Downmodulation of mitochondrial F0F1ATP synthase by diazoxide in cardiac myoblasts: a dual effect of the drug

2007 ◽  
Vol 292 (2) ◽  
pp. H820-H829 ◽  
Author(s):  
Marina Comelli ◽  
Giuliana Metelli ◽  
Irene Mavelli
Keyword(s):  
Atpase Activity ◽  
Atp Synthase ◽  
Transmembrane Potential ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Mitochondrial Transmembrane Potential ◽  
Mitochondrial Atpase ◽  
Inhibitor Protein ◽  
Atp Content ◽  
Intact Cells

Similar to ischemic preconditioning, diazoxide was documented to elicit beneficial bioenergetic consequences linked to cardioprotection. Inhibition of ATPase activity of mitochondrial F0F1ATP synthase may have a role in such effect and may involve the natural inhibitor protein IF1. We recently documented, using purified enzyme and isolated mitochondrial membranes from beef heart, that diazoxide interacts with the F1sector of F0F1ATP synthase by promoting IF1binding and reversibly inhibiting ATP hydrolysis. Here we investigated the effects of diazoxide on the enzyme in cultured myoblasts. Specifically, embryonic heart-derived H9c2 cells were exposed to diazoxide and mitochondrial ATPase was assayed in conditions maintaining steady-state IF1binding (basal ATPase activity) or detaching bound IF1at alkaline pH. Mitochondrial transmembrane potential and uncoupling were also investigated, as well as ATP synthesis flux and ATP content. Diazoxide at a cardioprotective concentration (40 μM cell-associated concentration) transiently downmodulated basal ATPase activity, concomitant with mild mitochondria uncoupling and depolarization, without affecting ATP synthesis and ATP content. Alkaline stripping of IF1from F0F1ATP synthase was less in diazoxide-treated than in untreated cells. Pretreatment with glibenclamide prevented, together with mitochondria depolarization, inhibition of ATPase activity under basal but not under IF1-stripping conditions, indicating that diazoxide alters alkaline IF1release. Diazoxide inhibition of ATPase activity in IF1-stripping conditions was observed even when mitochondrial transmembrane potential was reduced by FCCP. The results suggest that diazoxide in a model of normoxic intact cells directly promotes binding of inhibitor protein IF1to F0F1ATP synthase and enhances IF1binding indirectly by mildly uncoupling and depolarizing mitochondria.

Correlations between ATP hydrolysis, ATP synthesis, generation and utilization of ΔpH in mitochondrial ATPase-ATP synthase

1983 ◽  
Vol 725 (3) ◽  
pp. 464-471 ◽  
Author(s):  
Gilbert Deléage ◽  
François Penin ◽  
Catherine Godinot ◽  
Danièle C. Gautheron
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Mitochondrial Atpase

Synthesis and hydrolysis of ATP by the mitochondrial ATP synthase

1988 ◽  
Vol 66 (7) ◽  
pp. 677-682 ◽  
Author(s):  
M. Tuena de Gômez-Puyou ◽  
Orlando B. Martins ◽  
A. Gômez-Puyou
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Control Synthesis ◽  
Inhibitor Protein ◽  
Atpase Inhibitor ◽  
Mitochondrial Atp Synthase ◽  
High Concentrations ◽  
Hydrolysis Of

A brief summary of the factors that control synthesis and hydrolysis of ATP by the mitochondrial H+-ATP synthase is made. Particular emphasis is placed on the role of the natural ATPase inhibitor protein. It is clear from the existing data obtained with a number of agents that there is no correlation between variations of the rate of ATP hydrolysis and ATP synthesis as driven by respiration. The mechanism by which each condition differentially affects the two activities is not entirely known. For the case of the natural ATPase inhibitor protein, it appears that the protein controls the kinetics of the enzyme. This control seems essential for achieving maximal accumulation of ATP during electron transport in systems that contain relatively high concentrations of ATP.

scholarly journals Deleting the IF 1 -like ζ subunit from Paracoccus denitrificans ATP synthase is not sufficient to activate ATP hydrolysis

Open Biology ◽  
2018 ◽  
Vol 8 (1) ◽  
pp. 170206 ◽  
Author(s):  
Febin Varghese ◽  
James N. Blaza ◽  
Andrew J. Y. Jones ◽  
Owen D. Jarman ◽  
Judy Hirst
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Paracoccus Denitrificans ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Inhibitor Protein ◽  
Wild Type ◽  
Efficient Energy ◽  
Inverted Membrane Vesicles ◽  
Atp Synthases

In oxidative phosphorylation, ATP synthases interconvert two forms of free energy: they are driven by the proton-motive force across an energy-transducing membrane to synthesize ATP and displace the ADP/ATP ratio from equilibrium. For thermodynamically efficient energy conversion they must be reversible catalysts. However, in many species ATP synthases are unidirectional catalysts (their rates of ATP hydrolysis are negligible), and in others mechanisms have evolved to regulate or minimize hydrolysis. Unidirectional catalysis by Paracoccus denitrificans ATP synthase has been attributed to its unique ζ subunit, which is structurally analogous to the mammalian inhibitor protein IF 1 . Here, we used homologous recombination to delete the ζ subunit from the P. denitrificans genome, and compared ATP synthesis and hydrolysis by the wild-type and knockout enzymes in inverted membrane vesicles and the F 1 -ATPase subcomplex. ATP synthesis was not affected by loss of the ζ subunit, and the rate of ATP hydrolysis increased by less than twofold, remaining negligible in comparison with the rates of the Escherichia coli and mammalian enzymes. Therefore, deleting the P. denitrificans ζ subunit is not sufficient to activate ATP hydrolysis. We close by considering our conclusions in the light of reversible catalysis and regulation in ATP synthase enzymes.

scholarly journals Aerobic Growth of Escherichia coli Is Reduced, and ATP Synthesis Is Selectively Inhibited when Five C-terminal Residues Are Deleted from the ϵ Subunit of ATP Synthase

2015 ◽  
Vol 290 (34) ◽  
pp. 21032-21041 ◽  
Author(s):  
Naman B. Shah ◽  
Thomas M. Duncan
Keyword(s):  
Escherichia Coli ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Intrinsic Rate ◽  
Synthesis Rate ◽  
Final Segment ◽  
Rotary Catalysis

F-type ATP synthases are rotary nanomotor enzymes involved in cellular energy metabolism in eukaryotes and eubacteria. The ATP synthase from Gram-positive and -negative model bacteria can be autoinhibited by the C-terminal domain of its ϵ subunit (ϵCTD), but the importance of ϵ inhibition in vivo is unclear. Functional rotation is thought to be blocked by insertion of the latter half of the ϵCTD into the central cavity of the catalytic complex (F1). In the inhibited state of the Escherichia coli enzyme, the final segment of ϵCTD is deeply buried but has few specific interactions with other subunits. This region of the ϵCTD is variable or absent in other bacteria that exhibit strong ϵ-inhibition in vitro. Here, genetically deleting the last five residues of the ϵCTD (ϵΔ5) caused a greater defect in respiratory growth than did the complete absence of the ϵCTD. Isolated membranes with ϵΔ5 generated proton-motive force by respiration as effectively as with wild-type ϵ but showed a nearly 3-fold decrease in ATP synthesis rate. In contrast, the ϵΔ5 truncation did not change the intrinsic rate of ATP hydrolysis with membranes. Further, the ϵΔ5 subunit retained high affinity for isolated F1 but reduced the maximal inhibition of F1-ATPase by ϵ from >90% to ∼20%. The results suggest that the ϵCTD has distinct regulatory interactions with F1 when rotary catalysis operates in opposite directions for the hydrolysis or synthesis of ATP.

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.

scholarly journals Structure of a bacterial ATP synthase

2018 ◽  
Author(s):  
Hui Guo ◽  
Toshiharu Suzuki ◽  
John L. Rubinstein
Keyword(s):  
Atp Synthase ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Genetic Manipulation ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Complex ◽  
Rotational States ◽  
Membrane Region ◽  
Atp Synthases

AbstractATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed theBacillusPS3 ATP synthase inEschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunitεshows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

scholarly journals Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1456
Author(s):  
Amaravadhi Harikishore ◽  
Chui-Fann Wong ◽  
Priya Ragunathan ◽  
Dennis Litty ◽  
Volker Müller ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Α Subunit ◽  
Rotational States ◽  
C Terminus ◽  
Inverted Membrane Vesicles ◽  
Hydrolysis Of ◽  
Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

scholarly journals The structure of the catalytic domain of the ATP synthase fromMycobacterium smegmatisis a target for developing antitubercular drugs

2019 ◽  
Vol 116 (10) ◽  
pp. 4206-4211 ◽  
Author(s):  
Alice Tianbu Zhang ◽  
Martin G. Montgomery ◽  
Andrew G. W. Leslie ◽  
Gregory M. Cook ◽  
John E. Walker
Keyword(s):  
Atp Synthase ◽  
Mycobacterium Smegmatis ◽  
Catalytic Domain ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Geobacillus Stearothermophilus ◽  
Antitubercular Drugs ◽  
Atp Binding Site ◽  
E Coli ◽  
Catalytic Cycles

The crystal structure of the F1-catalytic domain of the adenosine triphosphate (ATP) synthase has been determined fromMycobacterium smegmatiswhich hydrolyzes ATP very poorly. The structure of the α3β3-component of the catalytic domain is similar to those in active F1-ATPases inEscherichia coliandGeobacillus stearothermophilus. However, its ε-subunit differs from those in these two active bacterial F1-ATPases as an ATP molecule is not bound to the two α-helices forming its C-terminal domain, probably because they are shorter than those in active enzymes and they lack an amino acid that contributes to the ATP binding site in active enzymes. InE. coliandG. stearothermophilus, the α-helices adopt an “up” state where the α-helices enter the α3β3-domain and prevent the rotor from turning. The mycobacterial F1-ATPase is most similar to the F1-ATPase fromCaldalkalibacillus thermarum, which also hydrolyzes ATP poorly. The βE-subunits in both enzymes are in the usual “open” conformation but appear to be occupied uniquely by the combination of an adenosine 5′-diphosphate molecule with no magnesium ion plus phosphate. This occupation is consistent with the finding that their rotors have been arrested at the same point in their rotary catalytic cycles. These bound hydrolytic products are probably the basis of the inhibition of ATP hydrolysis. It can be envisaged that specific as yet unidentified small molecules might bind to the F1domain inMycobacterium tuberculosis, prevent ATP synthesis, and inhibit the growth of the pathogen.

scholarly journals Structure of a bacterial ATP synthase

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Hui Guo ◽  
Toshiharu Suzuki ◽  
John L Rubinstein
Keyword(s):  
Atp Synthase ◽  
Biochemical Analysis ◽  
Atp Hydrolysis ◽  
Genetic Manipulation ◽  
Proton Translocation ◽  
Atp Synthesis ◽  
Mitochondrial Complex ◽  
Rotational States ◽  
Membrane Region ◽  
Atp Synthases

ATP synthases produce ATP from ADP and inorganic phosphate with energy from a transmembrane proton motive force. Bacterial ATP synthases have been studied extensively because they are the simplest form of the enzyme and because of the relative ease of genetic manipulation of these complexes. We expressed the Bacillus PS3 ATP synthase in Eschericia coli, purified it, and imaged it by cryo-EM, allowing us to build atomic models of the complex in three rotational states. The position of subunit ε shows how it is able to inhibit ATP hydrolysis while allowing ATP synthesis. The architecture of the membrane region shows how the simple bacterial ATP synthase is able to perform the same core functions as the equivalent, but more complicated, mitochondrial complex. The structures reveal the path of transmembrane proton translocation and provide a model for understanding decades of biochemical analysis interrogating the roles of specific residues in the enzyme.

A PLANT BIOCHEMIST'S VIEW OF H+-ATPases AND ATP SYNTHASES.

1992 ◽  
Vol 172 (1) ◽  
pp. 431-441
Author(s):  
RE Mccarty
Keyword(s):  
Plasma Membrane ◽  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Proton Gradient ◽  
Sequencing Analysis ◽  
Isolation And Purification ◽  
Membrane Atpases ◽  
Vacuolar Atpases ◽  
Atp Synthases

My twenty-five year fascination with membrane ATPases grew out of my experiences in the laboratories of André Jagendorf and Efraim Racker. André introduced me to photosynthetic phosphorylation and Ef, to whose memory this article is dedicated, convinced me that ATPases had much to do with ATP synthesis. Astounding progress has been made in the H+-ATPase field in just two decades. By the early 1970s, it was generally recognized that oxidative and photosynthetic ATP synthesis were catalyzed by membrane enzymes that could act as H+-ATPases and that the common intermediate between electron transport and phosphorylation is the electrochemical proton gradient. At that time, it had been shown that a cation-stimulated ATPase activity was associated with plasma membrane preparations from plant roots. The endomembrane or vacuolar ATPases were unknown. The application of improved biochemical methods for membrane isolation and purification, as well as membrane protein reconstitutions, led rapidly to the conclusion that there are three major classes of membrane H+-ATPases, P, V and F. P-ATPases, which will not be considered further in this article, are phosphorylated during their catalytic cycle and have a much simpler polypeptide composition than V- or F-ATPases. The plasma membrane H+-ATPase of plant, yeasts and fungal cells is one example of this class of enzymes (see Pedersen and Carafoli, 1987, for a comparison of plasma membrane ATPases). Biochemical and gene sequencing analysis have revealed that V- and F-ATPases resemble each other structurally, but are distinct in function and origin. The 'V' stands for vacuolar and the 'F' for F1Fo. F1 was the first factor isolated from bovine heart mitochondria shown to be required for oxidative phosphorylation. Fo was so named because it is a factor that conferred oligomycin sensitivity to soluble F1. Other F-ATPases are often named to indicate their sources. For example, chloroplast F1 is denoted CF1 (see Racker, 1965, for early work on F1). Recent successes in reconstitution of vacuolar ATPase have led to a V1Vo nomenclature for this enzyme as well. The term 'ATP synthase' is now in general use to describe F-ATPases. This term emphasizes the facts that although F-ATPases function to synthesize ATP, they do not catalyze, normally, ATP hydrolysis linked to proton flux. In contrast, V-ATPases are very unlikely to operate as ATP synthases. Thus, F-ATPases are proton gradient consumers, whereas V-ATPases generate proton gradients at the expense of hydrolysis. In this brief review, I will compare the structures of F- and V-ATPases. Also, I give some insight into the mechanisms that help prevent wasteful ATP hydrolysis by the chloroplast ATP synthase (CF1Fo).

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