Steady state measurements of the internal phosphorylation potential and the cross membrane electrochemical potential for proton in respiring mitochondria

Shen, Cynthia; Boens, Carmelita C.; Ogawa, Satoshi

doi:10.1016/s0006-291x(80)80272-5

Cited by 28 publications

(14 citation statements)

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“…assuming that a constant fraction (60%) of the accumulated TPMP + is bound). Similar corrections were made in studies which utilized TPP + for the measurement of Aq~ [13,28]. In these correction procedures no account is taken of external binding which can be substantial at low potentials, in particular at low salt concentrations (Fig.…”

Section: Resultsmentioning

confidence: 96%

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

1984

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The uptake and binding of the lipophilic cations ethidium+, tetraphenylphosphonium+ (TPP+), triphenylmethylphosphonium+ (TPMP+), and tetraphenylarsonium+ (TPA+) in rat liver mitochondria and submitochondrial particles were investigated. The effects of membrane potential, surface potentials and cation concentration on the uptake and binding were elucidated. The accumulation of these cations by mitochondria is described by an uptake and binding to the matrix face of the inner membrane in addition to the binding to the cytosolic face of the inner membrane. The apparent partition coefficients between the external medium and the cytosolic surface of the inner membrane (K'o) and the internal matrix volume and matrix face of the inner membrane (K'i) were determined and were utilized to estimate the membrane potential delta psi from the cation accumulation factor Rc according to the relation delta psi = RT/ZF ln [(RcVo - K'o)/(Vi + K'i)] where Vo and Vi are the volume of the external medium and the mitochondrial matrix, respectively, and Rc is the ratio of the cation content of the mitochondria and the medium. The values of delta psi estimated from this equation are in remarkably good agreement with those estimated from the distribution of 86Rb in the presence of valinomycin. The results are discussed in relation to studies in which the membrane potential in mitochondria and bacterial cells was estimated from the distribution of lipophilic cations.

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Section: Resultsmentioning

confidence: 96%

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

1984

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“…As an indication that problems may arise in systems other than B. subtilis, we may cite the observation by Shen et al (1980) that TPP + uptake in energized mitochondria exceeded Rb + uptake (in the presence of valinomycin) by a factor of 6.5 _+ 2 over a wide range of values of apparent potential.…”

Section: Discussionmentioning

confidence: 99%

“…U1-timately~ the only satisfactory criterion for deciding which is the most accurate is a direct measurement of the extent of binding in energized cells. Shen, Boens and Ogawa (1980) have attempted to measure TPP § binding in energized mitochondria, using 3~p NMR. The assumption was made that the peak deriving from bound TPP § would be too broad to be detected, and therefore that the extent of binding could be estimated from the missing intensity; however, since the result obtained was inconsistent with the extent of binding inferred indirectly from a comparison of TPP + and Rb § uptake data, Shen et al (1980) question the validity of the line-broadening assumption when applied to low-affinity, nonspecific binding.…”

Section: Discussionmentioning

confidence: 99%

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations, rubidium ion, and a cyanine dye as probes

1981

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Two of the commonly used probes for measuring membrane potential -lipophilic cations and the cyanine dye diS-C3(5 ) -indicated nominally opposite results when tetraphenylarsonium ion was added as a drug to suspensions of metabolizing Bacillus subtilis cells.[3H]-Triphenylmethylphosphonium uptake was enhanced by the addition, indicating hyperpolarization, yet fluorescence of diS-C3(5 ) was also enhanced, indicating depolarization. Evidence is presented that both effects are artifactual, and can occur without any change in membrane potential, as estimated by S6Rb+ uptake in lthe presence of valinomycin. The fluorescence studl es suggest that tetraphenylarsonium ion displaces he cyanine dye from the cell envelope, or other binding site, into the aqueous phase.The uptake characteristics of the radiolabeled lipophilic cations were quite unusual: At low concentrations (e.g., less than 10 gM for triphenylmethylphosphonium) there was potential-dependent uptake of the label to a stable level, but subsequent addition of nonradioactive lipophilic cation caused further uptake of label to a new stable level. Labeled triphenylmethylphosphonium ion taken up to the first stable level could be displaced by 10mM magnesium ion, whereas S6Rb+ uptake was unperturbed. Association of the lipophilic cations with the surface of de-energized cells was concentration-dependent, but there was no evidence for cooperative binding. This phenomenon of stimulated uptake in B. subtilis (which was not seen in Escherichia coli cells or vesicles) is consistent with a two-compartment model with access to the second compartment only being possible above a critical cation concentration. We * Work performed while on sabbatical leave from Department of Biology, Ben-Gurion University of the Negev, Beer-Sheva, Israel.tentatively propose such a model, in which these compartments are the cell surface and the cytoplasm, respectively.Triphenylmethylphosphonium up to 0.5 mM exhibited linear binding to de-energized cells; binding of tetraphenylphosphonium and tetraphenylarsonium was nonlinear but was not saturated at the highest concentration tested (lmM). The usual assumption, that association of the cation with cell surfaces is saturated and so can be estimated on de-energized cells, therefore leads to undercorrected estimates of cytoplasmic uptake in B. subtilis, and hence to overestimates of membrane potential. We describe a more realistic procedure, in which the estimate of extent of binding is based on a mean aqueous concentration related both to the external concentration and to the much higher internal concentration that exists in energized cells. Using this procedure we estimate the membrane potential in B. subtilis to be 120mV, inside-negative. The procedure is of general applicability, and should yield more accurate estimates of membrane potential in any system where there is significant potential-dependent binding.

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“…Our studies were done at 28°C and with more physiologic substrate levels (0.5-5 mM pyruvate rather than [10][11][12][13][14][15][16][17][18][19][20] mM (3)(4)(5)(6)(7)(8)(9)). Much less mitochondrial protein (7)(8)(9)(10) mg protein) was needed to obtain reasonable 31P NMR spectra, whereas other investigators used 25 to 120 mg mitochondrial protein (3)(4)(5)(6)(7)(8)(9). Our data demonstrate that mitochondria embedded in agarose beads are remarkably stable (no decrease in ATP production for more than 10 h), during which time they can be subjected to several interventions, followed by recovery periods.…”

Section: Discussionmentioning

confidence: 99%

“…The 679 composition of the medium used for these processes was as follows: 300 mM sucrose (heart mitochondria) or 250 mM sucrose (liver mitochondria), 10 mM HEPES (pH = 7.4), 1 mM EDTA, and 0.25% bovine serum albumin. After initial centrifugation (7 min at 480 g), the pellet containing nuclei and other cellular debris was discarded. The supernatant was centrifuged at 10,000 g for 15 min to obtain the mitochondrial fraction.…”

Section: Preparation Of Isolated Mitochondriamentioning

confidence: 99%

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with ³¹P‐NMR spectroscopy

Doliba

Wehrli

Babsky

et al. 1998

Magnetic Resonance in Med

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An NMR method to study on-line mitochondrial function was developed. Mitochondria were maintained in a stable physiologic state in agarose beads that were continuously superfused with oxygenated buffer at 28 degrees C. Oxidative function of both heart and liver mitochondria was evaluated with 31P NMR at 9.4 T using pyruvate plus malate as substrate. This method allows clear resolution of adenosine triphosphate-gamma (ATPgamma) and adenosine diphosphate-beta (ADPbeta) phosphate signals, whereas alpha signals of ATP and ADP overlap. ATP production by mitochondria was documented to be very sensitive to different interventions (hypoxia, ischemia, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP)) and depended on the ADP concentration in superfusion medium. These data demonstrate that the new application of NMR to study mitochondrial function can discriminate, on-line, between several physiologic and biochemical processes in intact physiologically stable mitochondria.

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Steady state measurements of the internal phosphorylation potential and the cross membrane electrochemical potential for proton in respiring mitochondria

Cited by 28 publications

References 5 publications

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations, rubidium ion, and a cyanine dye as probes

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with ³¹P‐NMR spectroscopy

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Steady state measurements of the internal phosphorylation potential and the cross membrane electrochemical potential for proton in respiring mitochondria

Cited by 28 publications

References 5 publications

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Membrane potential and surface potential in mitochondria: Uptake and binding of lipophilic cations

Measurement of membrane potential inBacillus subtilis: A comparison of lipophilic cations, rubidium ion, and a cyanine dye as probes

Encapsulation and perfusion of mitochondria in agarose beads for functional studies with 31P‐NMR spectroscopy

Contact Info

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Encapsulation and perfusion of mitochondria in agarose beads for functional studies with ³¹P‐NMR spectroscopy