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1.
Sci Rep ; 11(1): 13631, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211053

RESUMO

Bacterial energy metabolism is now recognized as a critical factor for the efficacy of antibiotics. The F-type ATPase/ATP synthase (FOF1) is a central player in cellular bioenergetics of bacteria and eukaryotes, and its potential as a selective antibiotic target has been confirmed by the success of bedaquiline in combatting multidrug-resistant tuberculosis. Venturicidin macrolides were initially identified for their antifungal properties and were found to specifically inhibit FOF1 of eukaryotes and bacteria. Venturicidins alone are not effective antibacterials but recently were found to have adjuvant activity, potentiating the efficacy of aminoglycoside antibiotics against several species of resistant bacteria. Here we discovered more complex effects of venturicidins on the ATPase activity of FOF1 in bacterial membranes from Escherichia coli and Pseudomonas aeruginosa. Our major finding is that higher concentrations of venturicidin induce time- and ATP-dependent decoupling of F1-ATPase activity from the venturicidin-inhibited, proton-transporting FO complex. This dysregulated ATPase activity is likely to be a key factor in the depletion of cellular ATP induced by venturicidins in prior studies with P. aeruginosa and Staphylococcus aureus. Further studies of how this functional decoupling occurs could guide development of new antibiotics and/or adjuvants that target the F-type ATPase/ATP synthase.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Escherichia coli/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Venturicidinas/farmacologia , Trifosfato de Adenosina/metabolismo , Antibacterianos/química , Escherichia coli/metabolismo , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Humanos , Macrolídeos/química , Macrolídeos/farmacologia , Modelos Moleculares , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Venturicidinas/química
2.
J Nat Prod ; 84(1): 110-119, 2021 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-33356258

RESUMO

Chemical screening of Streptomyces sp. NRRL S-4 with liquid chromatography-mass spectrometry (LC-MS) and the following chromatographic isolation led to the discovery of four 20-membered macrolides, venturicidin A (4) and three new congeners venturicidins D-F (1-3). Genome sequencing of strain S-4 revealed the presence of a biosynthetic gene cluster (BGC) encoding glycosylated type I polyketides (PKS). The BGC designated to venturicidin biosynthesis (ven) was supported by the proposed biosynthetic pathway and confirmed by inactivation of the core PKS gene of venK. Bioinformatic analyses on the conserved motifs and known stereospecificities in PKS modules are consistent with the structure and absolute configuration. This is the first report of venturicidin BGC since the discovery of the macrolide in 1961. In the biological assays, venturicidin A (4) and E (2) displayed a high selective cytotoxicity against acute monocytic leukemia MV-4-11 cells with IC50 values of 0.09 and 0.94 µM, respectively. Venturicidin A (4) also showed a weak inhibitory activity on FMS-like-tyrosine kinase.


Assuntos
Leucemia Monocítica Aguda/genética , Macrolídeos/farmacologia , Streptomyces/química , Venturicidinas/farmacologia , Vias Biossintéticas/genética , Biologia Computacional , Leucemia Monocítica Aguda/metabolismo , Macrolídeos/química , Macrolídeos/isolamento & purificação , Macrolídeos/metabolismo , Família Multigênica , Streptomyces/genética , Venturicidinas/química , Venturicidinas/isolamento & purificação , Venturicidinas/metabolismo
3.
Sci Rep ; 10(1): 8134, 2020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32424122

RESUMO

Despite the remarkable advances due to the discovery and development of antimicrobials agents, infectious diseases remain the second leading cause of death worldwide. This fact underlines the importance of developing new therapeutic strategies to address the widespread antibiotic resistance, which is the major contributing factor for clinical failures of the current therapeutics. In a screen for antibiotic adjuvants, we identified a natural product from actinomycetes, venturicidin A (VentA), that potentiates the aminoglycoside antibiotic gentamicin against multidrug-resistant clinical isolates of Staphylococcus, Enterococcus, and Pseudomonas aeruginosa. Furthermore, the combination of gentamicin and VentA was bactericidal and rapidly eradicated methicillin-resistant S. aureus (MRSA). The molecular mechanism of gentamicin potentiation activity is attributed to uncoupling of ATP synthesis by VentA from electron transport presumably by blocking the proton flow through ATP synthase, which results in an elevated concentration of extracellular protons and subsequent anticipated raise in gentamicin uptake. The disruption of the proton flux was characterized by perturbed membrane potential in MRSA. These results demonstrate that inhibition of ATP synthase along with the subsequent membrane dysregulation, as shown here with VentA, complements aminoglycoside antibiotics against MDR bacteria, and that this approach may be employed to combat bacterial resistance.


Assuntos
Complexos de ATP Sintetase/antagonistas & inibidores , Actinobacteria/química , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Venturicidinas/farmacologia , Complexos de ATP Sintetase/genética , Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoglicosídeos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/enzimologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/genética
4.
Talanta ; 205: 120083, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31450430

RESUMO

In vitro synthesis of ATP catalyzed by the ATP-synthase requires membrane vesicles, in which the ATP-synthase is present within the bilayer membrane. Inverted vesicle prepared from Gram negative cells (e.g., Escherichia coli or Pseudomonas putida) can be readily obtained and used for in vitro ATP-synthesis. Up to now, quantification of ATP synthesized by membrane vesicles has been mostly analyzed via bioluminescence-based assays. Alternatively, vesicle respiration and the associated ATP level can be determined using biosensors, which not only provide high selectivity, but allow ATP measurements without the sample being illuminated. Here, we present a microbiosensor for ATP in combination with scanning electrochemical microscopy (SECM) using an innovative two-compartment electrochemical cell for the determination of ATP levels at E.coli or P. putida inverted vesicles. For a protein concentration of 22 mg/ml, a total amount of 0.29 ±â€¯0.03 µM/µl ATP per vesicle was determined in case of E.coli; in turn, P. putida derived vesicles yielded 0.48 ±â€¯0.02 µM/µl ATP per vesicle at a total protein concentration of 25.2 mg/ml. Inhibition experiments with Venturicidin A clearly revealed that the respiratory chain enzyme complex responsible for ATP generation is effectively involved.


Assuntos
Trifosfato de Adenosina/análise , Técnicas Biossensoriais/métodos , Escherichia coli/citologia , Membrana Celular/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Medições Luminescentes , Microscopia Eletroquímica de Varredura , Microscopia de Fluorescência , Pseudomonas putida/citologia , Pseudomonas putida/metabolismo , Venturicidinas/farmacologia
5.
Biochim Biophys Acta Bioenerg ; 1858(11): 939-944, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28803911

RESUMO

Fo·F1H+-ATPase/synthase in coupled plasma membrane vesicles of Paracoccus denitrificans catalyzes ATP hydrolysis and/or ATP synthesis with comparable enzyme turnover. Significant difference in pH-profile of these alternative activities is seen: decreasing pH from 8.0 to 7.0 results in reversible inhibition of hydrolytic activity, whereas ATP synthesis activity is not changed. The inhibition of ATPase activity upon acidification results from neither change in ADP(Mg2+)-induced deactivation nor the energy-dependent enzyme activation. Vmax, not apparent KmATP is affected by lowering the pH. Venturicidin noncompetitively inhibits ATP synthesis and coupled ATP hydrolysis, showing significant difference in the affinity to its inhibitory site depending on the direction of the catalysis. This difference cannot be attributed to variations of the substrate-enzyme intermediates for steady-state forward and back reactions or to possible equilibrium between ATP hydrolase and ATP synthase Fo·F1 modes of the opposite directions of catalysis. The data are interpreted as to suggest that distinct non-equilibrated molecular isoforms of Fo·F1 ATP synthase and ATP hydrolase exist in coupled energy-transducing membranes.


Assuntos
Difosfato de Adenosina/química , Trifosfato de Adenosina/química , Membrana Celular/química , Paracoccus denitrificans/enzimologia , Subunidades Proteicas/química , ATPases Translocadoras de Prótons/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Concentração de Íons de Hidrogênio , Transporte de Íons , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Paracoccus denitrificans/química , Subunidades Proteicas/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Venturicidinas/química
6.
Med Hypotheses ; 83(2): 160-5, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24932580

RESUMO

A primary goal in antimicrobial drug design is to find molecules which inhibit key proteins in bacteria without affecting mammalian homologues. To this aim, structural differences between eukaryotic and prokaryotic enzyme proteins involved in life processes are widely exploited. The membrane-bound enzyme complex ATP synthase synthesizes the energy currency molecule of the cell. Due to its bioenergetic role, it represents "the enzyme of life" of all living beings. The enzyme complex has the unique bi-functional property of exploiting either the electrochemical transmembrane gradient to make ATP or, conversely, the free energy of ATP hydrolysis to build an electrochemical gradient across the membrane. The catalytic mechanism of ATP synthesis/hydrolysis, based on the coupling between the two rotary sectors FO and F1 is shared by eukaryotes and prokaryotes. However slight structural differences distinguish prokaryotic ATP synthases, embedded in cell membrane, from eukaryotic ones localized in the mitochondrial inner membrane. In spite of its fundamental task in living organisms, up to now the ATP synthase has been poorly exploited as target in antibacterial therapy, mainly due to harmful effects on patients. Recent advances shoulder the use of drugs targeting the ATP synthase to fight mycobacteria and treat human tuberculosis. Macrolide antibiotics and other antimicrobial drugs specifically bind to the c-ring of the membrane-embedded FO domain, thus blocking ion translocation through FO which is essential for both ATP synthesis and ATP hydrolysis. Our findings show that, once bound to the ATP synthase, probably through different binding sites on a common binding region on FO, the macrolide antibiotics oligomycin, venturicidin and bafilomycin behave as enzyme inhibitors. Interestingly, the c subunits of mitochondrial ATP synthase contain conserved cysteine residues which are absent in bacteria. We pointed out that when these crucial cysteine thiols are oxidized, the common drug binding site of the enzyme is somehow destabilized, thus weakening the enzyme-drug interactions and making the ATP synthase insensitive to drug inhibition. On these bases we hypothesize that the selective oxidation of these cysteine thiols can be exploited to desensitize the mitochondrial ATP synthase to drugs which target FO and maintain their inhibitory potency on bacterial ATP synthases. According to our hypothesis, this strategy could represent an intriguing tool to prevent adverse effects of antimicrobial drugs in mammals, thus enhancing the number of natural and synthetic compounds which can be used in therapy. To this aim studies should be addressed to the identification and formulation of compounds and/or treatments able to selectively oxidize the crucial cysteine thiols of c-subunits without affecting the overall functionality of the mitochondrial ATP synthase and other thiol containing proteins.


Assuntos
Anti-Infecciosos/metabolismo , Desenho de Fármacos , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Modelos Biológicos , Compostos de Sulfidrila/metabolismo , Anti-Infecciosos/efeitos adversos , Inibidores Enzimáticos/metabolismo , Humanos , Oligomicinas , Oxirredução , Venturicidinas
7.
J Antibiot (Tokyo) ; 61(6): 372-8, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18667785

RESUMO

More than 400 compounds isolated from soil microorganisms, and catalogued in the antibiotic library of the Kitasato Institute for Life Sciences, were screened against African trypanosomes. Ten compounds were found to have selective and potent antitrypanosomal activity in vitro: aureothin, cellocidin, destomycin A, echinomycin, hedamycin, irumamycin, LL-Z 1272beta, O-methylnanaomycin A, venturicidin A and virustomycin A. Results of the in vitro assays using the GUTat 3.1 strain of Trypanosomal brucei brucei and the STIB900 strain of T. b. rhodesiense are presented. Cytotoxicity was determined using a human MRC-5 cell line. This is the first report of antitrypanosomal activities of the 10 microbial metabolites listed above.


Assuntos
Tripanossomicidas/isolamento & purificação , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/efeitos dos fármacos , Alcinos/química , Alcinos/isolamento & purificação , Alcinos/metabolismo , Alcinos/farmacologia , Animais , Antraquinonas/química , Antraquinonas/isolamento & purificação , Antraquinonas/metabolismo , Antraquinonas/farmacologia , Linhagem Celular , Cromonas/química , Cromonas/isolamento & purificação , Cromonas/metabolismo , Cromonas/farmacologia , Equinomicina/química , Equinomicina/isolamento & purificação , Equinomicina/metabolismo , Equinomicina/farmacologia , Higromicina B/análogos & derivados , Higromicina B/química , Higromicina B/isolamento & purificação , Higromicina B/metabolismo , Higromicina B/farmacologia , Macrolídeos/química , Macrolídeos/isolamento & purificação , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Naftoquinonas/química , Naftoquinonas/isolamento & purificação , Naftoquinonas/metabolismo , Naftoquinonas/farmacologia , Microbiologia do Solo , Tripanossomicidas/química , Tripanossomicidas/metabolismo , Venturicidinas/química , Venturicidinas/isolamento & purificação , Venturicidinas/metabolismo , Venturicidinas/farmacologia
8.
J Biol Chem ; 276(18): 15269-74, 2001 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-11279047

RESUMO

ATP synthase (F0F1) transforms an electrochemical proton gradient into chemical energy (ATP) through the rotation of a subunit assembly. It has been suggested that a complex of the gamma subunit and c ring (c(10-14)) of F0F1 could rotate together during ATP hydrolysis and synthesis (Sambongi, Y., Iko, Y., Tanabe, M., Omote, H., Iwamoto-Kihara, A., Ueda, I., Yanagida, T., Wada, Y., and Futai, M. (1999) Science 286, 1722-1724). We observed that the rotation of the c ring with the cI28T mutation (c subunit cIle-28 replaced by Thr) was less sensitive to venturicidin than that of the wild type, consistent with the antibiotic effect on the cI28T mutant and wild-type ATPase activities (Fillingame, R. H., Oldenburg, M., and Fraga, D. (1991) J. Biol. Chem. 266, 20934-20939). Furthermore, we engineered F0F1 to see the alpha(3)beta(3) hexamer rotation; a biotin tag was introduced into the alpha or beta subunit, and a His tag was introduced into the c subunit. The engineered enzymes could be purified by metal affinity chromatography and density gradient centrifugation. They were immobilized on a glass surface through the c subunit, and an actin filament was connected to the alpha or beta subunit. The filament rotated upon the addition of ATP and generated essentially the same frictional torque as one connected to the c ring. These results indicate that the gammaepsilonc(10-14) complex is a mechanical unit of the enzyme and that it can be used as a rotor or a stator experimentally, depending on the subunit immobilized.


Assuntos
Escherichia coli/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Dicicloexilcarbodi-Imida/farmacologia , Inibidores Enzimáticos/farmacologia , Hidrólise , Plasmídeos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/química , Venturicidinas/farmacologia
9.
FEBS Lett ; 470(3): 244-8, 2000 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-10745076

RESUMO

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.


Assuntos
Artefatos , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Rotação , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Biopolímeros/metabolismo , Cromatografia em Gel , Detergentes/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Hidrólise/efeitos dos fármacos , Cinética , Sondas Moleculares/metabolismo , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reprodutibilidade dos Testes , Solubilidade/efeitos dos fármacos , Desacopladores/farmacologia , Venturicidinas/farmacologia
10.
Science ; 286(5445): 1722-4, 1999 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-10576736

RESUMO

F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Motores Moleculares/química , Proteínas Motores Moleculares/metabolismo , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Actinas/química , Actinas/metabolismo , Sítios de Ligação , Biotinilação , Transferência de Energia , Enzimas Imobilizadas , Escherichia coli/enzimologia , Hidrólise , Força Próton-Motriz , Desacopladores/metabolismo , Desacopladores/farmacologia , Venturicidinas/farmacologia , Gravação em Vídeo
11.
Int J Biochem Cell Biol ; 31(2): 319-30, 1999 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-10216964

RESUMO

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).


Assuntos
Mitocôndrias/enzimologia , Nucleotídeos/metabolismo , Placenta/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , Eletroforese/métodos , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Hidrólise , Técnicas In Vitro , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Nucleotídeos/farmacologia , Oligomicinas/farmacologia , Concentração Osmolar , Consumo de Oxigênio , Gravidez , ATPases Translocadoras de Prótons/antagonistas & inibidores , Fatores de Tempo , Tripsina/metabolismo , Tripsina/farmacologia , Vanadatos/farmacologia , Venturicidinas/farmacologia
12.
FEBS Lett ; 368(2): 253-6, 1995 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-7628616

RESUMO

The effect of tentoxin at high concentrations was investigated in thylakoids and proteoliposomes containing bacteriorhodopsin and CF0CF1. Venturicidin-sensitive ATP hydrolysis, ATP-generated delta pH and ATP synthesis were practically 100% inhibited at 2 microM tentoxin, and restored to various extents beyond 50 microM. With respect to the native enzyme, tentoxin-reactivated ATPase had the following properties: (i) a higher delta pH requirement to synthetise ATP; (ii) a decreased futile proton flow through CF0CF1 (without ADP), which remains 100% blocked by ADP. It is concluded that despite its altered kinetic performances, tentoxin-modified CF0CF1 preserves its mechanism and remains a tightly coupled proton pump.


Assuntos
Cloroplastos/enzimologia , Peptídeos Cíclicos/farmacologia , Bombas de Próton/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Bacteriorodopsinas/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Ativação Enzimática , Hidrólise/efeitos dos fármacos , Lipossomos/metabolismo , Proteolipídeos/efeitos dos fármacos , Proteolipídeos/metabolismo , Venturicidinas/farmacologia
13.
FEBS Lett ; 358(2): 142-4, 1995 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-7828724

RESUMO

ATP synthase (F-ATPase) of chloroplasts, CF0CF1, is both activated and driven by transmembrane protonmotive force. We dichotomized between activating and driving proton transfer by specific inhibitors, tentoxin and venturicidin. Thylakoids membranes were submitted to voltage steps (by flashing light) superimposed to a steady pH-difference. Transient proton intake, transfer and release by CF0CF1 was monitored by spectroscopic probes. Both activities, activation and catalysis, required all three partial reactions of the proton, however, activating proton transfer rose first (monophasically, tau 1/2 approximately 15 ms) followed by another phase of equal magnitude with a time lag of about 15 ms. Both types of consecutive proton transfer reactions contribute free energy for ATP synthesis.


Assuntos
ATPases Translocadoras de Prótons/metabolismo , Catálise , Cloroplastos/enzimologia , Ativação Enzimática , Peptídeos Cíclicos/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Prótons , Venturicidinas/farmacologia
14.
Biochem Biophys Res Commun ; 203(2): 1005-12, 1994 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-8093018

RESUMO

Purified F(o), isolated from bovine heart mitochondria was reconstituted into planar lipid bilayers. Two cation selective conductances could be identified. Most frequently, incorporation of F(o) resulted in a voltage sensitive K+ channel of 18 pS. Venturicidin drastically decreased the open probability of this channel. The second conductance of 47 pS had lost its voltage sensitivity but had retained the sensitivity to venturicidin. The F(o) preparation is very pure and will be used in crystallography studies. However, despite the high degree of biochemical purity, the preparation gives rise to several distinct conductances. Therefore, we conclude that for structure/function analysis of the F(o) part a biophysical characterization is required to assess the homogeneity of the preparation.


Assuntos
Bicamadas Lipídicas/metabolismo , Mitocôndrias Cardíacas/enzimologia , Potássio/farmacologia , ATPases Translocadoras de Prótons/metabolismo , Venturicidinas/farmacologia , Animais , Cátions , Bovinos , Cloretos/metabolismo , Condutividade Elétrica , Potássio/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/isolamento & purificação
16.
J Biol Chem ; 269(28): 18463-7, 1994 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8034593

RESUMO

Plastocyanin and the 33-kDa subunit of the oxygen-evolving complex (OE33) are two of several thylakoid lumen-located proteins that are made in the cytosol, imported into chloroplasts, and subsequently transported into thylakoids. Recently, competition studies showed that there are two pathways for protein transport into the thylakoid lumen and that plastocyanin and OE33 are on the same pathway (Cline, K., Henry, R., Li, C., and Yuan, J. (1993) EMBO J. 12, 4105-4114). Our expectation is that transport requirements reflect the steps of the process and that proteins on the same pathway share similar requirements. Unfortunately, the transport requirements for plastocyanin and OE33 are not well established. Here, we investigated transport in a reconstituted system with isolated thylakoids. Efficient transport of OE33 and plastocyanin was only obtained when stromal extract was included in the assay. Heat or protease treatment of stromal extract eliminated its ability to stimulate transport. Transport was abolished by treatments designed to deplete ATP or to prevent its formation and was greatly reduced in the presence of ionophores that dissipate the trans-thylakoidal proton gradient. These results show that transport of OE33 and plastocyanin requires ATP and is stimulated by stromal protein(s) and the trans-thylakoidal proton gradient. Taken together, these and previous results suggest that there are two mechanistically distinct pathways for protein transport into the thylakoid lumen.


Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Organelas/metabolismo , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Complexo de Proteína do Fotossistema II , Plastocianina/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte Biológico , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Ionóforos/farmacologia , Cinética , Substâncias Macromoleculares , Peso Molecular , ATPases Translocadoras de Prótons/antagonistas & inibidores , Venturicidinas/farmacologia
17.
Eur J Biochem ; 221(3): 1071-8, 1994 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-8181464

RESUMO

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (delta mu H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri-n-butyl tin and aurovertin), used at non-saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8. The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate-limiting step for ATPase deactivation.


Assuntos
Aurovertinas/farmacologia , Mitocôndrias/enzimologia , Plantas/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Compostos de Trialquitina/farmacologia , Venturicidinas/farmacologia , Trifosfato de Adenosina/metabolismo , Sítios de Ligação , Eletroquímica , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise , Oxirredução , ATPases Translocadoras de Prótons/antagonistas & inibidores
18.
FEBS Lett ; 338(2): 152-6, 1994 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-8307173

RESUMO

The ATP synthase from chloroplasts, CFo.F1, was reconstituted into liposomes, from which most of CF1 was removed by a short treatment with guanidinium chloride. ATP-dependent proton uptake was restored with these CFo-liposomes even better by the addition of the bacterial TF1-than of the related CF1-part. This proton uptake was prevented by tentoxin, a specific inhibitor of the CF1-ATPase, in these CFo.F1-liposomes, but not in the hybrid CFo.TF1-liposomes. Venturicidin, a specific inhibitor of proton flow through CFo, was able to block it in both the hybrid CFo.TF1-liposomes and reconstituted CFo.F1-liposomes. These results indicate that the bacterial TF1-part binds to the eukaryotic CFo-part of four subunits forming a functional CFo.TF1-ATPase.


Assuntos
Bactérias/enzimologia , Cloroplastos/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Lipossomos/metabolismo , Peptídeos Cíclicos/farmacologia , Multimerização Proteica , ATPases Translocadoras de Prótons/antagonistas & inibidores , Prótons , Venturicidinas/farmacologia
19.
FEBS Lett ; 336(1): 83-6, 1993 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-8262223

RESUMO

The effect of some F0F1 inhibitors on the activation of the H(+)-ATPase by the electrochemical proton gradient was investigated in mitochondria extracted from potato tubers. Transient activated state of the ATPase was revealed by addition of ATP and of the detergent lauryldimethylamine oxide (LDAO) to energized mitochondria. Venturicidin, tri-n-butyltin and aurovertin at high concentrations did not affect the process of delta mu H(+)-activation, whereas oligomycin fully blocked it. The results support the idea of separate pathways or binding sites for catalytic and activating protons.


Assuntos
Mitocôndrias/enzimologia , ATPases Translocadoras de Prótons/metabolismo , Solanum tuberosum/enzimologia , Aurovertinas/antagonistas & inibidores , Aurovertinas/farmacologia , Catálise , Detergentes/farmacologia , Dimetilaminas/farmacologia , Ativação Enzimática/efeitos dos fármacos , Oligomicinas/antagonistas & inibidores , Oligomicinas/farmacologia , ATPases Translocadoras de Prótons/antagonistas & inibidores , Prótons , Compostos de Trialquitina/antagonistas & inibidores , Compostos de Trialquitina/farmacologia , Venturicidinas/antagonistas & inibidores , Venturicidinas/farmacologia
20.
J Biol Chem ; 268(20): 14776-80, 1993 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-8325855

RESUMO

After incorporation of the purified Na(+)-translocating F0F1-ATPase from Propionigenium modestum into preformed phospholipid vesicles the synthesis of ATP from ADP and inorganic phosphate could be observed under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and/or a Na+ concentration gradient (delta pNa) were imposed. This reaction was not inhibited by the protonophore carbonyl cyanide p-tri-fluoromethoxyphenylhydrazone (FCCP). Furthermore, the delta pNa-driven ATP synthesis was stimulated by FCCP. In contrast, the addition of the Na+/H+ antiporter monensin or of the F0F1 inhibitors N,N'-dicyclohexylcarbodiimide and venturicidin abolished the synthesis of ATP completely. Finally, delta pNa alone was able to elicit ATP synthesis, when a Na+ concentration gradient of sufficient magnitude was applied. In this case ATP synthesis occurred above a threshold level of approximately 120 mV and, furthermore, delta psi and delta pNa appear to be equivalent as driving forces for this process. Therefore, the data provide firm evidence for the concept that delta"mu Na+ is the primary driving force for the synthesis of ATP in P. modestum.


Assuntos
Trifosfato de Adenosina/biossíntese , Bactérias Anaeróbias/enzimologia , Proteolipídeos/metabolismo , ATPases Translocadoras de Prótons/metabolismo , Trifosfato de Adenosina/antagonistas & inibidores , Transporte Biológico , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Dicicloexilcarbodi-Imida/farmacologia , Eletrofisiologia , Lipossomos , Monensin/farmacologia , Fosforilação , Potássio/metabolismo , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/isolamento & purificação , Sódio/metabolismo , Valinomicina/farmacologia , Venturicidinas/farmacologia
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