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At least 19 recordsLinked to original sources
PubMed1979

Uncoupling agents distinguish between the effects of metabolic inhibitors and transport inhibitors.

In studies with toad bladders, the uncoupling agent 2,4-dinitrophenol (DNP) reversed the inhibition of CO2 production produced by direct inhibition of transport. In contrast, DNP did not reverse the inhibition of CO2 production brought about by metabolic inhibitors. Therefore, the response to DNP distinguished between inhibition of transport and metabolism; this approach may be useful for the investigation of factors that regulate active transport.

Animals↗
PubMed1979

Circular dichroic evidence for a conformational change in a cytochrome b--c1 complex by uncoupling agents.

Circular dichroic spectra of the cytochrome b--c1 complex exhibit bilobe formation typical of exciton splitting in the presence of uncoupler. Bilobe formation occurs if both cytochrome c1 and cytochrome b are fully reduced. The fully oxidized and ascorbate-reduced complexes are not altered dichroically by uncouplers. The exciton splitting induced by uncoupler is consistent with heme--heme interaction: specifically, interaction between the two cytochromes b in the complex.

Circular Dichroism↗
PubMed1976

Effect of inhibitors on the substrate-dependent quenching of 9-aminoacridine fluorescence in inside-out membrane vesicles of Escherichia coli.

The effect of various inhibitors on the substrate-dependent quenching of the fluorescence of 9-aminoacridine was measured in inside-out membrane vesicles of Escherichia coli. The rate of fluorescence quenching in the presence of inhibitors was dependent on the rate of electron transfer through the respiratory chain with NADH, succinate, D-lactate or DL-glycerol 3-phosphate as substrates. Several patterns of response were given by the inhibitors. Inhibitors competitive with substrate, or those acting only on the dehydrogenases, gave a direct relationship between the extent of inhibition of oxidase activity and the rate of quenching. A biphasic relationship was given by 2-heptyl-4-hydroxyquinoline N-oxide and piericidin A which was due to these compounds acting both as inhibitors of the respiratory chain and, at higher concentrations, as uncoupling agents. Uncouplers inhibited fluorescence quenching with minimal inhibition of oxidase activity. The transmembrane pH difference was calculated from the extent of fluorescence quenching and the intravesicular volume. The maximum pH difference of 3.3--3.7 units was generated by each of the substrates tested.

Acridines↗
PubMed1976

The control of the adenosine triphosphatase of Rhodospirillum rubrum chromatophores by divalent cations and the membrane high energy state.

1. The rate of ATP hydrolysis, catalysed by Rhodospirilum rubrum chromatophores is accelerated by low concentrations and inhibited by high concentrations of uncoupling agent. 2. The inhibition at high concentrations of uncoupling agent is potentiated by the presence of free magnesium ions. At low uncoupler concentrations magnesium has no effect on the rate of ATP hydrolysis. 3. Inhibition of ATP hydrolysis by high concentrations of uncoupling agent and free magnesium ions is reversed by illumination. Illumination has less effect at low magnesium concentrations. 4. Free calcium ions inhibit ATP hydrolysis independently of the coupled state of the membrane. 5. Under coupled conditions, magnesium ions can overcome the inhibition induced by calcium. The two ions complete for the same site on the enzyme. 6. Inhibition by free magnesium in highly uncoupled chromatophores and inhibition by free calcium are both non-competitive with respect to the divalent cation-ATP substrate. 7. These data are consistent with a model in which divalent cations can bind to a site on the enzyme which is distinct from the substrate site. The regulation of the enzyme activity by the high energy state of the membrane is dependent on the occupant of this site.

Adenosine Triphosphatases↗
PubMed1976

Energy-conserving reactions in phosphorylating electron-transport particles from Nitrobacter winogradskyi. Activation of nitrite oxidation by the electrical component of the protonmotive force.

1. In electron-transport particles (ET particles) prepared from Nitrobacter winogradskyi, the uncoupling agent carbonyl cyanide phenylhydrazone increased the rate of NADH oxidation but decreased the rate of oxidation of NO2-. Its effectiveness in stimulating NADH oxidation closely paralleled its effectiveness in inhibiting NO2- oxidation. 2. In the presence of ADP and phosphate the oxidation of NADH was stimulated, whereas the oxidation of NO2- was inhibited. In the presence of excess of Pi the concentration dependence with respect to ADP was the same for acceleration of NADH oxidation and inhibition of NO2- oxidation. 3. Oligomycin inhibited NADH oxidation and stimulated the oxidation of NO2-. The concentration of oligomycin required to produce half-maximal effect in both systems was the same. 4. The apparent Km for NO2- was not affected by ADP together with Pi, by uncoupling agent or by oligomycin. 5. With NADH as substrate, classical respiratory control was observed. With NO2- as substrate the respiratory-control ratio was less than unity. 6. A reversible uptake of H+ accompanied the oxidation of NO2- by ET particles. 7. In the presence of NH4Cl or cyclohexylamine hydrochloride, H+ uptake was abolished and increased rates of NO2- oxidation were observed. When valinomycin was present in the reaction medium, low concentrations of NH4Cl inhibited NO2- oxidation. 8. Pretreatment of ET particles with oligomycin enhanced the stimulation of NO2- oxidation induced by NH4Cl or by cyclohexylamine hydrochloride. Pretreatment with the uncoupler carbonyl cyanide phenylhydrazone prevented these stimulations. 9. In the presence of dianemycin together with K+, the uptake of H+ was abolished and the rate of NO2- oxidation was increased. In contrast, in the presence of valinomycin together with K+, the uptake of H+ was increased, and the rate of NO2- oxidation decreased. 10. Sodium tetraphenylboron was found to be an inhibitor of NO2- oxidation, but caused a stimulation of NADH oxidation which was dependent on the presence of NH4Cl or cyclohexylamine hydrochloride. 11. It is concluded that the enhanced rate of NO2- oxidation observed in the absence of energy-dissipating processes clearly relates to some state before the involvement of adenine nucleotides, and it is suggested that the oxidation of NO2- generates a protonmotive force, the electrical component of which controls the rate of NO2- oxidation.

Adenosine Diphosphate↗
PubMed1975

Studies on energy-linked reactions: isolation, characterisation and genetic analysis of trialkyl-tin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to triethyl tin sulphate have been isolated and are cross-resistant to other trialkyl tin salts. Triethyl-tin-resistant mutants fall into two general phenotypic classes: class 1 and class 2. Class 1 mutants are cross-resistant to a variety of inhibitors and uncoupling agents which affect mitochondrial membranes (oligomycin, ossamycin, valinomycin, antimycin, erythromycin, chloramphenicol, '1799', tetrachlorotrifluoromethyl benzimidazole carbonylcyanide-m-chlorophenylhydrazone and cycloheximide). Class 2 mutants are specifically resistant to trithyl tin and the uncoupling agent "1799' [bis-(hexafluoroacetonyl)-acetone]. Triethyl tin at neutral pH values is a specific inhibitor of mitochondrial energy conservation reactions and prevents growth on oxidisable substrates such as glycerol and ethanol. Triethyl-tin-resistant mutants grow normally on glucose and ethanol in the presence of triethyl tin (10 muM). Biochemical studies indicate that the mutation involves a modification of the triethyl tin binding site on the mitochondrial inner membrane, probably the ATP-synthetase complex. Triethyl tin resistance/sensitivity in yeast is determined by cytoplasmic (mitochondrial) and nuclear genes. The mutants fall into a nuclear and a cytoplasmic (mitochondrial) class corresponding to the phenotypic cross-resistance classes 1 and 2. In the cytoplasmic mutants the triethyl tin resistance segregates mitotically and the resistance determinat is deleted by the action of ethidium bromide during petite induction. Recombination studies indicate that the triethyl tin mutations are not allelic with the other mitochondrial mutations at the loci RI, RIII and OLI. This indicates that the binding or inhibitory sites of oligomycin and triethyl tin are not identical and that the triethyl tin binding site is located on a different mitochondrial gene product to those which are involved in oligomycin binding. Interaction and cooperative effects between different binding sites on the mitochondrial inner membrane have been demonstrated in studies of the effect of the insertion of the TETr phenotype into mitochondrial oligomycin-resistant mutants and provide an experimental basis for complementation studies at the ATP-synthetase level.

Adenosine Triphosphatases↗
PubMed1977

Transport and oxidation of choline by liver mitochondria.

1. Rapid choline oxidation and the onset of P(i)-induced swelling by liver mitochondria, incubated in a sucrose medium at or above pH7.0, required the addition of both P(i) and an uncoupling agent. Below pH7.0, P(i) alone was required for rapid choline oxidation and swelling. 2. Choline oxidation was inhibited by each of several reagents that also inhibited P(i)-induced swelling under similar conditions of incubation, including EGTA, mersalyl, Mg(2+), the Ca(2+)-ionophore A23187, rotenone and nupercaine. None of these reagents had any significant effect on the rate of choline oxidation by sonicated mitochondria. There was therefore a close correlation between the conditions required for rapid choline oxidation and for P(i)-induced swelling to occur, suggesting that in the absence of mitochondrial swelling the rate of choline oxidation is regulated by the rate of choline transport across the mitochondrial membrane. 3. Respiratory-chain inhibitors, uncoupling agents (at pH6.5) and ionophore A23187 caused a loss of endogenous Ca(2+) from mitochondria, whereas nupercaine and Mg(2+) had no significant effect on the Ca(2+) content. Inhibition of choline oxidation and mitochondrial swelling by ionophore A23187 was reversed by adding Ca(2+), but not by Mg(2+). It is concluded that added P(i) promotes the Ca(2+)-dependent activation of mitochondrial membrane phospholipase activity in respiring mitochondria, causing an increase in the permeability of the mitochondrial inner membrane to choline and therefore enabling rapid choline oxidation to occur. Nupercaine and Mg(2+) appear to block choline oxidation and swelling by inhibiting phospholipase activity. 4. Choline was oxidized slowly by tightly coupled mitochondria largely depleted of their endogenous adenine nucleotides, suggesting that these compounds are not directly concerned in the regulation of choline oxidation. 5. The results are discussed in relation to the possible mechanism of choline transport across the mitochondrial membrane in vivo and the influence of this process on the pathways of choline metabolism in the liver.

Adenine Nucleotides↗
PubMed1977

[Proton translocation in membranes of submitochondrial particles].

Effect of an electrophilous inhibitor, chlorophenacyl, on energy-dependent functions of submitochondrial particles is studied. Chlorophenacyl at concentrations up to 1 mM is found practically not to affect the generation of membrane potential under NADH and succinate oxidation and ATP hydrolysis and to be a strong inhibitor of oxidative phosphorylation and reverse electron transport. The mechanism of the inhibition of energy-dependent functions of submitochondrial particles with chlorophenacyl is different from that of electron transport inhibitor, energy transport inhibitors and classical uncoupling agents--protonophors. The data obtained are suggested to be due to the existence of two ways of proton translocation in submitochondrial particle membrane, phosphorylating and non-phosphorylating, the effect of chlorophenacyl being directed on phosphorylating way only.

Biological Transport↗
PubMedDate not supplied

[The effect of oxidazable substrates and ATP on the sensitivity of certain energy-dependent functions submitochondrial particles to phospholipases A, C and D].

The effect of NADH, succinate and ATP on the sensitivity of a number of energy-dependent functions of submitochondrial particles ot phospholipases A, C and D has been studied. It has been shown that in the conditions of oxidation of NADH and succinate by oxygen and also of ATP hydrolysis, the decrease in the phosphorylating activity of the particles under the action of phospholipases C and D accelerates. No such acceleration has been observed with phospholipase A. For other two functions, i. e. reverse electron transfer (ATP-dependent NAD+ reduction by succinate) and ATP-dependent transhydrogenase reaction the results proved to be different. Oxidizable substrates and ATP promoted the maintenance of these functions in the presence of phospholipase A, but did not retard their suppression by phospholipases C and D. The effects of NADH, succinate and ATP on the sensitivity of different energy-dependent functions of submitochondrial particles to phospholipases A, C and D could be removed by the uncoupling agent carbonyl cyanide-m-chlorophenyl hydrazone. The conclusion is made that the effects revealed are associated with an increase in the sensitivity of coupling sites II PAND/OR III to phospholipases C and D and with a decrease in the sensitivity of sites I and IV to phospholipase A on energization of submitochondrial particles.

Adenosine Triphosphate↗
PubMed1975

Mitochondrial permeability to chloride ion.

It is generally accepted that the inner membrane of the mitchondrion is not penetrated by chloride ion, in contrast to other biological membranes which are chloride permeable. In this report mitochondrial permeablity to chloride ion has been reevaluated by the measurement of passive swelling in isotonic chloride-containing solutions in the presence of an uncoupling agent. Under these conditions, mitochondria prepared from rat liver or beef heart show a definite uptake of wide variety of chloride salts. Mitochondrial chloride transport appears to be electrogenic, as is the transmembrane movement of the other halides. Therefore, the mitochondrial inner membrane shares with other biological membranes a definite permeability to this ubiquitous anion.

Animals↗
PubMedDate not supplied

[Effect of growth conditions on substrate phosphorylation during sulfite oxidation in Thiocapsa roseopersicina].

Cell extracts of Thiocapsa roseopersicina, strains BBS and SL, grown in the light under anaerobic conditions and in the dark under aerobic conditions on a medium containing thiosulphate produce ATP during oxidation of sulphite in the presence of AMP cytochrome c and inorganic phosphate. The uncoupling agent (m-chlorocarbonylcyanidephenylhydrazone) has no effect on the rate of ATP production, this being an evidence of substrate phosphorylation. The rate of ATP synthesis is higher in extracts of the cells grown in the dark under autotrophic conditions than in extracts of the cells cultivated in the light under anaerobic conditions or in the dark in the presence of thiosulphate and glucose.

Adenosine Monophosphate↗
PubMed1975

Multiple forms of cytochrome b in Mycobacterium phlei: kinetics of reduction.

The kinetics of reduction of the b-type cytochromes in the electron transport particles (ETP) from Mycobacterium phlei were studied with nicotinamide adenine dinucleotide, reduced form (NADH) or succinate as electron donors. There appeared to be three active cytochromes b in the ETP,bS563 and bS559, which were reducible by either substrate, and bN563, which was reducible by NADH but not by succinate. In the presence of adenosine 5'-triphosphate, a substantial increase in b563 reduction was observed with succinate at anaerobiosis. This was followed by a decrease in absorption. Adenosine 5'-triphosphate did not effect an increase in cytochrome b563 reduction at transition with NADH, but the occurrence of a secondary decrease in absorption was reflected in a decrease in total enzymatic reduction. The adenosine 5'-triphosphate effect was altered in trypsin-treated ETP, and abolished by uncoupling agents or by removal of the coupling factor-latent adenosine triphosphatase. In the presence of a supernatant fraction obtained during the preparation of the ETP, b563 reduction with succinate was greatly increased. A smaller increase was observed with NADH. Cytochrome b reduction was also studied in ETP inhibited by 2-n-nonylhydroxyquinoline-N-oxide, which appears to inhibit at bS563. On the basis of these data the interrelationships among the b-type cytochromes can be described in relation to the M. phlei electron transport chain.

Adenosine Triphosphatases↗
PubMed1978

Adenosine triphosphate in the bovine chromaffin granule.

1. pH and potential gradients are generated across the membranes of chromaffin granule 'ghost' by incubating them with MgATP: the inside of the 'ghosts' is positive and acid with respect to the incubation medium. 2. The pH gradient is partially dissipated by inclusion of a substrate for the catecholamine pump, or a mitochondrial uncoupling agent, but is enhanced by reserpine. 3. An imposed pH gradient leads to amine uptake by the 'ghosts': a potential gradient leads to ATP uptake. Studies with inhibitors confirm that amine accumulation by chromaffin granules is dependent on the former, and that ATP uptake results from ATPase-induced potential difference generation. 4. ATP has two known roles in chromaffin granule structure: the first is as a substrate for a membrane-bound proton-translocating ATPase; the second is as a component of the intragranular catecholamine storage complex.

Adenosine Triphosphate↗
PubMed1976

Glucose transport in isolated prosthecae of Asticcacaulis biprosthecum.

Active transport of glucose in prosthecae isolated from cells of Asticcacaulis biprosthecum was stimulated by the non-physiological electron donor N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride. Glucose uptake was mediated by two transport systems; the apparent Km of the high-affinity system was 1.8 muM and that of the low-affinity system was 34 muM. Free glucose accumulated within prosthecae at a concentration 60 to 200 times above that present externally, depending on the Km of the system being observed. The glucose transport system in prosthecae was stereospecific for D-glucose, and neither methyl alpha-D-glucopyranoside nor 2-deoxyglucose was transported. Uptake of glucose was inhibited by N-ethylmaleimide (NEM) and p-chloromercuribenzoate (PCMB), and the inhibition by PCMB but not by NEM was reversed by dithiothreitol. Glucose uptake was also inhibited by the uncoupling agents 5-chloro-3-t-butyl-2'-nitrosalicylanilide (S-13), 5-chloro-3-(p-chlorophenyl)-4'-chlorosalicylanilide (S-6), and carbonyl-cyanide m-chlorophenylhydrazone (CCCP) and by the respiratory inhibitor KCN. Efflux of glucose from preloaded prosthecae was induced by PCMB and KCN, but not by S-13 or CCCP. Glucose uptake was not affected by arsenate or an inhibitor of membrane-bound adenosine triphosphatases, N, N'-dicyclohexylcarbodiimide. The lack of inhibition by these two compounds, combined with the extremely low levels of adenosine 5'-triphosphate present in prosthecae, indicates that adenosine 5'-triphosphate is not involved in the transport of glucose by prosthecae.

Adenosine Triphosphate↗
PubMed1976

Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli.

The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates. Pyruvate could not stimulate transport in the glucose-grown cells. Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca2+, Mg2+-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol. Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation. In contrast, in vesicles prepared from cytochrome-containing cells of E. coli SASX76 amino acid transport was energized under anaerobic conditions by this system. Inhibitors of the Ca2+, Mg2+-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system. Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome-containing cells under anaerobic conditions. It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under under anaerobic conditions is energized by ATP through the Ca2+, Mg2+-activated ATPase. In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.

Adenosine Triphosphatases↗
PubMed1975

The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The oxidized and reduced nicotinamide-adenine dinucleotide content of flight muscle and isolated mitochondria, the adenosine triphosphate and adenosine diphosphate content of mitochondria, and the energy status of the mitochondria during controlled respiration.

1. A study is presented of the mitochondrial NADH content during controlled (state 4) and active (state 3) pyruvate oxidation by blowfly flight-muscle mitochondria. The results confirm and extend those of an earlier study (Hansford, 1972), which indicated an increased reduction in state 3. Nicotinamide nucleotide is normally highly oxidized during state 4; however, there can be substantial reduction in the presence of carnitine or high concentrations of proline, or on lengthy incubation in the presence of either of the systems used to generate intramitochondrial tricarboxylate-cycle intermediate. 2. Omission of phosphate leads to substantial reduction and this can be reversed by adding phosphate or acetate. 3. Estimations of NAD-+ and NADH in fly thoraces show a marked increase in NADH on flight, tending to corroborate the results of mitochondrial experiments and testifying to the importance of dehydrogenase activation in this tissue. 4. Determination of intramitochondrial adenine nucleotides reveals a total of 4-5 nmol/mg of protein, and an ADP content of less than 0.1 nmol/mg during state 4 oxidation of pyruvate and proline. ATP content is found to increase slowly during state 4 and this is attributed to the net phosphorylation of AMP. 5. The uncoupling agent carbonyl cyanide p=trifluoromethoxyphenylhydrazone leads to hydrolysis of some, but not all, of the mitochondrial ATP. Studies of mitochondrial ATPase (adenosine triphosphatase), measured by external pH change, show that it is inactive unless the mitochondria are allowed to respire for several minutes in state 4 in the presence of phosphate before the addition of carbonyl cyanide p-trifluoromethoxyphenylhydrazone. It is suggested that phosphate uptake is essential for maximal ATPase activity. 6. Studies of the fluorescence of the fluorochrome 8-anilino-1-naphthalensulphonic acid suggest that the energy status of the mitochondrion is high during state 4-pyruvate oxidattion, and decrease slightly in state 3. The implications of these findings are discussed.

Adenosine Diphosphate↗