MAXIMAL OXIDATIVE CAPACITY OF MITOCHONDRIA IN LYMPHOMA NK/LY WITH METHYL ESTERS OF ENERGETIC SUBSTRATES
V. Hreniukh1, B. Manko1, O. Sidorova1, M. Holubyev2, J. Tsaryk1, A. Babsky1
This email address is being protected from spambots. You need JavaScript enabled to view it.
1Ivan Franko National Univresity of Lviv,
4 Hrushevsky Str., Lviv 79005, Ukraine
2National University of Life and Environmental Sciences of Ukraine,
15 Heroiv Oborony str., Kyiv 03041, Ukraine
The processes of respiration and oxidative phosphorylation in mitochondria are significantly determined by the transport of exogenous substrates through the plasma membrane. Methyl esters of the substrates of Krebs cycle better enter through the plasma membrane in normal and cancer cells. In polarographic chamber various substrates (2 mM) such as succinate, monomethyl-succinate, α-ketoglutarate, and dimethyl-α-ketoglutarate were added to 10 million NK/Ly cells. Rate of oxygen consumption was determined using polarographic method with Clark electrode at 37 °C. The maximum of oxidative capacity in lymphoma NK/Ly mitochondria was investigated by stimulation of oxygen uptake with different concentrations of protonophor FCCP (0.25–2 μM).
The maximum stimulation of respiration was caused by 0.5 and 1 μM FCCP depending on the substrate oxidation. Basal oxygen uptake rate (without FCCP) and maximum oxidative capacity with exogenous α-ketoglutarate or succinate did not differ significantly from control levels with energetic substrates indirectly indicating limited permeability of them through membrane. For the oxidation of dimethyl-α-ketoglutarate rate of FCCP-stimulated respiration significantly increased by 51 % and 132 % with 0.5 and 1 μM FCCP, respectively, compared to the non-methylated form. For the oxidation of monomethyl-succinate an increase in FCCP-stimulated respiration was not confirmed with parametric methods of statistics. Therefore, a methylated form of α-ketoglutarate can be successfully used as a substrate for evaluation of the maximum oxidative capacity in cancer cells without their permeabilization or mitochondria isolation.
Keywords: PERMEABILIZED CELLS, NK/LY, OXIDATION SUBSTRATES, THE MAXIMAL OXIDATIVE CAPACITY
