ATP Synthesis and Electrical Membrane Potential in Mitochondria

Rottenberg, Hagai

doi:10.1111/j.1432-1033.1970.tb00971.x

Cited by 78 publications

(39 citation statements)

References 19 publications

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“…At 150mM potassium, the AfiH amounted to 70 mV and consisted almost entirely of ApH component. The decrease in AfiH is probably due to the uncoupling activity of the high potassium concentration in the presence of valinomycin, as observed in other systems [28].…”

Section: Discussionsupporting

confidence: 62%

The Proton Electrochemical Gradient in Escherichia coli Cells

1976

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Respiring intact cells maintained an internal pH more alkaline by 0.63 -0.75 unit than that of the milieu at extracellular pH 7, both in growth medium and KCl solutions. The ApH decreased when respiration was inhibited by anaerobiosis or in the presence of KCN.The AGH, established by EDTA/valinomycin-treated cells, was constant (122 -129 mV) over extracellular potassium concentration of 0.01 mM -1 mM. At the lower potassium concentration A $ (110-120mV) was the predominant component, and at the higher concentration d p H increased to 0.7 units (42 mV). At 150 mM potassium A,EH was reduced to 70 mV mostly due to a d pH component of 0.89 (53 mV). The interchangeability of the A fiH components is consistent with an electrogenic proton pump and with potassium serving as a counter ion in the presence of valinomycin. Indeed both parameters of A ,EH decreased in the presence of carbonylcyanide p-trifluoromethoxyphenylhydrazone.The highest ApH of 2 units was observed in the intact cells at pH 6; increasing the extracellular pH decreased the ApH to 0 at pH 7.65 and to -0.51 at pH 9. A similar pattern of dependence of A pH on extracellular pH was observed in EDTA/valinomycin-treated cells but the d $ was almost constant over the whole range of extracellular pH values (6 -8) implying electroneutral proton movement.Potassium is specifically required for respiration of EDTA-treated E. coli K12 cells since other monovalent or divalent cations could not replace potassium and valinomycin was not required.It is established that an electrochemical proton gradient (dpH) is built up through energization across energy-conserving membranes of eucaryotic organelles -chloroplasts and mitochondria (reviews in [l] to [4]). The methods employed for determining AfiH in microscopic systems, recently critically reviewed [4], include determinations of the pH gradient (dpH) and the potential gradient (All/) across the membrane, since both parameters contribute to ApH according to the relation : Mitchell [5] suggested that this proton gradient is of primary importance in the mechanism of biological energy conversion.In the procaryotic cell, the cytoplasmic membrane is the site of energy coupling. Upon energization, outward translocation of protons from whole bacterial cells has been demonstrated showed that upon glycolysis intact cells of Strepto-COCCUS faecalis maintain a more alkaline internal pH (0.5-1 unit higher than the medium) and a potential of 150-200 mV across the membrane with the interior negative [lo]. Since both these parameters of dDH were not determined simultaneously, the magnitude of dpH and the relationship between its components can only be indirectly estimated for these cells. A membrane potential of about 140 mV was recently estimated in respiring E. coli cells [Ill but the ApH was not determined. While in chromatophore fractions obtained from photosynthetic bacteria A$ and dpH contribute equally to the ApH [12,13], in membraneous vesicles obtained from E. coli only A$ was detected [14,15].Recent studies showed that an a...

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Section: Discussionsupporting

confidence: 62%

The Proton Electrochemical Gradient in Escherichia coli Cells

1976

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“…This situation quantitively explains the distribution of K ÷ observed by Mitchell and Moyle [1] and by others in subsequent studies (e.g. [4][5][6]). They are not consistent with the electrogenic pump model of the chemiosmotic hypothesis.…”

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confidence: 81%

Absence of a metabolically induced electrical potential across the mitochondrial semipermeable membrane

Tedeschi

1975

FEBS Letters

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“…High pH protects against both the inhibition by ASPM and the incorporation of ASPM even in the absence of Pi. A most striking effect is the increase of inhibition by valinomycin which is known to increase the ApH across the membrane [13,14]. Malonate increases stimulation and counteracts protection (low dependence on pH) Fig.…”

Section: The Accessibility Of Sh-groups Knterpreted As An Influence Omentioning

confidence: 99%

Analysis of the Reactivity of SH-Reagents with the Mitochondrial Phosphate Carrier

Klingenberg¹,

Durand

Guérin

1974

Eur J Biochem

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The inhibition of mitochondrial Pi transport by SH-reagents was investigated together with incorporation of SH-reagents into mitochondrial membranes. The reactivity of the Pi carrier SH-group is influenced by several factors during the preincubation with the SH-reagent. By using more or less permeant maleimide derivatives, ethylmaleimide and N -(N-acetyl-4-sulfamoyIphenyl) maleimide (ASPM), different reactivities can be attributed to accessibility variations. Differences in reactivity become apparent only on short-term incubations with SH-reagents, which allow measurements of reaction rates. The following results were obtained :1. The reaction rate of ASPM with the Pi carrier is about three-times slower than that of ethylmaleimide. .With increasing pH the reactivity of the Pi carrier decreases with ASPM but increases with ethylmaleimide.3. Pi protects partially against inhibition both by ethylmaleimide and ASPM with a K , between 2 and 10 mM, slightly competitive with the SH-reagent. For protection Pi must be allowed to be taken up by the mitochondria indicating that endogenous Pi protects better than exogenous Pi.4. The incorporation of ethylmaleimide and ASPM into mitochondria shows smaller differences than inhibition of Pi transport. On titration with SH-reagents the inhibitor of the Pi carrier begins when 11 out of a total of 20 pmol SH-groups/g protein have been reacted. Inhibition is then complete when 15 pmoljg protein have been titrated. Thus 2-4 pmo1 SH-reagent cause the Pi carrier to pass from an active to an inactive form. This number probably includes more SHgroups than can be attributed to the Pi carrier.5. The SH-reactivity of the Pi carrier is strongly influenced by uncouplers, ionophores and respiratory inhibitors. The strongest increase of reactivity is obtained with valinomycin, less with uncoupler and none with nigericin. The protective effect of Pi is then completely abolished. On the other hand, valinomycin plus uncoupler does not increase the sensitivity to SH-reagents.6. Succinate and other respiratory substrates decrease reactivity and increase protection by Pi, whereas malonate increases inhibition. This shows that the removal of endogenous Pi by exchange with a nonrespiring dicarboxylate reverses the protective effect of Pi.The variability of SH-reactivity is interpreted in terms of accessibility of ASPM. The protection of the Pi carrier is mainly based on reorientation of the carrier inside and outside the membrane. A carrier model is derived from the data in which an equilibrium exists between a protonized immobile form (CH+) of the carrier and a dissociated mobile form (C). Only the CH+ form can bind with mono-anionic Pi-and gives the mobile Pi-carrier complex. Thus the Pi-carrier complex will distribute with d p H across the membrane opposite to Pi. The protection by Pi is mainly due to endogenous Pi moving Pi-carrier complex inside, the activation by H + mainly due to moving empty carrier outside. The model is in accordance with an electroneutral Pi transport. It explains the major...

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ATP Synthesis and Electrical Membrane Potential in Mitochondria

Cited by 78 publications

References 19 publications

The Proton Electrochemical Gradient in Escherichia coli Cells

The Proton Electrochemical Gradient in Escherichia coli Cells

Absence of a metabolically induced electrical potential across the mitochondrial semipermeable membrane

Analysis of the Reactivity of SH-Reagents with the Mitochondrial Phosphate Carrier

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