Golovach NG, Cheshchevik VT, Lapshina EA et al.
Department of Biochemistry, Yanka Kupala State University of Grodno, Blvd. Len. Kom. – 50, 230030, Grodno, Belarus.
The Journal of membrane biology. Mar 2017.
We evaluated the parameters of Ca(2+)-induced mitochondrial permeability transition (MPT) pore formations, Ca(2+) binding constants, stoichiometry, energy of activation, and the effect of oxidative agents, tert-butyl hydroperoxide (tBHP), and hypochlorous acid (HOCl), on Ca(2+) -mediated process in rat liver mitochondria. From the Hill plot of the dependence of MPT rate on Ca(2+) concentration, we determined the order of interaction of Ca(2+) ions with the mitochondrial sites, n = 3, and the apparent Kd = 60 ± 12 µM. We also found the apparent Michaelis-Menten constant, Km, for Ca(2+) interactions with mitochondria to be equal to 75 ± 20 µM, whereas that in the presence of 300 µM tBHP was 120 ± 20 µM. Using the Arrhenius plots of the temperature dependences of apparent mitochondrial swelling rate at various Ca(2+) concentrations, we calculated the activation energy of the MPT process. ΔEa was 130 ± 20 kJ/mol at temperatures below the break point of the Arrhenius plot (30-34 °C) and 50 ± 9 kJ/mol at higher temperatures. Ca(2+) ions induced rapid mitochondrial NADH depletion and membrane depolarization. Prevention of the pore formation by cyclosporin A inhibited Ca(2+)-dependent mitochondrial depolarization and Mg(2+) ions attenuated the potential dissipation. tBHP (10-150 µM) dose-dependently enhanced the rate of MPT opening, whereas the effect of HOCl on MPT depended on the ratio of HOCl/Ca(2+). The apparent Km of tBHP interaction with mitochondria in the swelling reaction was found to be Km = 11 ± 3 µM. The present study provides evidence that three calcium ions interact with mitochondrial site with high affinity during MPT. Ca(2+)-induced MPT pore formations due to mitochondrial membrane protein denaturation resulted in membrane potential dissipation. Oxidants with different mechanisms, tBHP and HOCl, reduced mitochondrial membrane potential and oxidized mitochondrial NADH in EDTA-free medium and had an effect on Ca(2+)-induced MPT onset.