0, whereas the pKa values of most protein Cys-SH residues are higher than 8.0. Proteins such as cysteine proteases (e.g. caspases) are known to contain an essential Cys-SH with a low pKa at their active sites (Ischiropoulos, 1998), and are thus potential candidates for reversible oxidation by intracellularly generated H2O2 (Rhee, 1999). Interestingly, H2O2 exposure activated Xcg caspase-3 activity in vitro. Two sites of electron leakage lead to ROS formation in the mitochondrial electron transport chain (Blackstone et al., 2004): site 1 on complex I [NADH-ubiquinone oxidoreductase]
and site 2 at the interface between the mobile lipid-soluble carrier, ubiquinone, and complex III (ubiquinone-cytochrome c oxidoreductase). Leakage of electrons during ETC is considered
to be an important route of free radical generation Ibrutinib purchase in obligate aerobic Selleckchem Epacadostat organisms (Flecha & Demple, 1995). The uncoupler DNP was used in this study to examine this possibility. Uncouplers abolish the link between oxidation and phosphorylation, allowing electron transport to proceed without coupled ATP synthesis (Brand, 2000). DNP is a lipid-soluble weak acid that can cross the membrane barrier in both protonated and unprotonated form, and sets up a catalytic cycle that dissipates the proton-motive force and thus results in the decrease in ROS generation during ETC. At high proton-motive force, respiration slows; hence, electron would accumulate on ubiquinone instead of passing down the ETC to oxygen. This would increase the steady-state concentration of QH•, leading to an increase in the rate of ROS production (Brand, 2000). Interestingly, in the Xcg cells grown in PIM in the presence of DNP, no H2O2 could be detected by the scopoletin assay. Additionally, when Xcg cells were grown in PIM fortified with DNP, the cell survival was found to be increased by one log cycle. Thus, the free radical generation in PIM-growing Xcg cells seems to be mediated through the electron leakage during ETC. This could result in the formation
of IKBKE superoxide radical (O2•−) upon incomplete reduction of oxygen, which could subsequently lead to the generation of other ROS such as H2O2 and the hydroxyl radical (OH•), eventually causing oxidative stress in the medium. Nalidixic acid, when used in sublethal concentrations, was found to inhibit PCD significantly. Nalidixic acid affects gyrase activity and could consequently reduce the rate of DNA replication and cell multiplication by maintaining DNA superhelicity. This ultimately may reduce the metabolic stress, leading to the inhibition of PCD (Crumplin & Smith, 1975; Syed, 1998). The role of gyrase in PCD has also been reported elsewhere (Hayes, 2003). Thus, while growing in a nutritionally rich PIM, Xcg cells acquire reducing potential, leading to the generation of ROS.