Tracking of proton flow during transition from anaerobiosis to steady state in rat liver mitochondria

(1) The hydrophobic pH indicator Bromthymol blue and the hydrophilic pH indicator Phenol red have been used to follow the redox-pump-linked proton flows during transition from anaerobiosis to static head. The domains monitored by the pH indicators, whether external or internal, and the localization of the dye, whether free or membrane bound, have been identified by recording the absorbance changes following addition of nigericin or valinomycin to anaerobic or aerobic mitochondria and the effects of permeant and impermeant buffers. (2) After addition of the H+/K+ exchanger, nigericin, to anaerobic mitochondria. Phenol red and Bromthymol blue record an alkalinization and an acidification, respectively, indicating that while the hydrophilic pH indicator faces an external domain, the hydrophobic pH indicator faces, at least partly, an internal domain. The latter effect is sensitive to phosphate and to phosphate carrier inhibitors. On the other hand, addition of nigericin to aerobic mitochondria leads to an increased Bromthymol blue absorbance, which reflects an alkalinization, indicating that the pH indicator faces an external domain. The reorientation of the dye from the internal to the external domain is a function of the uncoupler concentration and thus of the membrane potential (cf. Mitchell et al. (1968) Eur. J. Biochem. 4, 9-19). (3) The amount of oxygen required for the transition from anaerobiosis to static head has been determined by following in parallel the extent of oxidation of cytochrome aa3 and the rise of delta mu H+. With succinate as substrate, 50% levels of cytochrome oxidation are obtained at 0.125 ngatom oxygen/mg and 50% of Safranine response at about 0.2 ngatom oxygen/mg. These amounts of oxygen correspond to an H+ displacement of about 0.8-1.2 ngatom/mg on the basis of the H+/O stoichiometry. It is concluded that mitochondria are in presteady state below, and in static head above, displacement of 2-3 ngatom H+/mg. This figure is very close to the original calculation of Mitchell (Mitchell, P. (1966) Biol. Rev. 41, 445-502). (4) Transition, by oxygen pulses, of EGTA-supplemented mitochondria from anaerobiosis to either presteady state or static head state results in a response of the hydrophilic pH indicator, Phenol red, which is negligible in amount and/or kinetically unrelated to the delta mu H+ rise. The fact that H+ extrusion in the bulk aqueous phase is negligible also in presteady state excludes proton cycling as an explanation. Addition of oxygen pulses to Sr2(+)-supplemented anaerobic mitochondria results in an H+ extrusion whose amount and rate is proportional to the Sr2+ concentration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Comparison of the electrochemical proton gradient and phosphate potential maintained by Rhodospirillum rubrum chromatophores in the steady state.

A technique for the estimation of light-induced membrane potential in chromatophores is described. It is based on measurement of light-induced enhancement in fluorescence of 8-anilinonaphthalene sulfonic acid, which is calibrated by known K⁺ diffusion potentials. The electrochemical proton gradient (Δ_μH+^?) formed during lightinduced electron transport in Rhodospirillum rubrum chromatophores amounts to 250 mV, which is almost equally distributed between the membrane potential and the pH gradient as measured by changes in the fluorescence of anilinonaphthalene sulfonate and 9-amino acridine. Addition of the permeant anion, NaSCN, or of NH₄Cl reduces the overall Δ_μH+^? by less than 20% but changes its distribution between the pH gradient and the membrane potential so that with NaSCN it is composed mainly of the first and with NH₄Cl mainly of the second. Initiation of phosphorylation causes a drop of about 50 mV in the measured Δ_μH+^?. In the absence of salts, the drop is observed in both components, although two-thirds of it are reflected in the membrane potential. In the presence of NaSCN or NH₄Cl the 50-mV drop is exclusively recorded in the pH gradient or in the membrane potential, respectively. The steady-state phosphate potential maintained during electron transport was found to change in parallel to the Δ_μH+^?, but exceeded it by 60 to 80 mV when based on a stoichiometry of two protons translocated per ATP synthesized.     

Studies on the relationship between ATP synthesis and transport and the proton electrochemical gradient in rat liver mitochondria

The effect of ATP synthesis on delta mu H in rat liver mitochondria has been analyzed by separating the steps of adenine nucleotide translocation and ATP synthesis in the matrix. Either exchange of ATP, synthesized by substrate level phosphorylation in the matrix of oligomycin-treated mitochondria, for external ADP, or activity of the membrane-bound ATP synthase complex results in delta mu H depression with respect to resting state levels. This depression appears to be more pronounced, under strictly comparable conditions, when arsenate is used to stimulate ATP synthase activity than when the ornithine-citrulline conversion reaction is used for the same purpose.     

Rapid determination of L-lysine with an enzyme electrode by steady state and kinetic measurement

Fast and simple methods of determination of L-lysine by a potentiometric enzyme electrode based on a CO₂ electrode and L-lysine decarboxylase are described. Measuring devices for manual and automated operation for steady state response measurement as well as kinetic measurement are compared. Sample frequency may be increased by decreasing the time of a measuring cycle.     

Calcium transport and proton electrochemical potential gradient in mitochondria from guinea-pig cerebral cortex and rat heart

A method is described for the preparation of ;free' and ;synaptosomal' brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H(+) conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca(2+) at rates limited by the activity of the respiratory chain. Ca(2+) transport by ;free' brain mitochondria is compared with that of heart mitochondria. The Ca(2+) conductance of ;free' brain mitochondria was at least 20 times that for rat heart mitochondria. Ca(2+) uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca(2+) cycling decreased the H(+) electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca(2+) conductance of ;free' brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca(2+) concentration. In the presence of Ruthenium Red, a rapid Na(+)-dependent Ca(2+) efflux occurs. The H(+) electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ;free' brain and heart mitochondria. The Na(+) requirement for Ca(2+) efflux appears not to be related to the high Na(+)/H(+) exchange activity but may represent a direct exchange of Na(+) for Ca(2+).     

Increase of proton electrochemical potential and ATP synthesis in rat liver mitochondria irradiated in vitro by helium-neon laser

Particulate adenylate cyclase (AC) and guanylate cyclase (GC) activities localized in the ciliary membrane from Paramecium were solubilized by a two-step procedure using the detergents Brij 56 and Lubrol PX. The enzymes remained in the supernatant after a 100 000 × g centrifugation. Upon gel chromatography, AC and GC were almost completely separated proving that each enzyme is a distinct molecular entity. Solubilization of GC was achieved with the calmodulin subunit remaining firmly attached to the catalytic part. Antibodies against calmodulin inhibited the enzyme as did La³⁺ and EGTA. AC activity appeared to be regulated specifically by K⁺, enzyme activity being enhanced up to 100% by 15 mM K⁺. Na⁺ and Li⁺ were inactive.     

On the relationship between rate of ATP synthesis and H+ electrochemical gradient in rat-liver mitochondria

The relationship between rate of ATP synthesis, JATP, and value of the proton electrochemical gradient, delta mu H, has been analyzed in intact mitochondria. Onset of phosphorylation causes a depression of delta mu H of 1.5 kJ/mol. There is a close parallelism between inhibition of JATP and restoration of delta mu H to its state-4 value during titrations with oligomycin or atractyloside. Titrations with ionophores display the following features: (a) delta mu H can be depressed by 3-4 kJ/mol by valinomycin + K+ without affecting the rate of ATP synthesis; (b) uncouplers abolish JATP completely while depressing delta mu H by 3 kJ/mol; (c) complete abolition of ATP synthesis by inhibitors of electron transport is accompanied by a depression of delta mu H of only 1 kJ/mol. The results indicate that: (a) there is a close functional relationship between redox and ATPase H+ pumps, whereby inhibition of electron transfer is accompanied by simultaneous inhibition of the ATPase H+ pumps; and (b) uncoupling of oxidative phosphorylation is not due to depression of delta mu H per se. The consistence of the present data with either a chemiosmotic model where delta mu H is the sole and obligatory intermediate for energy coupling, or models where there is a direct transfer of energy between the two pumps is discussed.     

Relationship between phosphorylation potential and electrochemical H+ gradient during glycolysis in Streptococcus lactis.

Assays of intracellular ATP, ADP, and inorganic phosphate allowed calculation of the phosphorylation potential (delta G'ATP/F) maintained during glycolysis by Streptococcus lactis. At the same time, the electrochemical H+ gradient (delta mu-H+/F) was evaluated by distribution methods, using radioactive tetraphenylphosphonium bromide as a probe for the membrane potential and salicylic acid as a probe for the pH gradient. Detailed comparisons were made at pH 5, when the reaction mediated by the proton-translocating ATPase (BF0F1) was likely to have been poised near equilibrium; for those conditions, the ratio delta G'ATP/delta mu-H+ was used to estimate stoichiometry for BF0F1 during ATP hydrolysis. At an external pH of 5, in the presence or absence of valinomycin, this ratio was close to 3, over a range of 370 to 510 mV (8.5 to 11.7 kcal/mol) for delta G'ATP/F and a range of 128 to 167 mV for delta mu-H+/F. Other work suggested that delta G'ATP/delta mu-H+ increased from its minimum value of 3 to 4.3 as the external pH changed from pH 5 to 7.     

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Summary The uptake and binding of the lipophilic cations ethidium⁺, tetraphenylphosphonium⁺ (TPP⁺), triphenylmethylphosphonium⁺ (TPMP⁺), and tetraphenylarsonium⁺ (TPA⁺) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K' _o) and the internal matrix volume and matrix face of the inner membrane (K' _i) were determined and were utilized to estimate the membrane potential from the cation accumulation factorR _c according to the relation =RT/ZF ln [(R _cV_o–K'_o)/(V_i+K'_i)] whereV _o andV _i are the volume of the external medium and the mitochondrial matrix, respectively, andR _c is the ratio of the cation content of the mitochondria and the medium. The values of estimated from this equation are in remarkably good agreement with those estimated from the distribution of⁸⁶Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.     

The relationship between the electrochemical proton gradient and active transport in Escherichia coli membrane vesicles.

In the previous paper [ramos, S., and Kaback, H.R. (1977), Biochemistry 16 (preceding paper in this issue)], it was demonstrated that Escherichia coli membrane vesicles generate a large electrochemical proton gradient (delta-muH+) under appropriate conditions, and some of the properties of delta-muH+ and its component forces [i.e., the membrane potential (delta psi) and the chemical gradient of protons (deltapH)] were described. In this paper, the relationship between delta-muH+, delta psi, and deltapH and the active transport of specific solutes is examined. Addition of lactose or glucose 6-phosphate to membrane vesicles containing the appropriate transport systems results in partial collapse of deltapH, providing direct evidence for the suggestion that respiratory energy can drive active transport via the pH gradient across the membrane. Titration studies with valinomycin and nigericin lead to the conclusion that, at pH 5.5, there are two general classes of transport systems: those that are driven primarily by delta-muH+ (lactose, proline, serine, glycine, tyrosine, glutamate, leucine, lysine, cysteine, and succinate) and those that are driven primarily by deltapH (glucose 6-phosphate, D-lactate, glucuronate, and gluconate). Importantly, however, it is also demonstrated that at pH 7.5, all of these transport systems are driven by delta psi which comprises the only component of delta-muH+ at this external pH. In addition, the effect of external pH on the steady-state levels of accumulation of different solutes is examined, and it is shown that none of the pH profiles correspond to those observed for delta-muH+, delta psi, or deltapH. Moreover, at external pH values above 6.0-6.5, delta-muH+ is insufficient to account for the concentration gradients established for each substrate unless the stoichiometry between protons and accumulated solutes is greater than unity. The results confirm many facets of the chemiosmotic hypothesis, but they also extend the concept in certain important respects and allow explanations for some earlier observations which seemed to preclude the involvement of chemiosmotic phenomena in active transport.     

Membrane potential and surface potential in mitochondria. Binding of a cationic spin probe

The interaction of the cationic spin probe 4-(N,N-dimethyl-N-dodecyl)-ammonium-2,2,6,6-tetramethyl-piperidine-1-oxyl (Cat12) with intact mitochondria and submitochondrial particles was investigated as a function of salt concentration, pH and energization by ATP. In the presence of 1 mM Fe(CN)-36, which inhibits the probe reduction by the mitochondria, the probe signal is stable and shows both bound and free forms. The partition of the probe into mitochondrial membranes is decreased by various salts depending on the cation valency, indicating that the membrane is negatively charged (-10 to -15 mV at pH 7.0). The surface potential increases with pH from -3 mV at pH 5.0 to -18 mV at pH 8.0. Energization of intact mitochondria by ATP reduces the magnitude of both bound and free signals by more than 50%; the signal of the bound form slowly disappears on further incubation. The ATP effect is inhibited and also reversed by either oligomycin or CCCP. Similar effects of ATP were observed in mitoplasts but not in submitochondrial particles. In submitochondrial particles ATP has no effect on the probe signal or binding. These results suggest that the formation of membrane potential in mitochondria induces uptake and internal binding of the probe which results in broadening of the EPR signal of the internally bound probe. It is concluded that Cat12 is not a suitable probe for measurement of surface potential in energized mitochondria.     

Studies on the functional relationship between the phosphopyruvate synthesis and the substrate level phosphorylation in guinea-pig liver mitochondria

The PEP synthesis in guinea-pig liver mitochondria in the presence of -ketoglutarate and P_i was greatly increased by addition of 2,4-dinitrophenol, pentachlorophenol, dicumarol or the hexokinase-glucose-ATP system, but was hardly increased by exogenously added ATP. This result is in sharp contrast to those obtained with pigeon and chicken liver mitochondria.

By using ¹⁴C-labeled substrates, it was revealed that the ratio of the amounts of PEP formed to those of -ketoglutarate oxidized varied markedly according to the degree of stimulation of PEP synthesis, and when the PEP synthesis was maximally stimulated, the ratio reached the value of nearly 1.0. Under these conditions the oxidation of -ketoglutarate was considerably stimulated by the addition of malate or fumarate and in turn, the metabolism of fumarate was greatly increased by the addition of -ketoglutarate.

The nucleoside diphosphate kinase activity in guinea-pig liver mitochondria was found to be extremely low, while the activity in pigeon liver mitochondria was very high. Activities of GTP-AMP-phosphate transferase were low in both mitochondria.

It was concluded that in guinea-pig liver mitochondria guanine nucleotides involved in the substrate level phosphorylation are unable to interact easily with adenine nucleotides in the respiratory chain phosphorylation system because of the low level of nucleoside diphosphate kinase. It was also suggested that the -ketoglutarate oxidation in guinea pig liver might be supported to a considerable degree by coupling with the PEP synthesis even under physiological conditions.