Influence of nitric oxide donors on the intrinsic fluorescence of Na+,K+-ATPase and effects on the membrane lipids

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Preincubation of Na+,K+-ATPase with 0.4 mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein-lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols. (c) 2005 Published by Elsevier Inc.