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.     

Proton movements and electric potential generation in reconstituted ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716

ATP hydrolysis-induced proton translocation and electric potential generation have been studied in ATPase proteoliposomes by means of various optical probes. The proteoliposomes consisted of reconstituted ATPase complex and native lipid mixture isolated from the thermophilic cyanobacterium Synechococcus 6716 [Van Walraven et al. (1983) Eur. J. Biochem. 137, 101-106]. The native cartenoids and added oxonol VI served as probes for the electric membrane potential generated by the net charge separation (negative outside, positive inside). Their responses, with similar half-times as 9-tetradecylamino-6-chloro-2-methoxyacridine, are sensitive to valinomycin and stimulated by nigericin, as expected. The proton concentrations of extraliposomal and intraliposomal aqueous spaces were monitored by neutral red and cresol red; for internal measurements these pH indicators were trapped inside the vesicles during detergent dialysis. Internal acidification and external alkalinization induced by ATP hydrolysis are inhibited by nigericin and enhanced by valinomycin; at the commonly used higher valinomycin concentrations the neutral red response becomes transient, while the much slower cresol red response is diminished right from its onset. At smaller preset pH gradients both ATP hydrolysis activity and neutral red response are diminished. At increasing MgCl2 concentrations the neutral red responses are slowed down and the cresol red responses are slightly enhanced; this is observed for both internal and external dye responses. Neutral red permeation through the membrane is insignificant under our experimental conditions but is enhanced at temperatures below the lipid-phase transition. In the case of externally added neutral red the non-permeant buffer Hepes is only effective at high MgCl2 concentration, whereas some external cresol red response is visible only at high MgCl2 concentration in the presence of Hepes. The kinetics of the pH indicator and electric potential probe responses clearly distinguish fast interfacial and intra-membrane proton displacements from slow bulk proton equilibration. The data are summarized in a model that supports the importance of localized proton displacements for the primary energy-transducing events.     

Simultaneous stimulation of uric acid synthesis and gluconeogenesis in chicken hepatocytes by alpha-adrenergic action of epinephrine and calcium

Formation of a transmembrane electric potential coupled to ATP hydrolysis is demonstrated in chloroplast ATPase complex containing proteoliposomes. The ATP-induced signals were detected through absorbance changes of the membrane potential-responding dye oxonol VI. They were inhibited by the specific energy-transfer inhibitor, tentoxin and the ionophore valinomycin while stimulated by nigericin. Calibration of the transmembrane potential signal was possible by the application of a proton diffusion potential. The ATP-induced transmembrane potential was estimated to be 40–50 mV.     

Proton transport by gastric membrane vesicles

A highly purified membrane fraction was derived from hog gastric mucosa by a combination of differential and density gradient centrifugation and free flow electrophoresis. This final fraction was 35-fold enriched with respect to cation activated ouabain-insensitive ATPase. Antibody against this fraction was shown to be bound to the luminal surface of the gastric glands. The addition of ATP to this fraction or the density gradient fraction resulted in H⁺ uptake into an osmotically sensitive space. The apparent K_m for ATP was 1.7 · 10^?4 M in the absence of a K⁺ gradient similar to that found for ATPase activity. The reaction is specific for ATP and requires cation in the sequence K⁺ > Rb⁺ > Cs⁺ > Na⁺ > Li⁺ and is inhibited by ATPase inhibitors such as N,N′-dicylclohexylcarbodiimide. Maximal H⁺ uptake occurs with an outward K⁺ gradient but the minimal apparent K_A is found in the absence of a K⁺ gradient. The pH optimum for H⁺ uptake is between 5.8 and 6.2 which corresponds to the pH range for phosphorylation of the enzyme, but is considerably less than the pH maximum of the K⁺ dependent dephosphorylation. In the presence of an inward K^? gradient, protonophores such as tetrachlorsalicylanilide only partially abolish the H⁺ gradient but valinomycin dissipates 75% of the gradient, and nigericin abolishes the gradient. The vesicles therefore have a low K⁺ conductance but a measurable H⁺ conductance, hence a K⁺ gradient can produce an H⁺ gradient in the presence of valinomycin. The uptake and spontaneous leak of H⁺ are temperature sensitive skin with a similar transition temperature. Ultraviolet irradiation inactivates ATPase and proton transport at the same rate, approximately at twice the rate of p-nitrophenylphosphatase inactivation. It is concluded that H⁺ uptake by these vesicles is probably due to a dimeric (H⁺ + K⁺)-ATPase and is probably non-electrogenic.     

Factors affecting the development of the capacity for ATP formation in isolated chloroplasts

Full development of the capacity for ATP formation in isolated thylakoid membranes coincides with the beginning of illumination. Indeed, the yield of ATP per ms of illumination is about twice as great during the first 15 ms of high-intensity illumination as it is thereafter. The presence of valinomycin and K⁺ prevents the formation of a membrane potential (as indicated by the obliteration of most of the change in absorbance at 518 nm) and at the same time delays the formation of the capacity for ATP synthesis for many milliseconds. Presumably, phosphorylation is initially dependent on a rapidly formed membrane potential, whereas after a delay a ΔpH sufficient to drive ATP formation forms. The actual duration of this delay depends on the phosphoryl group transfer potential (i.e., ΔG_ATP) of the ATP-synthesizing reaction. If the delay in the presence of valinomycin and K⁺ represents the time required to develop a ΔpH capable of driving phosphorylation by itself, then the effect of ΔG_ATP on the duration of the delay suggests that the onset of phosphorylation is determined by the magnitude of the electrochemical potential of protons and not by factors affecting the activation of the coupling factor enzyme. The initial ATP formation, which is almost entirely dependent on the electrical potential, should not be affected by the electrically neutral exchange of cations catalyzed by nigericin. When the external pH is 7.0 this seems to be true, since the ATP synthesis which is initially sensitive to valinomycin and K⁺ is largely insensitive to nigericin and K⁺. However, when the external pH is 8.0 the response to nigericin is exactly the opposite and the ATP formation which is sensitive to valinomycin is also abolished by nigericin. These data suggest that there may be either an energetic requirement for both a ΔpH and membrane potential at alkaline pH or a non-energetic requirement for a minimum proton activity in the initiation of phosphorylation.     

Potassium-stimulated ATPase activity and hydrogen transport in gastric microsomal vesicles

The Mg²⁺-dependent, K⁺-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H⁺ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth).Valinomycin stimulated both dye uptake and K⁺-ATPase (valinomycin-stimulated K⁺-ATPase); stimulation by valinomycin was due to increased K⁺ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K⁺-ATPase and H⁺ accumulation as an index, the sequence for anion permeation was NO₃^? > Br^? > Cl^? > I^? > acetate ≈ isethionate. When permeability to both K⁺ and H⁺ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K⁺-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an ‘uncoupling’ of ATP hydrolysis from H⁺ accumulation.Thiocyanate interacts with valinomycin inhibiting the typical action of the K⁺ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN^? to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K⁺-ATPase, it appears that SCN^? is a very     

Subcellular localization of H+-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H⁺-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H⁺ pump was directly demonstrated. The H⁺ pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K⁺-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because -mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DES dethyltilbestrol     

An ATPase from dog gastric mucosa: changes of outer pH in suspensions of membrane vesicles accompanying ATP hydrolysis

A microsomal Mg-ATPase from the gastric mucosa of dog, cat and frog has a K_m for ATP in the region 20–25 μM and by the value of this coefficient can be differentiated from the mitochondrial Mg-ATPase. The microsomal Mg-ATPase from dog gastric mucosa can be stimulated by gramicidin, nigericin and valinomycin in a KCl medium. This Mg-ATPase seems to be located in the ion impermeable membrane of microsomal vesicles and ATP hydrolysis driven changes of the outer pH can be observed. The data are consistent with the ATP hydrolysis driven entry of H⁺ ions across the vesicle membrane.     

Demonstration of ΔpH- and Δψ-induced synthesis of inorganic pyrophosphate in chromatophores from Rhodospirillum rubrum

It is possible to obtain synthesis of PP_i by artifical ion potentials in Rhodospirillum rubrum chromatophores. PP_i can be formed by K⁺-diffusion gradients (Δψ), H⁺ gradients (ΔpH) or a combination of both. In contrast, ATP can only be synthesized by imposed Δψ or Δψ+ΔpH. For ATP formation there is also a threshold value of K⁺ concentration below which synthesis of ATP is not possible. Such a threshold is not found for PP_i formation. Both PP_i and ATP syntheses are abolished by addition of FCCP or nigericin and only marginally affected by electron transport inhibitors. The synthesis of PP_i can be monitored for several minutes before it ceases, while ATP production stops within 30 s. As a result the maximal yield of PP_i is 200 nmol PP_i/μmol BChl, while that of ATP is no more than 25 nmol ATP/μmol BChl. The initial rates of syntheses were 0.50 μmol PP_i/μmol BChl per min and 2.0 μmol ATP/μmol per min, respectively. These rates are approx. 50 and 20% of the respective photophosphorylation rates under saturating illumination.     

Adenosine triphosphate-generated transmembrane electric potential in chloroplasts

Utilizing oxonol VI as a transmembrane electric potential indicating dye, chloroplasts are shown to develop rapid transient light-induced and ATP-induced potentials. Following the large transient signal smaller steady-state potentials are maintained with either driving system. The ATP-induced potential in the dark depends upon preactivation of the light-triggered ATPase of the chloroplasts, and is inhibited by uncouplers, ionophores such as valinomycin, and energy-transfer inhibitors such as tentoxin, Dio-9 or DCCD. Nigericin increased the signal of both the light- and the ATP-induced reactions. The fact that relatively large transient membrane potentials are induced by either a dark-to-light transition or ATP in the dark provides an explanation for previously observed phenomena such as early kinetics of photophosphorylation and the ATP-induced luminescence.     

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

(1) Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-P_i exchange activity. (2) Aurovertin alone inhibits 50% of the oxonol shift at 2 μM, and no further inhibition occurs at up to 12 μM. In combination with even weakly effective uncouplers, 4 μM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. (3) No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10–27%, depending on the preparation. The extent of the oxonol response depends on the ATP / ADP ratio rather than the phosphorylation potential. (4) The dye response in the ATPase complex is 4–7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. (5) The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.     

Chemiosmotic coupling in Methanobacterium thermoautotrophicum: hydrogen-dependent adenosine 5''-triphosphate synthesis by subcellular particles.

Hydrogenase and the adenosine 5'-triphosphate (ATP) synthetase complex, two enzymes essential in ATP generation in Methanobacterium thermoautotrophicum, were localized in internal membrane systems as shown by cytochemical techniques. Membrane vesicles from this organism possessed hydrogenase and adenosine triphosphatase (ATPase) activity and synthesized ATP driven by hydrogen oxidation or a potassium gradient. ATP synthesis depended on anaerobic conditions and could be inhibited in membrane vesicles by uncouplers, nigericin, or the ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The presence of an adenosine 5'-diphosphate-ATP translocase was postulated. With fluorescent dyes, a membrane potential and pH gradient were demonstrated.     

Uptake of sulfate,sulfite and thiosulfate by proton-anion symport in Desulfovibrio desulfuricans

pH changes and sulfide production upon addition of sulfate, sulfite or thiosulfate to non-buffered H₂-saturated cell suspensions of Desulfovibrio desulfuricans were studied by means of electrodes. The addition of these electron acceptors resulted in a rapid alkalinization of the suspension which was accompanied by sulfide production. At-2° C, alkalinization without immediate sulfide production could be obtained. After addition of ³⁵S-labelled sulfate at-2° C, the label was found to be concentrated 7,500-fold in the cells, while 2 protons per sulfate molecule had disappeared from the outer bulk phase. Alkalinization and sulfide production from micromolar electron acceptor additions depended on the transmembraneous proton gradient ( pH), and were reversibly inhibited in alkaline solution (pH>8.0) or by the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP). Protonophore-inhibited sulfide production from sulfite or thiosulfate could be restored if the cell membranes were permeabilized by the detergent cetyltrimethylammonium bromide (CTAB), or if downhill transport was made possible by the addition of electron acceptors at millimolar concentrations. Sulfate was not reduced under these conditions, presumably because the cells did not contain ATP for its activation. K⁺-and Na⁺-ionophores such as nigericin, valinomycin or monensin appeared to be of limited efficiency in D. desulfuricans. In most experiments, sulfate reduction was inhibited by the K⁺–H⁺ antiporter nigericin in the presence of K⁺, but not by the thiocyanate anion or the K⁺-transporter valinomycin. The results indicate that sulfate, sulfite and thiosulfate are taken up by proton-anion symport, presumably as undissociated acids with an electroneutral mechanism, driven by the transmembraneous pH gradient ( pH) or by a solute gradient. Kinetics of alkalinization and sulfide production in cells grown with different electron acceptors revealed that D. desulfuricans has different specific uptake systems for sulfate and thiosulfate, and obviously also for sulfite. It is proposed that the electron acceptor transport finally will not consume net energy during growth in buffered medium: The protons taken up during active electron acceptor transport leave the cell with the reduced end-product by simple passive diffusion of H₂S.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - FCCP carbonyl cyanide p-trifluoromethoxy phenylhydrazone - CTAB cethyltrimethylammonium bromide     

Ion transport by heart mitochondria: XXVII. The relation of mercurial-dependent adenosine triphosphatase activity to ion movements

The properties of a mercurial-dependent adenosine triphosphatase activity have been examined in isolated beef heart mitochondria. The reaction differs from that induced by uncouplers in that it is associated with extensive ion uptake and osmotic swelling, is highly specific for K⁺ over Na⁺, and is enhanced by respiration. Evidence is presented which suggests that the following events can account for the observations: (1) The mercurial blocks the phosphate transporter so that phosphate hydrolyzed from ATP is trapped in the matrix. (2) This interior negative potential causes cations to move inward and swelling results. (3) Permeability to K⁺ but not to Na⁺ is enhanced greatly by the reaction of the mercurial with the membrane. The inward movement of K⁺ closely resembles that produced by valinomycin, in that it is accompanied by proton ejection into the medium and it rapidly establishes a condition in which ion gradients cannot be maintained. This marked increase in permeability may be related to the pH gradient and is manifest as additional passive swelling in the absence of sucrose and passive contraction when sucrose is present. A comparison of the kinetics of swelling and of ATP hydrolysis shows that the elevated rates of ATPase are correlated with this condition of high permeability. When a corresponding condition of high permeability to Na⁺ is established by treatment with gramicidin or EDTA, the mercurial-dependent ATPase is nearly as rapid in Na⁺ as in the K⁺ medium. It appears, therefore, that the K⁺ specificity resides at the level of membrane permeability and is not a feature of the ATPase reaction per se. (4) Respiration appears to affect the ATPase reaction by virtue of its ability to extrude ions from the matrix in the presence of the mercurial. p-Chloromercuriphenyl sulfonate causes a switch from respiration-dependent ion accumulation to respiration-dependent ion extrusion to occur. A model to explain these reactions is presented.     

Proton Transport Activity of the Purified Vacuolar H-ATPase from Oats : Direct Stimulation by Cl

To determine whether the detergent-solubilized and purified vacuolar H⁺-ATPase from plants was active in H⁺ transport, we reconstituted the purified vacuolar ATPase from oat roots (Avena sativa var Lang). Triton-solubilized ATPase activity was purified by gel filtration and ion exchange chromatography. Incorporation of the vacuolar ATPase into liposomes formed from Escherichia coli phospholipids was accomplished by removing Triton X-100 with SM-2 Bio-beads. ATP hydrolysis activity of the reconstituted ATPase was stimulated twofold by gramicidin, suggesting that the enzyme was incorporated into sealed proteoliposomes. Acidification of K⁺-loaded proteoliposomes, monitored by the quenching of acridine orange fluorescence, was stimulated by valinomycin. Because the presence of K⁺ and valinomycin dissipates a transmembrane electrical potential, the results indicate that ATP-dependent H⁺ pumping was electrogenic. Both H⁺ pumping and ATP hydrolysis activity of reconstituted preparations were completely inhibited by <50 nanomolar bafilomycin A₁, a specific vacuolar type ATPase inhibitor. The reconstituted H⁺ pump was also inhibited by N,N′-dicyclohexylcarbodiimide or NO₃⁻ but not by azide or vanadate. Chloride stimulated both ATP hydrolysis by the purified ATPase and H⁺ pumping by the reconstituted ATPase in the presence of K⁺ and valinomycin. Hence, our results support the idea that the vacuolar H⁺-pumping ATPase from oat, unlike some animal vacuolar ATPases, could be regulated directly by cytoplasmic Cl⁻ concentration. The purified and reconstituted H⁺-ATPase was composed of 10 polypeptides of 70, 60, 44, 42, 36, 32, 29, 16, 13, and 12 kilodaltons. These results demonstrate conclusively that the purified vacuolar ATPase is a functional electrogenic H⁺ pump and that a set of 10 polypeptides is sufficient for coupled ATP hydrolysis and H⁺ translocation.     

Characteristics of MgATP2--dependent electrogenic proton transport in tonoplast vesicles of the facultative crassulacean-acid-metabolism plant Mesembryanthemum crystallinum L.

Membrane vesicles were isolated from mesophyll cells of Mesembryanthemum crystallinum in the C₃ state and in the crassulacean acid metabolism (CAM) state. The distribution of ATP-hydrolysis and H⁺-transport activities, and the activities of hydroxypyruvate reductase and Antimycin-insensitive cytochrome-c-reductase on continuous sucrose gradients was studied. For isolations carried out routinely a discontinuous sucrose gradient (24%/37%/50%) was used. Nitrate-sensitive ATP-hydrolysis and H⁺-transport activities increased several-fold during the transition from C₃ photosynthesis to CAM. Nitrate-sensitive ATPase showed a substrate preference for ATP with an apparent K_m (MgATP^2-) of 0.19–0.37 mM. In both C₃ and CAM states the ATPase showed a concentration-dependent stimulation by the anions chloride and malate. However, the pH optima of the two states were different: the ATPase of C_3- M. crystallinum had an optimum of pH 7.4 and that of CAM-M. crystallinum an optimum of pH 8.4. The optical probe oxonol-VI was used to demonstrate the formation of MgATP^2--dependent electric-potential gradients in tonoplast vesicles.Abbreviations Bistris-Pronane 1,3-bis [tris(hydroxymethyl)-methylaminol propane - CAM Crassulacean acid metabolism - DIDS 4,4-dilsothiocyano-2,2-stilbene disulfonic acid: - DTT dithiothreitol - ER endoplasmic reticulum - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonic acid - HPR hydroxypyruvate reductase - IDPase inosine 5-diphosphatase - OX-VI oxonol VI - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Mussel and mammalian ATP synthase share the same bioenergetic cost of ATP

The molecular mechanism by which the membrane-embedded F_O sector of the mitochondrial ATP synthase translocates protons, thus dissipating the transmembrane protonmotive force and leading to ATP synthesis, involves the neutralization of the carboxylate residues of the c-ring. Carboxylates are thought to constitute the binding sites for ion translocation. In order to cast light on this mechanism, we exploited N,N’-dicyclohexylcarbodiimide, which covalently binds to F_O c-ring carboxylates, and ionophores which selectively modulate the transmembrane electric (Δφ) and chemical (ΔpH) gradients such as valinomycin, nigericin and dinitrophenol. ATP hydrolysis was evaluated in mitochondrial preparations and/or inside-out submitochondrial particles from mussel and mammalian tissues under different experimental conditions. The experiments pointed out striking similarities between mussel and mammalian mitochondrial ATP synthase. Our results support the hypothesis that the ATP synthase of Mytilus galloprovincialis induces intersubunit torque generation and translocates H⁺ by coordinating the hydronium ion (H₃O⁺) in the ion binding site of F_O. Our results are consistent with the hypothesis that in mussel mitochondria the main component of the electrochemical gradient driving proton flux and ATP synthesis is Δφ. Therefore, mussel F_O probably contains a small c-ring, which implies a low bioenergetic cost of making ATP as in mammals. These features which make mussel mitochondria as efficient in ATP production as mammalian ones may be especially advantageous in facultative aerobic species which intermittently exploit mitochondrial respiration to generate ATP.     

Use of 1-anilino-8-naphthalene-sulfonate as a probe of gastric vesicle transport

Summary The interaction of 1-anilino-8-naphthalene-sulfonate (ANS) with vesicles derived from hog fundic mucosa was studied in the presence of valinomycin and with the addition of ATP. Evidence was found for two classes of sites, those rapidly accessible to ANS with aK _D of 7.5 m and those slowly accessible, but rapidly accessed in the presence of valinomycin with aK _D of 2.5 m. ATP transiently increases the quantum yield of the latter ANS binding sites only in the presence of valinomycin, but does not alter the number ofK _D of those sites. The time course of this increase correlates with H⁺ uptake and Rb⁺ extrusion by those vesicles and H⁺ carriers such as tetrachlorsalicylanilide or nigericin abolish the ATP response. With ATP addition in the presence of SC¹⁴N and valinomycin there is transient uptake of SCN^–. It is concluded that ANS is acting as a probe of a structural change dependent on a potential and H⁺ gradient.     

Mechanism of uncoupling by uncouples of oxidative phosphorylation

Classical uncouplers duplicate exactly the uncoupling actions of the valinomycin-nigericin ionophoric combination in presence of K⁺ — a combination that mediates cyclical transport of K⁺ driven by electron transfer or pyrophosphorolysis of ATP in mitochondria. Evidence has been presented that uncouplers have the properties essential for mediating coupled cyclical transport of cations and that uncoupling of oxidative phosphorylation can be rationalized in terms of one coupled process being displaced and replaced by another. The critical demonstrations were first that uncoupling is a cation-dependent process and that only those cations that can undergo complexation with uncouplers are effective in restoring mitochondrial uncoupler action in a cation-deficient medium. The second demonstration was that uncouplers are ionophores, not only of the nigericin type but also of the valinomycin type (electrogenic). This combination in one molecule of electrogenic as well as non-electrogenic ionophoric activity for cations endows uncouplers with the capability for duplicating the uncoupling action of the valinomycin-nigericin combination and for mediating coupled cyclical transport of cations.     

Quantitation of corneal endothelial potentials using a carbocyanine dye

The carbocyanine dye, diS-C₃-(5) was used to quantitate the plasma membrane potential of the bullfrog corneal endothelium. It was shown that valinomycin hyperpolarized the endothelial cell and that in the presence of the ionophore the membrane potential largely reflected the K⁺ equilibrium potential. Using calibration curves constructed by changing medium K⁺ concentration in the presence of valinomycin, and nigericin and ouabain to abolish ion gradients and electrogenic pump activity, the cell membrane potential was calculated to be 28.6 ± 4.2 mV. The major source of this potential was a K⁺ diffusion potential, and the membrane Na⁺ conductance reduced the cell potential to less than the apparent K⁺ equilibrium potential of 51.5 ± 5.1 mV. About 20% of the cell potential could be ascribed to the rheogenic (Na⁺+K⁺)-ATPase.