Proapoptotic activity of cytochrome c in living cells: effect of K72 substitutions and species differences

  • Rita V. Chertkova
  • George V. Sharonov
  • Alexei V. Feofanov
  • Ol’ga V. Bocharova
  • Ramil F. Latypov
  • Boris V. Chernyak
  • Alexander S. Arseniev
  • Dmitry A. DolgikhEmail author
  • Mikhail P. Kirpichnikov


Cytochrome c is one of the key proteins involved in the programmed cell death, and lysine 72 is known to be required for its apoptogenic activity. We have engineered a number of horse and murine cytochrome c single-point mutants with various substitutions at position 72 and compared quantitatively their proapoptotic activity in living cells. Apoptosis was activated by transferring exogenous cytochrome c into the cytoplasm of cells via a nontraumatic electroporation procedure. All mutant proteins studied exhibited significantly reduced proapoptotic activities in comparison with those for the wild type cytochromes. Relative activity of the horse (h(K72X)) and murine (m(K72W)) mutant proteins diminished in the order: h(K72R) > h(K72G) > h(K72A) > h(K72E) > h(K72L) ≫ h(K72W) > m(K72W). As estimated, the horse and murine K72W mutants were at least 200- and 500-fold less active than corresponding wild type proteins. Thus, the K72W-substituted cytochrome c can serve as an adequate candidate for knock-in studies of cytochrome c-mediated apoptosis. The proapoptotic activity of wild-type cytochrome c from different species in murine monocytic WEHI-3 cells reduced in the order: murine cytochrome c > human cytochrome c ≈ horse cytochrome c, thus indicating that apoptotic effect of cytochrome c depends on the species compatibility.


Apoptosis Cytochrome c Mutagenesis Electroporation Fluorescence microscopy Confocal spectral imaging 



Apoptotic protease-activating factor


Recombinant human annexin V conjugated with R-phycoerythrin


Confocal spectral imaging


Fetal calf serum

h(K72A), h(K72E), h(K72L), and h(К72W)

Horse heart cytochrome c mutants K72A, K72E, K72L, and К72W, respectively


Murine cytochrome c mutant К72W


Propidium iodide


Rhodamine 123





We gratefully acknowledge Professor V. P. Skulachev for his encouragement and fruitful discussions. Thе research was supported by the RAS MCB Programme and by the grant SS-1061.2008.4.


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Copyright information

© Springer Science+Business Media, LLC. 2008

Authors and Affiliations

  • Rita V. Chertkova
    • 1
  • George V. Sharonov
    • 1
  • Alexei V. Feofanov
    • 1
    • 2
  • Ol’ga V. Bocharova
    • 1
  • Ramil F. Latypov
    • 3
    • 4
  • Boris V. Chernyak
    • 5
  • Alexander S. Arseniev
    • 1
  • Dmitry A. Dolgikh
    • 1
    • 2
    Email author
  • Mikhail P. Kirpichnikov
    • 1
    • 2
  1. 1.Shemyakin-Ovchinnikov Institute of Bioorganic ChemistryRussian Academy of SciencesMoscowRussia
  2. 2.Bioengineering Department, Biological FacultyMoscow State UniversityMoscowRussia
  3. 3.Basic Science DivisionFox Chase Cancer CenterPhiladelphiaUSA
  4. 4.Amgen IncSeattleUSA
  5. 5.A.N.Belozersky Institute of Physico-Chemical BiologyMoscow State UniversityMoscowRussia

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