Comparative analysis of proapoptotic activity of cytochrome <Emphasis Type="Italic">c</Emphasis> mutants in living cells |
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Authors: | G?V?Sharonov Email author" target="_blank">A?V?FeofanovEmail author O?V?Bocharova M?V?Astapova V?I?Dedukhova B?V?Chernyak D?A?Dolgikh A?S?Arseniev V?P?Skulachev M?P?Kirpichnikov |
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Institution: | (1) Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russia;(2) A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, 119899, Russia |
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Abstract: | A non-traumatic electroporation procedure was developed to load exogenous cytochrome c into the cytoplasm and to study the apoptotic effect of cytochrome c, its K72-substitued mutants and “yeast → horse” hybrid cytochrome c in living WEHI-3 cells. The minimum apoptosis-activating intracellular concentration of horse heart cytochrome c was estimated to be 2.7 ± 0.5 μM (47 ± 9 fg/cell). The equieffective concentrations of the K72A-, K72E- and K72L-substituted mutants of cytochrome c were five-, 15- and 70-fold higher. The “yeast → horse” hybrid created by introducing S2D, K4E, A7K, T8K, and K11V substitutions (horse protein numbering) and deleting five N-terminal residues in yeast cytochrome c did not evoke apoptotic activity in mammalian cells. The apoptotic function of cytochrome c was abolished by the K72W substitution. The K72W-substituted cytochrome c possesses reduced affinity to the apoptotic protease activating factor-1 (Apaf-1) and forms an inactive complex. This mutant is competent as a respiratory-chain electron carrier and well suited for knock-in studies of cytochrome c-mediated apoptosis. |
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Keywords: | apoptosis confocal spectral imaging cytochrome c electroporation fluorescence microscopy mutagenesis |
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