Influence of aurovertin on affinity of mitochondrial adenosine triphosphatase for ATP and ADP

Mitchell, Peter; Moyle, Jennifer

doi:10.1016/0014-5793(70)80085-0

Cited by 28 publications

(13 citation statements)

References 16 publications

(3 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The data were corrected for the back-decay of the H+ gradient and for the scalar H+ production exactly as they described. In a large number of experiments we observed a value of 2.00 ± 0.09 (SD) for the corrected H+/ATP ratio, in close agreement with Mitchell and Moyle (7,(12)(13)(14) ADP3-+ (1-n)H2PO4-+ nHPO42-+ nH+ [1] where the value of n increases with pH above about 6.1. They estimated the scalar H+ production by carrying out a parallel ATP pulse in the presence of a proton-conducting uncoupling agent, which eliminates the H+ gradient generated by the ejection of the vectorial protons.…”

Section: Resultssupporting

confidence: 70%

“…by Mitchell and Moyle (7,(12)(13)(14), oversimplifies a complex set of circumstances and cannot correct quantitatively for scalar H+ production. [This criticism does not apply to experiments on inverted inner membrane vesicles (14,15), since in this case reaction 1 occurs in the medium phase; moreover, those experiments were carried out at a low pH, where the value of n is negligibly small.…”

Section: Resultsmentioning

confidence: 99%

“…Mitochondria (10 mg of protein) were incubated in 2.0 ml of an anaerobic medium of 120 mM KC1, 3 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 1 mM EDTA, 5 AM rotenone, 1 ,M antimycin A, 100 ng of valinomycin per mg of protein, and 25 ng of aurovertin at pH 7.10 and 28°. Valinomycin was present to prevent formation of transmembrane potentials by allowing K+ movements across the membrane, EDTA to chelate Mg2+ and thus inhibit adenylate kinase, and aurovertin to ensure rapid and complete hydrolysis of added ATP (13,14,24). At zero time 5…”

Section: Methodsmentioning

confidence: 99%

“…9). Since measurements of the H+/ATP ratio during hydrolysis of added ATP also gave the value 2.0 in both mitochondria (7,(10)(11)(12)(13)(14) and submitochondrial particles (14, 15), the postulated condition of equality (H+/site = H+/ATP) was apparently fulfilled. However, we have recently demonstrated that the original oxygen-pulse experiments of Mitchell and Moyle (7,8) t To whom reprint requests should be addressed.…”

mentioning

confidence: 99%

“…Are the previously reported (7,(10)(11)(12)(13)(14) values of 2.0 for the H+/ATP ratio correct or have they also been underestimated? Second, is the equality of the H+/site and H+/ATP ratios postulated in the chemiosmotic theory necessarily valid?…”

mentioning

confidence: 99%

See 4 more Smart Citations

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

Brand¹,

Lehninger²

1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The stoichiometry of H+ ejection by mitochondria during hydrolysis of a small pulse of ATP (the H+/ATP ratio) has been reexamined in the light of our recent observation that the stoichiometry of H+ ejection during mitochondrial electron transport (the H+/site ratio) was previously underestimated. We show that earlier estimates of the H+/ATP ratio in intact mitochondria were based upon an invalid correction for scalar H+ production and describe a modified method for determination of this ratio which utilizes mersalyl or N-ethylmaleimide to prevent complicating transmembrane movements of phosphate and H+. This method gives a value for the H+/ATP ratio of 2.0 without the need for questionable corrections, compared with a value of 3.0 for the H+/site ratio also obtained by pulse methods.A modified version of the chemiosmotic theory is presented, in which 3 H+ are ejected per pair of electrons traversing each energy-conserving site of the respiratory chain. Of these, 2 H+ return to the matrix through the ATPase to form ATP from ADP and phosphate, and 1 H+ returns through the combined action of the phosphate and adenine nucleotide exchange carriers of the inner membrane to allow the energy-requiring influx of Pi and ADP3-and efflux of ATP4-. Thus, up to one-third of the energy input into synthesis of extramitochondrial ATP may be required for transport work. Since other methods suggest that the H+/site significantly exceeds 3.0, an alternative possibility is that 4 H+ are ejected per site, followed by return of 3 H+ through the ATPase and 1 H+ through the operation of the proton-coupled membrane transport systems.The passage of electrons from substrates to oxygen via the respiratory chain can develop an electrochemical gradient of H+ across the inner membrane of intact mitochondria (1-4). The number of H+ equivalents ejected during passage of a pair of electrons through each of the energy-conserving sites is designated the H+/site ratio (5). According to the chemiosmotic theory of oxidative phosphorylation (1-3, 6), the electrochemical H+ gradient so produced may drive H+ back into the mitochondrial matrix through the mitochondrial ATPase, causing coupled synthesis of ATP. The number of H+ equivalents translocated inward per ATP synthesized is the H+/ATP ratio. Since there is no net production or utilization of H+ during oxidative phosphorylation in vivo, the chemiosmotic theory postulates that the H+/site and the H+/ATP ratios are equal.The original measurements of the H+/site ratio by Mitchell and Moyle (7, 8) yielded the value 2.0 (see also ref. 9). Since measurements of the H+/ATP ratio during hydrolysis of added ATP also gave the value 2.0 in both mitochondria (7,(10)(11)(12)(13)(14) and submitochondrial particles (14, 15), the postulated condition of equality (H+/site = H+/ATP) was apparently fulfilled. However, we have recently demonstrated that the original oxygen-pulse experiments of Mitchell and Moyle (7,8) t To whom reprint requests should be addressed. movements were suppressed, H+/site ratios of ...

show abstract

Section: Resultssupporting

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

Brand¹,

Lehninger²

1977

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

Mechanistic basis for differential inhibition of the F₁F_o‐ATPase by aurovertin

et al. 2009

View full text Add to dashboard Cite

The mitochondrial F 1 F o -ATPase performs the terminal step of oxidative phosphorylation. Small molecules that modulate this enzyme have been invaluable in helping decipher F 1 F o -ATPase structure, function, and mechanism. Aurovertin is an antibiotic that binds to the β subunits in the F 1 domain and inhibits F 1 F o -ATPase-catalyzed ATP synthesis in preference to ATP hydrolysis. Despite extensive study and the existence of crystallographic data, the molecular basis of the differential inhibition and kinetic mechanism of inhibition of ATP synthesis by aurovertin has not been resolved. To address these questions, we conducted a series of experiments in both bovine heart mitochondria and E. coli membrane F 1 F o -ATPase. Aurovertin is a mixed, noncompetitive inhibitor of both ATP hydrolysis and synthesis with lower K i values for synthesis. At low substrate concentrations, inhibition is cooperative suggesting a stoichiometry of two aurovertin per F 1 F 0 -ATPase. Furthermore, aurovertin does not completely inhibit the ATP hydrolytic activity at saturating concentrations. Single-molecule experiments provide evidence that the residual rate of ATP hydrolysis seen in the presence of saturating concentrations of aurovertin results from a decrease in the binding change mechanism by hindering catalytic site interactions. The results from these studies should further the understanding of how the F 1 F o -ATPase catalyzes ATP synthesis and hydrolysis.

show abstract

Dynamics of Membrane-Associated Energy-Transducing Catalysts. A Study with Mitochondrial Adenosine Triphosphatase Inhibitor

Ernster

Asami

Juntti

et al. 1977

Structure of Biological Membranes

View full text Add to dashboard Cite

Influence of aurovertin on affinity of mitochondrial adenosine triphosphatase for ATP and ADP

Cited by 28 publications

References 16 publications

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

Mechanistic basis for differential inhibition of the F₁F_o‐ATPase by aurovertin

Dynamics of Membrane-Associated Energy-Transducing Catalysts. A Study with Mitochondrial Adenosine Triphosphatase Inhibitor

Contact Info

Product

Resources

About

Influence of aurovertin on affinity of mitochondrial adenosine triphosphatase for ATP and ADP

Cited by 28 publications

References 16 publications

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

H+/ATP ratio during ATP hydrolysis by mitochondria: modification of the chemiosmotic theory.

Mechanistic basis for differential inhibition of the F1Fo‐ATPase by aurovertin

Dynamics of Membrane-Associated Energy-Transducing Catalysts. A Study with Mitochondrial Adenosine Triphosphatase Inhibitor

Contact Info

Product

Resources

About

Mechanistic basis for differential inhibition of the F₁F_o‐ATPase by aurovertin