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Kinetics of hydroperoxide degradation by NADP-glutathione system in mitochondria
K Kurosawa1, H Shibata, N Hayashi
1Department of Physiological Chemistry, Osaka University Medical School.
Abstract:
Hydroperoxide decomposition by the NADP-glutathione system in rat liver mitochondria was analyzed. Mitochondria were found to contain high concentrations of the reduced form of glutathione (GSH) (4.32 +/- 0.50 nmol/mg) and NADPH (4.74 +/- 0.64 nmol/mg), and high activities of glutathione peroxidase and reductase. In the initial phase of the reaction, the rate of hydroperoxide decomposition was proportional to both the GSH level and the activity of GSH peroxidase. However, in the later steady state, the step of NADP reduction was rate-limiting, and the overall reaction rate was independent of the initial concentration of GSH, and activities of glutathione peroxidase and reductase. Some GSH was released from mitochondria during incubation, but the rate of the decomposition could be simply expressed as kappa [GSH]/2, where kappa is the first-order rate constant of the peroxidase and [GSH] is the intramitochondrial level of GSH in the steady state. The rate of the reaction in the steady state was also dependent on the NADPH level, its reciprocal being linearly correlated with [NADPH]-1. The rate of decomposition of hydroperoxide was influenced by the respiratory state. During state 3 respiration, the rate was greatly depressed, but was still considered to exceed by far the rate of physiological generation of hydroperoxide.
Insights
The NADP-glutathione system in rat liver mitochondria efficiently decomposes hydroperoxides. In steady state, NADP reduction becomes rate-limiting, with the reaction rate dependent on NADPH levels, not initial glutathione or peroxidase activity.
Area of Science:
- Biochemistry
- Mitochondrial Function
- Oxidative Stress
Background:
- Mitochondria utilize the NADP-glutathione system for hydroperoxide decomposition.
- Rat liver mitochondria possess high levels of reduced glutathione (GSH) and NADPH, along with significant glutathione peroxidase and reductase activities.
Purpose of the Study:
- To analyze hydroperoxide decomposition kinetics by the NADP-glutathione system in rat liver mitochondria.
- To elucidate the rate-limiting steps and dependencies of this system under varying conditions.
Main Methods:
- Mitochondrial incubation and measurement of hydroperoxide decomposition rates.
- Analysis of glutathione (GSH) and NADPH concentrations.
- Assay of glutathione peroxidase and reductase activities.
- Investigation of reaction kinetics under different respiratory states.
Main Results:
- Initial hydroperoxide decomposition rate is proportional to GSH levels and GSH peroxidase activity.
- In steady state, NADP reduction becomes rate-limiting, making the overall rate independent of initial GSH and enzyme activities.
- The steady-state rate is dependent on NADPH concentration and influenced by the mitochondrial respiratory state.
- Despite depression during state 3 respiration, the decomposition rate significantly exceeds physiological hydroperoxide generation.
Conclusions:
- The NADP-glutathione system is a robust antioxidant defense in rat liver mitochondria.
- NADP reduction is a critical control point for hydroperoxide decomposition in the steady state.
- Mitochondrial hydroperoxide decomposition capacity surpasses endogenous production, even under inhibited respiration.