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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 microM; the Kh was equal to 800 microM. 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 Vm, 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.

The results suggest that ROS-induced inhibition of Na+, K+-ATPase activity is involved in the mechanism of postischemic contractile dysfunction and support the view that singlet oxygen may be one of the major causes of oxidative injury during ischemia and reperfusion.

The aims of this study were to determine concentrations of total homocysteine, cysteine, cysteinylglycine and glutathione in spermatozoa, seminal fluid and blood plasma and to analyse their relationships with sperm parameters. For this reason, a new highly effective method of spermatozoa lysis was developed, using methanol, freezing and subsequent thawing in ultrasonic bath. An HPLC-FD assay was conducted on thiols concentrations in lysed spermatozoa, seminal fluid and blood plasma. Concentrations of thiols in spermatozoa were significantly lower in men with normozoospermia than in samples with pathological semen parameters. Statistical analysis found significant correlations between thiol concentrations in spermatozoa and semen parameters, while the same analysis with thiol concentrations in seminal fluid was substantially less powerful. Only cysteinylglycine concentrations in seminal fluid significantly correlated with pathological semen parameters. No significant differences or correlations were found with blood plasma concentrations.