Kinetics of Pi-Pi exchange in rat liver mitochondria. Rapid filtration experiments in the millisecond time range

E. Ligeti, Gérard Brandolin, Yves Dupont, Pierre V. Vignais

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Abstract

Phosphate-phosphate exchange through the inorganic phosphate (Pi) carrier of rat liver mitochondria was investigated by a new rapid filtration technique, which does not require the use of transport inhibitors to stop the reaction and offers high time resolution (starting from 10 ms), thus allowing kinetic measurements on a fine time scale even at room temperature. At approximately 22°C, isotopic equilibrium of [32P]Pi is achieved within 0.8-2.5 s - depending on the Pi concentration - and an initial linear phase, lasting for 400-500 ms, is observed. Complete inhibition of Pi exchange by an excess (33 nmol/mg) of mersalyl, a well-known organomercurial inhibitor, required 200 ms, pointing to the insufficiency of this reagent for effective inhibitor stop. On the other hand, investigation of the effect of mersalyl (allowed to react with mitochondria for at least 20 s) on the initial rate of Pi exchange supports earlier observations on the protective effect of this inhibitor; i.e., up to 3 nmol of mersalyl/mg of protein does not decrease the transport rate whereas these low concentrations protect approximately 50% of the transport capacity from irreversible inactivation by N-ethylmaleimide. In nonrespiring mitochondria, at pH 7.3, Pi exchange exhibited a Km of 1.6 mM and a Vmax of 3.0 μmol min-1 (mg of mitochondrial protein)-1. The increase of the membrane potential without any concomitant change of ΔpH had no significant influence on the kinetic parameters. The maximal velocity of Pi transport is significantly higher than the maximal velocity of all the other components of oxidative phosphorylation at comparable temperatures. The possible physiological significance of this excess capacity is discussed.

Original languageEnglish
Pages (from-to)4423-4428
Number of pages6
JournalBiochemistry
Volume24
Issue number16
Publication statusPublished - 1985

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Mersalyl
Mitochondria
Liver Mitochondrion
Liver
Rats
Ion exchange
Phosphates
Kinetics
Phosphate Transport Proteins
Ethylmaleimide
Temperature
Experiments
Mitochondrial Proteins
Oxidative Phosphorylation
Kinetic parameters
Membrane Potentials
Membranes
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Kinetics of Pi-Pi exchange in rat liver mitochondria. Rapid filtration experiments in the millisecond time range. / Ligeti, E.; Brandolin, Gérard; Dupont, Yves; Vignais, Pierre V.

In: Biochemistry, Vol. 24, No. 16, 1985, p. 4423-4428.

Research output: Contribution to journalArticle

Ligeti, E. ; Brandolin, Gérard ; Dupont, Yves ; Vignais, Pierre V. / Kinetics of Pi-Pi exchange in rat liver mitochondria. Rapid filtration experiments in the millisecond time range. In: Biochemistry. 1985 ; Vol. 24, No. 16. pp. 4423-4428.
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