Absence Of Uncoupler Research Articles

Resistência a antimicrobianos dependente do sistema de efluxo multidrogas em Escherichia coli isoladas de leite mastítico

Identificaram-se e caracterizaram-se a resistência e a multirresistência aos principais antimicrobianos usados no tratamento de mastite bovina causada por Escherichia coli. A concentração inibitória mínima (MIC) e o sistema de efluxo foram detectados pelas curvas de crescimento, com base na densidade óptica, em diferentes concentrações da droga e na presença e na ausência do desacoplador da força próton-motora (PMF). E. coli 1 foi resistente à neomicina e à gentamicina; E. coli 3 e 4, à tetraciclina e à estreptomicina; e E. coli 2 e 6 à gentamicina. E. coli 5 apresentou modelo de sensibilidade. Observou-se que MICs de todos os antimicrobianos dos multirresistentes (E. coli 1, 3 e 4) diminuíram na presença do desacoplador, o que sugere sistema de efluxo multidrogas. Após cura, apenas E. coli 1 apresentou modelo de sensibilidade, porém não houve alterações das MICs, antes e após adição do desacoplador. Os resultados indicam possível presença de mecanismo de resistência dependente da PMF codificado, ou parte dele, em plasmídeo.

Unusual Slow Gating of Gap Junction Channels in Oocytes Expressing Connexin32 or Its COOH-Terminus Truncated Mutant

Gap junction channels are gated by a chemical gate and two transjunctional voltage (V (j))-sensitive gates: fast and slow. Slow V (j) gate and chemical gate are believed to be the same. The slow gate closes at the negative side of V (j) and is mostly inactive without uncouplers or connexin (Cx) mutations. In contrast, our present data indicate otherwise. Oocytes expressing Cx32 were subjected to series of -100 mV V (j) pulses (12-s duration, 30-s intervals). Both peak (PK) and steady-state (SS) junctional conductances (G (j)), measured at each pulse, decreased exponentially by 50-60% (tau = approximately 1.2 min). G (j)PK dropped more dramatically, such that G (j)SS/G (j)PK increased from 0.4 to 0.6, indicating a drop in V (j) sensitivity. Less striking effects were obtained with -60 mV pulses. During recovery, G (j), measured by applying 20 mV pulses (2-s duration, 30-s intervals), slowly returned to initial values (tau = approximately 7 min). With reversal of V (j) polarity, G (j)PK briefly increased and G (j)SS/G (j)PK decreased, suggesting that V (j)-dependent hemichannel reopening is faster than hemichannel closing. Similar yet more dramatic results were obtained with COOH-terminus truncated Cx32 (Cx32-D225), a mutant believed to lack fast V (j) gating. The data indicate that the slow gate of Cx32 is active in the absence of uncouplers or mutations and displays unusual V (j) behavior. Based on previous evidence for direct calmodulin (CaM) involvement in chemical/slow gating, this may also be CaM-mediated.

Energy-dependent conformational change in the TolA protein of Escherichia coli involves its N-terminal domain, TolQ, and TolR.

TolQ, TolR, and TolA inner membrane proteins of Escherichia coli are involved in maintaining the stability of the outer membrane. They share homology with the ExbB, ExbD, and TonB proteins, respectively. The last is involved in energy transduction between the inner and the outer membrane, and its conformation has been shown to depend on the presence of the proton motive force (PMF), ExbB, and ExbD. Using limited proteolysis experiments, we investigated whether the conformation of TolA was also affected by the PMF. We found that dissipation of the PMF by uncouplers led to the formation of a proteinase K digestion fragment of TolA not seen when uncouplers are omitted. This fragment was also detected in Delta tolQ, Delta tolR, and tolA(H22P) mutants but, in contrast to the parental strain, was also seen in the absence of uncouplers. We repeated those experiments in outer membrane mutants such as lpp, pal, and Delta rfa mutants: the behavior of TolA in lpp mutants was similar to that observed with the parental strain. However, the proteinase K-resistant fragment was never detected in the Delta rfa mutant. Altogether, these results suggest that TolA is able to undergo a PMF-dependent change of conformation. This change requires TolQ, TolR, and a functional TolA N-terminal domain. The potential role of this energy-dependent process in the stability of the outer membrane is discussed.

Formation and decay of monodehydroascorbate radicals in illuminated thylakoids as determined by EPR spectroscopy

H 2O 2 is reduced by an ascorbate-specific peroxidase (APX) in chloroplasts, generating the monodehydroascorbate (MDA) radical as the primary oxidation product. Using EPR spectroscopy we have measured the light-driven formation and decay of this species in thylakoids containing active APX. Illumination caused a rapid exponential rise in the steady-state MDA radical concentration in the absence of added electron acceptors other than O 2. This increase was sensitive to KCN and catalase and was prevented by anaerobic conditions, demonstrating the requirement for APX activity and endogenously generated H 2O 2, i.e., the Mehler reaction. When the illumination was removed, a second, transient increase in the radical signal was observed, indicating that photoreduction of the MDA radical and O 2 were occurring simultaneously in the light. This interpretation is also supported by the sigmoidal behavior of the chlorophyll dependence of MDA radical formation in illuminated thylakoids. Ferredoxin lowered the light-induced, steady-state MDA radical concentration, and is thus implicated as the physiological photoreductant for this Hill acceptor. In the absence of uncoupler, NADP + prevented formation of the MDA radical by lowering the flux to molecular O 2. However, in the presence of uncoupler (5 mM NH 4Cl) this constraint was apparently overcome, i.e., net formation of the radical occurred. The EPR method represents a novel approach to investigating the interaction of O 2 and ascorbate metabolism in chloroplasts under a variety of physiologically relevant conditions, to be applied in future studies of plant response to environmental stress.

The regulation of respiration in cell suspensions of Petunia hybrida, studied by inhibiting mitochondrial protein synthesis with chloramphenicol

When Petunia hybrida L. cv. Rosy Morn Fertile suspension cells were inoculated in fresh medium with chloramphenicol (CAP), the activity of cytochrome C oxidase (EC 1. 9. 3. 1), and the respiration via the cytochrome pathway of isolated mitochondria decreased, while in untreated cells these parameters more than doubled in 2–3 days. However, the in vivo respiratory activity of the cytochrome pathway of CAP‐treated cells showed a similar course in time as that of untreated cells, even in the presence of an uncoupler, a large rise during the first 2–3 days followed by a decline. This leads to the conclusion that the respiration via the cytochrome pathway, even when measured in the presence of an uncoupler, is not the capacity of this pathway. Furthermore, the results suggest that, although new‐synthesis of proteins occurs directly after in‐oculation, a large overcapacity must be present of cytochrome pathway elements (at least of those that are mitochondrial encoded). CAP had little effect on the uninhibited respiration and the cyanide‐resistant, alternative pathway of the Petunia cells. However, the engagement of the alternative pathway (in the presence or absence of uncoupler) was increased in CAP‐treated cells, especially after day 3 of the batch cycle, possibly as an effect of higher sugar degradation in combination with substrate phosphorylation to compensate the loss of ATP‐synthesizing ability of the cytochrome pathway. It will be discussed that in general one should be careful using the term ‘capacity’ for the respiratory pathways.

Immunology, biosynthesis and in vivo assembly of the branched‐chain 2‐oxoacid dehydrogenase complex from bovine kidney

Specific, polyclonal antisera have been raised to the native branched-chain 2-oxoacid dehydrogenase complex (BCOADC) from bovine kidney and each of its three constituent enzymes: E1, the substrate-specific 2-oxoacid dehydrogenase; E2, the multimeric dihydrolipoamide acyltransferase 'core' enzyme and E3, dihydrolipoamide dehydrogenase. Purified BCOADC, isolated by selective poly(ethyleneglycol) precipitation and hydroxyapatite chromatography, contains only traces of endogenous E3 as detected by a requirement for this enzyme in assaying overall complex activity and by immunoblotting criteria. A weak antibody response was elicited by the E1 beta subunit relative to the E2 and E1 alpha polypeptides employing either purified E1 or BCOADC as antigens. Anti-BCOADC serum showed no cross-reaction with high levels of pig heart E3 indicating the absence of antibody directed against this component. However, immunoprecipitates of mature BCOADC from detergent extracts of NBL-1 (bovine kidney) or PK-15 (porcine kidney) cell lines incubated for 3-4 h in the presence of [35S]methionine contained an additional 55,000-Mr species which was identified as E3 on the basis of immunocompetition studies. Accumulation of newly synthesised [35S]methionine-labelled precursors for E2, E1 alpha and E3 was achieved by incubation of PK-15 cells for 4 h in the presence of uncouplers of oxidative phosphorylation. Pre-E2 exhibited an apparent Mr value of 56,500, pre-E1 alpha, 49,000 and pre-E3, 57,000 compared to subunit Mr values of 50,000, 46,000 and 55,000, respectively, for the mature polypeptides. Thus, like the equivalent lipoate acyltransferases of the mammalian pyruvate dehydrogenase (PDC) and 2-oxoglutarate dehydrogenase (OGDC) complexes, pre-E2 of BCOADC characteristically contains an extended presequence. In NBL-1 cells, pre-E2 was found to be unstable since no cytoplasmic pool of this precursor could be detected; moreover, processed E1 alpha was not assembled into intact BCOADC as evidenced by the absence of E2 or E3 in immunoprecipitates with anti-(BCOADC) serum after a 45-min 'chase' period in the absence of uncoupler. Dihydrolipoamide dehydrogenase (E3), in its precursor state, was not present in immune complexes with anti-(BCOADC) serum, indicating that its co-precipitation with mature complex is by virtue of its high affinity for assembled complex in vivo whereas no equivalent interaction of pre-E3 with its companion precursors occurs prior to mitochondrial import.

Steady-state redox behavior of cytochrome c, cytochrome a, and CuA of cytochrome c oxidase in intact rat liver mitochondria

We have examined the steady-state redox behavior of cytochrome c (Fec), Fea, and CuA of cytochrome c oxidase during steady-state turnover in intact rat liver mitochondria under coupled and uncoupled conditions. Ascorbate was used as the reductant and TMPD (N,N,N',N'-tetramethyl-1,4-phenylenediamine) as the redox mediator. After elimination of spectroscopic interference from the oxidized form of TMPD, we found that Fea remains significantly more oxidized than previously thought. During coupled turnover, CuA always appears to be close to redox equilibrium with Fec. By increasing the amount of TMPD, both centers can be driven to fairly high levels of reduction while Fea remains relatively oxidized. The reduction level at Fea is close to a linear function of the enzyme turnover rate, but the levels at Fec and CuA do not keep pace with enzyme turnover. This behavior can be explained in terms of a redox equilibrium among Fec, CuA, and Fea, where Fea is the electron donor to the oxygen reduction site, but only if Fea has an effective Em (redox midpoint potential) of 195 mV. This is too low to be accounted for on the basis of nonturnover measurements and the effects of the membrane potential. However, if there is no equilibrium, the internal CuA----Fea electron-transfer rate constant must be slow in the time average (about 200 s-1). Other factors which might contribute to such a low Em are discussed. In the presence of uncoupler, this situation changes dramatically. Both Fec and CuA are much less reduced; within the resolution of our measurements (about 10%), we were unable to measure any reduction of CuA. Fea and CuA remain too oxidized to be in redox equilibrium with Fec during steady-state turnover. Furthermore, our results indicate that, in the uncoupled system, the (time-averaged) internal electron-transfer rate constants in cytochrome oxidase must be of the order of 2500 s-1 or higher. When turnover is slowed by azide, the relative redox levels at Fea and Fec are much closer to those predicted from nonturnover measurements. In presence of uncouplers, Fea is always more reduced than Fec, but in the absence of uncouplers, the two centers track together. Unlike the uninhibited, coupled system, the redox behavior here is consistent with the known effect of the electrical membrane potential on electron distribution in the enzyme. Interestingly, in these circumstances (azide and uncoupler present), Fea behaves as if it were no longer the kinetically controlling electron donor to the bimetallic center.

31P and 39K nuclear magnetic resonance spectroscopy studies of halobacterial bioenergetics

The bioenergetics of two extreme halophiles, Halobacterium halobium and Halobacterium marismortui, have been investigated by respiratory studies and in vivo nuclear magnetic resonance (NMR) spectroscopy. The phosphorus resonances of both species were very broad under anaerobic conditions, but sharpened somewhat on initiation of respiration and considerably upon cooling; changes attributable in part to slow chemical exchange processes. Transmembrane pH gradients (ΔpH) of H. marismortui were somewhat lower than those of H. halobium. Uncoupling agents caused ATP to disappear and inhibited respiration in both species, but dissipated ΔpH at best only partially. Uptake of Mn 2+ by H. halobium was unaffected by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), whereas H. marismortui appeared not to transport the ion, even in the absence of uncoupler. FCCP caused a slow efflux of K + from H. halobium, but no K + was lost from H. marismortui. These findings are discussed with particular reference to the intracellular environments of the two organisms.

Regulation of the imbalance in light excitation between Photosystem II and Photosystem I by cations and by the energized state of the thylakoid membrane

The imbalance in photoactivity between the two photosystems in broken chloroplasts during steady-state electron transport was investigated using modulated chlorophyll a fluorimetry and oxygen evolution. No imbalance in favor of PS II (imbalance term equals zero) was found at low cation concentration (e.g., 10 mM NaCl) where the membranes are unstacked, while some imbalance in favor of PS II (imbalance term about 0.1–0.2) could be observed at ‘high’ cation medium (e.g., 100 mM for univalent, 5 mM for divalent and 100 μM for trivalent cations) where the membranes are stacked. At the high cation concentration the imbalance was particularly noticeable at a pH range 6–7.5 under conditions where the membranes were non-energized, e.g., in the presence of a range of uncouplers and ionophores (an imbalance term of between 0.44 and 1.1). In the absence of uncouplers or ionophores the imbalance term was initially high, decreasing to a low steady-state value during the light-induced energization of the membranes. The increase in the imbalance measured with the addition of gramicidin D was wavelength-dependent, implying changes in the allocation of excitation energy to the photosystems rather than any other mechanism. This effect was reversed at higher pH: At a pH higher than about 8, the imbalance in absence of uncouplers or ionophores was stronger than in their presence. The relation between the state of imbalance and the cross-membrane proton gradient (ΔpH) was not straightforward or simple, as follows: (i) Imbalance was induced by uncouplers and ionophores also at sufficiently low light intensities which produce only very small ΔpH. (ii) Valinomycin (+KCl) had the same effect as uncouplers like gramicidin D, nigericin, NH4Cl and others, although it presumably abolishes membrane potential but not ΔpH. (iii) The effect of gramicidin D and NH4Cl was close to saturation at concentrations which affect ΔpH still minutely. (iv) The same level of large imbalance could be achieved even without uncouplers or ionophores when a much higher cation concentration (approx. 10-fold) than that considered normal to achieve membrane stacking was used. It is therefore concluded that: (a) High cationic levels change the allocation of excitation energy, probably via the screening of negative charges on the thylakoid surface. (b) The effect of uncouplers and ionophores in the presence of high cationic levels most probably reflects, at least in part, the effect on external surface charges, their exposure by membrane energization and their screening by cations.

Influence of non-esterified fatty acids on respiratory control of reconstituted cytochrome- c oxidase

Bovine heart cytochrome- c oxidase was reconstituted in liposomes (asolectin) and the activity measured in the presence and absence of uncoupler at increasing concentrations of non-esterified fatty acids. Palmitic and stearic acids resulted in a decrease of about 40% in the respiratory control ratio at a concentration of 1 νM, when measured using a spectrophotometric procedure but not with a polarographic assay method. At higher fatty acid concentrations no further change was found. A 50% decrease in respiratory control was determined when the enzyme was reconstituted in pure phosphatidylcholine containing 2% cardiolipin. The respiratory control of reconstituted cytochrome- c oxidase from bovine liver was not influenced by fatty acids.

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — energy-dependent switching between two different active states of the proton-translocation adenosine triphosphatase

Single-turnover flash-induced ATP synthesis coupled to natural cyclic electron flow in Photosystem I-enriched subchloroplast vesicles (from spinach) was continuously followed by the luciferin-luciferase luminescence. The ATP yield per flash was maximal (1 ATP per s per 1000 Chl) around a flash frequency of 0.5–2 Hz. It decreased both at lower and higher flash frequencies. The decrease at high flash frequency was due to limitation by the electron-transfer rate, while the decrease at low flash frequency was directly due to intrinsic properties of the ATPase itself. Carbonylcyanide- p-trifluoromethoxyphenylhydrazone (FCCP) decreased the yield at low frequency more than at high frequency. The same behaviour was observed if electron transfer was artificially mediated by pyocyanin. If the ADP concentration was increased from 40 to at least 80 μM, or if the vesicles were preincubated with 5 mM dithiothreitol (DTT), the decrease of the yield at flash frequencies below 0.5 Hz was no longer observed. Incubation with DTT increased the rates of ATP hydrolysis and synthesis at any flash frequency. The decrease of the yield could be elicited again by addition of 50 nM FCCP. It is concluded that at low levels of the protonmotive force (Δ \\ ̃ gm H + ), the ATPase is converted into an active ATP-hydrolyzing state in which ATP synthesis activity is decreased due to a decreased affinity towards ADP and/or to a decreased release of newly synthesized ATP, that can be cancelled by increasing the ADP concentration or by addition of DTT in the absence of uncoupler.

A mechanism of respiratory control: studies with proteoliposomes containing cytochrome oxidase and bacteriorhodopsin.

Both beef heart cytochrome oxidase and bacteriorhodopsin of Halobacterium halobium were reconstituted into liposomes by the sonication-cholate dialysis method. The proteoliposomes showed the respiratory control ratio of 4.2, and steady-state illumination of the vesicles lead to the 2.7-fold stimulation of the oxidase activity in the absence of uncouplers. The light-stimulated state 4 respiration increased with light intensity, but light had no effect on the oxidase activity that had been relieved by addition of uncouplers. Proteoliposomes with the photosensitive oxidase activity were also obtained when cytochrome oxidase vesicles were fused with bacteriorhodopsin vesicles in the presence of calcium chloride, and the extent of photoactivation was maximally 1.4-fold. The light-induced respiratory release was observed even in the presence of valinomycin or nigericin, indicating that the oxidase activity was sensitive to both the membrane potential and the pH gradient. We propose as a mechanism of the respiratory control that the process of proton transport to the reaction center for water formation is the rate limiting step for the cytochrome oxidase activity.

Short-term regulation of the nitrogenase activity in Rhodopseudomonas sphaeroides.

The nitrogenase activity in whole cells of Rhodopseudomonas sphaeroides could be inhibited by lowering the electrical potential across the cytoplasmic membrane. The membrane potential was partly dissipated either by lowering the light intensity or by the addition of a lipophilic cation, tetraphenylphosphonium. Under these circumstances, it was shown that the intracellular ATP/ADP ratio was not affected and that the inhibition of the whole cell nitrogenase activity was not due to an inactivation of the nitrogenase enzyme. From these results it is concluded that electron transport to nitrogenase in Rps. sphaeroides is dependent on a high membrane potential. The nitrogenase enzyme in whole cells could be inactivated by lowering the membrane potential across the cytoplasmic membrane by incubating the cells in the dark or in the light in the presence of uncouplers. Nitrogenase could be reactivated in the light in the absence of uncouplers. Some possible mechanisms of action of NH+4 inhibition of whole cell nitrogenase activity could be excluded. Inhibition by NH4Cl of whole cell nitrogenase activity in Rps. sphaeroides could neither be explained by a rapid inactivation of the nitrogenase enzyme, nor by an effect on the intracellular ATP/ADP ratio or the membrane potential. NH+4 inhibits whole cell nitrogenase activity not directly but probably after being assimilated by glutamine synthetase. The role of glutamine, glutamate and 2-oxoglutarate on the regulation of electron transport to nitrogenase will be discussed.

On the extent of localization of the energized membrane state in chromatophores from Rhodopseudomonas capsulata N22.

1. The principle of the double-inhibitor titration method for assessing competing models of electron transport phosphorylation is expounded. 2. This principle is applied to photophosphorylation by chromatophores from Rhodopseudomonas capsulata N22. 3. It is found that, in contrast to the predictions of the chemiosmotic coupling model, free energy transfer is confined to individual electron transport chain and ATP synthase complexes. 4. This conclusion is not weakened by arguments concerning, the degree of uncoupling in the native chromatophore preparation or the relative number of electron transport chain and ATP synthase complexes present. 5. Photophosphorylation is completely inhibited by the uncoupler SF 6847 at a concentration corresponding to 0.31 molecules per electron transport chain. 6. The apparent paradox is solved by the proposal, consistent with the available evidence on the mode of action of uncouplers, that uncoupler binding causes a co-operative conformation transition in the chromatophore membrane, which leads to uncoupling and which is not present in the absence of uncoupler.

Membrane-potential- and surface-potential-induced absorbance changes of merocyanine dyes added to chromatophores from Rhodopseudomonas sphaeroides

(1) Three analogs of merocyanine dyes added to suspensions of chromatophore vesicles showed absorbance changes responding to the change in surface potential induced by salt addition and to the change in membrane potential induced by illumination. (2) The extent of the light-induced absorbance changes of the dyes was linearly related, in the presence and absence of uncouplers, to that of carotenoid spectral shift which is an intrinsic probe of the intramembrane electric field. (3) Comparison of the merocyanine absorbance changes induced by salt addition with those induced by illumination indicated that the surface potential change in the outer surface of chromatophore membranes during illumination was very small. (4) Judging from the spectra of these absorbance and from the low permeabilities of the dyes to membrane, the absorbance change are attributed to change in distribution of the dyes between the medium and the outer surface region in chromatophore membranes. The extent of the light-induced absorbance changes of merocyanine dyes depended on the salt concentration of the medium. The types of dependence were different among three merocyanine analogs. This is explained by the mechanism mentioned above assuming appropriate parameters. It is suggested that, under continuous illumination, an equilibrium of the electrochemical potential of H + is reached between the bulk aqueous phase and the outer surface region in the membrane where the merocyanine dyes are distributed.

Bovine heart mitochondrial transhydrogenase catalyzes an exchange reaction between NADH and NAD+.

Reconstitution of homogeneous bovine heart mitochondrial transhydrogenase into phosphatidylcholine liposomes results in a greater than 80% inhibition of NADPH leads to 3-acetylpyridine adenine dinucleotide (AcPyAD+) transhydrogenation. This coupled rate is stimulated 5-fold by addition of protonophore to the rate observed with unreconstituted enzyme. In the absence of uncoupler, addition of low concentrations (10 microM) of NADH promotes by about 3-fold the rate of reduction of AcPyAD+ in the presence of NADPH. In the absence of NADPH, no reduction of AcPyAD+ by NADH occurs. Addition of valinomycin to K+-loaded proteoliposomes stimulated NADPH leads to AcPyAD+ transhydrogenation to the uncoupled rate and allowed the uptake of protons from the medium. In the absence of valinomycin, the rapid reduction of AcPyAD+ seen on addition of NADH was not coupled to proton translocation. In the presence of valinomycin, NADH addition neither equilibrated protons across the liposomal membrane nor affected the stoichiometry of proton uptake to hydride ion transfer (H+:H- ratio). Addition of NADH to proteoliposomes in the presence of AcPyAD+ and NADPH resulted in an initial rapid formation of reduced 3-acetylpyridine adenine dinucleotide comparable to the amount of NADH added. Thereafter, the rate of AcPyAD+ reduction returned to that seen in the absence of NADH. These results suggest that the NADH requirement for AcPyAD+ reduction is stoichiometric rather than catalytic. Stereospecificity studies demonstrate that reconstituted transhydrogenase catalyzes a direct transfer of a hydride ion equivalent from the 4A locus of NADH to the 4A locus of the NADH product in the presence, but not in the absence, of NADPH. It is proposed that NADH leads to NAD+ transhydrogenation represents a partial reaction of NADPH leads to NAD+ transhydrogenation which involves the participation of a reduced-enzyme intermediate.

Specificity of nucleotide binding and coupled reactions utilising the mitochondrial ATPase

1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min. 2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP. 3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site. 4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate- 32P i exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites. 5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism. 6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way. 7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD + by succinate. 8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.

Relative Contribution of Cytochrome-mediated and Cyanide-resistant Electron Transport in Fresh and Aged Potato Slices

The respiration of fresh potato (Solanum tuberosum, var. Russet Burbank) slices is predominantly cyanide-sensitive whether in the presence or absence of uncoupler. By contrast, the wound-induced respiration which develops in thin slices with aging is cyanide-resistant, and in the presence of cyanide, sensitive to chlorobenzhydroxamic acid, a selective inhibitor of the cyanide-resistant respiration. Titration of the alternate path in coupled slices with chlorobenzhydroxamic acid, in the presence and absence of cyanide, shows that the contribution of the cyanide-resistant pathway to the wound-induced respiration is zero. Similar titrations with uncoupled slices reveal that the alternate path is engaged and utilized extensively.The maximal capacity of the cytochrome path (V(cyt)) has been estimated in fresh and aged slices in the presence of the uncoupler carbonyl-cyanide m-chlorophenyl hydrazone. It has been found that V(cyt) of aged slices is but 30 to 40% higher than that of fresh slices. The results suggest that the bulk of the wound-induced respiration is mediated through the cytochrome pathway which exists in fresh slices in suppressed form, and which is fully expressed by slice aging. The engagement of the alternate path by uncouplers in aged slices is attributed to an increase in substrate mobilization, with the result that the electron transport capacity of the cytochrome chain is exceeded.

Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits

The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.The maximal capacities of the cytochrome and alternate paths, V(cyt) and V(alt), respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V(alt) decreases slightly with ripening.The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises the alternate as well as the cytochrome path. The ripening of intact fruits is seemingly independent of the nature of the electron transport path.Uncouplers are thought to stimulate glycolysis to the point where the glycolytic flux exceeds the oxidative capacity of the cytochrome path, with the result that the alternate path is engaged.

Control of respiration in proteoliposomes containing cytochrome aa3. I. Stimulation by valinomycin and uncoupler

1. Both valinomycin and p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP) are required for full release of respiration by cytochrome c oxidase-containing proteoliposomes (prepared by sonicating beef heart cytochrome aa 3 in salt solution with 4 parts phosphatidylcholine, 4 parts phosphatidylethanolamine and 2 parts cardiolipin) in the presence of external ascorbate and cytochrome c. In the absence of valinomycin the response to FCCP is rather sluggish, as reported by Wrigglesworth et al. (1976) (Abstracts, 10th Int. Congr. Biochem., No. 06-6-230). 2. The K m for cytochrome c in 67 mM, pH 7.4, phosphate buffer with ascorbate as substrate, was 9 μM in both absence and presence of valinomycin and FCCP. Energization thus acts non-competitively towards cytochrome c oxidation. 3. The apparent K m for oxygen is greater in the energized than in the deenergized state; double reciprocal plots of respiration rate versus oxygen concentration are concave downward in the absence of uncouplers, as found with intact mitochondria. Energization thus acts “competitively” towards oxygen. 4. Despite the lack of a functional ATPase system, all the kinetic features of energization found in intact mitochondria can be mimicked in the reconstituted liposomes. This supports the chemiosmotic idea that electrical and perhaps H + gradients modify the oxidase activity in reconstituted vesicles.