Sensitivity of some marine bacteria, a moderate halophile, and Escherichia coli to uncouplers at alkaline pH.

The inhibitory effects of uncouplers on amino acid transport into three marine bacteria, Vibrio alginolyticus 118, Vibrio parahaemolyticus 113, and Alteromonas haloplanktis 214, into a moderate halophile, Vibrio costicola NRC 37001, and into Escherichia coli K-12 were found to vary depending upon the uncoupler tested, its concentration, and the pH. Higher concentrations of all of the uncouplers were required to inhibit transport at pH 8.5 than at pH 7.0. The protonophore carbonyl cyanide m-chlorophenylhydrazone showed the greatest reduction in inhibitory capacity as the pH was increased, carbonyl cyanide p-trifluoromethoxyphenylhydrazone showed less reduction, and 3,3',4',5-tetrachlorosalicylanilide was almost as effective as an inhibitor of amino acid transport at pH 8.5 as at pH 7.0 for all of the organisms except A. haloplanktis 214. Differences between the protonophores in their relative activities at pHs 7.0 and 8.5 were attributed to differences in their pK values. 3,3',4',5-Tetrachlorosalicylanilide, carbonyl cyanide m-chlorophenylhydrazone, 2-heptyl-4-hydroxyquinoline-N-oxide, and NaCN all inhibited Na+ extrusion from Na+-loaded cells of V. alginolyticus 118 at pH 8.5. The results support the conclusion that Na+ extrusion from this organism at pH 8.5 occurs as a result of Na+/H+ antiport activity. Data are presented indicating the presence in V. alginolyticus 118 of an NADH oxidase which is stimulated by Na+ at pH 8.5.     

Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences.

The transport of precursor proteins into mitochondria requires an energized inner membrane. We report here that the import of various precursor proteins showed a differential sensitivity to treatment of the mitochondria with the uncoupler carbonyl cyanide m-chlorophenylhydrazone. The differential inhibition by carbonyl cyanide m-chlorophenylhydrazone was not influenced by the length of the precursor, the presence of mature protein parts, or the folding state of the precursor but was specific for the presequence. Moreover, only the membrane potential delta psi and not the total proton motive force was required for the transport of precursors, indicating that protein translocation across the inner membrane is not driven by a movement of protons. We conclude that delta psi (negative inside) is needed for the translocation of the positively charged presequences, possibly via an electrophoretic effect.     

Respiration-driven Na+ pump and Na+ circulation in Vibrio parahaemolyticus.

Sodium circulation in Vibrio parahaemolyticus was investigated. We observed respiration-driven Na+ extrusion from cells by using a Na+ electrode. The Na+ extrusion was insensitive to a proton conductor, carbonyl cyanide m-chlorophenylhydrazone, and sensitive to a respiratory inhibitor, CN-. These results support the idea of the existence of a respiratory Na+ pump in V. parahaemolyticus. The respiration-driven Na+ extrusion was observed only under alkaline conditions.     

Respiratory oscillations and heat evolution in synchronous cultures of Candida utilis

Synchronous cultures of the budding yeast Candida utilis prepared by continuous-flow size selection showed respiratory oscillations when the energy source was either glucose, acetate or glycerol. The period of the oscillations was about one-third of the cell cycle time (i.e. about 0.5 h). No fluctuations in heat evolution could be detected. In organisms growing with acetate or glycerol, the effects of cyanide, N,N'-dicyclohexylcarbodi-imide and carbonyl cyanide m-chlorophenylhydrazone (maximum inhibition of respiration at respiratory maxima, maximum uncoupling of energy conservation at respiratory minima) suggest that the control mechanism responsible for the oscillations is mitochondrial respiratory control in vivo. The effects of cyanide and N,N'-dicyclohexylcarbodi-imide on the respiration of cultures growing synchronously with glucose were different from those for cultures growing with the non-fermentable substrates; this suggests that the mitochondrial respiratory system interacts with the early reactions of glucose utilization.     

Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism.

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the 'binding protein' for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.     

Studies on the role of calcium ions in the stimulation by adrenaline of amylase release from rat parotid

1. Mitochondrial and microsomal fractions were prepared from rat parotid glands. Both fractions were able to take up (45)Ca. The mitochondrial (45)Ca-uptake system could be driven by ATP (energy-coupled Ca(2+) uptake) or by ADP+succinate (respiration-coupled Ca(2+) uptake). Energy-coupled Ca(2+) uptake was blocked by oligomycin but not by carbonyl cyanide m-chlorophenylhydrazone; respiration-coupled Ca(2+) uptake was blocked by carbonyl cyanide m-chlorophenylhydrazone but not by oligomycin. Microsomal Ca(2+) uptake was dependent on the presence of ATP; the ATP-dependent Ca(2+) uptake was not affected by oligomycin or carbonyl cyanide m-chlorophenylhydrazone. Ca(2+) uptake by both fractions was inhibited by Ni(2+). 2. Incubation of parotid pieces with adrenaline increased the rate of release of amylase and the uptake of (45)Ca. The adrenaline-stimulated release of amylase was not dependent on the presence of extracellular Ca(2+). 3. The effect of adrenaline on the subcellular distribution of (45)Ca in parotid pieces incubated with (45)Ca was studied. In parotid tissue incubated with (45)Ca, both mitochondrial and microsomal fractions contained (45)Ca. Incubation with adrenaline increased the amount of (45)Ca incorporated into the mitochondrial fraction but not the microsomal fraction. In parotid tissue preloaded with (45)Ca subsequent incubation with adrenaline caused a decrease in the amount of (45)Ca found in both the mitochondrial and microsomal fractions. 4. From these data we conclude that the regulation of the cytosolic Ca(2+) concentration in the parotid may involve both mitochondrial and microsomal Ca(2+)-uptake systems. We suggest that the action of adrenaline on the parotid may be to increase the movement of Ca(2+) to the cytosol by increasing the flux of Ca(2+) across mitochondrial, microsomal and plasma membranes.     

Electrochemical study of proton translocation function of hydrogenase 3

The oxidation of formate associated with fast acidification of medium by whole Escherichia coli cells lacking both hya and hyb hydrogenases was studied. The extent of acidification was dependent on the amount of formate added. An average H+/formate ratio of 1.3 was obtained. The proton release was inhibited by carbonyl cyanide m-chlorophenylhydrazone. Inverted vesicles of E. coli were found to translocate protons upon oxidation of formate at pH 6.5. The extent of alkalization was also dependent on the amount of formate added. The maximum H+/formate ratio for this reaction was close to 0.6. Formate oxidation by inverted vesicles from E. coli (delta hya delta hyb) was sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone. It was supposed that the hydrogenase 3 (hyc) component of E. coli formate hydrogen lyase is responsible for the translocation of protons at low pH.     

Interaction of respiration and luminescence in a common marine bacterium

Investigators have proposed for some time that bacterial luciferase forms a shunt around the pathway of respiratory electron transport. Certain physiologic evidence for coupling between luminescence and respiration has supported such a view. In this study, Vibrio harveyi cells were monitored for luminescent responses to artificial manipulation of respiratory electron flow. The effects of cyanide under aerobic and anaerobic conditions confirmed that luminescence and respiration compete for oxygen. The effects of an uncoupler of oxidative phosphorylation indicated that luminescence and respiration compete for a common reductant. Treatment with uncoupler also induced aldehyde deficiency in vivo.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - Tris tris(hydroxymethyl) aminomethane     

The role of mitochondria in the regulation of calcium influx into Jurkat cells.

In electrically nonexcitable cells the activity of the plasma membrane calcium channels is controlled by events occurring in mitochondria, as well as in the lumen of the endoplasmic reticulum. Thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+-ATPase, produces the release of calcium from the endoplasmic reticulum and thus, activation of store-operated calcium channels in the plasma membrane. However, thapsigargin failed to produce significant activation of the channels in Jurkat cells that had been pretreated with mitochondria-directed agents: an uncoupler (carbonyl cyanide m-chlorophenylhydrazone) and oligomycin. This is in spite of the fact that Jurkat cells pretreated with carbonyl cyanide m-chlorophenylhydrazone plus oligomycin are otherwise energetically competent, due to a high rate of glycolysis and the inhibition of mitochondrial F1Fo-ATPase by oligomycin. The pool of intracellular ATP was found not to be influenced by the pretreatments of cells with oligomycin or with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone. In the control cells, we found that the ATP pool amounted to 23.2 +/- 1.9 nmoles per 107 cells (n = 4). In cells pretreated with oligomycin the level of ATP was 21.8 +/- 1.9 nmoles per 107 cells (n = 4), and in cells pretreated with both oligomycin and an uncoupler the level of ATP was 22.1 +/- 0.2 nmoles per 107 cells (n = 3). Moreover, in cells pretreated with oligomycin plus carbonyl cyanide m-chlorophenylhydrazone and suspended in a nominally calcium-free medium, thapsigargin produces transient increases in cytosolic calcium identical to those in the control cells. Thus, this pretreatment does not modify either the content of intracellular calcium stores and/or the activity of calcium ATPase in the plasma membrane. Similar results were obtained when Jurkat cells were challenged by myxothiazol, a potent inhibitor of mitochondrial cytochrome bc1 oxidoreductase. Thapsigargin, although producing calcium release from intracellular stores, was ineffective in triggering the activation of calcium channels in the plasma membrane in the case of cells pretreated with myxothiazol and oligomycin. Our results suggest that coupled mitochondria participate directly in the control of calcium channel activity in the plasma membrane of Jurkat cells. When the mitochondrial protonmotive force is collapsed, either by carbonyl cyanide m-chlorophenylhydrazone or myxothiazol, the channel remains inactive even under conditions of empty intracellular calcium stores.     

Mechanism of control of adenylate cyclase activity in yeast by fermentable sugars and carbonyl cyanide m-chlorophenylhydrazone

The phosphorylation of fructose-1,6-bisphosphatase is preceded by a transient increase in the intracellular level of cyclic AMP which activates a cyclic AMP-dependent protein kinase (Pohlig, G., and Holzer, H. (1985) J. Biol. Chem. 260, 13818-13823). Possible mechanisms by which sugars or ionophores might activate adenylate cyclase and thereby lead to an increase in cyclic AMP concentrations were studied. Studies with permeabilized yeast cells demonstrated that neither sugar intermediates nor carbonyl cyanide m-chlorophenylhydrazone are able to increase adenylate cyclase activity. In the light of striking differences of the effects of fermentable sugars and of carbonyl cyanide m-chlorophenylhydrazone on parameters characterizing the membrane potential, it seems not reasonable that the activity of adenylate is under control of the membrane potential. Rapid quenching of 9-aminoacridine fluorescence after addition of fermentable sugars to starved yeast cells indicated an intracellular acidification. The 31P NMR technique showed a fast drop of the intracellular pH from 6.9 to 6.55 or 6.4 immediately after addition of glucose or carbonyl cyanide m-chlorophenylhydrazone. The time course of the decrease of the cytosolic pH coincides with the transient increase of cyclic AMP concentration and the 50% inactivation of fructose-1,6-bisphosphatase under the conditions of the NMR experiments. Kinetic studies of adenylate cyclase activity showed an approximately 2-fold increase of activity when the pH was decreased from 7.0 to 6.5, which is the result of a decrease in the apparent Km for ATP with no change in Vmax. These studies suggest that activation of adenylate cyclase by decrease in the cytosolic pH starts a chain of events leading to accumulation of cyclic AMP and phosphorylation of fructose-1,6-bisphosphatase.     

Effects of Protonophores on the Synthesis of Catecholamines and the Intracellular pH in Cultured Bovine Adrenal Medullary Cells

The protonophores carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) stimulated the synthesis of 14C-catecholamines from [14C]tyrosine in cultured bovine adrenal medullary cells. The stimulatory effect of CCCP but not of FCCP was partially dependent on extracellular Ca2+. CCCP but not FCCP increased the influx of 45Ca2+ to the cells. When cells were incubated with either CCCP or FCCP (0.01-0.2 microgram/ml), the intracellular pH fell from 7.2 to 6.3-6.5 and catecholamine synthesis increased. Tyrosine hydroxylase activity in a soluble fraction prepared from cultured adrenal medullary cells was measured after incubation of the cells with FCCP or CCCP. Although FCCP did not affect the activity of the enzyme, CCCP caused a stable activation of it which was dependent on extracellular Ca2+. Since the optimal pH of soluble tyrosine hydroxylase is around 6.0 in adrenal medullary cells, FCCP may increase the synthesis of catecholamines by shifting the intracellular pH toward it. In addition to this mechanism, CCCP may enhance the synthesis of catecholamines by a Ca2+-dependent mechanism.     

Purification and Ligand Binding of EmrR, a Regulator of a Multidrug Transporter

EmrR, the repressor of the emrRAB operon of Escherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump-2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone-with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.     

The sodium cycle. I. Na+-dependent motility and modes of membrane energization in the marine alkalotolerant vibrio Alginolyticus

Respiration, membrane potential generation and motility of the marine alkalotolerant Vibrio alginolyticus were studied. Subbacterial vesicles competent in NADH oxidation and delta psi generation were obtained. The rate of NADH oxidation by the vesicles was stimulated by Na+ in a fashion specifically sensitive to submicromolar HQNO (2-heptyl-4-hydroxyquinoline N-oxide) concentrations. The same amounts of HQNO completely suppressed the delta psi generation. Delta psi was also inhibited by cyanide, gramicidin D and by CCCP + monensin. CCCP (carbonyl cyanide m-chlorophenylhydrazone) added without monensin exerted a much weaker effect on delta psi. Na+ was required to couple NADH oxidation with delta psi generation. These findings are in agreement with the data of Tokuda and Unemoto on Na+-motive NADH oxidase in V. alginolyticus. Motility of V. alginolyticus cells was shown to be (i) Na+-dependent, (ii) sensitive to CCCP + monensin combination, whereas CCCP and monensin, added separately, failed to paralyze the cells, (iii) sensitive to combined treatment by HQNO, cyanide or anaerobiosis and arsenate, whereas inhibition of respiration without arsenate resulted only in a partial suppression of motility. Artificially imposed delta pNa, i.e., addition of NaCl to the K+ -loaded cells paralyzed by HQNO + arsenate, was shown to initiate motility which persisted for several minutes. Monensin completely abolished the NaCl effect. Under the same conditions, respiration-supported motility was only slightly lowered by monensin. The artificially-imposed delta pH, i.e., acidification of the medium from pH 8.6 to 6.5 failed to activate motility. It is concluded that delta mu Na+ produced by (i) the respiratory chain and (ii) an arsenate-sensitive anaerobic mechanism (presumably by glycolysis + Na+ ATPase) can be consumed by an Na+ -motor responsible for motility of V. alginolyticus.     

Exochelin-mediated iron acquisition by the leprosy bacillus, Mycobacterium leprae

Exochelins, water-soluble siderophores of mycobacteria, were isolated and partially purified from culture filtrates of iron-deficiently grown cultures of Mycobacterium neoaurum NCTC 10439 and an armadillo-derived Mycobacterium (ADM 8563). Two biologically active fractions mediating iron uptake were isolated from each bacterium which not only were able to transport iron into the producing organism but also into suspensions of Mycobacterium leprae isolated from armadillo liver. The rate of exochelin-mediated iron uptake into M. leprae was about 1.5% of the rate observed into the producing organisms. The process of iron uptake appears to be by facilitated diffusion as it was not inhibited by HgCl2, NaN3, KCN, dinitrophenol or carbonyl cyanide m-chlorophenylhydrazone. Since no uptake of iron occurred into iron-sufficient ADM cells, this may indicate that M. leprae, as recovered from an animal tissue, had been growing iron-deficiently in order for iron uptake to have been demonstrated in vitro.     

cAMP-mediated catabolite repression and electrochemical potential-dependent production of an extracellular amylase in Vibrio alginolyticus

Vibrio alginolyticus, a halophilic marine bacterium, produced an extracellular amylase with a molecular mass of approximately 56,000, and the amylase appeared to be subject to catabolite repression mediated by cAMP. The production of amylase at pH 6.5, at which the respiratory chain-linked H+ pump functions, was inhibited about 75% at 24 hours following the addition of 2 microM carbonyl cyanide m-chlorophenylhydrazone (CCCP), while the production at pH 8.5, at which the respiratory chain-linked Na+ pump functions, was only slightly inhibited by the addition of 2 microM CCCP. In contrast, the production of amylase in a mutant bacterium defective in the Na+ pump was almost completely inhibited even at pH 8.5 as well as pH 6.5 by the addition of 2 microM CCCP.     

pH effects on cystine transport in lysosome-rich leucocyte granular fractions.

Inhibitors of lysosomal acidification (4,4'-di-isothiocyanostilbene-2,2'-disulphonate, NN'-dicyclohexylcarbodi-imide, carbonyl cyanide m-chlorophenylhydrazone, NH4Cl and methylamine hydrochloride) did not alter cystine egress or countertransport in polymorphonuclear-leucocyte lysosome-rich granular fractions at pH 7.0. Together, 2 mM-MgCl2/MgATP and 90 mM-KCl stimulated cystine egress 2-fold, but this effect also was not influenced by inhibitors of ATP-dependent lysosomal acidification. MgCl2/MgATP stimulated cystine transport at pH 5.5, but the effect also occurred with MgCl2, MgSO4 or MnCl2 alone, was prevented by chelation, and was not seen with NaATP; therefore, it was considered a bivalent-cation, not an ATP, effect. Proton-pump-mediated acidification of lysosomes does not appear to be required for cystine transport in normal polymorphonuclear-leucocyte granular fractions, as reported for lymphoblast lysosomes.     

Proton motive force-dependent and -independent protein translocation revealed by an efficient in vitro assay system of Escherichia coli

Inverted membrane vesicles prepared from Escherichia coli spheroplasts were fractionated by means of sucrose gradient centrifugation, and a vesicle preparation exhibiting efficient and quantitative translocation of secretory proteins was obtained. The translocation of OmpA and an uncleavable model protein, uncleavable OmpF-Lpp, took place almost completely in 2-3 min, whereas that of OmpF-Lpp, a chimeric secretory protein, required 20 min for completion. The requirement of the proton motive force (delta muH+) for in vitro translocation was then examined with these three proteins. The translocation of all these proteins was significantly inhibited by the addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP) or when stripped membrane vesicles lacking F1-ATPase were used, suggesting that delta muH+ generally participates in the translocation reaction. The inhibition was complete with OmpF-Lpp, whereas significant amounts of uncleavable OmpF-Lpp and OmpA were translocated at a slower rate even with the stripped membrane vesicles in the presence of a high concentration of carbonyl cyanide m-chlorophenylhydrazone. The delta muH+-independent translocation was inhibited by a nonhydrolyzable ATP analogue. These results indicate that although translocation of OmpF-Lpp obligatory requires delta muH+, the latter two proteins can be translocated in not only a delta muH+-dependent manner but also a delta mu H+-independent manner.     

The effect of uncoupler on the distribution of the electron flow between the terminal acceptors oxygen and nitrite in the cells of Paracoccus denitrificans

The preferential utilization of oxygen, the terminal acceptor, in anaerobically grown cells of Paracoccus denitrificans was abolished in the presence of uncoupler (3 microM carbonyl cyanide m-chlorophenylhydrazone) which brought about a switch to the reduction of nitrite. It has been proved by measuring the redox state of cytochromes that this effect is due to the inhibition of the electron flow to oxygen caused by nitrite, which attains the site of its inhibitory action when the membrane potential is lowered.     

Mutual inactivation of valinomycin and protonophores by complex formation in liposomal membranes

The stimulation presence of a protonophore [3,5-di(ter-butyl)-4-hydroxybenzylidenemalononitrile or carbonyl cyanide m-chlorophenylhydrazone] and valinomycin in a liposome suspension results in time-dependent inactivation of ion transport by both the protonophore and valinomycin. Correlation of the inactivation with spectrophotometric observations on the formation of a complex between the protonophore and valinomycin strongly suggests that the complex observed has no (or very low) activity for the transport of either H+ or K+. The stoichiometry of valinomycin and the protonophore in the inactive complex is shown to be 1:1.     

Longitudinal component of trans-root electrical potential difference: evidence from application of metabolic inhibitors to the cut end of excised maize roots

Changes in trans-root electrical potential induced by application of metabolic inhibitors (carbonyl cyanide m-chlorophenylhydrazone, vanadate, diethylstilbestrol, N-ethyl maleimide, p-chloromercuribenzene sulfonic acid, KCN, salicylhydroxamic acid) and electron acceptors (hexachloroiridate IV and hexacyanoferrate III) to the cut end of excised roots of maize demonstrated existence of a longitudinal component of trans-root electrical potential. It was probably associated with redox plasma membrane bound system(s) and coupled to the cyanide sensitive and alternative respiration pathways. This revised version was published online in July 2006 with corrections to the Cover Date.