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. Pre-incubation of Na+,K+-ATPase with 0.4mM 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.
de Lourdes Barriviera
Maria
M
Departamento de Física, Pontifícia Universidade Católica do Rio de Janeiro, PUC-Rio, Rio de Janeiro, Brazil.
Fontes
Carlos Frederico L
CF
Hassón-Voloch
Aída
A
Louro
Sonia Renaux W
SR
D000818:Animals; D004578:Electron Spin Resonance Spectroscopy; D008560:Membrane Fluidity / Q000187:drug effects*; D008563:Membrane Lipids; D020030:Nitric Oxide Donors / Q000494:pharmacology*; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D026403:S-Nitrosothiols / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013552:Swine
D008563:Membrane Lipids; D020030:Nitric Oxide Donors; D026403:S-Nitrosothiols; C110881:S-nitro-N-acetylpenicillamine; D013113:Spin Labels; D000254:Sodium-Potassium-Exchanging ATPase; D010396:Penicillamine