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 $\text{Δ}\text{}\text{?}\text{gm}{}_{\mathrm{<mtext>H</mtext>}}$.     

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 .     

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.     

Complete stoichiometry of free NADPH oxidation in liver microsomes

The stoichiometry of free NADPH oxidation in phenobarbital induced rabbit liver microsomes was measured by means of registering the rates of NADPH, H⁺ and O₂ consumption and $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ and H₂O₂ production. $\text{ΔO}{}_2{}^{\mathrm{<mtext>?</mtext>}}\text{:ΔH}{}_2\text{O}{}_2$ ratio is approximately I indicating that about half H₂O₂ results from $\text{O}{}_2{}^{\mathrm{<mtext>?</mtext>}}$ dismutation, the second half being formed directly. ΔNADPH:ΔH₂O₂ and ΔO₂:ΔH₂O₂ ratios exceed I and therefore another product of the reaction is water. The fact that the ratio (ΔNADPH-ΔH₂O₂):(ΔO₂-ΔH₂O₂) is 2 allows one to consider direct 4-electron O₂ reduction as the major way of water formation rather than endogenous substrate hydroxylation.     

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.     

Kinetics of bidirectional active sodium fluxes in the toad bladder

The kinetics of isotopic Na⁺ flows was studied in urinary bladders of toads from the Dominican Republic. Initial studies of the potential dependence of passive serosal to mucosal ²²Na⁺ efflux demonstrated the absence of isotope interaction and/or other coupling with passive Na⁺ flow. The electrical current $\text{I}$ and mucosal to serosal ²²Na⁺ influx were then measured with transmembrane potential clamped at $\text{Δψ = 0, 25, 50, 75 or 100}\text{mV}$. Subsequent elimination of active Na⁺ transport mucosal amiloride permitted calculation of the rates of active Na⁺ transport $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and active and passive influx and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}$ and $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>p</mtext>}}$. The results indicate that for Dominican toad bladders mounted in chambers only Na⁺ contributes significantly to transepithelial active ion transport; hence $\text{J}{}_{\mathrm{<mtext>Na</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= J}{}^{\mathrm{<mtext>a</mtext>}}$. $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was abolished at $\text{Δψ = E = 96.3 ± 1.9}\text{(S.E.) mV}$. As Δψ approached $\text{E}$, active efflux $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ became demonstrable. At $\text{Δ = 100}\text{mV}\text{,}\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ exceeded $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$, so that $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ was negative. Experimental values of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ agreed well with theoretical values predicted by a thermodynamic formulation: $\text{J}{}_{\mathrm{<mtext>exp</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{= 0.985}\text{J}{}_{\mathrm{<mtext>theor</mtext>}}{}^{\mathrm{<mtext>a</mtext>}}\text{(r = 0.993)}$. The dependence of $\text{J}{}^{\mathrm{<mtext>a</mtext>}}$ on Δψ is curvilinear.     

Kinetic studies on the reaction of ethylenediaminetetraacetatochromium(III) complex with hydrogen peroxide

The rate of reaction of [Cr(III)Y]_aq (Y is EDTA anion) with hydrogen peroxide was studied in aqueous nitrate media [μ = 0.10 M (KNO₃)] at various temperatures. The general rate equation, Rate = $\text{k}{}_1\text{+}\text{k}{}_2\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}\text{1 +}\text{K}{}_1\text{[H}{}^{\mathrm{+}}\text{]}{}^{\mathrm{?1}}$ [Cr(III)Y]_aq[H₂O₂] holds over the pH range 5–9. The decomposition reaction of H₂O₂ is believed to proceed via two pathways where both the aquo and hydroxo-quinquedentate EDTA complexes are acting as the catalyst centres. Substitution-controlled mechanisms are suggested and the values of the second-order rate constants k₁ and k₂ were found to be 1.75 × 10^?2 M^?1 s^?1 and 0.174 M^?1 s^?1 at 303 K respectively, where k₂ is the rate constant for the aquo species and k₂ is that for the hydroxo complex. The respective activation enthalpies (ΔH^*₁ = 58.9 and ΔH^*₂ = 66.5 KJ mol^?1) and activation entropies (ΔS^*₁ = ?85 and ΔS^*₂ = ?40 J mol^?1 deg^?1) were calculated from a least-squares fit to the Eyring plot. The ionisation constant pK₁, was inferred from the kinetic data at 303 K to be 7.22. Beyond pH 9, the reaction is markedly retarded and ceases completely at pH ? 11. This inhibition was attributed in part to the continuous loss of the catalyst as a result of the simultaneous oxidation of Cr(III) to Cr(VI).     

d-Gluconate transport in Arthrobacter pyridinolis. Metabolic trapping of a protonated solute

d-Gluconate uptake was studied in whole cells of Arthrobacter pyridinolis; the uptake activity was inducible, mutable and showed saturation kinetics ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= 5 μM}$). Uptake of d-gluconate was not mediated by a phosphoenolpyruvate: hexose phosphotransferase system, nor was it directly energized by ATP. A transmembrane pH gradient, ΔpH, of ?63 mV was generated by A. pyridinolis cells at pH 6.5, while at pH 7.5, $\text{ΔpH = 0}$. Addition of 8 μM d-gluconate significantly reduced the ΔpH. The transmembrane electrical potential, Δψ, which was ?87 mV over a range of pH from 5.5 to 7.5, was unaffected by the presence of substrate. d-Gluconate accumulated at the same rate and as the protonated solute, at both pH 6.5 and 7.5. Experiments in which a diffusion potential was generated in cyanide-treated cells, indicated that the Δψ did not energize transport. Rather, the rate of d-gluconate uptake correlated with and appeared to be determined by the rate of d-gluconate metabolism: (a) treatment of cells with valinomycin or nigericin, under conditions in which there was a loss of intracellular potassium, inhibited both d-gluconate uptake and the metabolism of pre-accumulated d-gluconate; (b) the effects of cyanide and azide on d-gluconate uptake were much more severe at pH 6.5 than pH 7.5, a pattern which paralleled the effects of these inhibitors on d-gluconate metabolism; (c) extraction and chromatography of intracellular label from d-gluconate uptake revealed that accumulation of unaltered d-gluconate was negligible; (d) a series of mutant strains with lower d-gluconate kinase activities also exhibited low rates of d-gluconate uptake; (e) spontaneous revertants of these mutant strains consistently regained both d-gluconate kinase activity and wild type levels of uptake.     

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.     

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.     

Active K+ transport in Mycoplasma mycoides var. Capri. Net and unidirectional K+ movements

Analysis of the cation composition of growing Mycoplasma mycoides var. Capri indicates that these organisms have a high intracellular K⁺ concentration ($\text{K}{}_{\mathrm{<mtext>i</mtext>}}\text{: 200–300}\text{mM}$) which greatly exceeds that of the growth medium, and a low Na⁺ concentration ($\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{: 20}\text{mM}$). Unlike $\text{Na}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}\text{, K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ varies with cell aging.The K⁺ transport properties studied in washed organisms resuspended in buffered saline solution show that cells maintain a steady and large K⁺ concentration gradient across their membrane at the expense of metabolic energy mainly derived from glycolysis. In starved cells, $\text{K}{}_{\mathrm{<mtext>i</mtext>}}{}^{\mathrm{+}}$ decreases and is partially compensated by a gain in Na⁺. This substitution completely reverses when metabolic substrate is added (K⁺ reaccumulation process). Kinetic analysis of K⁺ movement in cells with steady K⁺ level shows that most of K⁺ influx is mediated by an autologous K⁺-K⁺ exchange mechanism. On the other hand, during K⁺ reaccumulation by K⁺-depleted cells, a different mechanism (a K⁺ uptake mechanism) with higher transport capacity and affinity drives the net K⁺ influx. Both mechanisms are energy-dependent.Ouabain and anoxia have no effect on K⁺ transport mechanisms; in contrast, both processes are completely blocked by dicyclohexylcarbodiimide, an inhibitor of the Mg²⁺-dependent ATPase activity.     

Effect of alkali ions on the active transport of neutral amino acids into Streptomyces hydrogenans

The active transport of neutral amino acids into Streptomyces hydrogenans is inhibited by external Na⁺. There is no indication that in these cells amino acid accumulation is driven by an inward gradient of Na⁺. The extent of transport inhibition by Na⁺ depends on the nature of the amino acid. It decreases with increasing chain length of the amino acid molecules i.e. with increasing non-polar properties of the side chain. Kinetic studies show that Na⁺ competes with the amino acid for a binding site at the amino acid carrier. There is a close relation between the $\text{K}{}_{\mathrm{<mtext>i</mtext>}}$ values for Na⁺ and the number of C atoms of the amino acids. Other cations also inhibit neutral amino acid uptake competitively; the effectiveness decreases in the order $\text{Li}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Rb}{}^{\mathrm{+}}\text{> Cs}{}^{\mathrm{+}}$. Anions do not have a significant effect on the uptake of neutral amino acids. After prolonged incubation of the cells with 150 mM Na⁺, in addition to the competitive inhibition of transport Na⁺ induces an increase in membrane permeability for amino acids.     

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.     

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.     

Equilibrium constants under physiological conditions for the reactions of L-phosphoserine phosphatase and pyrophosphate: L-serine phosphotransferase

The observed equilibrium constants (K_obs) for the l-phosphoserine phosphatase reaction [EC 3.1.3.3] have been determined under physiological conditions of temperature (38 °C) and ionic strength (0.25 m) and physiological ranges of pH and free [Mg²⁺]. Using Σ and square brackets to indicate total concentrations $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{Σ L-serine][Σ P}{}_{\mathrm{i}}\text{]}\text{Σ L-phosphoserine]H}{}_2\text{O]}\text{, K =}\text{L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O]}$. The value of K_obs has been found to be relatively sensitive to pH. At 38 °C, K⁺] = 0.2 m and free [Mg²⁺] = 0; K_obs = 80.6 m at pH 6.5, 52.7 m at pH 7.0 [ΔG_obs⁰ = ?10.2 kJ/mol (?2.45 kcal/mol)], and 44.0 m at pH 8.0 ([H₂O] = 1). The effect of the free [Mg²⁺] on K_obs was relatively slight; at pH 7.0 ([K⁺] = 0.2 m) K_obs = 52.0 m at free [Mg²⁺] = 10^?3, m and 47.8 m at free [Mg²⁺] = 10^?2, m. K_obs was insignificantly affected by variations in ionic strength (0.12–1.0 m) or temperature (4–43 °C) at pH 7.0. The value of K at 38 °C and I = 0.25 m has been calculated to be 34.2 ± 0.5 m [ΔG_obs⁰ = ?9.12 kJ/mol (?2.18 kcal/ mol)]([H₂O] = 1). The K for the phosphoserine phosphatase reaction has been combined with the K for the reaction of inorganic pyrophosphatase [EC 3.6.1.1] previously estimated under the same physiological conditions to calculate a value of 2.04 × 10⁴, m [ΔG_obs⁰ = ?28.0 kJ/mol (?6.69 kcal/mol)] for the K of the pyrophosphate:l-serine phosphotransferase [EC 2.7.1.80] reaction. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{]}\text{[}\text{Σ L-phosphoserine}\text{][}\text{H}{}_2\text{O}\text{]}\text{, K =}\text{[L-H · serine}{}^{\mathrm{\pm }}\text{]HPO}{}_4{}^{\mathrm{2?}}\text{]}\text{[L-H · phosphoserine}{}^{\mathrm{2?}}\text{]H}{}_2\text{O}$. Values of K_obs for this reaction at 38 °C, pH 7.0, and I = 0.25 m are very sensitive to the free [Mg²⁺], being calculated to be 668 [ΔG_obs⁰ = ?16.8 kJ/mol (?4.02 kcal/mol)] at free [Mg²⁺] = 0; 111 [ΔG_obs⁰ = ?12.2 kJ/mol (?2.91 kcal/mol)] at free [Mg²⁺] = 10^?3, m; and 9.1 [ΔG_obs⁰ = ?5.7 kJ/mol (?1.4 kcal/mol) at free [Mg²⁺] = 10^?2, m). K_obs for this reaction is also sensitive to pH. At pH 8.0 the corresponding values of K_obs are 4000 [ΔG_obs⁰ = ?21.4 kJ/mol (?5.12 kcal/mol)] at free [Mg²⁺] = 0; and 97.4 [ΔG_obs⁰ = ?11.8 kJ/ mol (?2.83 kcal/mol)] at free [Mg²⁺] = 10^?3, m. Combining K_obs for the l-phosphoserine phosphatase reaction with K_obs for the reactions of d-3-phosphoglycerate dehydrogenase [EC 1.1.1.95] and l-phosphoserine aminotransferase [EC 2.6.1.52] previously determined under the same physiological conditions has allowed the calculation of K_obs for the overall biosynthesis of l-serine from d-3-phosphoglycerate. $\text{K}{}_{\mathrm{<mtext>obs</mtext>}}\text{=}\text{[}\text{Σ L-serine}\text{][}\text{Σ NADH}\text{][}\text{Σ}\text{P}{}_{\mathrm{<mtext>i</mtext>}}\text{][}\text{Σ}\text{α-}\text{ketoglutarate}\text{]}\text{[Σ d-3-}\text{phosphoglycerate}\text{][Σ NAD}{}^{\mathrm{+}}\text{][Σ L-glutamat0]}$ The value of K_obs for these combined reactions at 38 °C, pH 7.0, and I = 0.25 m (K⁺ as the monovalent cation) is 1.34 × 10^?2, m at free [Mg²⁺] = 0 and 1.27 × 10^?2, m at free [Mg²⁺] = 10^?3, m.     

The permeability of the lysosomal membrane to small ions

The permeability of the lysosomal membrane to small anions and cations was studied at 37°C and pH 7.0 in a lysosomal-mitochondrial fraction isolated from the liver of untreated rats. The extent of osmotic lysis following ion influx was used as a measure of ion permeancy. In order to preserve electroneutrality, anion influx was coupled to an influx of K⁺ in the presence of valinomycin, and cation influx was coupled to an efflux of H⁺ using the protonophore $\text{3-tert-}\text{butyl-5,2′-dichloro-4′-nitrosalicilylanilide}$. Lysosomal lysis was monitored by observing the loss of latency of two lysosomal hydrolases.The order of permeability of the lysosomal membrane to anions was found to be $\text{SCN}{}^{\mathrm{?}}\text{> I}{}^{\mathrm{?}}\text{> CH}{}_3\text{COO}{}^{\mathrm{?}}\text{> Cl}{}^{\mathrm{?}}\text{≈ HCO}{}^{\mathrm{?}}{}_3\text{≈ P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2?}}$ and that to cations $\text{Cs}{}^{\mathrm{+}}\text{> K}{}^{\mathrm{+}}\text{> Na}{}^{\mathrm{+}}\text{> H}{}^{\mathrm{+}}$. These orders are largely in agreement with the lyotropic series of anions and cations.The implications of these findings for the mechanism by means of which a low intralysosomal pH is produced and maintained are discussed.     

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.     

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.