Cytochromes and gastric acid secretion. A reevaluation of mucosal acidification experiments.

Using an improved spectrophotometer, we have reinvestigated the report (Hersey, S.J. (1974) Biochim. Biophys. Acta 344, 157--203) that acidification of the mucosal surface of frog gastric mucosa produces a crossover point between flavoprotein and cytochrome b, thus identifying a site of energy coupling between the cytochrome and H+ transport systems. While we find spectrophotometric changes upon addition of HCl to the mucosal solution, we find similar changes upon addition of NaCl without pH change, but no changes when the pH is lowered by substitution of H+ for Na+ at constant osmolality. We show that osmolality changes, with consequent alteration in tissue light scattering, are responsible for these effects. Further, we can show that the pH changes used do not inhibit acid secretion, and that one cannot do so without osmolality increase. We conclude that the imputed crossover point is not demonstrated, and that models based on its existence must be revised.     

A non-alkalophilic mutant of Bacillus alcalophilus lacks the Na+/H+ antiporter.

A non-alkalophilic mutant strain of $\text{Bacillus}\text{alcalophilus}$ grows on L-malate over a pH range from 5.0 to 9.0. The mutant does not exhibit the energy-dependent efflux of Na⁺ that has been used to assay a $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ antiporter in the wild type organism. The mutant also fails to transport α-aminoisobutyric acid, at pH 9.0, either in the presence or absence of Na⁺; at pH 5.5, the amino acid analogue is taken up by a Na⁺-independent mechanism. The properties of the mutant constitute strong evidence that the $\text{Na}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ antiporter is involved in maintaining an acidified cytoplasm in $\text{B}\text{.}\text{alcalophilus}$.     

Oxidative phosphorylation by membrane vesicles from Bacillus alcalophilus

ADP and P_i-loaded membrane vesicles from l-malate-grown Bacillus alcalophilus synthesized ATP upon energization with $\text{ascorbate}\text{N,N,N′,N′-}\text{tetramethyl}\text{-p-}\text{phenylenediamine}$. ATP synthesis occurred over a range of external pH from 6.0 to 11.0, under conditions in which the total protonmotive force was as low as ?30 mV. The phosphate potentials (ΔG_p) were calculated to be 11 and 12 kcal/mol at pH 10.5 and 9.0, respectively, whereas the values in vesicles at these two pH values were quite different (?40 ± 20 mV at pH 10.5 and ?125 ± 20 mV at pH 9.0). ATP synthesis was inhibited by KCN, gramicidin, and by N,N′-dicyclohexylcarbodiimide. Inward translocation of protons, concomitant with ATP synthesis, was demonstrated using direct pH monitoring and fluorescence methods. No dependence upon the presence of Na⁺ or K⁺ was found. Thus, ATP synthesis in B. alcalophilus appears to involve a proton-translocating ATPase which functions at low .     

Effects of pH on β-hydroxybutyrate transport in rat erythrocytes and thymocytes

Entry of β-hydroxybutyrate into erythrocytes and thymocytes is facilitated by a carrier (C), as judged from temperature dependence, saturation kinetics, stereospecificity, competition with lactate and pyruvate, and inhibition by moderate concentrations of methylisobutylxanthine, phloretin, or α-cyanocinnamate. We studied the dependence of influx and efflux on internal and external pH and [β-hydroxybutyrate]. Lowering external pH from 8.0 to 7.3 to 6.6 enhanced influx into erythrocytes by lowering entry $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ from 29 to 16 to 10 mM, entry $\text{V}$ being independent of external pH. Lowering external pH inhibited efflux. At low external pH, external β-hydroxybutyrate enhanced efflux slightly. At high external pH, external β-hydroxybutyrate inhibited efflux. Internal acidification inhibited influx and internal alkalization enhanced influx. Internal β-hydroxybutyrate (βHB) enhanced influx more in acidified than alkalized cells. These data are compatible with coupled βHB^?/OH^? exchange, βHB^? and OH^? competing for influx, C : OH^? moving faster than C : βHB^?, empty C being immobile. They are also compatible with coupled βHB^?/H⁺ copermeation, empty C moving inward faster than H⁺ : C : βHB^?, H⁺ : C being immobile, and C : βHB^? (without H⁺) being so unstable as not to be formed in significant amounts (relative to C, H⁺ : C, and H⁺ : C : βHB^?).     

Involvement of the proton electrochemical gradient in genetic transformation in Escherichia coli

Plasmid pIY2 DNA which encodes for ampicillin-resistance was used to study the energetics of Ca⁺⁺-induced transformation in Escherichia coli. When cells are exposed to DNA in the presence of carbonylcyanide-m-chlorophenylhydrazone or 2,4-dinitrophenol, two protonophores that collapse the proton electrochemical gradient across the cell membrane ($\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$), transformation to ampicillin-resistance is drastically reduced with little or no effect on viability. Furthermore, when the components of $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$ are altered by varying ambient pH or by performing transformation in the presence of valinomycin or nigericin, the efficiency of transformation is directly correlated with the magnitude of the membrane potential and changes in the pH gradient have no significant effect. It is concluded that $\text{Δ}\text{μ}{}_{\mathrm{H}}\text{+}$, more specifically the membrane potential, plays a critical role in Ca⁺⁺-induced transformation.     

H+ transport in the evolution of photosynthesis

Extant photosynthetic organisms all appear to use transmembrane H⁺ fluxes as the coupling agent in the use of light energy in ATP synthesis. In the steady-state there is a large H⁺ free energy difference across the coupling membrane, and when this is reflected as a light-induced change in pH of the phase (cytosol or stroma) containing the enzymes of carbon assimilation, the H⁺ transport can have an informational role in activating and inactivating enzymes.The earliest organisms probably lived fermentatively (substrate-level phosphorylation) in an anaerobic environment provided with organic solutes synthesised abiotically. There are good reasons for believing that one of the earliest primary active transport systems (interconverting chemical and electrical/osmotic energy) was an H⁺ extrusion pump powered by ATP or PP_i. Its initial function was extrusion of excess H⁺ from the fermenting cells, and the support of a number of co-transport processes. The earliest energetic use of light energy is envisaged as being the energization of an alternative H⁺ extrusion pump, with bacteriorhodopsin or (bacterio-) chlorophyll as the pigment. The former type of cyclic photoredox system (Halobacterium-type) is simpler than the latter: a “pre-respiratory” chemical redox H⁺ pump may have preceded the (bacterio-) chlorophyll-based process. Any of these H⁺ pumps could spare the use of fermentative ATP in powering active H⁺ efflux and would thus have been favoured as fermentative substrates became scarce; eventually the larger generated by the light-powered H⁺ pump was used to drive the ATP-powered H⁺ pump backwards and thus generate ATP with light as the ultimate energy source.Scarcity of suitable reductants for biosynthesis as life proliferated provided a selective impetus for a non-cyclic photoredox system which could use light energy to generate a low-potential reductant at the expense of more readily available higher-potential reductants. The non-cyclic photoredox system is not possible in its simplest form (with all the redox energy coming from excitation energy of one or more photoreactions) in the bacteriorhodopsin line of evolution. Such a simple photoredox system is found in the Chlorobiaceae; even if (as seems likely) the non-cyclic photoredox process generates a (and thus, potentially, ATP), some of the ATP needed for CO₂ fixation and cell growth must be generated by a cyclic photoredox system.In the extant purple bacteria the generation of low-potential reductant involves a non-cyclic photoredox pathway which produces a reductant unable to reduce NAD⁺; the “energy gap” is spanned by “reverse electron transfer” which uses energy from a . It is not clear if this energetic requirement for the H⁺ gradient can be quantitatively satisfied from a non-cyclic photoredox H⁺ transport; it is certain that there is a major requirement for cyclic photoredox H⁺ pumping in these organisms.The photosynthetic bacteria are today restricted to reducing (low E_h) environments similar to those found in the early, anoxic earth; they are unable to use very weak reductants as donors for non-cyclic photoredox processes. As the sources of even weakly reducing donors (other than H₂O) on the primitive earth were depleted the two photoreactions scheme of extant O₂-producers evolved by modification of the bacterial photoreaction. This non-cyclic photoredox process is definitely H⁺-translocating and the role of cyclic photoredox processes in ATP generation in O₂-evolvers is smaller than in photosynthetic bacteria.In parallel with the biochemical and biophysical changes in the photosystems there was a morphological evolution, with an increasing tendency for “internalisation” of the photoredox processes (originally present in the plasma membrane, as in extant Chlorobineae) into thylakoids (as in most Rhodospirillineae, Cyanobacteria and in all eukaryotes). With a plasmalemma-located photoredox system, and the constraints of a fixed, alkaline external pH and the cytoplasmic pH of 7–8, the would be generated largely as an electrical P.D. The presence of a phase (intrathylakoid space) with a “negotiable pH” would permit the generation and use of a largely present as a pH gradient.In both cases illumination can cause an increase in cytoplasmic (stromal) pH over the dark value; this is an important aspect of the regulation of “phototrophic” and “heterotrophic” enzyme systems in the light and in the dark. However, it is argued that these differences in pH are not absolutely light-dependent unless they depend upon some more uniquely light-dependent signal, probably based on a redox component only generated in the light.     

The accumulation of superoxide radical during the aerobic action of xanthine oxidase A requiem for H2O4−

The action of xanthine oxidase upon acetaldehyde or xanthine at pH 10.2 has been shown to be accompanied by substantial accumulation of O₂^? during the first few minutes of the reaction. H₂O₂ decreases this accumulation of O₂^? presumably because of the Haber-Weiss reaction ($\text{H}{}_2\text{O}{}_2\text{+}\text{O}{}_2{}^{\mathrm{?}}\text{→}\text{OH}{}^{\mathrm{?}}\text{+}\text{OH}\text{+}\text{O}{}_2$) and very small amounts of superoxide dismutase eliminate it. This accumulation of O₂^? was demonstrated in terms of a burst of reduction of cytochrome $\text{c}$, seen when the latter compound was added after aerobic preincubation of xanthine oxidase with its substrate. The kinetic peculiarities of the luminescence seen in the presence of luminol, which previously led to the proposal of H₂O₄^?, can now be satisfactorily explained entirely on the basis of known radical intermediates.     

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated.Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H⁺ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K⁺. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons.The proton ejection is the result of true translocation of H⁺ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system.Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions.It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H⁺.     

Electrogenic proton ejection coupled to electron transport through the energy-conserving site 2 and K+/H+ exchange in yeast mitochondria

The proton ejection coupled to electron flow from succinate and/or endogenous substrate(s) to cytochrome c using the impermeable electron acceptor ferricyanide is studied in tightly coupled mitochondria isolated from two strains of the yeast Saccharomyces cerevisiae. (1) The observed H⁺ ejection/2e^? ratio approaches an average value of 3 when K⁺ (in the presence of valinomycin) is used as charge-compensating cation. (2) In the presence of the proton-conducting agent carbonyl cyanide m-chlorophenylhydrazone, an H⁺ ejection/2e^? ratio of 2 is observed. (3) The low stoichiometry of 3H⁺ ejected (instead of 4) per 2e^? and the high rate of H⁺ back-decay (0.1615 $\text{ln}\text{δ-}\text{(}\text{ngatom}\text{)}\text{H}{}^{\mathrm{+}}\text{s}$ and a half-time of 4.6 s for 10 mg protein) into the mitochondrial matrix are related to the presence of an electroneutral K⁺/H⁺ antiporter which is demonstrated by passive swelling experiments in isotonic potassium acetate medium.     

Reconstitution of b-type cytochrome oxidase from Rhodopseudomonas capsulata in liposomes and turnover studies of proton translocation

Purified b-type cytochrome oxidase from Rhodopseudomonas capsulata was incorporated into phospholipid vesicles to measure proton extrusion with pulses of ferrocytochrome c for one oxidase turnover. In accordance with the pH shift of its midpoint potential, the purified oxidase showed a proton extrusion of 0.24 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ with uptake of 1 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$ from the liposomes for the reduction of oxygen to water. This proton translocation could only be observed in the presence of valinomycin +K⁺ and was not inhibited by DCCD. Oxidase preparations from the first purification step, which contain other protein compounds especially a membrane-bound cytochrome c but not the ubiquinol-cytochrome c₂-oxidoreductase showed a pumping activity of 0.9 $\text{H}{}^{\mathrm{+}}\text{e}{}^{\mathrm{?}}$, which was inhibited by DCCD for nearly 75%. Inhibition of the electron transfer was not observed, which could be explained by a ‘molecular slipping’ of proton extrusion and electron transfer. Proton extrusion from two oxidase-turnovers was only 80% of that from one turnover. The proton pumping of the b-type oxidase strongly depended on the enzyme/phospholipid ratio.     

A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Respiratory control and mitochondrial monovalent cation permeability of isolated liver cells

Uncoupling agent releases the respiratory control of rat hepatocytes to approximately the same degree as in isolated mitochondria indicating that mitochondria $\text{in situ}$ possess a low H⁺ conductance as $\text{in vitro}$. Mitochondria also have no detectable natural K⁺ conductance since the ionophore, valinomycin, is required for K⁺ ions to uncouple. Na⁺ but not K⁺ or choline inhibits the uncoupled respiration of liver cells. This is consistent with operation of neutral mitochondrial Na⁺ for H⁺ exchange $\text{in vivo}$. These results indicate a considerable similarity between certain functional and permeability properties of mitochondria $\text{in vitro}$ and $\text{in situ}$. These similarities form the basis for discussion of the role of mitochondrial ion transport in metabolic regulation.     

Lactose transport in Escherichia coli cells Dependence of kinetic parameters on the transmembrane electrical potential difference

We determine the kinetic parameters $\text{V}$ and $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ of lactose transport in Escherichia coli cells as a function of the electrical potential difference (Δψ) at pH 7.3 and $\text{Δ}\text{pH}\text{= 0}$. We report that transport occurs simultaneously via two components: a component which exhibits a high $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ (larger than 10 mM) and whose contribution is independent of Δψ, a component which exhibits a low $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ independent of Δψ (0.5 mM) but whose $\text{V}$ increases drastically with increasing Δψ. We associate these components of lactose transport with facilitated diffusion and active transport, respectively. We analyze the dependence upon Δψ of $\text{K}{}_{\mathrm{<mtext>T</mtext>}}$ and $\text{V}$ of the active transport component in terms of a mathematical kinetic model developed by Geck and Heinz (Geck, P. and Heinz, E. (1976) Biochim. Biophys. Acta 443, 49–63). We show that within the framework of this model, the analysis of our data indicates that active transport of lactose takes place with a H⁺/lactose stoichiometry greater than 1, and that the lac carrier in the absence of bound solutes (lactose and proton(s)) is electrically neutral. On the other hand, our data relative to facilitated diffusion tend to indicate that lactose transport via this mechanism is accompanied by a H⁺/lactose stoichiometry smaller than that of active transport. We discuss various implications which result from the existence of H⁺/lactose stoichiometry different for active transport and facilitated diffusion.     

Phosphate transport in Neurospora: Kinetic characterization of a constitutive,low-affinity transport system

Log-phase cells of Neurospora crassa, grown in standard minimal medium, possess an energy-dependent transport system for inorganic phosphate, with a $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ (at pH 5.8) of 0.123 mM and a J_max of 1.64 mmoles/l cell water per min. Like the PO₄^3? transport system in yeast, the Neurospora system is stimulated by high intracellular K⁺. In addition, it is inhibited by high extracellular salt concentrations, an effect which may be related to the known depolarization of the Neurospora plasma membrane at high salt concentrations.The most striking property of the system is its strong dependence upon the extracellular pH. From pH 4.0 to pH 7.3, the J_max remains essentially constant but the $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ increases nearly 400-fold, from 0.01 to 3.62 mM. The increase cannot be accounted for by a single system with a preference for H₂PO₄^? (which would show only a 3-fold increase in apparent $\text{K}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ over this pH range) nor by two systems with different affinities and pH optima (which would display nonlinear double-reciprocal plots at intermediate pH values). It can be explained, however, by a model in which OH^? or H⁺ is assumed to act as a modifier of the transport system, altering its affinity for substrate.     

Unmasking the mitochondrial K/H exchanger: tetraethylammonium-induced K+-loss.

Mitochondria respiring in media containing 80 mM tetraethylammonium ions lose all of their endogenous K⁺ within 7 minutes. K⁺-loss is associated with uptake of tetraethylammonium ions. K⁺ efflux under these conditions is energy-dependent and electroneutral. It is concluded that tetraethylammonium uptake unmasks the endogenous $\text{K}\text{H}$ exchanger. Considered in relation to the chemiosmotic theory, these results support the existence of a “carrier-brake” mechanism which modulates $\text{K}\text{H}$ exchange to maintain volume homeostasis $\text{in vivo}$.     

Phosphate transport and the stoicheiometry of respiratory driven proton translocation in Escherichia coli.

The stoicheiometry of respiratory driven proton translocation associated with the oxidation of endogenous substrates has been measured in an organic phosphate auxotrophic mutant of $\text{Escherichia coli}$. The results obtained indicate that movements of inorganic phosphate do not result in an experimental underestimation of the observed H⁺/site ratio in $\text{E. coli}$, as has previously been suggested for mitochondria.     

Cation/proton antiport systems in Escherichia coli.

Three distinct systems which function as proton/cation antiports have been identified in $\text{E.}\text{coli}$ by the ability of the ions to dissipate the ΔpH component of the protonmotive force in everted vesicles. System I exchanges H⁺ for K⁺, Rb⁺ or Na⁺; System II has Na⁺ and Li⁺ as substrates; and System III catalyzes proton exchange for Ca²⁺, Mn²⁺ or Sr²⁺.     

Stimulation of respiration-linked proton efflux in Escherichia coli by carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP).

The proton efflux from intact, anaerobic $\text{Escherichia}\text{coli}$ cells following a small oxygen pulse is both slow ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>M</mtext><mtext>2</mtext>}}\text{～-10s}$) and inefficient ($\text{H}{}^{\mathrm{+}}\text{O}\text{～-0.5}$. Very low levels (<80 nM) of the proton ionophore carbonylcyanide-$\text{p}$-trifluoromethoxyphenylhydrazone (FCCP), which have no detectable effect upon active transport, cause a 3–5 fold stimulation in the extent of proton efflux without affecting the efflux rate. At slightly higher concentrations of FCCP (80 nM to 0.5 μM), a sharp inhibition of this increased proton efflux occurs, with the $\text{H}{}^{\mathrm{+}}\text{O}$ ratio obtained in the presence of 0.5 μM FCCP approximately equal to that obtained in the absence of FCCP. Still higher concentrations of FCCP (> 1 μM), which inhibit active transport, cause a further gradual decrease in the $\text{H}{}^{\mathrm{+}}\text{O}$ ratio. The unusual increase in the apparent efficiency of H⁺ efflux by <80 nM FCCP is not accompanied by an increase in the rate of membrane deenergization following an O₂ pulse, although such an increase is seen with the higher (uncoupling) FCCP concentrations.