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.     

Na+-driven Ca2+ transport in alkalophilic Bacillus

Ca²⁺ transport was studied in membrane vesicles of alkalophilic Bacillus. When Na⁺-loaded membrane vesicles were suspended in KHCO₃/KOH buffer (pH 10) containing Ca²⁺, rapid uptake of Ca²⁺ was observed. The apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for Ca²⁺ measured at pH 10 was about 7 μM, and the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value shifted to 24 μM when measured at pH 7.4. The efflux of Ca²⁺ was studied with Ca²⁺-loaded vesicles. Ca²⁺ was released when Ca²⁺-loaded vesicles were suspended in medium containing 0.4 M Na⁺.Ca²⁺ was also transported in membrane vesicles driven by an artificial pH gradient and by a membrane potential generated by K⁺-valinomycin in the presence of Na⁺.These results indicate the presence of Ca²⁺/Na⁺ and H⁺/Na⁺ antiporters in the alkalophilic Bacillus A-007.     

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 .     

Determination of delta psi, delta pH and the proton electrochemical gradient in isolated cholinergic synaptic vesicles

The electrical potential (Δψ) of intact cholinergic synaptic vesicles was measured in the presence and absence of the proton translocator carbonyl cyanide p-trifluoromethoxy-phenylhydrazone (FCCP), and the results were utilized to calculate the vesicular proton chemical gradient (ΔpH) and proton electrochemical potential $\text{(Δ}\text{μ}{}_{\mathrm{<mtext>H</mtext>}}\text{+)}$. At external pH = 7.4 the vesicles maintain a proton electrochemical gradient of $\text{?+20}\text{mV}$ (positive inside) which is composed of $\text{Δψ??80}\text{mV}$ (negative inside) and $\text{Δ}\text{pH}\text{?1.6}$ (acidic inside). The proton chemical gradient (ΔpH) increases as a function of pH_out whereas the vesicular electrical potential (Δψ) is only slightly affected by the external pH. Consequently, $\text{Δ}\text{μ}{}_{\mathrm{<mtext>H</mtext>}}\text{+}$ is larger at basic external pH values ($\text{?+40}\text{mV}$ at pH_out = 9.0) and smaller at acidic external pH values ($\text{Δ}\text{μ}{}_{\mathrm{<mtext>H</mtext>}}\text{+?0}$ at (pH_out = 5.6). The possible physiological role of the electrochemical potentials in maintaining high concentrations of acetylcholine within the cholinergic synaptic vesicle is discussed.     

Characterization of sodium and proton flows in sub-bacterial particles of Halobacterium halobium in terms of nonequilibrium thermodynamics

A thermodynamic characterization of the Na⁺-H⁺ exchange system in Halobacterium halobium was carried out by evaluating the relevant phenomenological parameters derived from potential-jump measurements. The experiments were performed with sub-bacterial particles devoid of the purple membrane, in 1 M NaCl, 2 M KCl, and at pH 6.5–7.0. Jumps in either pH or pNa were brought about in the external medium, at zero electric potential difference across the membrane, and the resulting relaxation kinetics of protons and sodium flows were measured. It was found that the relaxation kinetics of the proton flow caused by a pH-jump follow a single exponential decay, and that the relaxation kinetics of both the proton and the sodium flows caused by a pNa-jump also follow single exponential decay patterns. In addition, it was found that the decay constants for the proton flow caused by a pH-jump and a pNa-jump have the same numerical value. The physical meaning of the decay constants has been elucidated in terms of the phenomenological coefficients (mobilities) and the buffering capacities of the system. The phenomenological coefficients for the Na⁺-H⁺ flows were determined as differential quantities. The value obtained for the total proton permeability through the particle membrane via all available channels, $\text{L}{}_{\mathrm{<mtext>H</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H\; +</mtext>}}\text{?Δ}\text{pH}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pNa</mtext>}}$, was in the range of 850–1150 nmol H⁺·(mg protein)^?1·h^?1·(pH unit)^?1 for four different preparations; for the total Na⁺ permeability, $\text{L}{}_{\mathrm{<mtext>Na</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 1620–2500 nmol Na⁺·(mg protein)^?1·h^?1·(pNa unit)^?1; and for the proton ‘cross-permeability’, $\text{L}{}_{\mathrm{<mtext>HNa</mtext>}}\text{= (}\text{?J}{}_{\mathrm{<mtext>H</mtext>}}{}^{\mathrm{+}}\text{?Δ}\text{pNa}\text{)}{}_{\mathrm{<mtext>\Delta \psi ,\Delta </mtext><mtext>pH</mtext>}}$, it was 220–580 nmol H⁺·(mg protein)^?1·h^?1·(pNa unit)^?1, for different preparations. From the above phenomenological parameters, the following quantities have been calculated: the degree of coupling (q), the maximal efficiency of Na⁺-H⁺ exchange (η_max), the flow and force efficacies (?) of the above exchange, and the admissible range for the values of the molecular stoichiometry parameter (r). We found q ? 0.4; η_max ? 5%; 0.36 ? r ? 2; ?_JNa⁺ ? 1.3 · 10⁵μmol · (RT unit)^?1 at J_Na = 1 μmolNa⁺ · (mgprotein)^?1 · h^?1; and ?_ΔpNa ? 5 · 10⁴ ΔpNa · (mg protein) · h · (RT unit)^?1 at ΔpNa = 1 unit, for different preparations.     

Proton electrochemical gradient and phosphate potential in submitochondrial particles

1. The aerobic uptake of inorganic ions, such as ⁸⁶Rb⁺ or ¹²⁵I^?, by submitochondrial particles, is about one order of magnitude lower than the uptake of organic ions, such as acridines or 8-anilino-1-naphthalene sulphonate. The values of ΔpH, the transmembrane pH differential, and Δψ, the transmembrane membrane potential are between 60 and 100 mV when calculated on the inorganic ions and between 150 and 240 mV when calculated on the organic ions. The discrepancy between the ΔpH and Δψ values from organic and inorganic ions is large at high but not at low ion/protein ratios.2. In the absence of weak bases and strong acids the values of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$, the proton electrochemical potential difference, are close to 100 mV and the magnitude of ΔpH and Δψ are similar. Weak bases decrease ΔpH and enhance Δψ. Strong acids decrease Δψ and enhance ΔpH. Interchangeability of ΔpH with Δψ occurs at low concentrations of weak bases and strong acids. High concentrations of weak bases and strong acids cause depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.3. Concentrations of weak bases capable of abolishing ΔpH, do not affect ATP synthesis. Concentrations of strong acids capable of abolishing Δψ affect only slightly ATP synthesis. Concentrations of weak bases and strong acids capable of causing a decline of ΔpH + Δψ inhibit ATP synthesis.4. Depression of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is paralleled by inhibition of ATP synthesis and decline of ΔGp, the phosphate potential. Abolition of ATP synthesis occurs only when $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ is below 20 mV. The $\text{ΔG}\text{p}\text{/Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$ ratio increases hyperbolically with the decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.     

A chloride requirement for Na+-dependent amino-acid transport by brush border membrane vesicles isolated from the intestine of a mediterranean teleost (Boops salpa)

The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na⁺ electrochemical gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{C}{}_{\mathrm{<mtext>in</mtext>}}$). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN^?, increases the Na⁺-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na⁺ gradient, the presence of Cl^? on the external side of the vesicles. In the absence of Cl^?, amino acid uptake is not stimulated by the Na⁺ gradient and is not influenced by an electrical membrane potential generated by SCN^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) or by a K⁺ diffusion potential ($\text{C}{}_{\mathrm{<mtext>in</mtext>}}\text{>C}{}_{\mathrm{<mtext>out</mtext>}}$). This Cl^? requirement differs from the Na⁺ requirement, since a Cl^? gradient ($\text{C}{}_{\mathrm{<mtext>out</mtext>}}\text{>C}{}_{\mathrm{<mtext>in</mtext>}}$) does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na⁺ gradient.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.     

Triple helix formation and disulphide bonding during the biosynthesis of glomerular basement membrane collagen.

White erythrocyte membranes, or ghosts, were monoconcave discocytes when incubated in 50mM N-tris (hydroxymethyl) methyl-2-aminoethane sulfonic acid titrated to pH 7.4 with triethanolamine. If 3mM MgCl₂ was included in the incubation medium, the ghosts were predominantly echinocytes. The echinocytic form could also be induced by Co⁺⁺, Ni⁺⁺, Li⁺, Na⁺, K⁺, $\text{NH}{}_4{}^{\mathrm{+}}$ and tetramethylammonium ion, all as chloride salts. The concentration of cation necessary for 50% of the ghosts to be echinocytes was correlated with the hydrated charge density of the cation with the most highly charged cations being the most effective. The cations Ca⁺⁺, Sr⁺⁺, Ba⁺⁺ and La⁺⁺⁺, (also as chloride salts) did not induce the normal echinocytic form, but at high levels induced a few misshapen forms with some resemblance to echinocytes. Instead Ca⁺⁺, Sr⁺⁺, Ba⁺⁺ and La⁺⁺⁺ suppressed the formation of echinocytes in the presence of Mg⁺⁺ and other ions. This suggests the presence of a specific Ca⁺⁺ binding site important to shape control in the erythrocyte membrane.     

Differences in uncoupling effects associated with the uptake of lactose and dansyl-galactoside in Escherichia coli membrane: Active transport versus specific binding

Cytoplasmic membrane vesicles isolated from Escherichia coli take up dansyl-galactoside, a fluorescent competitive inhibitor of lactose transport, to much lower levels than lactose. An initial interpretation, based on the study of the fluorescent changes accompanying the energy-dependent uptake, was that it represented a one-to-one specific binding to the lac carrier protein which was not followed by transport. Recently, on the basis of a new estimation of the number of lac carrier in the membrane, it has been advanced that the uptake of dansyl-galactoside represents a nonspecific binding on the inner surface of the membrane following transport. We discriminate between the two interpretations by comparing the effects of lactose and dansyl-galactoside uptake on the electrochemical gradient of protons ($\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$), generated by the oxidation of substrates, and on the uptake of proline. Indeed, it is known that the rate of lactose transport is such that it leads, as a consequence of the lactose/H⁺ symport, to an observable decrease of $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{H}}\text{+}$, and secondary to this decrease to an inhibition of the uptake of proline transported at much lower rate. We show that the rates of uptake of lactose and dansyl-galactoside by the membrane vesicles are similar; yet the uptake of dansyl-galactoside does not lead to the uncoupling effects which are associated with the uptake of lactose. We discuss the possible reasons for the absence of this uncoupling effect, and we conclude that our data are incompatible with the notion that the energy-dependent uptake of dansyl-galactoside is associated with an active transport involving a dansyl-galactoside/H⁺ symport. On the contrary, the data substantiate the initial interpretation that the energy-dependent uptake of dansyl-galactoside reflects the binding to the lac carrier not followed by transport.     

The basic asymmetry of Na+-dependent glycine transport in Ehrlich cells

Influx and efflux of glycine have been examined as a function of external and internal Na⁺ concentrations, respectively, when $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$ it was found that at comparable external and cellular Na⁺ levels, the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for efflux was larger by an order of magnitude than the value for influx and the $\text{V}$ for efflux was several times greater than the $\text{V}$ for influx. For both fluxes the major effect of Na⁺ was to decrease the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value. The observations are consistent with the conclusion that the Na⁺-dependent transport system is asymmetric per se. Influx and efflux of glycine were increased in a near linear manner by increasing the Na⁺ concentration from 13 to 100 mM, the half-time for glycine equilibration being a function of the Na⁺ concentration in absence of an electrochemical potential difference for Na⁺. In Na⁺-free media ($\text{[}\text{Na}{}^{\mathrm{+}}\text{] < 5}\text{mM}$) equilibration of glycine between cells and medium was not achieved after 60 min at 25°C. With $\text{Δ}\text{μ}{}_{\mathrm{<mtext>Na</mtext>}}\text{= 0}$, efflux (or uptake) of glycine was not affected by internal (or external) K⁺ between 20 and 120 mM suggesting that K⁺ plays no direct role in Na⁺-dependent transport of glycine in Ehrlich cells.     

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium⁺ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model.     

Relationship between H+, anion, and monovalent cation movements and Ca2+ transport in sarcoplasmic reticulum: further proof of a cation exchange mechanism for the Ca2+-Mg2+-ATPase pump

Two spectroscopic probes of free internal Ca²⁺ were used to determine the influence of H⁺ and anion permeation on the active transport of Ca²⁺ by skeletal sarcoplasmic reticulum. The studies were carried out on a well-characterized Ca²⁺-Mg²⁺-ATPase-rich sarcoplasmic reticulum fraction. Studies of D. McKinley and G. Meissner (1977, FEBS Lett., 82, 47–50) show that this fraction consists of two populations of vesicles: type I which has an electrically active monovalent cation (M⁺) permeability and type II which lacks it. The present study distinguishes between electrically active (charge-carrying) and electrically silent (e.g., countertransport) mechanisms of ion permeation in the two vesicles and shows how the active transport of Ca²⁺ is influenced by these permeabilities. The major results are as follows: (1) Both type I and II vesicles have an electrically active H⁺ permeability. (2) Type I vesicles have electrically active anion (A^?) permeabilities; type II vesicles do not. (3) At low concentrations of nonpenetrating buffers, ion imbalances across the membrane can create pH imbalances. This is due to the coupling of M⁺ and A^? movements with H⁺ movements. Following a jump in KCl concentration internal acidification is observed in type I vesicles while internal alkalinization is observed in type II vesicles. These pH gradients are dissipated on a time scale of seconds and tens of minutes for type I and II vesicles, respectively. (4) Tris(hydroxymethyl)aminomethane (Tris) was shown to be effective in dissipating pH gradients in type II vesicles. A model is proposed whereby HCl is equilibrated across the membrane by a Tris-catalyzed transport cycle involving transport of an ion pair between Tris-H⁺ and Cl^? and return of the unprotonated form of the buffer. (5) The permeabilities of several physiological and nonphysiological anions were determined for type I and II vesicles. Electrically active permeability was demonstrated for Cl^? and phosphate in type I vesicles. Type II vesicles lacked electrically active mechanisms for these two anions. Evidence is given for slow $\text{Cl}{}^{\mathrm{?}}\text{OH}{}^{\mathrm{?}}$ exchange and for rapid $\text{Cl}{}^{\mathrm{?}}\text{HCO}{}_3{}^{\mathrm{?}}$ exchange in type II vesicles. Electrically silent phosphate influx probably occurs by $\text{H}{}_2\text{PO}{}_4{}^{\mathrm{?}}\text{OH}{}^{\mathrm{?}}$ exchange. (6) Under normal conditions the Ca²⁺ uptake of type II vesicles is masked. It can be unmasked by addition of nigericin in the presence of Tris. The combination of ionophore and penetrating buffer render the type II vesicles KCl permeable, allowing the replenishment of internal K⁺ during active transport. The results are analyzed and shown to be in agreement with the Ca²⁺-Mg²⁺-ATPase pump acting as a $\text{Ca}{}^{\mathrm{2+}}\text{K}{}^{\mathrm{+}}$ exchanger. The results are shown to be in disagreement with electrogenic models of pump function.     

Flash-induced photophosphorylation in chloroplasts with activated ATPase

Chloroplasts which were rapidly isolated from illuminated leaves showed activity of ATP hydrolysis at a level much higher than that of the dark control. Under the high-intensity illumination or under repetitive flash excitation, the activated chloroplasts synthesized more ATP than those with a low ATP hydrolysis activity. formed under repetitive flashes was smaller in the activated chloroplasts than in the inactive chloroplasts. The inhibition of ATP yield per flash by valinomycin or nigericin in the presence of K⁺ was stronger in the inactive chloroplasts than in the activated chloroplast. ATP synthesis in the activated chloroplasts seems to have a lower threshold.     

Light-driven sodium transport in sub-bacterial particles of Halobacterium halobium

Light-induced Na⁺ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na⁺ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na⁺ efflux, the inhibition being complete at low light intensity. The Na⁺ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na⁺ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na⁺ transport. Proton influx in the dark followed the artificial formation of a sodium gradient ($\text{[Na}{}^{\mathrm{+}}\text{]}{}_{\mathrm{in}}\text{> [Na}{}^{\mathrm{+}}\text{]}{}_{\mathrm{out}}$). These results may be explained by a Na⁺/H⁺ antiport mechanism. The fluxes of Na⁺ and H⁺ were of comparable magnitude, but the initial rate of Cl^? efflux in the same experiment was one-third of the initial rate of Na⁺ efflux. Consequently Cl^? is not regarded as a participant in the Na⁺ efflux mechanism.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Ultracentrifugation studies on early stage polymerization of tobacco mosaic virus protein

The effects of absolute temperature (T), ionic strength (μ), and pH on the polymerization of tobacco mosaic virus protein from the 4 S form (A) to the 20 S form (D) were investigated by the method of sedimentation velocity. The loading concentration in grams per liter (C) was determined at which a just-detectable concentration (β) of 20 S material appeared. It was demonstrated experimentally that under the conditions employed herein, an equilibrium concentration of 20 S material was achieved in 3 h at the temperature of the experiment and that 20 S material dissociated again in 4 h or less to 4 S material either upon lowering the temperature or upon dilution. Thus, the use of thermodynamic equations for equilibrium processes was shown to be valid. The equation used to interpret the results, log ($\text{C?β) =}\text{constant}\text{+ (}\text{ΔH}{}^1\text{2.3RT}$) + ($\text{Δ}\text{W}{}^1{}_{\mathrm{<mtext>el</mtext>}}\text{2.3RT}$) ? K′_sμ + ζpH, was derived from three separate models of the process, the only difference being in the anatomy of the constant; thus, the method of analysis is essentially independent of the model. $\text{ΔH}{}^1$ and ΔW¹_el are the enthalpy and the change in electrical work per mole of A protein (the trimer of the polypeptide chain), K^′_s is the salting-out constant on the ionic strength basis, ζ is the number of moles of hydrogen ion bound per mole of A protein in the polymerization, and R is the gas constant. The three models leading to this equation are: a simple 11th-order equilibrium between A₁ (the trimer of the polypeptide chain) and D, either the double disk or the double spiral of approximately the same molecular weight, designated model A; a second model, designated B, in which A₁ was assumed to be in equilibrium with D at the same time that it is in equilibrium with A₂, A₃, etc., dimers and trimers, etc., of A₁ in an isodesmic system; and a phase-separation model, designated model C, in which A protein is treated as a soluble material in equilibrium with D, considered as an insoluble phase. From electrical work theory, $\text{Δ}\text{W}{}_{\mathrm{el}}{}^1\text{/T}$ was shown to be essentially independent of T; therefore, in experiments at constant μ and constant pH the equation of log (C ? β) versus 1/T is linear with a slope of $\text{ΔH}{}^1\text{/2.3R}$. The results fit such an equation over nearly a 20 °C-temperature range with a single value of $\text{Δ}\text{H}{}^1$ of +32 kcal/mol A₁. Results obtained when T and pH were held constant but μ was varied did not fit a straight line, which shows that more than simple salting-out is involved. When the effect of ionic strength on the electrical work contribution was considered in addition to salting-out, the data were interpreted to indicate a value of $\text{Δ}\text{W}{}^1{}_{\mathrm{el}}$ of 1.22 kcal/mol A₁ at pH 6.7 and a value of 4.93 for K_s^′. When μ and T were held constant but pH was varied, and when allowance was made for the effect of pH changes on the electrical work contribution, a value of 1.1 was found for ζ. This means that something like 1.1 mol of hydrogen ion must be bound per mole of A₁ protein in the formation of D. When this is added to the small amount of hydrogen ion bound per A₁ before polymerization, at the pH values used, it turned out that for D to be formed, 1.5 H⁺ ions must be bound per A₁ or 0.5 per protein polypeptide chain. This amounts to 1 H⁺ ion per polypeptide chain for half of the protein units, presumably those in one but not the other layer of the double disk or turn of the double spiral. When polymerization goes beyond the D stage, as shown by previously published data, additional H⁺ ions are bound. Simultaneous osmotic pressure studies and sedimentation studies were carried out, in both cases as a function of loading concentration C. These results were in complete disagreement with models A and C but agreed reasonably well with model B. The sedimentation studies permitted evaluation of the constant, β, to be 0.33 g/liter.     

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.     

Enthalpy and volume changes accompanying electron transfer from P-870 to quinones in Rhodopseudomonas sphaeroides reaction centers

A capacitor microphone was used to measure the enthalpy and volume changes that accompany the electron transfer reactions, $\text{PQ}{}_{\mathrm{A}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{A}}\text{and PQ}{}_{\mathrm{A}}\text{Q}{}_{\mathrm{B}}\text{→}\text{hv}\text{P}{}^{\mathrm{+}}\text{Q}{}_{\mathrm{A}}\text{Q}{}^{\mathrm{?}}{}_{\mathrm{B}}$, following flash excitation of photosynthetic reaction centers isolated from Rhodopseudomonas sphaeroides. P is a bacteriochlorophyll dimer (P-870), and Q_A and Q_B are ubiquinones. In reaction centers containing only Q_A, the enthalpy of P⁺Q^?_A is very close to that of the PQ_A ground state (ΔH_r = 0.05 ± 0.03 eV). The free energy of about 0.65 eV that is captured in the photochemical reaction evidently takes the form of a substantial entropy decrease. In contrast, the formation of P⁺Q_AQ^?_B in reaction centers containing both quinones has a ΔH_r of 0.32 ± 0.02 eV. The entropy change must be near zero in this case. In the presence of o-phenanthroline, which blocks electron transfer between Q^?_A and Q_B, ΔH_r for forming P⁺Q^?_AQ_B is 0.13 ± 0.03 eV. The influence of flash-induced proton uptake on the results was investigated, and the ΔH_r values given above were measured under conditions that minimized this influence. Although the reductions of Q_A and Q_B involve very different changes in enthalpy and entropy, both reactions are accompanied by a similar volume decrease of about 20 ml/mol. The contraction probably reflects electrostriction caused by the charges on P⁺ and Q^?_A or Q^?_B.