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Hiroyuki Noji - One of the best experts on this subject based on the ideXlab platform.
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correlation between the numbers of rotation steps in the atpase and proton conducting domains of f and v atpases
Biophysical Reviews, 2020Co-Authors: Hiroyuki Noji, Hiroshi Ueno, Ryohei KobayashiAbstract:This letter reports the correlation in the number of distinct rotation steps between the F1/V1 and Fo/Vo domains that constitute common rotary F- and V-ATP synthases/ATPases. Recent single-molecule studies on the F1-ATPase revealed differences in the number of discrete steps in rotary catalysis between different organisms-6 steps per turn in bacterial types and mitochondrial F1 from yeast, and 9 steps in the mammalian mitochondrial F1 domains. The number of rotational steps that Fo domain makes is thought to correspond to that of proteolipid subunits within the rotating c-ring present in Fo. Structural studies on Fo and in the whole ATP synthase complex have shown a large diversity in the number of proteolipid subunits. Interestingly, 6 steps in F1 are always paired with 10 steps in Fo, whereas 9 steps in F1 are paired with 8 steps in Fo. The correlation in the number of steps has also been revealed for two types of V-ATPases: one having 6 steps in V1 paired with 10 steps in Vo, and the other one having 3 steps in V1 paired with 12 steps in Vo. Although the abovementioned correlations await further confirmation, the results suggest a clear trend; ATPase motors with more steps have proton-conducting motors with less steps. In addition, ATPases with 6 steps are always paired with proton-conducting domains with 10 steps.
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Catalytic robustness and torque generation of the F1-ATPase
Biophysical reviews, 2017Co-Authors: Hiroyuki Noji, Hiroshi Ueno, Duncan G. G. McmillanAbstract:The F1-ATPase is the catalytic portion of the FoF1 ATP synthase and acts as a rotary molecular motor when it hydrolyzes ATP. Two decades have passed since the single-molecule rotation assay of F1-ATPase was established. Although several fundamental issues remain elusive, basic properties of F-type ATPases as motor proteins have been well characterized, and a large part of the reaction scheme has been revealed by the combination of extensive structural, biochemical, biophysical, and theoretical studies. This review is intended to provide a concise summary of the fundamental features of F1-ATPases, by use of the well-described model F1 from the thermophilic Bacillus PS3 (TF1). In the last part of this review, we focus on the robustness of the rotary catalysis of F1-ATPase to provide a perspective on the re-designing of novel molecular machines.
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Rotary catalysis of the stator ring of F1-ATPase
Biochimica et biophysica acta, 2012Co-Authors: Ryota Iino, Hiroyuki NojiAbstract:Abstract F1-ATPase is a rotary motor protein in which 3 catalytic β-subunits in a stator α3β3 ring undergo unidirectional and cooperative conformational changes to rotate the rotor γ-subunit upon adenosine triphosphate hydrolysis. The prevailing view of the mechanism behind this rotary catalysis elevated the γ-subunit as a “dictator” completely controlling the chemical and conformational states of the 3 catalytic β-subunits. However, our recent observations using high-speed atomic force microscopy clearly revealed that the 3 β-subunits undergo cyclic conformational changes even in the absence of the rotor γ-subunit, thus dethroning it from its dictatorial position. Here, we introduce our results in detail and discuss the possible operating principle behind the F1-ATPase, along with structurally related hexameric ATPases, also mentioning the possibility of generating hybrid nanomotors. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
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Pressure Sensitive Reaction of F1-ATPase
Biophysical Journal, 2012Co-Authors: Daichi Okuno, Masayoshi Nishiyama, Hiroyuki NojiAbstract:F1-ATPase is a rotational stepping molecular motor in which γ subunit rotates 120o against the α3β3 cylinder upon one ATP molecule hydrolysis. This 120o step is further divided into 80o and 40o substeps and each substep is triggered by ATP binding and ADP release and by ATP hydrolysis and Pi release, respectively. The stepping motion was sensitive against physical and chemical conditions, such as temperature and load Hydrostatic pressure is also a physical parameter to modulate the structure and function of protein molecules. Here, we developed a novel assay that monitored the stepping motion of single F1-ATPase molecules under various pressure conditions [1]. At ambient conditions, F1-ATPases derived from thermophilic Bacillus PS3 smoothly rotated with 9 Hz in the presence of 2 mM ATP. The rotational rate decreased with increased pressure, and then reached to 3 Hz and became stepping rotation by slowing a certain dwell at 140 MPa In order to identify which chemical state this dwell corresponds, the mutant F1(βE190D) which shows the pause of ATP catalytic dwell due to extremely slow ATP hydrolysis even under Vmax condition [2] was used. This pressure dependent dwell became obvious at +40o from catalytic dwell with applying pressure, i.e., it is the same position as ATP binding, where F1 executes ATP binding and ADP release on different catalytic sites. Thus, applied pressures seem to inhibit the ATP binding and/or ADP release reactions.[1] Nishiyama et al., Biophys J. 96(3) 1142-1150 (2009).[2] Shimabukuro et al.,Proc. Natl. Acad. Sci. U.S.A. 100, 14731–14736 (2003).
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Rotation of Escherichia coli F1-ATPase
Biochemical and biophysical research communications, 1999Co-Authors: Hiroyuki Noji, Masasuke Yoshida, Katrin Häsler, Wolfgang Junge, Kazuhiko Kinosita, Siegfried EngelbrechtAbstract:Abstract By applying the same method used for F1-ATPase (TF1) from thermophilic Bacillus PS3 (Noji, H., Yasuda, R., Yoshida, M., and Kinosita, K., Jr. (1997) Nature 386, 299–302), we observed ATP-driven rotation of a fluorescent actin filament attached to the γ subunit in Escherichia coli F1-ATPase. The torque value and the direction of the rotation were the same as those observed for TF1. F1-ATPases seem to share common properties of rotation irrespective of the sources.
Masamitsu Futai - One of the best experts on this subject based on the ideXlab platform.
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Proton Translocating ATPases
Reviews in Cell Biology and Molecular Medicine, 2006Co-Authors: Masamitsu Futai, Ge-hong Sun-wada, Yoh WadaAbstract:Proton-translocating ATPases synthesize or hydrolyze ATP coupling with electrochemical proton gradient, and have important roles in animal and plant physiology. The ion transporting ATPases are classified as F-ATPases (F-type ATPases), V-ATPases (V-type ATPases), and P-ATPases (P-type ATPase). The F-ATPase, also called ATP synthase, is found in chloroplasts, mitochondria, or bacterial membranes, and synthesizes most of the ATP, the biological energy currency. The enzyme is formed basically from eight subunits, a-, b-, and c-subunits forming membrane intrinsic F0 sector with ab 2 c 10 stoichiometry, and α-, β-, γ-, δ-, and ɛ-subunits forming peripheral F1 sector with α3β3γδɛ stoichiometry. Three catalytic β-subunits forming a hexamer with α-subunit (α3β3) have catalytic cooperativity. The γɛc 10 subunits complex, formed from peripheral (γɛ) and intrinsic membrane (c) subunits is a cential stalk, rotating during catalysis. The V-ATPase forms inside acidic pH in endomembrane organelles such as lysosomes, endosomes, synaptic vesicles, and so on, and is similar to F-ATPase in subunit organization and structure. The same enzyme is found in plasma membrane of special cells such as osteoclast and kidney intercalated cell. Gastric proton pump H+/K+ATPase or plant plasma membrane H+ATPase belongs to P-ATPases forming acyl phosphate intermediate, and is not discussed in this article. Keywords: F0F1 ; F-ATPase; P-type ATPase; Rotational Catalysis; γ-Subunit; V-ATPase
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mouse proton pump atpase c subunit isoforms c2 a and c2 b specifically expressed in kidney and lung
Journal of Biological Chemistry, 2003Co-Authors: Gehong Sunwada, Yoh Wada, Akitsugu Yamamoto, Yoshiko Murata, Miwako Namba, Masamitsu FutaiAbstract:Abstract The vacuolar-type H+-ATPases (V-ATPases) are multimeric proton pumps involved in a wide variety of physiological processes. We have identified two alternative splicing variants of C2 subunit isoforms: C2-a, a lungspecific isoform containing a 46-amino acid insertion, and C2-b, a kidney-specific isoform without the insert. Immunohistochemistry with isoform-specific antibodies revealed that V-ATPase with C2-a is localized specifically in lamellar bodies of type II alveolar cells, whereas the C2-b isoform is found in the plasma membranes of renal α and β intercalated cells. Immunoprecipitation combined with immunohistological analysis revealed that C2-b together with other kidney-specific isoforms was selectively assembled to form a unique proton pump in intercalated cells. Furthermore, a chimeric yeast V-ATPase with mouse the C2-a or C2-b isoform showed a lower Km(ATP) and lower proton transport activity than that with C1 or Vma5p (yeast C subunit). These results suggest that V-ATPases with the C2-a and C2-b isoform are involved in luminal acidification of lamellar bodies and regulation of the renal acid-base balance, respectively.
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a proton pump atpase with testis specific e1 subunit isoform required for acrosome acidification
Journal of Biological Chemistry, 2002Co-Authors: Gehong Sunwada, Yoh Wada, Yoko Imaisenga, Akitsugu Yamamoto, Yoshiko Murata, Tomoyuki Hirata, Masamitsu FutaiAbstract:Abstract The vacuolar-type H+-ATPases (V-ATPases) are a family of multimeric proton pumps involved in a wide variety of physiological processes. We have identified two novel mouse genes, Atp6e1 and Atp6e2, encoding testis-specific (E1) and ubiquitous (E2) V-ATPase subunit E isoforms, respectively. TheE1 transcript appears about 3 weeks after birth, corresponding to the start of meiosis, and is expressed specifically in round spermatids in seminiferous tubules. Immunohistochemistry with isoform-specific antibodies revealed that the V-ATPase withE1 and a2 isoforms is located specifically in developing acrosomes of spermatids and acrosomes in mature sperm. In contrast, the E2 isoform was expressed in all tissues examined and present in the perinuclear compartments of spermatocytes. The E1 isoform exhibits 70% identity with theE2, and both isoforms functionally complemented a null mutation of the yeast counterpart VMA4, indicating that they are bona fide V-ATPase subunits. The chimeric enzymes showed slightly lower K m ATP than yeast V-ATPase. Consistent with the temperature-sensitive growth of Δvma4-expressing E1 isoform, vacuolar membrane vesicles exhibited temperature-sensitive coupling between ATP hydrolysis and proton transport. These results suggest thatE1 isoform is essential for energy coupling involved in acidification of acrosome.
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vacuolar type h atpase in mouse bladder epithelium is responsible for urinary acidification
FEBS Letters, 1997Co-Authors: Ken Ichi Tomochika, Yoshinori Moriyama, Sumio Shinoda, Hiromi Kumon, Masaharu Mori, Masamitsu FutaiAbstract:Abstract The urine in the mouse bladder was found to be acidic, ranging from pH 5.3 to 5.5 in the daytime and pH 6.0 to 6.3 at night. Administration of bafilomycin A1 or concanamycin A, specific inhibitors of vacuolar-type H+-ATPase, into bladder lumen caused neutralization of urinary pH at least for 36 h, whereas inhibitors of mitochondrial ATP synthase (F-type H+-ATPase) or P-type H+-ATPases did not. Bafilomycin A1-sensitive proton secretion from isolated inside-out bladder was also observed. Immuno-electron microscopy with antibodies against vacuolar H+-ATPase revealed that vacuolar-type H+-ATPase is rich in luminal plasma membrane and endosomes of superficial cells of the bladder epithelium. These results indicate that vacuolar-type H+-ATPases present in luminal plasma membrane of the superficial epithelial cells secrete protons so as to acidify the urine in mouse bladder. © 1997 Federation of European Biochemical Societies.
Carsten A Wagner - One of the best experts on this subject based on the ideXlab platform.
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The ammonia transporter RhCG modulates urinary acidification by interacting with the vacuolar proton-ATPases in renal intercalated cells
Kidney International, 2017Co-Authors: Soline Bourgeois, Lisa Bounoure, Isabelle Mouro-chanteloup, Yves Colin, Dennis Brown, Carsten A WagnerAbstract:Ammonium, stemming from renal ammoniagenesis, is a major urinary proton buffer and is excreted along the collecting duct. This process depends on the concomitant secretion of ammonia by the ammonia channel RhCG and of protons by the vacuolar-type proton-ATPase pump. Thus, urinary ammonium content and urinary acidification are tightly linked. However, mice lacking Rhcg excrete more alkaline urine despite lower urinary ammonium, suggesting an unexpected role of Rhcg in urinary acidification. RhCG and the B1 and B2 proton-ATPase subunits could be co-immunoprecipitated from kidney. In ex vivo microperfused cortical collecting ducts (CCD) proton-ATPase activity was drastically reduced in the absence of Rhcg. Conversely, overexpression of RhCG in HEK293 cells resulted in higher proton secretion rates and increased B1 proton-ATPase mRNA expression. However, in kidneys from Rhcg -/- mice the expression of only B1 and B2 subunits was altered. Immunolocalization of proton-ATPase subunits together with immuno-gold detection of the A proton-ATPase subunit showed similar localization and density of staining in kidneys from Rhcg +/+ and Rhcg -/- mice. In order to test for a reciprocal effect of intercalated cell proton-ATPases on Rhcg activity, we assessed Rhcg and proton-ATPase activities in microperfused CCD from Atp6v1b1 -/- mice and showed reduced proton-ATPase activity without altering Rhcg activity. Thus, RhCG and proton-ATPase are located within the same cellular protein complex. RhCG may modulate proton-ATPase function and urinary acidification, whereas proton-ATPase activity does not affect RhCG function. This mechanism may help to coordinate ammonia and proton secretion beyond physicochemical driving forces.
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BASIC RESEARCH www.jasn.org Angiotensin II Stimulates Vacuolar H �-ATPase Activity in Renal Acid-Secretory Intercalated Cells from the Outer Medullary Collecting Duct
2013Co-Authors: Florina Rothenberger, Ana Velic, Paul A. Stehberger, Jana Kovacikova, Carsten A WagnerAbstract:Final urinary acidification is mediated by the action of vacuolar H �-ATPases expressed in acid-secretory type A intercalated cells (A-IC) in the collecting duct. Angiotensin II (AngII) has profound effects on renal acid-base transport in the proximal tubule, distal tubule, and collecting duct. This study investigated the effects on vacuolar H �-ATPase activity in A-IC in freshly isolated mouse outer medullary collecting ducts. AngII (10 nM) stimulated concanamycin-sensitive vacuolar H �-ATPase activity in A-IC in freshly isolated mouse outer medullary collecting ducts via AT 1 receptors, which were also detected immunohistochemically in A-IC. AngII increased intracellular Ca 2 � levels transiently. Chelation of intracellular Ca 2 � with BAPTA and depletion of endoplasmic reticulum Ca 2 � stores prevented the stimulatory effect on H �-ATPase activity. The effect of AngII on H �-ATPase activity was abolished by inhibitors of small G proteins and phospholipase C, by blockers of Ca 2 �-dependent and-independent isoforms of protein kinase C and extracellular signal–regulated kinase 1/2. Disruption of the microtubular network and cleavage of cellubrevin attenuated the stimulation. Finally, AngII failed to stimulate residual vacuolar H �-ATPase activity in A-IC from mice that were deficient for the B1 subunit of the vacuolar H �-ATPase. Thus, AngII presents a potent stimulus for vacuolar H �-ATPase activity in outer medullary collecting duct IC and requires traffickin
Eiro Muneyuki - One of the best experts on this subject based on the ideXlab platform.
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basic properties of rotary dynamics of the molecular motor enterococcus hirae v1 atpase
Journal of Biological Chemistry, 2013Co-Authors: Yoshihiro Minagawa, Hiroshi Ueno, Mayu Hara, Yoshiko Ishizukakatsura, Noboru Ohsawa, T Terada, Mikako Shirouzu, Shigeyuki Yokoyama, Ichiro Yamato, Eiro MuneyukiAbstract:Abstract V-ATPases are rotary molecular motors that generally function as proton pumps. We recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1) and proposed a model for its rotation mechanism. Here, we characterized the rotary dynamics of EhV1 using single-molecule analysis employing a load-free probe. EhV1 rotated in a counterclockwise direction, exhibiting two distinct rotational states, namely clear and unclear, suggesting unstable interactions between the rotor and stator. The clear state was analyzed in detail to obtain kinetic parameters. The rotation rates obeyed Michaelis-Menten kinetics with a maximal rotation rate (Vmax) of 107 revolutions/s and a Michaelis constant (Km) of 154 μm at 26 °C. At all ATP concentrations tested, EhV1 showed only three pauses separated by 120°/turn, and no substeps were resolved, as was the case with Thermus thermophilus V1-ATPase (TtV1). At 10 μm ATP (⪡Km), the distribution of the durations of the ATP-waiting pause fit well with a single-exponential decay function. The second-order binding rate constant for ATP was 2.3 × 106 m−1 s−1. At 40 mm ATP (⪢Km), the distribution of the durations of the catalytic pause was reproduced by a consecutive reaction with two time constants of 2.6 and 0.5 ms. These kinetic parameters were similar to those of TtV1. Our results identify the common properties of rotary catalysis of V1-ATPases that are distinct from those of F1-ATPases and will further our understanding of the general mechanisms of rotary molecular motors.
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thermodynamic efficiency and mechanochemical coupling of f1 atpase
Proceedings of the National Academy of Sciences of the United States of America, 2011Co-Authors: Shoichi Toyabe, Takahiro Watanabenakayama, Tetsuaki Okamoto, Seishi Kudo, Eiro MuneyukiAbstract:F1-ATPase is a nanosized biological energy transducer working as part of FoF1-ATP synthase. Its rotary machinery transduces energy between chemical free energy and mechanical work and plays a central role in the cellular energy transduction by synthesizing most ATP in virtually all organisms. However, information about its energetics is limited compared to that of the reaction scheme. Actually, fundamental questions such as how efficiently F1-ATPase transduces free energy remain unanswered. Here, we demonstrated reversible rotations of isolated F1-ATPase in discrete 120° steps by precisely controlling both the external torque and the chemical potential of ATP hydrolysis as a model system of FoF1-ATP synthase. We found that the maximum work performed by F1-ATPase per 120° step is nearly equal to the thermodynamical maximum work that can be extracted from a single ATP hydrolysis under a broad range of conditions. Our results suggested a 100% free-energy transduction efficiency and a tight mechanochemical coupling of F1-ATPase.
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V-ATPase of Thermus thermophilus Is Inactivated during ATP Hydrolysis but Can Synthesize ATP
Journal of Biological Chemistry, 1998Co-Authors: Ken Yokoyama, Eiro Muneyuki, Toyoki Amano, Seiji Mizutani, Masasuke Yoshida, Masami Ishida, Shouji OhkumaAbstract:Abstract The ATP hydrolysis of the V1-ATPase of Thermus thermophilus have been investigated with an ATP-regenerating system at 25 °C. The ratio of ATPase activity to ATP concentration ranged from 40 to 4000 μm; from this, an apparent K m of 240 ± 24 μm and a V max of 5.2 ± 0.5 units/mg were deduced. An apparent negative cooperativity, which is frequently observed in case of F1-ATPases, was not observed for the V1-ATPase. Interestingly, the rate of hydrolysis decayed rapidly during ATP hydrolysis, and the ATP hydrolysis finally stopped. Furthermore, the inactivation of the V1-ATPase was attained by a prior incubation with ADP-Mg. The inactivated V1-ATPase contained 1.5 mol of ADP/mol of enzyme. Difference absorption spectra generated from addition of ATP-Mg to the isolated subunits revealed that the A subunit can bind ATP-Mg, whereas the B subunit cannot. The inability to bind ATP-Mg is consistent with the absence of Walker motifs in the B subunit. These results indicate that the inactivation of the V1-ATPase during ATP hydrolysis is caused by entrapping inhibitory ADP-Mg in a catalytic site. Light-driven ATP synthesis by bacteriorhodopsin-VoV1-ATPase proteoliposomes was observed, and the rate of ATP synthesis was approximately constant. ATP synthesis occurred in the presence of an ADP-Mg of which concentration was high enough to induce complete inactivation of ATP hydrolysis of VoV1-ATPase. This result indicates that the ADP-Mg-inhibited form is not produced in ATP synthesis reaction.
Anne Abot - One of the best experts on this subject based on the ideXlab platform.
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Pharmacological inhibition of the F 1 -ATPase/P2Y 1 pathway suppresses the effect of apolipoprotein A1 on endothelial nitric oxide synthesis and vasorelaxation
Acta Physiologica, 2019Co-Authors: Cendrine Cabou, Paula Honorato, Luis Briceño, Lamia Ghezali, Thibaut Duparc, Marcelo León, Guillaume Combes, Laure Frayssinhes, Audren Fournel, Anne AbotAbstract:AIM: The contribution of apolipoprotein A1 (APOA1), the major apolipoprotein of high-density lipoprotein (HDL), to endothelium-dependent vasodilatation is unclear, and there is little information regarding endothelial receptors involved in this effect. Ecto-F1 -ATPase is a receptor for APOA1, and its activity in endothelial cells is coupled to adenosine diphosphate (ADP)-sensitive P2Y receptors (P2Y ADP receptors). Ecto-F1 -ATPase is involved in APOA1-mediated cell proliferation and HDL transcytosis. Here we investigated the effect of lipid-free APOA1 and the involvement of ecto-F1 -ATPase and P2Y ADP receptors on nitric oxide (NO) synthesis and the regulation of vascular tone. METHOD: NO synthesis was assessed in human endothelial cells from umbilical veins (HUVECs) and isolated mouse aortas. Changes in vascular tone were evaluated by isometric force measurements in isolated human umbilical and placental veins and by assessing femoral artery blood flow in conscious mice. RESULTS: Physiological concentrations of lipid-free APOA1 enhanced endothelial NO synthesis, which was abolished by inhibitors of endothelial nitric oxide synthase (eNOS) and of the ecto-F1 -ATPase/P2Y1 axis. Accordingly, APOA1 inhibited vasoconstriction induced by thromboxane A2 receptor agonist and increased femoral artery blood flow in mice. These effects were blunted by inhibitors of eNOS, ecto-F1 -ATPase and P2Y1 receptor. CONCLUSIONS: Using a pharmacological approach, we thus found that APOA1 promotes endothelial NO production and thereby controls vascular tone in a process that requires activation of the ecto-F1 -ATPase/P2Y1 pathway by APOA1. Pharmacological targeting of this pathway with respect to vascular diseases should be explored. This article is protected by copyright. All rights reserved.
