scholarly journals F1F0-ATP synthase from bovine heart mitochondria: development of the purification of a monodisperse oligomycin-sensitive ATPase

1993 ◽  
Vol 295 (3) ◽  
pp. 799-806 ◽  
Author(s):  
R Lutter ◽  
M Saraste ◽  
H S van Walraven ◽  
M J Runswick ◽  
M Finel ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Gel Filtration ◽  
Primary Objective ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
F1 Atpase ◽  
Ammonium Sulphate Precipitation ◽  
F1f0 Atp Synthase ◽  
Hydrolysis Activity

A new procedure for the isolation of ATP synthase from bovine mitochondria has been developed, with the primary objective of producing enzyme suitable for crystallization trials. Proteins were extracted from mitochondrial membranes with dodecyl-beta-D-maltoside, and the ATP synthase was purified from the extract in the presence of the same detergent by a combination of ion-exchange and gel-filtration chromatography and ammonium sulphate precipitation. This simple and rapid procedure yields 20-30 mg of highly pure and monodisperse enzyme, evidently consisting of 14 different subunits, amongst them, in apparently stoichiometric amounts with the established subunits, subunit e, a recently discovered subunit of unknown function. The enzyme preparation has an oligomycin-sensitive ATP hydrolysis activity, and so the F1 domain is functionally associated with the membrane domain, F0. In contrast with the N-termini of some of the subunits of bovine mitochondrial F1-ATPase, those of the F1F0-ATP synthase are not degraded by proteolysis during the isolation procedure. This preparation therefore satisfies prerequisites for crystallization trials.

scholarly journals Purification and Biochemical Characterization of the F1Fo-ATP Synthase from Thermoalkaliphilic Bacillus sp. Strain TA2.A1

2003 ◽  
Vol 185 (15) ◽  
pp. 4442-4449 ◽  
Author(s):  
Gregory M. Cook ◽  
Stefanie Keis ◽  
Hugh W. Morgan ◽  
Christoph von Ballmoos ◽  
Ulrich Matthey ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Biochemical Characterization ◽  
Atp Hydrolysis ◽  
Sodium Dodecyl ◽  
Electrical Potential ◽  
Atp Synthesis ◽  
Intrinsic Property ◽  
Protein Sequencing ◽  
Hydrolysis Activity

ABSTRACT We describe here purification and biochemical characterization of the F1Fo-ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F1Fo-ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F1 subunits α, β, γ, δ, and ε and Fo subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F1Fo holoenzyme or the isolated F1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F1. After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (Δψ). A transmembrane proton gradient or sodium ion gradient in the absence of Δψ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na+/H+ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na+ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N′-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

F0 Membrane Domain of ATP Synthase from Bovine Heart Mitochondria: Purification, Subunit Composition, and Reconstitution with F1-ATPase

Biochemistry ◽  
1994 ◽  
Vol 33 (25) ◽  
pp. 7971-7978 ◽  
Author(s):  
Ian R. Collinson ◽  
Michael J. Runswick ◽  
Susan K. Buchanan ◽  
Ian M. Fearnley ◽  
J. Mark Skehel ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Subunit Composition ◽  
Heart Mitochondria ◽  
Membrane Domain ◽  
F1 Atpase ◽  
Bovine Heart

scholarly journals Interface mobility between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes

2021 ◽  
Vol 118 (8) ◽  
pp. e2021012118
Author(s):  
Tobias E. Spikes ◽  
Martin G. Montgomery ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
Angular Variation ◽  
Physiological Conditions ◽  
Catalytic Domains ◽  
Cryo Electron Microscopy ◽  
Lipid Structures

The ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle, and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. Disruption of the interface between dimers by mutation affects the morphology of the cristae severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76 and 95°. These particles represent active dimeric ATP synthase. Some angular variations arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer–monomer interaction is mediated mainly by j subunits attached to the surface of wedge-shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other interwedge contacts, molding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.

scholarly journals α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites

1999 ◽  
Vol 343 (1) ◽  
pp. 135 ◽  
Author(s):  
Toyoki AMANO ◽  
Tadashi MATSUI ◽  
Eiro MUNEYUKI ◽  
Hiroyuki NOJI ◽  
Kiyotaka HARA ◽  
...  
Keyword(s):  
Steady State ◽  
Atp Hydrolysis ◽  
Thermophilic Bacillus ◽  
F1 Atpase ◽  
Catalytic Sites ◽  
Hydrolysis Activity

scholarly journals Amputation of a C-terminal helix of the γ subunit increases ATP-hydrolysis activity of cyanobacterial F1 ATP synthase

2018 ◽  
Vol 1859 (5) ◽  
pp. 319-325 ◽  
Author(s):  
Kumiko Kondo ◽  
Yu Takeyama ◽  
Ei-ichiro Sunamura ◽  
Yuka Madoka ◽  
Yuki Fukaya ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Γ Subunit ◽  
Hydrolysis Activity

scholarly journals α3β3γ complex of F1-ATPase from thermophilic Bacillus PS3 can maintain steady-state ATP hydrolysis activity depending on the number of non-catalytic sites

1999 ◽  
Vol 343 (1) ◽  
pp. 135-138 ◽  
Author(s):  
Toyoki AMANO ◽  
Tadashi MATSUI ◽  
Eiro MUNEYUKI ◽  
Hiroyuki NOJI ◽  
Kiyotaka HARA ◽  
...  
Keyword(s):  
Steady State ◽  
Atp Hydrolysis ◽  
Catalytic Site ◽  
Wild Type ◽  
Thermophilic Bacillus ◽  
F1 Atpase ◽  
Type Complex ◽  
Catalytic Sites ◽  
Phase Activity ◽  
Hydrolysis Activity

Homogeneous preparations of α3β3γ complexes with one, two or three non-competent non-catalytic site(s) were performed as described [Amano, Hisabori, Muneyuki, and Yoshida (1996) J. Biol. Chem. 271, 18128-18133] and their properties were compared with those of the wild-type complex. The ATPase activity of the complex with three non-competent non-catalytic sites decayed rapidly to an inactivated state, as reported previously [Matsui, Muneyuki, Honda, Allison, Dou, and Yoshida (1997) J. Biol. Chem. 272, 8215-8221]. In contrast, the complex with one or two non-competent non-catalytic sites displayed a substantial steady-state phase activity depending on the number of non-competent non-catalytic sites in the complex. This result indicates that one competent non-catalytic site can maintain the continuous catalytic turnover of the enzyme and can potentially relieve all three catalytic sites from inhibition by MgADP-. Furthermore, the results suggest that the interaction between three non-catalytic sites might not be as strong as that between catalytic sites, which are all strictly required for a continuous catalytic turnover.

scholarly journals The mobility of interfaces between monomers in dimeric bovine ATP synthase participates in the ultrastructure of inner mitochondrial membranes

2020 ◽  
Author(s):  
Tobias E. Spikes ◽  
Martin G. Montgomery ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Catalytic Mechanism ◽  
Membrane Domains ◽  
Membrane Domain ◽  
Mitochondrial Membranes ◽  
Angular Variation ◽  
Physiological Conditions ◽  
Catalytic Domains ◽  
Cryo Electron Microscopy ◽  
Lipid Structures

SUMMARYThe ATP synthase complexes in mitochondria make the ATP required to sustain life by a rotary mechanism. Their membrane domains are embedded in the inner membranes of the organelle and they dimerize via interactions between their membrane domains. The dimers form extensive chains along the tips of the cristae with the two rows of monomeric catalytic domains extending into the mitochondrial matrix at an angle to each other. When the interaction between membrane domains is disrupted in living cells, the morphology of the cristae is affected severely. By analysis of particles of purified dimeric bovine ATP synthase by cryo-electron microscopy, we have shown that the angle between the central rotatory axes of the monomeric complexes varies between ca. 76° and ca. 95°. Some variations in this angle arise directly from the catalytic mechanism of the enzyme, and others are independent of catalysis. The monomer-monomer interaction is mediated mainly by j-subunits attached to the surface of wedge shaped protein-lipid structures in the membrane domain of the complex, and the angular variation arises from rotational and translational changes in this interaction, and combinations of both. The structures also suggest how the dimeric ATP synthases might be interacting with each other to form the characteristic rows along the tips of the cristae via other inter-wedge contacts, moulding themselves to the range of oligomeric arrangements observed by tomography of mitochondrial membranes, and at the same time allowing the ATP synthase to operate under the range of physiological conditions that influence the structure of the cristae.

scholarly journals Rotor subunits adaptations in ATP synthases from photosynthetic organisms

2021 ◽  
Vol 49 (2) ◽  
pp. 541-550
Author(s):  
Anthony Cheuk ◽  
Thomas Meier
Keyword(s):  
Time Scales ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Primary Source ◽  
Thylakoid Membranes ◽  
Photosynthetic Organisms ◽  
Long Time ◽  
Domains Of Life ◽  
Hydrolysis Activity ◽  
Atp Synthases

Driven by transmembrane electrochemical ion gradients, F-type ATP synthases are the primary source of the universal energy currency, adenosine triphosphate (ATP), throughout all domains of life. The ATP synthase found in the thylakoid membranes of photosynthetic organisms has some unique features not present in other bacterial or mitochondrial systems. Among these is a larger-than-average transmembrane rotor ring and a redox-regulated switch capable of inhibiting ATP hydrolysis activity in the dark by uniquely adapted rotor subunit modifications. Here, we review recent insights into the structure and mechanism of ATP synthases specifically involved in photosynthesis and explore the cellular physiological consequences of these adaptations at short and long time scales.

scholarly journals The Unique C-Terminal Extension of Mycobacterial F-ATP Synthase Subunit α Is the Major Contributor to Its Latent ATP Hydrolysis Activity

2020 ◽  
Vol 64 (12) ◽  
Author(s):  
Chui-Fann Wong ◽  
Gerhard Grüber
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Proton Motive Force ◽  
Terminal Deletion ◽  
Deletion Mutants ◽  
Major Contributor ◽  
Hydrolysis Activity ◽  
Binding Subunit

ABSTRACT Mycobacterial F1Fo-ATP synthases (α3:β3:γ:δ:ε:a:b:b′:c9) are incapable of ATP-driven proton translocation due to their latent ATPase activity. This prevents wasting of ATP and altering of the proton motive force, whose dissipation is lethal to mycobacteria. We demonstrate that the mycobacterial C-terminal extension of nucleotide-binding subunit α contributes mainly to the suppression of ATPase activity in the recombinant mycobacterial F1-ATPase. Using C-terminal deletion mutants, the regions responsible for the enzyme’s latency were mapped, providing a new compound epitope.

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