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The polarity and stoichiometry of respiration-driven proton translocation was studied by electrometric and spectrophotometric techniques inMicrococcus denitrificans in the context of the energy transduction mechanism in bacterial oxidative phosphorylation.
- Protons are ejected through the plasma membrane during respiratory pulses and thereafter diffuse slowly back.
- In presence of ionic species mobile across the membrane (K+-valinomycin, K+-gramicidin, or SCN?), limiting→H+/O quotients of 8 were obtained with endogenous respiratory substrates, and the rate of translocation (14·3 μg ions of H+/sec g cell dry weight) was commensurate with that of respiration optimally stimulated by FCCP at an →H+/O quotient of 8.
- The rate of decay of the proton pulses was greatly increased by FCCP, but there was little or no effect on the →H+/O quotient characteristic of the respiratory system.
- Various interpretations of the observations are discussed, and it is concluded that respiration is probably coupled directly or indirectly to electrogenic proton translocation. The observations are compatible with the chemiosmotic hypothesis of coupling between respiration and phosphorylation.
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Respiration-driven proton translocation by yeast mitochondria with differing efficiencies of oxidative phosphorylation 总被引:5,自引:4,他引:1
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Measurements were made of the stoicheiometry of proton translocation coupled to respiration in mitochondria from Candida utilis where the number of functional energy-conservation sites between intramitochondrial NADH and oxygen was one in a mutant with a novel oxidase (Downie & Garland, 1972), two in sulphate-deficient cells (Haddock & Garland, 1971) or three in glycerol-limited cells (Light & Garland, 1971). The stoicheiometries of protons translocated per atom of oxygen utilized (i.e. -->H(+)/2e(-) ratio; Mitchell, 1966) were close to 2.0, 4.0 and 6.0 respectively. Thus by using the same substrate (intramitochondrial NADH) and oxygen throughout, the -->H(+)/2e(-) ratio is shown to be 2.0 per energy-conservation site when the number of such sites is varied from one to three. 相似文献
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Respiration-driven proton translocation in Escherichia coli (Short Communication) 总被引:11,自引:0,他引:11
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Measurements were made of the stoicheiometry of respiration-driven proton translocation coupled to the oxidation of NAD(P)-linked or flavin-linked substrates in intact cells of Escherichia coli. Observed stoicheiometries (-->H(+)/O quotient; Mitchell, 1966) were approx. 4 with l-malate as substrate and approx. 2 for succinate, d-lactate and glycerol oxidation. It is concluded that the potential number of equivalent energy-conservation sites associated with the respiratory chain is 2 in aerobically grown cells of E. coli harvested during the exponential phase of growth. 相似文献
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(1) +/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 , , for reduction to N2 and 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. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of 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 acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
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Sodium/proton antiporter of rat liver mitochondria 总被引:2,自引:0,他引:2
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Proton translocation coupled to ATP hydrolysis in rat liver mitochondria 总被引:15,自引:0,他引:15
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The periplasmic location of enzymes A and B of the thiosulphate-oxidizing multienzyme system of Thiobacillus versutus has been further confirmed by differential radiolabelling of periplasmic and cytoplasmic proteins. The stoichiometries of respiration-driven proton translocation in T. versutus were determined using the oxygen pulse and the initial rate methods. A value for the H+/O quotient (number of protons translocated per oxygen atom reduced) of about 2.8 was found for the oxidation of thiosulphate, and of about 2.5 for sulphite. The H+/O quotient for endogenous respiration was about 5.7. The data are shown to be in good agreement with the scheme proposed previously for thiosulphate oxidation by this organism. Proton generation during the oxidation of thiosulphate or sulphite is indicated to occur in the periplasm rather than by pumping across the cytoplasmic membrane. The results also suggest that a H+/O quotient of six occurs during NADH oxidation (from endogenous metabolism measurements) and that the terminal cytochrome oxidase, aa3, does not function as a proton pump.Abbreviations DCCD
dicyclohexyl carbodiimide
- FCCP
carbonyl cyanide p-trifluoromethoxyphenylhydrazone
- HQNO
2-n-heptyl-4-hydroxyquinoline N-oxide
- TMPD
N,N,N,N-tetramethyl-p-phenylenediamine
- IEF
isoelectric focusing
- HIC
hydrophobic interaction chromatography
- EAI
ethyl acetimidate hydrochloride
- IAI
isethionyl acetimidate 相似文献
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Inhibition by local anaesthetics of adenine nucleotide translocation in rat liver mitochondria 总被引:3,自引:3,他引:0
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1. The mechanism of adenine nucleotide translocation in mitochondria isolated from rat liver was further examined by using the local anaesthetics procaine, butacaine, nupercaine and tetracaine as perturbators of lipid-protein interactions. Each of these compounds inhibited translocation of ADP and of ATP; butacaine was the most effective with 50% inhibition occurring at 30mum for 200mum-ATP and at 10mum for 200mum-ADP. The degree of inhibition by butacaine of both adenine nucleotides was dependent on the concentration of adenine nucleotide present; with low concentrations of adenine nucleotide, low concentrations of butacaine-stimulated translocation, but at high concentrations (greater than 50mum) low concentrations of butacaine inhibited translocation. Butacaine increased the affinity of the translocase for ATP to a value which approached that of ADP. 2. Higher concentrations of nupercaine and of tetracaine were required to inhibit translocation of both nucleotides; 50% inhibition of ATP translocation occurred at concentrations of 0.5mm and 0.8mm of these compounds respectively. The pattern of inhibition of ADP translocation by nupercaine and tetracaine was more complex than that of ATP; at very low concentrations (less than 250mum) inhibition ensued, followed by a return to almost original rates at 1mm. At higher concentrations inhibition of ADP translocation resulted. 3. That portion of ATP translocation stimulated by Ca(2+) was preferentially inhibited by each of the local anaesthetics tested. In contrast, inhibition by the anaesthetics of ADP translocation was prevented by low concentrations of Ca(2+). 4. The data provide further support for our hypothesis that lipid-protein interactions are important determinants in the activity of the adenine nucleotide translocase in mitochondria. 相似文献
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Modification by calcium ions of adenine nucleotide translocation in rat liver mitochondria 总被引:6,自引:6,他引:0
1. Added Ca(2+) stimulates the translocation of ATP by isolated rat liver mitochondria. 2. The apparent K(m) for added Ca(2+) in stimulating the translocation of 200mum-ATP is approx. 160mum (75mum ;free' Ca(2+)). 3. The greatest stimulation of ATP translocation by Ca(2+) occurs at the lower concentrations of ATP. 4. Sr(2+) (and to a lesser extent Ba(2+)) can replace Ca(2+) whereas Mg(2+) and Mn(2+) have only little ability to stimulate ATP translocation. 5. Translocation of dATP is also stimulated by Ca(2+) whereas that of ADP is stimulated to only a relatively small degree. 6. Studies with metabolic inhibitors and uncouplers provide evidence that stimulation by Ca(2+) and by uncouplers is additive and that the mechanism of Ca(2+) stimulation does not seem to involve the high-energy intermediate of oxidative phosphorylation. 7. In the presence of Ca(2+), ATP is able to effectively compete with ADP for translocation. 8. Added K(+) further enhances the ability of Ca(2+) to stimulate ATP translocation. 9. These findings are discussed in relation to the potential involvement of Ca(2+) in modifying enzymic reactions involved in the regulation of cell metabolism. 相似文献
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Some features of H+-ATPase function in intact mitochondria of rat liver were studied. Simultaneously the activities of ATPase and proton translocase were measured, using a previously described technique. The proton translocation coefficient of H+-ATPase has been found to be equal to 3.6. The protonophore 3.5-di-tert-butyl-4-hydroxybenzylidenemalononitrile diminishes the proton translocation coefficient. It was concluded that when considering the mechanism of proton translocation by H+-ATPase, it is necessary to assume the possibility of transport of 3 or 4 protons per every hydrolyzed molecule of ATP allowing a changeable efficiency of the process. The decrease of the translocase coefficient in the presence of the protonophore appears to result from the ability of this uncoupler to return the transferred protons to the mitochondrial matrix. 相似文献
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Factors affecting the translocation of oxaloacetate and l-malate into rat liver mitochondria 总被引:5,自引:5,他引:0
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1. The rates of translocation of oxaloacetate and l-malate into rat liver mitochondria were measured by a direct spectrophotometric assay. 2. Penetration obeyed Michaelis-Menten kinetics, and apparent K(m) values were 40mum for oxaloacetate and 0.13mm for l-malate. 3. Arrhenius plots of the temperature-dependence of rates of penetration gave activation energies of +10kcal./mole for oxaloacetate and +8kcal./mole for l-malate. 4. The translocation of both oxaloacetate and l-malate was competitively inhibited by d-malate, succinate, malonate, meso-tartrate, maleate and citraconate. The K(i) values of these inhibitors were similar for the penetration of both oxaloacetate and l-malate. 5. Rates of penetration were stimulated by NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate under aerobic conditions or by ATP under anaerobic conditions. 6. The energy-dependent stimulation of translocation was abolished by uncouplers of oxidative phosphorylation. Oligomycin A, aurovertin, octyl-guanidine and atractyloside prevented the stimulation by ATP, but did not inhibit the stimulation by NNN'N'-tetramethyl-p-phenylenediamine dihydrochloride plus ascorbate. 7. Mitochondria prepared in the presence of ethylene-dioxybis(ethyleneamino)tetra-acetic acid did not exhibit the energy-dependent translocation, but this could be restored by the addition of 50mum-calcium chloride. 8. Valinomycin or gramicidin plus potassium chloride enhanced the energy-dependent translocation of oxaloacetate and l-malate. 9. Addition of oxaloacetate stimulated the adenosine triphosphatase activity of the mitochondria, and the ratio of ;extra' oxaloacetate translocation to ;extra' adenosine triphosphatase activity was 1.6:1. 10. Possible mechanisms for the energy-dependent entry of oxaloacetate and l-malate into mitochondria are discussed in relation to the above results. 相似文献