Rate Of Proton Uptake Research Articles

Regulation of proton leakage from broken chloroplasts by CF 0

Measurements of proton translocation in CF 1-depleted, N, N′-dicyclohexylcarbodiimide-resealed broken chloroplasts were made under different light intensities. Kinetic analysis of the data shows that the outward leakage of accumulated protons through CF 0 is still dependent on light intensity with a first-order rate constant equal to mR 0, where R 0 is the initial rate of proton uptake which normally increases with light intensity and m is a characteristic constant which is independent of proton gradient and light intensity. Measurements of proton translocation in these modified chloroplasts cross-linked with glutaraldehyde under illumination and in the dark respectively suggest that the light-dependent proton leakage through CF 0 is regulated by conformation change in the membrane. It is proposed that the ovserved regulation of proton leakage through the CF 1.CF 0 complex in native chloroplasts is for optimizing the steady state synthesis of ATP under different light intensities.

Factors controlling the binding of two protons per electron transferred through the ubiquinone and cytochrome [formula omitted] segment of Rhodopseudomonas sphaeroides chromatophores

1. 1. On every turnover, 2.0 protons can be bound by the membrane for each single electron moving through the Q-b c 2 oxidoreductase. 2. 2. One proton (H + II) binding reaction is, and one (H + I) is not, sensitive to antimycin. 3. 3. The redox states of electron transfer components other than the proton binding agents can affect both the rate of proton uptake and the apparent p K values on the agents binding the protons. 4. 4. The presence of valinomycin under certain well-defined conditions can strongly influence the value of the measured p K on the H + II binding agent.

Mechanism of 3,5-di- tert-butyl-4-hydroxybenzylidenemalononitrile-mediated proton uptake in liposomes. Kinetics of proton uptake compensated by valinomycin-induced k +-efflux

1. Kinetic studies were made on proton translocation across the phospholipid bilayer membrane in liposomes; this proton translocation is mediated by the uncoupler 3,5-di- tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847) and compensated by valinomycin-induced potassium efflux. The initial rate of proton uptake was directly proportional to the concentration of SF6847 in the presence of excess valinomycin, indicating that the monomer of SF6847, at least, is a proton carrier and participates in limiting the rate of the proton uptake cycle. 2. The partition constant of SF6847 between the liposomal membranes, prepared from Escherichia coli phospholipids, and the aqueous phase was determined kinetically to be 21 mM −1 in medium of pH 7.0 and 4.5 mM −1 in medium of pH 8.0. The turnover rates of SF6847 across the bilayer membrane at pH 7.0 and 8.0 were determined as 140 and 550 times/molecule per s, respectively. 3. The initial rate of proton uptake depended hyperbolically on the concentration of valinomycin in the presence of a limiting amount of SF6847. It also depended on the concentration of potassium ion (K +) inside the liposomes. These results suggest strongly that a ternary complex valinomycin · K + · SF − (SF −, anionic form of SF6847) is a direct intermediate, participating in limiting the rate of the proton uptake cycle. 4. The equilibrium constants of the following equilibria, valinomycin + K + ▪ valinomycin · K + and valinomycin · K + + SF − ▪ valinomycin · K + · SF − were determined: K 1 was 3.8 · 10 −1M and K 2 A ( A, constant; see text) was 4.0 · 10 −9M under the conditions employed. From these results a model is proposed for the mechanism of the proton uptake cycle mediated by SF6847 in liposomes. 5. The mechanism of proton uptake mediated by carbonylcyanide m-chlorophenylhydrazone (CCCP) was also studied. It was concluded that the mechanism was essentially the same as that mediated by SF6847.

Active transport of alanine by thermostable membrane vesicles isolated from a thermophilic bacterium.

1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3. 2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine methosulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60 degrees. 3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55 degrees in 0.5 M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4 mM. 4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1 : 1. 5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient. 6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.

Control of proton translocation induced by ATPase activity in chloroplasts

1. 1. Proton uptake was induced by ATP in the dark following light triggering of ATPase activity in chloroplasts. The accumulated protons were released when ATPase activity was inhibited by the energy transfer inhibitor DIO-9. 2. 2. Approximately two protons were taken up for each ATP hydrolyzed at pH 8. A drop in H +/ATP ratio was caused by uncouplers such as NH 4Cl and carbonyl cyanide p-trifluoromethoxyphenylhydrazone. These uncouplers caused an increase in the rate of ATP hydrolysis without a corresponding increase in proton uptake. 3. 3. The energy transfer inhibitor dicyclocarbodiimide inhibited both ATPase activity and the rate of proton uptake without changing the H +/ATP ratio. 4. 4. The antibiotic valinomycin caused an increase in the rate of both proton uptake and ATP hydrolysis without altering the ratio of H +/ATP. The H +/ATP ratio varied with changes in the external pH. The results were discussed in view of the chemiosmotic theory of oxidative and photosynthetic phosphorylation.

The electric generator in the photosynthesis of green plants. II. Kinetic correlation between protolytic reactions and redox reactions

In a preceding paper (Junge, W. and Ausländer, W. (1974) Biochim. Biophys. Acta 333, 59–70), we attributed the four protolytic reactions at the outer and the inner side of the functional membrane of photosynthesis to the protolytic properties of the redox components, water, plastoquinone and the terminal acceptor. The experimental evidence presented was conclusive except for one argument. The rate of the protolytic reactions as detected by the dye cresol red after a short flash of light was considerably slower than the rate of the corresponding redox reactions. In this communication it is demonstrated that the rate of proton uptake from the outer phase of the functional membrane is slowed down by a diffusion barrier for protons which shields the redox reaction sites at the outer side of the membrane against the outer aqueous phase. This barrier can be lowered by sand grinding the chloroplasts, by digitonin treatment and by uncoupling agents. At the extreme the barrier can be practically eliminated to yield rates of proton uptake matching the rates of the corresponding redox reactions. This gives conclusive evidence that the electrochemical potential difference which light induces across the functional membrane of photosynthesis is generated by a vectorial electron-hydrogen transport system as postulated by Mitchell (e.g. (1966) Biol. Rev. 41, 445–502).

Interaction of biguanides with mitochondrial and synthetic membranes. Effects on ion conductance of mitochondrial membranes and electrical properties of phospholipid bilayers.

Primary reactions of the energy‐conserving pathway in oxidative phosphorylation are inhibited by biguanides and alkylguanidines. The inhibitors bind to phospholipids of mitochondria and synthetic membranes with identical affinities and are assumed to modify the physical properties of membranes, as for example cation conductance.The study compares effects of biguanides on respiratory‐linked ion fluxes across mitochondrial membranes with those on the conductance and the electrical capacitance of phospholipid bilayers. The following observations have been made:1. The rate of energy‐linked mitochondrial swelling and the rate of proton uptake during phosphorylation of ADP is inhibited in presence of biguanides. The activity of different compounds is proportional to their binding affinity to phospholipids.2. Oscillatory volume changes of mitochondria are largely abolished by biguanides.3. Biguanides cause a small increase of electrical conductance of phospholipid bilayers. This is only in part due to charge transfer by the lypophilic biguanide cations. It is suggested that also structural changes are induced by biguanides altering the surface area per molecule of phospholipid. At high biguanide concentrations structural reorganization is also expressed by changes in electrical capacitance. Both effects are saturable and proportional to binding affinity.4. Biguanides affect the electrical conductance of bilayers in the opposite way but to the same extent with positively charged species (K+/ionophore) and with negatively charged species (uncouplers of oxidative phosphorylation). This is explained by generation of a positive surface potential at phospholipid containing membranes.5. The data fit excellently the application of diffuse double‐layer theory to lipid bilayers. The relative effectiveness of different biguanides on model membranes is identical to that on ion fluxes with mitochondrial membranes. From this it is concluded that a similar mechanism of action applies to synthetic and natural membranes as well, suggesting that screening of functional negative sites interferes with the translocation of cations linked to electron transport and the primary reactions in conservation of respiratory energy.

The hexose-proton symport system of Chlorella vulgaris. Specificity, stoichiometry and energetics of sugar-induced proton uptake.

The specificity of the hexose transport system of Chlorella for sugars is the same as the specificity of sugar‐induced proton uptake. The Km‐values of various sugars for the rate of proton uptake agree well with the respective Km‐values for the rate of uptake of these sugars. Under anaerobic conditions in the dark, when the rate of sugar uptake is very low, sugar‐stimulated proton uptake is also hardly observable. Under the same conditions in the light [712 nm; O2 evolution is inhibited by 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea] sugar again stimulates proton uptake. The uncoupler carbonylcyanide‐p‐trifluoromethoxyphenylhydrazone, which inhibits sugar uptake, also prevents sugar induced proton uptake. The Stoichiometry for 6‐deoxyglucose and for glucose under various energetic conditions is one proton taken up per sugar molecule. For other glucose analogues the Stoichiometry has been found to be larger than one. Potassium and sodium ions cannot replace protons in the hexoseproton symport system.

Kinetic studies of the acylation of pig muscle D-glyceraldehyde 3-phosphate dehydrogenase by 1,3-diphosphoglycerate and of proton uptake and release in the overall enzyme mechanism.

In the presence of NAD(+) the acylation by 1,3-diphosphoglycerate of the four active sites of pig muscle d-glyceraldehyde 3-phosphate dehydrogenase can be monitored at 365nm by the disappearance of the absorption band present in the binary complex of NAD(+) and the enzyme. A non-specific salt effect decreased the acylation rate 25-fold when the ionic strength was increased from 0.10 to 1.0. This caused acylation to be the rate-limiting process in the enzyme-catalysed reductive dephosphorylation of 1,3-diphosphoglycerate at high ionic strength at pH8. The salt effect permitted investigation of the acylation over a wide range of conditions. Variation of pH from 5.4 to 8.6 produced at most a two-fold change in the acylation rate. One proton was taken up per site acylated at pH8.0. By using a chromophoric H(+) indicator the rate of proton uptake could be monitored during the acylation and was also almost invariant in the pH range 5.5-8.5. Transient kinetic studies of the overall enzyme-catalysed reaction indicated that acylation was the process involving proton uptake at pH8.0. The enzyme mechanism is discussed in the light of these results.

The absorption of protons with specific amino acids and carbohydrates by yeast.

1. Proton uptake in the presence of various amino acids was studied in washed yeast suspensions containing deoxyglucose and antimycin to inhibit energy metabolism. A series of mutant strains of Saccharomyces cerevisiae with defective amino acid permeases was used. The fast absorption of glycine, l-citrulline and l-methionine through the general amino acid permease was associated with the uptake of about 2 extra equivalents of protons per mol of amino acid absorbed, whereas the slower absorption of l-methionine, l-proline and, possibly, l-arginine through their specific permeases was associated with about 1 proton equivalent. l-Canavanine and l-lysine were also absorbed with 1-2 equivalents of protons. 2. A strain of Saccharomyces carlsbergensis behaved similarly with these amino acids. 3. Preparations of the latter yeast grown with maltose subsequently absorbed it with 2-3 equivalents of protons. The accelerated rate of proton uptake increased up to a maximum value with the maltose concentration (K(m)=1.6mm). The uptake of protons was also faster in the presence of alpha-methylglucoside and sucrose, but not in the presence of glucose, galactose or 2-deoxyglucose. All of these compounds except the last could cause acid formation. The uptake of protons induced by maltose, alpha-methylglucoside and sucrose was not observed when the yeast was grown with glucose, although acid was then formed both from sucrose and glucose. 4. A strain of Saccharomyces fragilis that both fermented and formed acid from lactose absorbed extra protons in the presence of lactose. 5. The observations show that protons were co-substrates in the systems transporting the amino acids and certain of the carbohydrates.

Proton uptake and quenching of bacteriochlorophyll fluorescence in Rhodopseudomonas spheroides

The addition of the cyclic cofactor 2,3,5,6-tetramethyl- p-phenylenediamine (diaminodurene) to a suspension of chromatophores of Rhodopseudomonas spheroides causes a light-dependent quenching of bacteriochlorophyll fluorescence. This effect is similar to one observed in chloroplasts and related to proton uptake. It is distinct from the quenching operative through the redox state of the primary electron donor and acceptor, as shown by its sensitivity to uncouplers and ionophorous antibiotics. The quenching is dependent on light intensity and diaminodurene concentration, and has a pH optimum at 7.1 where up to 70% of the fluorescence could be quenched in the presence of 0.33 mM diaminodurene. Measurements of this light-induced fluorescence quenching and of light-induced proton uptake were performed on similar solutions containing diaminodurene and various uncouplers and ionophorous antibiotics. All of these substances affected the fluorescence quenching and proton uptake in a similar fashion. The nigericin-like antibiotic, X-464, inhibited the proton uptake by 85% and reversed or prevented at least 95% of the fluorescence quenching. Qualitatively similar results were obtained with NH 4Cl, (NH 4) 2SO 4 and carbonylcyanide 3-chlorophenylhydrazone (CCCP). However, those compounds such as valinomycin and gramicidin which cause an increase in the rate of proton uptake, precipitated a rapid quenching of fluorescence, which differed kinetically from the usual quenching. It is concluded from these studies that the fluorescence quenching is related to the uptake of protons, and may be useful as a probe to monitor proton accumulation in chromatophores.

Interaction of biguanides with mitochondrial and synthetic membranes.

Primary reactions of the energy-conserving pathway in oxidative phosphorylation are inhibited by biguanides and alkylguanidines. The inhibitors bind to phospholipids of mitochondria and synthetic membranes with identical affinities and are assumed to modify the physical properties of membranes, as for example cation conductance. The study compares effects of biguanides on respiratory-linked ion fluxes across mitochondrial membranes with those on the conductance and the electrical capacitance of phospholipid bilayers. The following observations have been made: 1. The rate of energy-linked mitochondrial swelling and the rate of proton uptake during phosphorylation of ADP is inhibited in presence of biguanides. The activity of different compounds is proportional to their binding affinity to phospholipids. 2. Oscillatory volume changes of mitochondria are largely abolished by biguanides. 3. Biguanides cause a small increase of electrical conductance of phospholipid bilayers. This is only in part due to charge transfer by the lypophilic biguanide cations. It is suggested that also structural changes are induced by biguanides altering the surface area per molecule of phospholipid. At high biguanide concentrations structural reorganization is also expressed by changes in electrical capacitance. Both effects are saturable and proportional to binding affinity. 4. Biguanides affect the electrical conductance of bilayers in the opposite way but to the same extent with positively charged species (K+/ionophore) and with negatively charged species (uncouplers of oxidative phosphorylation). This is explained by generation of a positive surface potential at phospholipid containing membranes. 5. The data fit excellently the application of diffuse double-layer theory to lipid bilayers. The relative effectiveness of different biguanides on model membranes is identical to that on ion fluxes with mitochondrial membranes. From this it is concluded that a similar mechanism of action applies to synthetic and natural membranes as well, suggesting that screening of functional negative sites interferes with the translocation of cations linked to electron transport and the primary reactions in conservation of respiratory energy.

Proton translocation induced by ATPase activity in chloroplasts

Proton uptake by chloroplasts was induced by light-triggered ATPase activity. A quotient of two was obtained when the initial rate of proton uptake was divided by the rate of P i released from ATP. Gramicidin accelerated the rate of ATPase activity and reduced the H +/P i ratio to 1.4. The results were found to be consistent with the chemiosmotic theory.