Using F0F1-ATPase motors as micro-mixers accelerates thrombolysis

We have developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer was constructed from arrays of chromatophore-embedded δ-free F₀F₁-ATPases, where the δ-free F₁ part acted as a rotator to mix solutions, and the F₀ part was driven by light. Confocal microscope studies indicated that the micro-mixer did not touch directly on the fibrin labeled with FITC. The nanomechanical force generated by the motor induced drug movement in the solution and accelerated the fibrinolysis process. All results strongly suggest that the micro-mixers generated a nanomechanical force which accelerated the fibrinolysis process in the presence of lower concentrations of lumbrokinase.     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Mechanically driven proton conduction in single delta-free F0F1-ATPase

In order to observe mechanically driven proton flux in F(0)F(1)-ATPase coupled with artificial driven rotation on F(1) simultaneously, a double channel observation system was established. An artificial delta-free F(0)F(1)-ATPase was constructed with alpha(3), beta(3), epsilon, gamma, and c(n) subunits as rotator and a, b(2) as stator. The chromatophore was immobilized on the glass surface through biotin-streptavidin-biotin system, and the magnetic bead was attached to the beta subunit of delta-free F(0)F(1)-ATPase. The mechanically driven proton flux was indicated by the fluorescence intensity change of fluorescein reference standard (F1300) and recorded by a cooled digital CCD camera. The mechanochemical coupling stoichiometry between F(0) and F(1) is about 4.15 +/- 0.2H(+)/rev when the magnetic field rotated at 0.33 Hz (rps).     

F0F1-ATPase as biosensor to detect single virus

F(0)F(1)-ATPase within chromatophore was constructed as a biosensor (immuno-rotary biosensor) for the purpose of capturing single virus. Capture of virus was based on antibody-antigen reaction. The detection of virus based on proton flux change driven by ATP-synthesis of F(0)F(1)-ATPase, which was indicated by F1300, was directly observed by a fluorescence microscope. The results demonstrate that the biosensor loading of virus particles has remarkable signal-to-noise ratio (3.8:1) compared to its control at single molecular level, and will be convenient, quick, and even super-sensitive for detecting virus particles.     

Caenorhabditis elegans MAI-1 protein, which is similar to mitochondrial ATPase inhibitor (IF1), can inhibit yeast F0F1-ATPase but cannot be transported to yeast mitochondria

In Caenorhabditis elegans, two proteins that are similar to mitochondrial ATPase inhibitor protein (IF₁) have been found and named MAI-1 and MAI-2. In this study, we overexpressed and purified both the proteins and examined their properties. Circular dichroism spectra indicated that both the MAI-1 and MAI-2 predominantly consisted of β- and random structure, and in contrast to mammalian IF₁, α-helixes were barely detected. Both MAI-1 and MAI-2 could inhibit yeast F₀F₁-ATPase, but the inhibition by MAI-1 was pH-independent. MAI-2-GFP fusion protein was transported to yeast mitochondria, but MAI-1-GFP was not. These results indicate that the MAI-2 is _{C. elegans} IF₁. MAI-1 seems to be a cytosolic protein and may regulate cytosolic ATPase(s).     

Activation and Deactivation of F0F1-ATPase in Yeast Mitochrondia

The regulation of membrane-bound proton F₀F₁ATPase by the protonmotive force and nucleotides was studied in yeastmitochondria. Activation occurred in whole mitochondria and the ATPaseactivity was measured just after disrupting the membranes with Triton X-100.Deactivation occurred either in whole mitochondria uncoupled with FCCP, or indisrupted membranes. No effect of Triton X-100 on the ATPase was observed,except a slow reactivation observed only in the absence of MgADP. BothAMPPNP and ATP increased the ATPase deactivation rate, thus indicating thatoccupancy of nucleotidic sites by ATP is more decisive than catalyticturnover for this process. ADP was found to stimulate the energy-dependentATPase activation. ATPase deactivated at the same rate in uncoupled anddisrupted mitochondria. This suggests that deactivation is not controlled byrebinding of some soluble factor, like IF1, but rather by the conversion ofthe F₁.IF1 complex into an inactive form.     

Formate increases the F0F1-ATPase activity in Escherichia coli growing on glucose under anaerobic conditions at slightly alkaline pH

Escherichia coli growing on glucose under anaerobic conditions at slightly alkaline pH carries out a mixed-acid fermentation resulting in the production of formate among the other products that can be excreted or further oxidized to H(2) and CO(2). H(2) production is largely dependent on formate dehydrogenase H and hydrogenases 3 and 4 constituting two formate hydrogen lyases, and on the F(0)F(1)-ATPase. In this study, it has been shown that formate markedly increased ATPase activity in membrane vesicles. This activity was significantly (1.8-fold) stimulated by 100mM K(+) and inhibited by N,N(')-dicyclohexylcarbodiimide and sodium azide. The increase in ATPase activity was absent in atp, trkA, and hyf but not in hyc mutants. ATPase activity was also markedly increased by formate when bacteria were fermenting glucose with external formate (30mM) in the growth medium. However this activity was not stimulated by K(+) and absent in atp and hyc but not in hyf mutants. The effects of formate on ATPase activity disappeared when cells were performing anaerobic (nitrate/nitrite) or aerobic respiration. These results suggest that the F(0)F(1)-ATPase activity is dependent on K(+) uptake TrkA system and hydrogenase 4, and on hydrogenase 3 when cells are fermenting glucose in the absence and presence of external formate, respectively.     

The Inhibitor Protein (IF1) of the F1F0-ATPase Modulates Human Osteosarcoma Cell Bioenergetics

The bioenergetics of IF₁ transiently silenced cancer cells has been extensively investigated, but the role of IF₁ (the natural inhibitor protein of F₁F₀-ATPase) in cancer cell metabolism is still uncertain. To shed light on this issue, we established a method to prepare stably IF₁-silenced human osteosarcoma clones and explored the bioenergetics of IF₁ null cancer cells. We showed that IF₁-silenced cells proliferate normally, consume glucose, and release lactate as controls do, and contain a normal steady-state ATP level. However, IF₁-silenced cells displayed an enhanced steady-state mitochondrial membrane potential and consistently showed a reduced ADP-stimulated respiration rate. In the parental cells (i.e. control cells containing IF₁) the inhibitor protein was found to be associated with the dimeric form of the ATP synthase complex, therefore we propose that the interaction of IF₁ with the complex either directly, by increasing the catalytic activity of the enzyme, or indirectly, by improving the structure of mitochondrial cristae, can increase the oxidative phosphorylation rate in osteosarcoma cells grown under normoxic conditions.     

Modulation at a Distance of Proton Conductance through the Saccharomyces cerevisiae Mitochondrial F1F0-ATP Synthase by Variants of the Oligomycin Sensitivity-Conferring Protein Containing Substitutions near the C-Terminus

We have sought to elucidate how the oligomycin sensitivity-conferring protein (OSCP) of the mitochondrial F₁F₀-ATP synthase (mtATPase) can influence proton channel function. Variants of OSCP, from the yeast Saccharomyces cerevisiae, having amino acid substitutions at a strictly conserved residue (Gly166) were expressed in place of normal OSCP. Cells expressing the OSCP variants were able to grow on nonfermentable substrates, albeit with some increase in generation time. Moreover, these strains exhibited increased sensitivity to oligomycin, suggestive of modification in functional interactions between the F₁ and F₀ sectors mediated by OSCP. Bioenergetic analysis of mitochondria from cells expressing OSCP variants indicated an increased respiratory rate under conditions of no net ATP synthesis. Using specific inhibitors of mtATPase, in conjunction with measurement of changes in mitochondrial transmembrane potential, it was revealed that this increased respiratory rate was a result of increased proton flux through the F₀ sector. This proton conductance, which is not coupled to phosphorylation, is exquisitely sensitive to inhibition by oligomycin. Nevertheless, the oxidative phosphorylation capacity of these mitochondria from cells expressing OSCP variants was no different to that of the control. These results suggest that the incorporation of OSCP variants into functional ATP synthase complexes can display effects in the control of proton flux through the F₀ sector, most likely mediated through altered protein—protein contacts within the enzyme complex. This conclusion is supported by data indicating impaired stability of solubilized mtATPase complexes that is not, however, reflected in the assembly of functional enzyme complexes in vivo. Given a location for OSCP atop the F₁-₃₃ hexamer that is distant from the proton channel, then the modulation of proton flux by OSCP must occur at a distance. We consider how subtle conformational changes in OSCP may be transmitted to F₀.     

Atrazine binds to F1F0-ATP synthase and inhibits mitochondrial function in sperm

Atrazine is a widely used triazine herbicide. Although controversy still exists, a number of recent studies have described its adverse effects on various animals including humans. Of particular interest is its effects on reproductive capacity. In this study, we investigated the mechanisms underlying the adverse effects of atrazine, with a focus on its effects on sperm. Here we show evidence that mitochondrial F₁F₀-ATP synthase is a molecular target of atrazine. A series of experiments with sperm and isolated mitochondria suggest that atrazine inhibits mitochondrial function through F₁F₀-ATP synthase. Moreover, affinity purification using atrazine as a ligand demonstrates that F₁F₀-ATP synthase is a major atrazine-binding protein in cells. The inhibitory activity against mitochondria and F₁F₀-ATP synthase is not limited to atrazine but is likely to be applicable to other triazine-based compounds. Thus, our findings may have wide relevance to pharmacology and toxicology.     

Relevance of Divalent Cations to ATP-Driven Proton Pumping in Beef Heart Mitochondrial F0F1-ATPase

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F₀F₁-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K _m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K _m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F₀F₁-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.     

Probing conformations of the beta subunit of F0F1-ATP synthase in catalysis

A subcomplex of F0F1-ATP synthase (F0F1), alpha3beta3gamma, was shown to undergo the conformation(s) during ATP hydrolysis in which two of the three beta subunits have the "Closed" conformation simultaneously (CC conformation) [S.P. Tsunoda, E. Muneyuki, T. Amano, M. Yoshida, H. Noji, Cross-linking of two beta subunits in the closed conformation in F1-ATPase, J. Biol. Chem. 274 (1999) 5701-5706]. This was examined by the inter-subunit disulfide cross-linking between two mutant beta(I386C)s that was formed readily only when the enzyme was in the CC conformation. Here, we adopted the same method for the holoenzyme F0F1 from Bacillus PS3 and found that the CC conformation was generated during ATP hydrolysis but barely during ATP synthesis. The experiments using F0F1 with the epsilon subunit lacking C-terminal helices further suggest that this difference is related to dynamic nature of the epsilon subunit and that ATP synthesis is accelerated when it takes the pathway involving the CC conformation.     

Cloning, sequence analysis and expression of the F1F0-ATPase beta-subunit from wine lactic acid bacteria

The nucleotide sequences of the genes encoding the F1F0-ATPase beta-subunit from Oenococcus oeni, Leuconostoc mesenteroides subsp. mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Lactobacillus brevis and Lactobacillus hilgardii were determined. Their deduced amino acid sequences showed homology values of 79-98%. Data from the alignment and ATPase tree indicated that O. oeni and L. mesenteroides subsp. mesenteroides formed a group well-separated from P. damnosus and P. parvulus and from the group comprises L. brevis and L. hilgardii. The N-terminus of the F1F0-ATPase beta-subunit of O. oeni contains a stretch of additional 38 amino acid residues. The catalytic site of the ATPase beta-subunit of the investigated strains is characterized by the two conserved motifs GGAGVGKT and GERTRE. The amplified atpD coding sequences were inserted into the pCRT7/CT-TOPO vector using TA-cloning strategy and transformed in Escherichia coli. SDS-PAGE and Western blot analyses confirmed that O. oeni has an ATPase beta-subunit protein which is larger in size than the corresponding molecules from the investigated strains.     

Interaction of beef-heart mitochondrial F1-ATPase with immobilized ATP in the presence of dimethylsulfoxide

Dimethylsulfoxide [Me₂SO, 30% (v/v)] promotes the formation of ATP from ADP and phosphate catalyzed by soluble mitochondrial F₁-ATPase. The effects of this solvent on the interaction of beef-heart mitochondrial F₁ with the immobilized ATP of Agarose-hexane-ATP were studied. In the presence of Me₂SO, F₁ bound less readily to the immobilized ATP, but once bound was more difficult to elute with exogenous ATP. This suggests that not only was the binding affinity for adenine nucleotide at the first binding site affected but that adenine nucleotide binding affinity at the second and/or third sites, which interact cooperatively with the first site to release bound nucleotide, was also affected. A reduction in the binding of [³H]ADP to these sites was shown. A change in the conformation of F₁ in 30% (v/v) Me₂SO was demonstrated by crosslinking and by the increased resistance of the enzyme to cold denaturation.     

Hypothyroidism Leads to a Decreased Expression of Mitochondrial F0F1-ATP Synthase in Rat Liver

In liver mitochondria isolated from hypothyroid rats, the rate of ATP synthesis is lower than in mitochondria from normal rats. Oligomycin-sensitive ATP hydrolase activity and passive proton permeability were significantly lower in submitochondrial particles from hypothyroid rats compared to those isolated from normal rats. In mitochondria from hypothyroid rats, the changes in catalytic activities of F₀F₁-ATP synthase are accompanied by a decrease in the amount of immunodetected -F₁, F₀1-PVP, and OSCP subunits of the complex. Northern blot hybridization shows a decrease in the relative cytosolic content of mRNA for -F₁ subunit in liver of hypothyroid rats. Administration of 3,5,3-triodo-L-thyronine to the hypothyroid rats tends to remedy the functional and structural defects of F₀F₁-ATP synthase observed in the hypothyroid rats. The results obtained indicate that hypothyroidism leads to a decreased expression of F₀F₁-ATP synthase complex in liver mitochondria and this contributes to the decrease of the efficiency of oxidative phosphorylation.     

The nucleotide-independent Fe(III)-binding site is located on beta subunit of the mitochondrial F(1)-ATPase

The need for methods to identify disease biomarkers is underscored by the survival-rate of patients diagnosed at early stages of cancer progression. Surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) is a novel approach to biomarker discovery that combines two powerful techniques: chromatography and mass spectrometry. One of the key features of SELDI-TOF MS is its ability to provide a rapid protein expression profile from a variety of biological and clinical samples. It has been used for biomarker identification as well as the study of protein-protein, and protein-DNA interaction. The versatility of SELDI-TOF MS has allowed its use in projects ranging from the identification of potential diagnostic markers for prostate, bladder, breast, and ovarian cancers and Alzheimer's disease, to the study of biomolecular interactions and the characterization of posttranslational modifications. In this minireview we discuss the application of SELDI-TOF MS to protein biomarker discovery and profiling.     

The coupling of the relative movement of thea andc subunits of the F0 to the conformational changes in the F1-ATPase

F₀F₁-ATPase structural information gained from X-ray crystallography and electron microscopy has activated interest in a rotational mechanism for the F₀F₁-ATPase. Because of the subunit stoichiometry and the involvement of both thea- andc-subunits in the mechanism of proton movement, it is argued that relative movement must occur between the subunits. Various options for the arrangement and structure of the subunits involved are discussed and a mechanism proposed.     

Temperature-sensitive reaction intermediate of F1-ATPase

F(1)-ATPase is a rotary molecular motor that makes 120 degrees stepping rotations, with each step being driven by a single-ATP hydrolysis. In this study, a new reaction intermediate of F(1)-ATPase was discovered at a temperature below 4 degrees C, which makes a pause at the same angle in its rotation as when ATP binds. The rate constant of the intermediate reaction was strongly dependent on temperature with a Q(10) factor of 19, implying that the intermediate reaction accompanies a large conformational change. Kinetic analyses showed that the intermediate state does not correspond to ATP binding or hydrolysis. The addition of ADP to the reaction mixture did not alter the angular position of the intermediate state, but specifically lengthened the time constant of this state. Conversely, the addition of inorganic phosphate caused a pause at an angle of +80 degrees from that of the intermediate state. These observations strongly suggest that the newly found reaction intermediate is an ADP-releasing step.     

Inhibition of mitochondrial ATPase by dicarbopolyborate,a new enzyme inhibitor

Polyborate anions were found to inhibit mitochondrial ATPase. Mercapto and chloro derivatives of dicarbononaborates showed full inhibition of the enzyme activity at 0.5–0.8 mM. The inhibitory effect of dodecaborates was lower. The inhibition was of competitive type with respect to ATP. The inhibition of soluble F₁-ATPase indicates a direct interaction of the polyborate anion with the catalytic part of the enzyme molecule.