Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E.
The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.
Thomas
C E
CE
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331-6503.
Reed
D J
DJ
D000818:Animals; D004285:Dogs; D005609:Free Radicals; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006878:Hydroxides / Q000494:pharmacology*; D017665:Hydroxyl Radical; D007501:Iron / Q000494:pharmacology; D007668:Kidney / Q000201:enzymology; D015227:Lipid Peroxidation / Q000187:drug effects; D008081:Liposomes; D008563:Membrane Lipids / Q000378:metabolism*; D008567:Membranes, Artificial; D010084:Oxidation-Reduction; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D014810:Vitamin E / Q000494:pharmacology*
C045076:Fenton's reagent; D005609:Free Radicals; D006878:Hydroxides; D008081:Liposomes; D008563:Membrane Lipids; D008567:Membranes, Artificial; D014810:Vitamin E; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D007501:Iron; D000254:Sodium-Potassium-Exchanging ATPase