Oxidative resistance of Na/K-ATPase.

1. Oxidative modification of Na/K-ATPase from brain and kidney has been studied. Brain enzyme has been found to be more sensitive than kidney enzyme to inhibition by both H2O2 and NaOCl. 2. The inhibition of Na/K-ATPase correlates well with the decrease in a number of SH groups, suggesting that the latter belong mainly to ATPase protein and are essential for the enzyme activity. We suggest that the differences in the number, location, and accessibility of SH groups in Na/K-ATPase isozymes predict their oxidative stability. 3. The hydrophilic natural antioxidant carnosine, the hydrophobic natural antioxidant alpha-tocopherol, and the synthetic antioxidant ionol as well as the ferrous ion chelating agent deferoxamine were found to protect Na/K-ATPase from oxidation by different concentrations of H2O2. The data suggest that these antioxidants are effective due to their ability to neutralize or to prevent formation of hydroxyl radicals.

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 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.

Quenching of singlet molecular oxygen by carnosine and related antioxidants. Monitoring 1270-nm phosphorescence in aqueous media.

In order to elucidate the biochemical roles of imidazol-containing dipeptides, we have studied quenching of singlet molecular oxygen (1O2) by carnosine (beta-alanyl-L-histidine), its structural components (L-histidine, imidazole, and beta-alanine), and related natural free-radical scavengers-L-anserine (beta-alanyl-1-methyl-histidine), ergothioneine (2-thiol-L-histidine-betaine), and taurine (2-aminoethanesulfonic acid) in aqueous (D2O, pD 7) solutions by using monitoring of 1O2-phosphorescence (1270-nm). The rate constants of 1O2 quenching (Kq) by carnosine, anserine, and ergothioneine were shown to be similar [(3 +/- 1) x 10(7) M-1s-1]. Their values resembled those of free-L-histidine [Kq = (4 +/- 1) x 10(7) M-1s-1] and imidazole [Kq = (2 +/- 1) x 10(7) M-1s-1]. Non-aromatic amino acids-taurine and beta-alanine-showed very low quenching activities (Kq < 3 x 10(3) M-1c-1). The Kq values did not correlate with the literature data on abilities of the tested compounds to stimulate muscle working capacities and inhibit myeloperoxidase-catalyzed oxygenation. Thus, the dipeptides can be used as potent water-soluble protectors against 1O2 attack whereas their natural biochemical functions are most probably determined by the processes of different nature.

Mechanism of oxidative damage of dog kidney Na/K-ATPase.

Oxidative modification of kidney Na/K-ATPase was found to be accompanied by a decrease in the amount of sulfhydryl groups accessible for Elmann reagent with subsequent transformation of kinetic behavior of the enzyme. Oxidation of Na/K-ATPase with 20 mM hydrogen peroxide during 20 min results in about 50% inhibition of its activity and subsequent transformation of complex substrate-velocity dependence into the simple hyperbolic curve. In terms of kinetic analysis the suggestion was made that partial oxidation of Na/K-ATPase by hydrogen peroxide results in disordering of interprotomer interaction in the oligomeric complex of Na/K-ATPase.

Inhibition of Na+/K(+)-ATPase by phenoxyl radicals of etoposide (VP-16): role of sulfhydryls oxidation.

In the present work, we studied the effects of phenoxyl radicals, generated by tyrosinase-catalyzed oxidation of a phenolic antitumor drug, Etoposide (VP-16), on a purified dog kidney Na+/K(+)-ATPase by characterizing interactions of VP-16 phenoxyl radicals with the enzyme's SH-groups by ESR and correlating the loss of the enzymatic activity with the oxidation of its SH-groups, and oxidation of VP-16. VP-16/tyrosinase caused inhibition of Na+/K(+)-ATPase which was dependent on the incubation time and concentration of tyrosinase. The inhibition of Na+/K(+)-ATPase was accompanied by a decrease of DTNB (5,5'-dithiobis-(2-nitrobenzoic acid)-titratable SH-groups. In the presence of Na+/K(+)-ATPase, a typical ESR signal of the VP-16 phenoxyl radical could be observed only following a lag period the duration of which was proportional to the concentration of the Na+/K(+)-ATPase added. Our HPLC measurements demonstrated that Na+/K(+)-ATPase protected VP-16 against tyrosinase-catalyzed oxidation. Combined these results suggest that redox-cycling of VP-16/VP-16 phenoxyl radical by SH-groups of Na+/K(+)-ATPase occurred. Ascorbate which is known to reduce the VP-16 phenoxyl radicals, protected the enzyme against inactivation, prevented oxidation of the enzyme's SH-groups. Reduction of VP-16 phenoxyl radicals by ascorbate was directly observed by the semidehydroascorbyl radical signal in the ESR spectra. VP-16 phenoxyl radical-induced oxidation of sulfhydryls and inhibition of the Na+/K(+)-ATPase may be responsible for at least some of its clinical side effects (e.g., cardiotoxicity) which can be prevented by ascorbate.

[The effect of hydrogen peroxide and hypochlorite on brain Na,K-ATPase activity]. / Vliianie peroksida vodoroda i gipokhlorita na aktivnost' Na,K-ATPazy mozga.

Brain Na,K-ATPase is subjected to direct modifying influence of the hypochlorite anion and hydrogen peroxide resulting in irreversible inactivation of the enzyme. The K0.5 values for these compounds are 20-40 microM and 1 mM, respectively. The inhibiting effect of hydrogen peroxide is suppressed by desferrioxamine (by 20-30%) which indicates that the H2O2-induced inhibition is partly due to the generation of the hydroxyl radical. Carnosine, homocarnosine and the hydrophobic antioxidants, alpha-tocopherol and ionol, protect the enzyme from H2O2: their effective concentrations are to 10 mM (carnosine), 5 microM (alpha-tocopherol), and 0.5 microM (ionol). The data obtained suggest that inhibition of Na,K-ATPase by the antioxidants is caused by their direct action on the protein molecule, while the protective effect of natural hydrophilic antioxidants are due to the quenching of the hydroxyl radical.

[A comparison of the antioxidative activity of carnosine by using chemical and biological models]. / Sravnenie antiokislitel'noi aktivnosti karnozina s ispol'zovaniem khimicheskikh i biologicheskikh modelei.

The difference in the efficiency of carnosine as an antioxidant was found to be explained both by the source of carnosine and the specificity of models used to achieve visualization. Commercial carnosine samples were contaminated with compound (s) absorbing at 255-332 nm. At the same time they possessed better antioxidant activity in the models with Fe2-induced peroxidation process. In the case of chemical models for generation of active forms of oxygen (several modifications of the Fenton reaction) or during burst of superoxide generation by leucocytes, the antioxidant effect of carnosine did not depend of the source of the compound under study.

Natural histidine-containing dipeptide carnosine as a potent hydrophilic antioxidant with membrane stabilizing function. A biomedical aspect.

A review on the distribution and biological effects of carnosine and a hypothesis for its biological mechanisms of action are presented. Carnosine and its structural and functional relative, anserine, were found in skeletal muscles at the beginning of the century. Their effects on muscle-working capacity, on the stability of membrane-bound enzymes, as well as their potent immunomodulating property, could not be explained by their pH-buffering capacity or formation of the secondary metabolites histidine and beta-alanine alone. This article suggests that the basis for the biological activities of carnosine and relative compounds is their potent antioxidant and membrane-protecting activity. The plausible chemical mechanism of this activity is discussed, and data regarding the usage of carnosine as a drug for treatment of immunodeficiency are summarized.

[Direct measurement of the interaction of carnosine and its analogs with free radicals]. / Priamoe izmerenie vzaimodeistviia karnozina i ego analogov so svobodnymi radikalami.

An ESR study of interactions of carnosine and its derivatives with free radicals has been carried out. In model systems the spin trap OH. radical adduct generation has been shown to decrease significantly in the presence of carnosine in a pronounced concentration-dependent manner. A comparative study of effects of some other histidine-containing dipeptides on this process has revealed a similarity in anserine, homocarnosine, and acetylcarnosine actions.

[Stimulating effects of carnosine on hemopoietic stem cells]. / Stimuliruiushchee deistvie karnozina na gemopoéticheskie stvolovye kletki.

It was shown that intake of carnosine in a dose of 50-100 mg/kg of body weight before X-ray irradiation resulted in an increase of the survival of experimental mice. The protective effect of carnosine was manifested, when it was injected either before or after irradiation, but the effect was more pronounced in the case of shortening time between irradiation and injection. An enhancement of colony forming index of bound cells in spleen was also observed simultaneously with protective action of carnosine. These effects are supposed to be the result of immunomodulating activity of carnosine.