Na/K-ATPase under Oxidative Stress: Molecular Mechanisms of Injury

1. The authors compare oxidative injury to brain and kidney Na/K-ATPase using in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide modification. A computer simulation model was used for calculating kinetic parameters.2. The substrate dependence curve for the unmodified endogenous enzyme displayed a typical curve with an intermediate plateau, adequately described by the sum of hyperbolic and sigmoidal components.3. The modified enzyme demonstrated a dependent curve that closely approximates normal hyperbola. The estimated ATP K_m value for the endogenous enzyme was about 85 M; the K_h was equal to 800 M. The maximal number of protomers interacting was 8. Following oxidative modification, the enzyme substrate dependence curve did not show a significant change in the maximal protomer rate V_m, while the K_m was increased slightly and interprotomer interaction was abolished.4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in specific activity. The maximal rate of ATP hydrolysis by an enzyme protomer changed slightly, but the sigmoidal component, characterizing the enzyme's ability to form oligomers was abolished completely. The K_m value was almost unchanged, but the Hill coefficient fell to 1. These data show that Na/K-ATPase molecules isolated from the ischemic brain have lost the ability to interact with one another.5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis rate suppression.