Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Streptococcus faecalis proton gradients and tetracycline transport.

The transport of chlortetracycline by Streptococcus faecalis is energy dependent. Addition of glucose to energy-depleted cells enhances both the transport rates and accumulation levels. Transport rates can be altered independently of glucose by treating cells with ionophores that increase or decrease the proton gradient. The transport of the antibiotic is linked only to the transmembrane pH difference, delta pH, and not the transmembrane electrical potential, delta psi. This conclusion was verified by quantitative measurements of delta pH, delta psi, and tetracycline accumulation levels. A linear correlation between delta pH and the tetracycline electrochemical potential was observed. Tetracycline most likely accumulates by the symport of protons in which the protons are bound to an anionic form of the antibiotic to form an uncharged molecule.     

Inhibition of bacterial transport by uncouplers of oxidative phosphorylation. Effects of pentachlorophenol and analogues in Bacillus subtilis.

Analogues of the potent uncoupler of oxidative phosphorylation pentachlorophenol were tested as inhibitors of proline and glycine transport by Bacillus subtilis. These analogues included less highly substituted chlorophenols and pentachlorothiophenol. Like pentachlorophenol, they are non-competitive inhibitors of proline transport and uncompetitive inhibitors of glycine transport. However, the less highly substituted chlorophenols are weaker acids than pentachlorophenol and also weaker inhibitors. Analysis indicated that the anionic form of the uncouplers is the inhibiting species. Pentachlorothiophenol, a water-insoluble anion, is also a potent inhibitor. These results support previous studies that concluded that uncouplers of oxidative phosphorylation inhibit amino acid transport by binding at specific sites on proteins, the free energy of interaction stabilizing 'unproductive' conformations. Such specific interactions of uncoupler with protein are probably commonplace.     

Inhibition of nitrite assimilation by uncouplers of phosphorylation

Summary Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and pentachlorophenol (PCP), two powerful uncouplers of phosphorylation, specifically inhibit the assimilation of nitrite in the course of nitrate reduction. These results support our former conclusion that high-energy phosphate is involved in the metabolism of nitrite.     

Quantitative structure-activity relationship of carbonylcyanide phenylhydrazones as uncouplers of mitochondrial oxidative phosphorylation

The dependence of the uncoupling activity in the series of 16 carbonylcyanide phenylhydrazones on their physico-chemical properties (partition coefficient, dissociation constant and rate constant for reaction with thiols) is investigated using two physiologically based models, one for protonophoric mechanism of uncoupling and the other assuming the covalent modification of a membrane constituent to be the key step in this process. As indicated by uptake experiments, at the given conditions a lipophilic-hydrophilic equilibrium is attained without any loss of the compounds via chemical reactions. Using this fact to reduce the number of adjustable parameters, a better fit to the data on stimulation of respiration is obtained with the former (protonophoric) model.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.     

Effects of growth temperature on transport and membrane viscosity in Streptococcus faecalis

A shift in the growth temperature of Streptococcus faecalis from 37 to 10°C resulted in an 18% increase in the proportion of unsaturated fatty acids. Electron spin resonance spectra of spin-labeled membranes and extracted phospholipids indicated viscosity changes consistent with the alterations in fatty acid composition. Growth temperature had no significant effect on the active transport of leucine and alanine; uptake rates assayed at 10 or 35°C were essentially the same in cells grown at either 10 or 37°C. The relative rapidity of amino acid transport, which presumably contributes to the ability of S. faecalis to thrive in cold environments, is evidently unrelated to adaptive changes in the viscosity of membrane lipids.Abbreviations doxyl 4-4-dimethyloxazolidine-N-oxyl - proxyl 2,2-disubstituted 5,5-dimethylpyrrolidine-N-oxyl     

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane.From titration experiments of the rate of O₂ uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.     

Physiological adaptation of Euglena gracilis to uncouplers and inhibitors of oxidative phosphorylation

Cells of Euglena gracilis which have adapted to growth in the presence of 2,4-dinitrophenol are at the same time resistant to the inhibitory effect on growth of carbonylcyanide m-chlorophenylhydrazone, 1,7-hexafluoro-2,6-dihydroxy-2,6-bis(trifluoromethyl)-4 hep-tanone (“1799”), oligomycin, and tri-n-butylchlorotin. Cells adapted to any of the latter are at the same time resistant to dinitrophenol.The adaptation is a response to the primary effects of the uncouplers, i.e., high levels of ADP, and not to the uncouplers per se, since arsenate induced uncoupler resistance while nonuncoupling substituted phenols did not.No evidence for any protein in the cells which can bind uncouplers tightly could be found, and no modification of reactions of isolated mitochondria could be detected. Substrate utilization by adapted cells was only slightly less efficient than in normal cells.The inhibitors were accumulated by and not excluded from the cells. Thus, the cross-resistance implies either the formation of a modified mitochondrial membrane, impermeable to these hydrophobic effectors, or an alternative or greatly modified pathway for ATP synthesis, insensitive to these effectors.     

Mutants of Bacillus megaterium resistant to uncouplers of oxidative phosphorylation.

Mutants of Bacillus megaterium displaying malate-stimulated ATP synthesis resistant to uncouplers of oxidative phosphorylation were isolated and partially characterized. ATP synthesis in such mutants was resistant to carbonyl cyanide m-chlorophenyl hydrazone as well as to other uncouplers including 2,4-dinitrophenol, pentachlorophenol, and sodium azide. ATP synthesis in the wild type and in resistant mutants was sensitive to N,N'-dicyclohexylcarbodiimide, tributyltin, valinomycin plus potassium, and potassium cyanide. Active transport of glycine and glutamine which are sensitive to uncouplers in the wild type was also uncoupler-sensitive in the mutants.     

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.