Reactive oxygen species production by potato tuber mitochondria is modulated by mitochondrially bound hexokinase activity.

Potato tuber (Solanum tuberosum) mitochondria (PTM) have a mitochondrially bound hexokinase (HK) activity that exhibits a pronounced sensitivity to ADP inhibition. Here we investigated the role of mitochondrial HK activity in PTM reactive oxygen species generation. Mitochondrial HK has a 10-fold higher affinity for glucose (Glc) than for fructose (KMGlc=140 microM versus KMFrc=1,375 microM). Activation of PTM respiration by succinate led to an increase in hydrogen peroxide (H2O2) release that was abrogated by mitochondrial HK activation. Mitochondrial HK activity caused a decrease in the mitochondrial membrane potential and an increase in oxygen consumption by PTM. Inhibition of Glc phosphorylation by mannoheptulose or GlcNAc induced a rapid increase in H2O2 release. The blockage of H2O2 release sustained by Glc was reverted by oligomycin and atractyloside, indicating that ADP recycles through the adenine nucleotide translocator and F0F1ATP synthase is operative during the mitochondrial HK reaction. Inhibition of mitochondrial HK activity by 60% to 70% caused an increase of 50% in the maximal rate of H2O2 release. Inhibition in H2O2 release by mitochondrial HK activity was comparable to, or even more potent, than that observed for StUCP (S. tuberosum uncoupling protein) activity. The inhibition of H2O2 release in PTM was two orders of magnitude more selective for the ADP produced from the mitochondrial HK reaction than for that derived from soluble yeast (Saccharomyces cerevisiae) HK. Modulation of H2O2 release and oxygen consumption by Glc and mitochondrial HK inhibitors in potato tuber slices shows that hexoses and mitochondrial HK may act as a potent preventive antioxidant mechanism in potato tubers.

Mitochondrial creatine kinase activity prevents reactive oxygen species generation: antioxidant role of mitochondrial kinase-dependent ADP re-cycling activity.

As recently demonstrated by our group (da-Silva, W. S., Gómez-Puyou, A., Gómez-Puyou, M. T., Moreno-Sanchez, R., De Felice, F. G., de Meis, L., Oliveira, M. F., and Galina, A. (2004) J. Biol. Chem. 279, 39846-39855) mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP re-cycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP re-cycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H(2)O(2) production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H(2)O(2) generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H(2)O(2) production in a Cr-sensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both DeltaPsi(m) and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.