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  • Photo-activation of mitochondrial ATP synthesis
    Publication . Khmelinskii, Igor; Makarov, Vladimir I.
    ATP production by mitochondria isolated from Saccharomyces cerevisiae cells was accelerated upon both direct and indirect mitochondrial photo-activation (MPA). The extent of direct MPA was dependent on the wavelength of excitation light. Direct MPA was created by light in cytochrome c spectral absorption bands (440, 520 and 550 nm), this light was absorbed producing electronically excited cytochrome c, and the excitation energy of the latter was used in the ATP production chain. The activity of cytochrome c was tested with 600 nm light, where cytochrome c does not absorb, and thus ATP production rate remained the same as in darkness. Note that ATP production rates were significantly larger under light at 550, 520 and 440 nm. Therefore, photo-activation of cytochrome c was the first step of MPA synthesis of ATP. Indirect MPA of ATP production also proceeded via electronically excited cytochrome c, by energy transfer from electronically excited Co/BN film to cytochrome c located in the inner mitochondrial membrane (IMM). Co/BN excitons were generated by photons absorbed by the Co/BN film, which was not in contact with the mitochondrial sample. Next, these excitons propagated along the Co/BN film to the part of the film that was in contact with the mitochondrial sample. There the exciton energy was transferred to cytochrome c located in the IMM, producing electronically excited cytochrome c. Thus, excited cytochrome c was generated in a way different from that of direct MPA. Next, the energy of excited cytochrome c was used in activated ATP synthesis, with virtually the same effect for 519 and 427 nm excitation. Thus, the first step of ATP synthesis in indirect MPA was the exciton energy transfer from Co/BN film to cytochrome c located in the IMM, producing an electronically excited cytochrome c molecule. A phenomenological mechanism of direct and indirect MPA was proposed, and the model parameters were obtained by fitting the model to the experimental data. However, more information is needed before the detailed mechanism of ATP synthesis activation by electronically excited cytochrome c could be understood. The present results support the earlier proposed hypothesis of indirect MPA of ATP production in vertebrate retina in daylight.
  • Mitochondrial ATP synthesis activated by exciton energy transfer from Müller cell intermediate filaments
    Publication . Khmelinskii, Igor; Makarov, Vladimir I.
    ATP production by mitochondria isolated from yeast cells (S. cerevisiae) was accelerated by exciton energy transfer from intermediate filaments (IFs). The effect was dependent on mitochondrial concentration and the intensity of light used to generate excitons. Presumably, mitochondrial cytochrome C (CC) was activated by IF exciton energy transfer to CC in the inner mitochondrial membrane (IMM), generating electronically excited CC, and the respective excitation energy was then used in the ATP production chain. A qualitative model was proposed describing observations, although more experiments are required before the detailed mechanisms could be deduced. The present results support the earlier proposed hypothesis of ATP production in vertebrate retina by excess photons in daytime, when retina needs extra energy for recovering the used opsins. Additionally, it shows that at early stages of evolution mitochondria could have been using solar radiation to produce ATP.
  • Energy transfer along Müller cell intermediate filaments isolated from porcine retina: II. Excitons at 2500 cm−1 produced by ADH1A upon hydrolysis of one ATP molecule
    Publication . Khmelinskii, Igor; Makarov, Vladimir I.
    Low-energy (IR -2500 cm-1) exciton transfer was explored in Muller cell (MC) intermediate filaments (IFs) isolated from porcine retina and filling a capillary matrix. IR excitons were generated either by absorption of IR radiation at 4 mu m, or by ATP hydrolysis energy transfer to the MC IFs. ATP hydrolysis was catalyzed by human alcohol dehydrogenase (ADH1A) in ADH1A...NAD+...IF complexes. Each exciton was generated by hydrolysis of one ATP molecule. Exciton energy was independent on ATP concentration. The emission spectra of IR excitons arriving to the other side of the capillary matrix were dependent on the exciton generation method. Timeresolved experiments with direct exciton generation by pulsed IR radiation allowed to measure exciton travel velocity, along with the exciton emission time. The results obtained support new ideas on ATP energy transfer along protein filaments in vivo. These unique experimental results provide for detailed understanding of the electronic state structure of the MC IFs. This study reports that MC IFs isolated from porcine retina have a longliving low-lying electronic excited state at 2500 cm-1 above the ground state.