Phosphorylation without protonmotive force

Kinnally, Kathleen W.; Tedeschi, Henry

doi:10.1016/0014-5793(76)80012-9

Cited by 28 publications

(19 citation statements)

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confidence: 93%

“…We have found that in the giant mitochondria, the ApH is generally not higher than 0.3. These results are in close agreement with our previous experiments with rat liver mitochondria using 5,5-dimethyloxazolidine-2,4-dione [13].Furthermore, this estimate is a t least in approximate agreement with preliminary studies using pH-sensitive microelectrodes (carried out in conjunction with H. Yamaguchi and C. Bowman and not reported here). Together with our previous studies, the present findings show that the total protonmotive force is too low to play a role in mitochondrial oxidative phosphorylation.…”

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confidence: 93%

“…The viability of the individual mitochondria after these manipulations can be assayed either by examining their ability to accumulate calcium phosphate [4] or to synthesize ATP from ADP and Pi [4,7 -91. Previous studies and the accompanying paper [8,9] have shown that neither the impalement with microelectrodes or the electrophoretic microinjection of dyes interfere with the ability of mitochondria or mitoplasts to carry out oxidative phosphorylation.Our previous reports have presented evidence for the absence of a significant membrane potential in biochemically functioning giant mitochondria using microelectrodes [3,4,7-111 and electrochromic dyes in giant [13] or conventional rat liver mitochondria [12,13].A number of studies using a variety of techniques have shown that at least in rat liver mitochondria the metabolically dependent proton concentration gradient ( A pH) is relatively minor in the absence of valinomycin [I3 -271 and therefore would only play a minor role in a chemiosmotic mechanism of oxidative phosphorylation.In the present study we have estimated the d p H using the fluorescent pH indicator, pyranine (8-hydroxy-l,3,6-pyrenetrisulfonate). The dye was microinjected into giant mitochondria and the changes in fluorescence accompanying changes in metabolic states were monitored.…”

mentioning

confidence: 99%

“…Our previous reports have presented evidence for the absence of a significant membrane potential in biochemically functioning giant mitochondria using microelectrodes [3,4, and electrochromic dyes in giant [13] or conventional rat liver mitochondria [12,13].…”

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confidence: 99%

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Protonmotive force in giant mitochondria

Campo

Tedeschi

1984

European Journal of Biochemistry

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The accompanying communication [Eur. J . Biochem. 141 (1984) 1 -41 indicates that the microinjection of the pH fluorescent indicator pyranine (8-hydroxy-I ,2,6-pyrenesulfonate) into giant mitochondria or mitoplasts does not affect their ability to carry out oxidative phosphorylation. The dye can therefore be used as a quantitative indicator of internal mitochondrial pH. We found that activation of metabolism in rotenone-inhibited giant mitochondria by the addition of succinate produces an internal pH change corresponding to a pH shift of 0.3 to the alkaline range, approximately the same value found previously for conventional rat liver mitochondria.The availability of giant mitochondria 5 -10 l m in diameter [I -51 has permitted the study of mitochondrial function using micromanipulation procedures. The mitochondrial membrane potential [3,4] or the pH of the inner mitochondrial space can be measured directly after impalement using the appropriate microelectrodes. The location of the micropipette tips in the mitoplasts or the inner mitochondrial space has been demonstrated most clearly by the electrophoretic microinjection of dyes and measuring the rate of efflux of Lucifer Yellow C H from these vesicles [5,6]. The microinjection of water-soluble dyes also allows monitoring the mitochondrial lumen with pH indicators. The viability of the individual mitochondria after these manipulations can be assayed either by examining their ability to accumulate calcium phosphate [4] or to synthesize ATP from ADP and Pi [4,7 -91. Previous studies and the accompanying paper [8,9] have shown that neither the impalement with microelectrodes or the electrophoretic microinjection of dyes interfere with the ability of mitochondria or mitoplasts to carry out oxidative phosphorylation.Our previous reports have presented evidence for the absence of a significant membrane potential in biochemically functioning giant mitochondria using microelectrodes [3,4,7-111 and electrochromic dyes in giant [13] or conventional rat liver mitochondria [12,13].A number of studies using a variety of techniques have shown that at least in rat liver mitochondria the metabolically dependent proton concentration gradient ( A pH) is relatively minor in the absence of valinomycin [I3 -271 and therefore would only play a minor role in a chemiosmotic mechanism of oxidative phosphorylation.In the present study we have estimated the d p H using the fluorescent pH indicator, pyranine (8-hydroxy-l,3,6-pyrenetrisulfonate). The dye was microinjected into giant mitochondria and the changes in fluorescence accompanying changes in metabolic states were monitored. Pyranine has been previously used to measure the A p H in liposomal preparations [18,19]. We have found that in the giant mitochondria, the ApH is generally not higher than 0.3. These results are in close agreement with our previous experiments with rat liver mitochondria using 5,5-dimethyloxazolidine-2,4-dione [13].Furthermore, this estimate is a t least in approximate agreement with preliminary studies using...

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supporting

confidence: 93%

supporting

confidence: 93%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Protonmotive force in giant mitochondria

Campo

Tedeschi

1984

European Journal of Biochemistry

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“…In all tables and the figure, results are expressed as means ± standard deviation of at least four determinations, except where noted. (10)]. This disappearance approximately matched the ATP synthesized, as determined with the luciferin-luciferase reaction (Table 1).…”

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confidence: 63%