarization led to a decrease and depolarization to an increase in fluorescent intensity.2. The change in fluorescence with the addition of valinomycin was dependent on the concentration of K in both the cells and the medium. Changes in fluorescence were not observed when valinomycin was added to suspensions of frozen-thawed, haemoglobin-free ghosts with dye in KCl or NaCl solutions. Such changes were observed with reconstituted ghosts provided that there was a K concentration gradient across the membrane.3. From values of cellular K and the corresponding external K concentrations for which there were no changes in fluorescence with valinomycin, estimations of membrane potentials were made. The potential was -5 to -8 mV for the human red cell and -19 mV for Amphiuma. These values are in good agreement with the potentials estimated from the Cl ratios (-9 mV for human and -17 to -20 mV for Amphiuma) and from those obtained by direct electrical measurements (-15 mV for Amphiuma).4. Fluorescent intensity of the dye in suspensions of human red cells was shown to be a linear function of the log Cl(c)/Cl(0).5. The dye (2.9 x 10(-6)M) increased the inward rate constants for (24)Na (3-4-fold) and (42)K (0.5-2-fold) for human red cells. In addition, the dye was found to be haemolytic (5-6% in 1 hr) at 22 degrees C.6. In contrast, the dye did not alter the rate of (35)SO(4) efflux at 37 degrees C from human red cells previously equilibrated with a Cl-free SO(4) medium.7. The dye was also seen to interact with certain impermeant anions and other compounds, e.g. inhibitors of anion permeability, of interest. These interactions and other limitations of the use of this dye are discussed.

Animals↗
PubMed2001

Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis.

We investigated cytochrome c release kinetics in response to three apoptosis-inducing agents (tumor necrosis factor-alpha, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent protein (EGFP)-tagged cytochrome c. All three agents induced significant caspase activation in the cultures determined by monitoring the cleavage of fluorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cells, albeit the valinomycin-induced activation was less pronounced. Time-lapse confocal microscopy showed that tumor necrosis factor-alpha and staurosporine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochrome c-EGFP release occurred slowly over several hours. Unlike staurosporine, the valinomycin-induced cytochrome c release was not associated with translocation of the proapoptotic Bax protein to the mitochondria, and was not accompanied by co-release of the proapoptotic Smac protein. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apoptosis.

Apoptosis↗
PubMed1977

Phototaxis and membrane potential in the photosynthetic bacterium Rhodospirillum rubrum.

Cells of the photosynthetic bacterium Rhodospirillum rubrum cultivated anaerobically in light show phototaxis. The behavior of individual cells in response to the phenomenon is reversal(s) of the swimming direction when the intensity of the light available to them abruptly decreases. The tactic response was inhibited by antimycin, an inhibitor of the photosynthetic electron transfer system. The inhibitory effect of antimycin was overcome by phenazine methosulfate. Motility of the cells was not impaired by antimycin under aerobic conditions. Valinomycin plus potassium also inhibited their phototactic response; however, valinomycin or potassium alone had no effect. A change in membrane potential of the cells was measured as an absorbance change of carotenoid. Changes in the membrane potential caused by "on-off" light were prevented by antimycin and by valinomycin plus potassium, but not by antimycin plus phenazine methosulfate nor valinomycin or potassium alone. The results indicated that the phototactic response of R. rubrum is mediated by a sudden change in electron flow in the photosynthetic electron transfer system, and that the membrane potential plays an important role in manifestation of the response.

Aerobiosis↗
PubMed1986

Solubilization and reconstitution of a chloride transporter from tracheal apical membrane.

Electrogenic Cl- transport in bovine tracheal membrane vesicles was studied by measuring the kinetics of Cl- efflux. Efflux from KCl-loaded vesicles was assayed as the appearance of Cl- in the medium using an anion-specific electrode. Maximal sensitivity was obtained by passage of the vesicles through a Sephadex column immediately before the assay to remove external Cl-. Two components of Cl- transport could be detected. Vesicles exhibited a characteristic spontaneous Cl- efflux that appears to be limited by the cation permeability of the membrane. Addition of the K+-conducting ionophore to increase selectively the permeability to K+ resulted in a stimulation of Cl- efflux. The electrically neutral ionophore nigericin caused only a slight transient increase in Cl- efflux. The valinomycin-dependent flux may be assumed to represent flux through a parallel conductive pathway. Valinomycin-dependent electrogenic Cl- flux was 240 +/- 36 nmol X mg protein-1 X min-1 (n = 4). Soluble extracts obtained with the nonionic detergent octyl glucoside were reconstituted by the freeze-thaw procedure. The reconstituted vesicles exhibited a number of similarities to the native vesicles. Efflux was maximally stimulated with the ionophore valinomycin in the presence of an outwardly directed K+ gradient. Nigericin had no effect on efflux, suggesting the reconstituted transporter is a conductive rather than exchange mechanism. The initial rate of the valinomycin-dependent component was 896 +/- 63 nmol X mg protein-1 X min-1 (n = 5), representing approximately a fourfold increase in specific activity compared with the native membrane vesicle.

Animals↗
PubMed2005

The direct physiological effects of mitoK(ATP) opening on heart mitochondria.

The mitochondrial ATP-sensitive K+ channel (mitoK(ATP)) has been assigned multiple roles in cell physiology and in cardioprotection. Each of these roles must arise from basic consequences of mitoK(ATP) opening that should be observable at the level of the mitochondrion. MitoK(ATP) opening has been proposed to have three direct effects on mitochondrial physiology: an increase in steady-state matrix volume, respiratory stimulation (uncoupling), and matrix alkalinization. Here, we examine the evidence for these hypotheses through experiments on isolated rat heart mitochondria. Using perturbation techniques, we show that matrix volume is the consequence of a steady-state balance between K+ influx, caused either by mitoK(ATP) opening or valinomycin, and K+ efflux caused by the mitochondrial K+/H+ antiporter. We show that increasing K+ influx with valinomycin uncouples respiration like a classical uncoupler with the important difference that uncoupling via K+ cycling soon causes rupture of the outer mitochondrial membrane and release of cytochrome c. By loading the potassium binding fluorescent indicator into the matrix, we show directly that K+ influx is increased by diazoxide and inhibited by ATP and 5-HD. By loading the fluorescent probe BCECF into the matrix, we show directly that increasing K+ influx with either valinomycin or diazoxide causes matrix alkalinization. Finally, by comparing the effects of mitoK(ATP) openers and blockers with those of valinomycin, we show that four independent assays of mitoK(ATP) activity yield quantitatively identical results for mitoK(ATP)-mediated K+ transport. These results provide decisive support for the hypothesis that mitochondria contain an ATP-sensitive K+ channel and establish the physiological consequences of mitoK(ATP) opening for mitochondria.

Adenosine Triphosphate↗
PubMed1981

Electrogenic proton translocation coupled to ATP hydrolysis by the plasma membrane Mg2+-dependent ATPase of yeast in reconstituted proteoliposomes.

The purified plasma membrane Mg2+-dependent ATPase of the yeast Schizosaccharomyces pombe was incorporated in liposomes using a cholate-dialysis method. The ATPase activity of the incorporated enzyme was stimulated by the H+-conducting agent carbonyl cyanide m-chlorophenylhydrazone and to a much lower extent of the K+-ionophore valinomycin in the presence of potassium. The K+/H+ exchanger nigericin (plus K+) did not stimulate ATPase activity, whereas the combined addition of both nigericin plus valinomycin was strongly stimulatory. The incorporated ATPase activity was controlled by the generated electrochemical H+ gradient since only conditions which collapse both the membrane potential and the pH gradient stimulated fully the ATPase activity of the incorporated enzyme. Direct measurement of proton movement with a pH glass electrode showed a fast and transient proton entry into the proteoliposomes upon addition of MgATP in the presence of the charge-compensating cation K+ (plus valinomycin). Moreover, during the steady state ATP hydrolysis, a H+ entry was again observed when the membrane potential was collapsed upon addition of valinomycin in the presence of K+. These data demonstrate that the plasma membrane ATPase of yeast cells is involved in electrogenic H+ translocation coupled to ATP hydrolysis since the purified enzyme incorporated in the liposomes is virtually free of mitochondrial F1F0-ATPase contaminant.

Adenosine Triphosphatases↗