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1.
In order to get insight into the origin of apparent negative cooperativity observed for F1-ATPase, we compared ATPase activity and ATPMg binding of mutant subcomplexes of thermophilic F1-ATPase, α(W463F)3β(Y341W)3γ and α(K175A/T176A/W463F)3β(Y341W)3γ. For α(W463F)3β(Y341W)3γ, apparent Km's of ATPase kinetics (4.0 and 233 μM) did not agree with apparent Km's deduced from fluorescence quenching of the introduced tryptophan residue (on the order of nM, 0.016 and 13 μM). On the other hand, in case of α(K175A/T176A/W463F)3β(Y341W)3γ, which lacks noncatalytic nucleotide binding sites, the apparent Km of ATPase activity (10 μM) roughly agreed with the highest Km of fluorescence measurements (27 μM). The results indicate that in case of α(W463F)3β(Y341W)3γ, the activating effect of ATP binding to noncatalytic sites dominates overall ATPase kinetics and the highest apparent Km of ATPase activity does not represent the ATP binding to a catalytic site. In case of α(K175A/T176A/W463F)3β(Y341W)3γ, the Km of ATPase activity reflects the ATP binding to a catalytic site due to the lack of noncatalytic sites. The Eadie-Hofstee plot of ATPase reaction by α(K175A/T176A/W463F)3β(Y341W)3γ was rather linear compared with that of α(W463F)3β(Y341W)3γ, if not perfectly straight, indicating that the apparent negative cooperativity observed for wild-type F1-ATPase is due to the ATP binding to catalytic sites and noncatalytic sites. Thus, the frequently observed Km's of 100-300 μM and 1-30 μM range for wild-type F1-ATPase correspond to ATP binding to a noncatalytic site and catalytic site, respectively.  相似文献   

2.
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.  相似文献   

3.
Xiao-Min Gong  Tal Lev  Chanoch Carmeli 《BBA》2009,1787(2):97-104
Photosystem I (PS I) mediates light-induced electron transfer from P700 through a chlorophyll a, a quinone and a [4Fe-4S] iron-sulfur cluster FX, located on the core subunits PsaA/B to iron-sulfur clusters FA/B on subunit PsaC. Structure function relations in the native and in the mutant (psaB-C565S/D566E) of the cysteine ligand of FX cluster were studied by X-ray absorption spectroscopy (EXAFS) and transient spectroscopy. The structure of FX was determined in PS I lacking clusters FA/B by interruption of the psaC2 gene of PS I in the cyanobacterium Synechocystis sp PCC 6803. PsaC-deficient mutant cells assembled the core subunits of PS I which mediated electron transfer mostly to the phylloquinone. EXAFS analysis of the iron resolved a [4Fe-4S] cluster in the native PsaC-deficient PS I. Each iron had 4 sulfur and 3 iron atoms in the first and second shells with average Fe-S and Fe-Fe distances of 2.27 Å and 2.69 Å, respectively. In the C565S/D566E serine mutant, one of the irons of the cluster was ligated to three oxygen atoms with Fe-O distance of 1.81 Å. The possibility that the structural changes induced an increase in the reorganization energy that consequently decreased the rate of electron transfer from the phylloquinone to FX is discussed.  相似文献   

4.
Efrapeptins (EF), a family of fungal peptides, inhibit proteasomal enzymatic activities and the in vitro and in vivo growth of HT-29 cells. They are also known inhibitors of F1F0-ATPase, a mitochondrial enzyme that functions as an Hsp90 co-chaperone. We have previously shown that treatment of cancer cells with EF results in disruption of the Hsp90:F1F0-ATPase complex and inhibition of Hsp90 chaperone activity. The present study examines the effect of EF on breast cancer growth in vitro and in vivo. As a monotherapy, EF inhibited cell proliferation in vitro with an IC50 value ranging from 6 nM to 3.4 μM. Inhibition of Hsp90 chaperone function appeared to be the dominant mechanism of action and the factor determining cellular sensitivity to EF. In vitro inhibition of proteasome became prominent in the absence of adequate levels of Hsp90 and F1F0-ATPase as in the case of the relatively EF-resistant MDA-MB-231 cell line. In vivo, EF inhibited MCF-7 and MDA-MB-231 xenograft growth with a maximal inhibition of 60% after administration of 0.15 and 0.3 mg/kg EF, respectively. 2-Deoxyglucose (2DG), a known inhibitor of glycolysis, acted synergistically with EF in vitro and antagonistically in vivo. In vitro, the synergistic effect was attributed to a prolonged endoplasmic reticulum (ER) stress. In vivo, the antagonistic effect was ascribed to the downregulation of tumoral and/or stromal F1F0-ATPase by 2DG.  相似文献   

5.
Mitochondria are central to heart function and dysfunction, and the pathways activated by different cardioprotective interventions mostly converge on mitochondria. In a context of perspectives in innate and acquired cardioprotection, we review some recent advances in F0F1ATPsynthase structure/function and regulation in cardiac cells. We focus on three topics regarding the mitochondrial F0F1ATPsynthase and the plasma membrane enzyme, i.e.: i) the crucial role of cardiac mitochondrial F0F1ATPsynthase regulation by the inhibitory protein IF1 in heart preconditioning strategies; ii) the structure and function of mitochondrial F0F1ATPsynthase oligomers in mammalian myocardium as possible endogenous factors of mitochondria resistance to ischemic insult; iii) the external location and characterization of plasma membrane F0F1 ATP synthase in search for possible actors of its regulation, such as IF1 and calmodulin, at cell surface.  相似文献   

6.
The role of αPhe-291 residue in phosphate binding by Escherichia coli F1F0-ATP synthase was examined. X-ray structures of bovine mitochondrial enzyme suggest that this residue resides in close proximity to the conserved βR246 residue. Herein, we show that mutations αF291D and αF291E in E. coli reduce the ATPase activity of F1F0 membranes by 350-fold. Yet, significant oxidative phosphorylation activity is retained. In contrast to wild-type, ATPase activities of mutants were not inhibited by MgADP-azide, MgADP-fluoroaluminate, or MgADP-fluoroscandium. Whereas, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) inhibited wild-type ATPase essentially completely, ATPase in mutants was inhibited maximally by ∼75%, although reaction still occurred at residue βTyr-297, proximal to αPhe-291 in the phosphate-binding pocket. Inhibition characteristics supported the conclusion that NBD-Cl reacts in βE (empty) catalytic sites, as shown previously by X-ray structure analysis. Phosphate protected against NBD-Cl inhibition in wild-type but not in mutants. In addition, our data suggest that the interaction of αPhe-291 with phosphate during ATP hydrolysis or synthesis may be distinct.  相似文献   

7.
F1-ATPase is an ATP-driven motor in which γε rotates in the α3β3-cylinder. It is attenuated by MgADP inhibition and by the ε subunit in an inhibitory form. The non-inhibitory form of ε subunit of thermophilic Bacillus PS3 F1-ATPase is stabilized by ATP-binding with micromolar Kd at 25 °C. Here, we show that at [ATP] > 2 μM, ε does not affect rotation of PS3 F1-ATPase but, at 200 nM ATP, ε prolongs the pause of rotation caused by MgADP inhibition while the frequency of the pause is unchanged. It appears that ε undergoes reversible transition to the inhibitory form at [ATP] below Kd.  相似文献   

8.
The effect of nanomolar concentrations of PBR/TSPO ligands—Ro 5-4864, PK11195, and PPIX—on Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria was investigated. PBR/TSPO agonist Ro 5-4864 (100 nM) and endogenous ligand PPIX (1 μM) were shown to stimulate PTP opening, while antagonist PK11195 (100 nM) suppressed this process. Correlation between PBR ligand action on PTP opening and phosphorylation of a 3.5 kDa polypeptide was investigated. In intact brain mitochondria, incorporation of [γ-32P]ATP into 3.5 kDa peptide was decreased in the presence of Ro 5-4864 and PPIX and increased in the presence of PK11195. At threshold Ca2+ concentrations leading to PTP opening, PBR/TSPO ligands were found to stimulate dephosphorylation of the 3.5 kDa peptide. Specific anti-PBR/TSPO antibody prevented both PTP opening and dephosphorylation of the 3.5-kDa peptide. The peptide was identified as subunit c of FoF1-ATPase by Western blot using specific anti-subunit c antibody. The results suggest that subunit c of FoF1-ATPase could be an additional target for PBR/TSPO ligands action, is subjected to Ca2+- and TSPO-dependent phosphorylation/dephosphorylation, and is involved in PTP operation in mitochondria.  相似文献   

9.
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.  相似文献   

10.
The insertion of inner membrane proteins in Escherichia coli occurs almost exclusively via the SecYEG pathway, while some membrane proteins require the membrane protein insertase YidC. In vitro analysis demonstrates that subunit a of the F1F0 ATP synthase (F0a) is strictly dependent on Ffh, SecYEG and YidC for its membrane insertion but independent of the proton motive force. The insertion of the first transmembrane segment of F0a also depends on Ffh and SecYEG but not on YidC, whereas the insertion is strongly dependent on the proton motive force, unlike the full-length F0a protein. These data demonstrate an extensive role of YidC in the assembly of the F0 sector of the F1F0 ATP synthase.  相似文献   

11.
The ATP synthase consists of two opposing rotary motors, F0 and F1, coupled to each other. When the F1 motor is not coupled to the F0 motor, it can work in the direction hydrolyzing ATP, as a nanomotor called F1-ATPase. It has been reported that the stiffness of the protein varies nonlinearly with increasing load. The nonlinearity has an important effect on the rotating rate of the F1-ATPase. Here, considering the nonlinearity of the γ shaft stiffness for the F1-ATPase, a nonlinear chemo-mechanical coupled dynamic model of F1 motor is proposed. Nonlinear vibration frequencies of the γ shaft and their changes along with the system parameters are investigated. The nonlinear stochastic response of the elastic γ shaft to thermal excitation is analyzed. The results show that the stiffness nonlinearity of the γ shaft causes an increase of the vibration frequency for the F1 motor, which increases the motor’s rotation rate. When the concentration of ATP is relatively high and the load torque is small, the effects of the stiffness nonlinearity on the rotating rates of the F1 motor are obvious and should be considered. These results are useful for improving calculation of the rotating rate for the F1 motor and provide insight about the stochastic wave mechanics of F1-ATPase.  相似文献   

12.
猪心线粒体Fo的纯化、重建及其质子转运功能   总被引:1,自引:0,他引:1  
比较了猪心线粒体FoF1-ATPase膜部分Fo的四种纯化方法.结果表明,用NaBr从亚线粒体除去FoF1-ATPase的水溶性部分F1-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的Fo.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的Fo含有b、OSCP(寡霉素敏感授予蛋白)、d、a、e、F6、IF1、A6L和c等9种亚基.用去污剂稀释法将纯化的Fo在脂质体上重建后,重建Fo表现较高的被动转运质子活性.这为在体外深入研究Fo的活性、构象与膜脂的关系,以及Fo与F1-ATPase的组装等提供了很好的实验模型.  相似文献   

13.
Motor enzymes such as F1-ATPase and kinesin utilize energy from ATP for their motion. Molecular motions of these enzymes are critical to their catalytic mechanisms and were analyzed thoroughly using a single molecule observation technique. As a tool to analyze and control the ATP-driven motor enzyme motion, we recently synthesized a photoresponsive ATP analog with a p-tert-butylazobenzene tethered to the 2′ position of the ribose ring. Using cis/trans isomerization of the azobenzene moiety, we achieved a successful reversible photochromic control over a kinesin-microtubule system in an in vitro motility assay. Here we succeeded to control the hydrolytic activity and rotation of the rotary motor enzyme, F1-ATPase, using this photosensitive ATP analog. Subsequent single molecule observations indicated a unique pause occurring at the ATP binding angle position in the presence of cis form of the analog.  相似文献   

14.
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.  相似文献   

15.
Tributyltin (TBT), a persistent lipophilic contaminant found especially in the aquatic environment, is known to be toxic to mitochondria with the F1F0-ATPase as main target. Recently our research group pointed out that in mussel digestive gland mitochondria TBT, apart from decreasing the catalytic efficiency of Mg-ATPase activity, at concentrations ≥1.0 μM in the ATPase reaction medium lessens the enzyme inhibition promoted by the specific inhibitor oligomycin. The present work aims at casting light on the mechanisms involved in the TBT-driven enzyme desensitization to inhibitors, a poorly explored field. The mitochondrial Mg-ATPase desensitization is shown to be confined to inhibitors of transmembrane domain F0, namely oligomycin and N,N′-dicyclohexylcarbodiimide (DCCD). Accordingly, quercetin, which binds to catalytic portion F1, maintains its inhibitory efficiency in the presence of TBT. Among the possible mechanisms involved in the Mg-ATPase desensitization to oligomycin by ≥1.0 μM TBT concentrations, a structural detachment of the two F1 and F0 domains does not occur according to experimental data. On the other hand TBT covalently binds to thiol groups on the enzyme structure, which are apparently only available at TBT concentrations approaching 20 μM. TBT is able to interact with multiple sites on the enzyme structure by bonds of different nature. While electrostatic interactions with F0 proton channel are likely to be responsible for the ATPase activity inhibition, possible changes in the redox state of thiol groups on the protein structure due to TBT binding may promote structural changes in the enzyme structure leading to the observed F1F0-ATPase oligomycin sensitivity loss.  相似文献   

16.
Mitochondrial ATP synthase (F1F0-ATPase) is regulated by an intrinsic ATPase inhibitor protein. In this study, we overexpressed and purified human and bovine ATPase inhibitors and their properties were compared with those of a yeast inhibitor. The human and bovine inhibitors inhibited bovine ATPase in a similar way. The yeast inhibitor also inhibited bovine F1F0-ATPase, although the activity was about three times lower than the mammalian inhibitors. All three inhibitors inhibited yeast F1F0-ATPase in a similar way. The activities of all inhibitors decreased at higher pH, but the magnitude of the decrease was different for each combination of inhibitor and ATPase. The results obtained in this study show that the inhibitory mechanism of the inhibitors was basically shared in yeast and mammals, but that mammalian inhibitors require unique residues, which are lacking in the yeast inhibitor, for their maximum inhibitory activity. Common inhibitory sites of mammalian and yeast inhibitors are suggested.  相似文献   

17.
The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF1 has been determined at 2.5 Å resolution. The inhibitory region of IF1 from residues 1 to 36 is entrapped between the C-terminal domains of the αDP- and βDP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F1-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic βE-subunits. The nucleotide binding site in βE-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.  相似文献   

18.
Vladimir V. Bulygin 《BBA》2009,1787(8):1016-1023
Nucleotide binding to nucleotide-depleted F1-ATPase from Escherichia coli (EcF1) during MgATP hydrolysis in the presence of excess ? subunit has been studied using a combination of centrifugal filtration and column-centrifugation methods. The results show that nucleotide-binding properties of catalytic sites on EcF1 are affected by the state of occupancy of noncatalytic sites. The ATP-concentration dependence of catalytic-site occupancy during MgATP hydrolysis demonstrates that a bi-site mechanism is responsible for the positive catalytic cooperativity observed during multi-site catalysis by EcF1. The results suggest that a bi-site mechanism is a general feature of F1 catalysis.  相似文献   

19.
Rotary catalysis in F1F0 ATP synthase is powered by proton translocation through the membrane-embedded F0 sector. Proton binding and release occur in the middle of the membrane at Asp-61 on the second transmembrane helix (TMH) of subunit c, which folds in a hairpin-like structure with two TMHs. Previously, the aqueous accessibility of Cys substitutions in the transmembrane regions of subunit c was probed by testing the inhibitory effects of Ag+ or Cd2+ on function, which revealed extensive aqueous access in the region around Asp-61 and on the half of TMH2 extending to the cytoplasm. In the current study, we surveyed the Ag+ and Cd2+ sensitivity of Cys substitutions in the loop of the helical hairpin and used a variety of assays to categorize the mechanisms by which Ag+ or Cd2+ chelation with the Cys thiolates caused inhibition. We identified two distinct metal-sensitive regions in the cytoplasmic loop where function was inhibited by different mechanisms. Metal binding to Cys substitutions in the N-terminal half of the loop resulted in an uncoupling of F1 from F0 with release of F1 from the membrane. In contrast, substitutions in the C-terminal half of the loop retained membrane-bound F1 after metal treatment. In several of these cases, inhibition was shown to be due to blockage of passive H+ translocation through F0 as assayed with F0 reconstituted into liposomes. The results suggest that the C-terminal domain of the cytoplasmic loop may function in gating H+ translocation to the cytoplasm.  相似文献   

20.
Extracellular ATP formation from ADP and inorganic phosphate, attributed to the activity of a cell surface ATP synthase, has so far only been reported in cultures of some proliferating and tumoral cell lines. We now provide evidence showing the presence of a functionally active ecto-FoF1-ATP synthase on the plasma membrane of normal tissue cells, i.e. isolated rat hepatocytes. Both confocal microscopy and flow cytometry analysis show the presence of subunits of F1 (α/β and γ) and Fo (FoI-PVP(b) and OSCP) moieties of ATP synthase at the surface of rat hepatocytes. This finding is confirmed by immunoblotting analysis of the hepatocyte plasma membrane fraction. The presence of the inhibitor protein IF1 is also detected on the hepatocyte surface. Activity assays show that the ectopic-ATP synthase can work both in the direction of ATP synthesis and hydrolysis. A proton translocation assay shows that both these mechanisms are accompanied by a transient flux of H+ and are inhibited by F1 and Fo-targeting inhibitors. We hypothesise that ecto-FoF1-ATP synthase may control the extracellular ADP/ATP ratio, thus contributing to intracellular pH homeostasis.  相似文献   

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