Na/K-ATPase under oxidative stress: Molecular mechanisms of injury

1. The authors compare oxidative injury to brain and kidney Na/K-ATPase usi ng in vitro and in vivo approaches. The substrate dependence of dog kidney Na/K-ATPase was examined both before and after partial hydrogen peroxide mo dification. A computer simulation model was used for calculating kinetic pa rameters. 2, The substrate dependence curve for the unmodified endogenous enzyme disp layed 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 approxim ates normal hyperbola. The estimated ATP K-m value for the endogenous enzym e was about 85 mu M; the K-h was equal to 800 mu M. The maximal number of p rotomers interacting was 8. Following oxidative modification, the enzyme su bstrate dependence curve did not show a significant change in the maximal p rotomer rate V-m, while the K-m was increased slightly and interprotomer in teraction was abolished. 4. Na/K-ATPase from an ischemic gerbil brain showed a 22% decrease in speci fic activity. The maximal rate of ATP hydrolysis by an enzyme protomer chan ged slightly, but the sigmoidal component, characterizing the enzyme's abil ity to form oligomers was abolished completely. The K-m value was almost un changed, but the Hill coefficient fell to I. These data show that Na/K-ATPa se molecules isolated from the ischemic brain have lost the ability to inte ract with one another. 5. We suggest that the most important consequence of oxidative modification is Na/K-ATPase oligomeric structure formation and subsequent hydrolysis ra te suppression.