Detection of the local H+ gradients on the internal mitochondrial membrane

FEBS Letters

Krasinskaya, Inna P¹  Lapin, Mikhail V¹  Yaguzhinsky, Lev S¹ 

[1]A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia

关键词: Mitochondrion;    Submitochondrial particle;    Fluorescein isothiocyanate;    Respiratory proton pump;    Local H+ activity gradient;    BSA;    bovine serum albumin;    DMSO;    dimethylsulfoxide;    EDTA;    ethylenediaminetetraacetate;    FCCP;    carbonyl cyanide p-trifluoromethoxyphenylhydrazone;    FITC;    fluorescein isothiocyanate;    HEPES;    N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid;    MES;    2-(N-morpholino)ethanesulfonic acid;    SMP;    submitochondrial particles;    TTFA;    tenoyltrifluoroacetone;    Tris;    tris(hydroxymethyl)aminomethane;   

DOI  :  10.1016/S0014-5793(98)01463-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Respiration-dependent responses of a pH probe (fluorescein isothiocyanate, FITC), covalently bound to the membrane proteins of mitochondria and submitochondrial particles (SMP) have been studied. A spectral shift indicating FITC deprotonation was observed when respiration was activated in coupled mitochondria. Such a response was increased by valinomycin and reduced by uncoupler. Some FITC deprotonation was detected in the presence of excess of an uncoupler, but the response was smaller and insensitive to valinomycin. FITC deprotonation was also observed in submitochondrial particles after succinate addition. In this case it was not affected by uncoupler. Increase in the buffer concentration was found to (i) decrease the FITC response and (ii) increase the rate of uncoupled respiration in both mitochondria and submitochondrial particles. The results are consistent with the assumption that respiration initiates appearance of local H⁺ activity gradients on the inner side of the internal mitochondrial membrane during the steady-state H⁺ pumping. We suggest that the formation of this gradient is due to kinetic barrier to proton transfer from the bulk phase to the respiratory proton pump vicinity.

【 授权许可】

Unknown