engleski

Na+, K+-ATPase submembrane localization and activity reflects redistribution of gangliosides and cholesterol following thermally induced membrane reorganization

Na+/K+-ATPase (NKA), an enzyme expressed by all mammalian cells, is crucial for maintenance of ion homeostasis. Being a membrane protein, its positioning as well as different functions of active and inactive pools depend on interactions with neighboring membrane lipids. Ganglioside and cholesterol enriched lipid rafts (LR) are housing active, pumping pool, while the bulk membrane (non-LR) contains the non-pumping pool1. Aim of this study was to ascertain NKA activity and submembrane localization after multiple cycles of thermally induced membrane reorganization. Homogenates from mouse brain cortices were subjected to multiple freeze-thaw cycles. NKA activity was measured spectrophotometrically, followed by Western blot quatification of protein amount for each corresponding cycle. Submembrane localization of NKA was analyzed in time points with biggest difference in activity by conducting LR isolation utilizing ultracentrifugation in discontinous sucrose density gradients. Gangliosides from LR and non-LR fractions were purified by newly developed method including organic extraction, DEAE exchange chromatography, gel-filtration, separation by high performance thin layered chromatography and visualization by immunoblot. Cholesterol concentration in submembrane fractions was determined spectrophotometrically. Our results indicate that oscillations in NKA activity are attributed to different lipid environment, even though the total amount of NKA was constant through all cycles. Subfractionation of the membrane showed that NKA is shifted trough different lipid environments, with different cycles having distinct distribution of NKA between LR and non-LR fractions. Analysis of ganglioside and cholesterol content led us to conclude that lipid environment aid NKA positioning within the membrane and therefore optimal function of the pump. 1. Puljko B, Stojanović M, Ilic K, i ostali. Redistribution of gangliosides accompanies thermally induced Na+, K+-ATPase activity alternation and submembrane localisation in mouse brain. Biochim Biophys Acta - Biomembr 2021. ; 1863: 183475.

lipid rafts ; glycosphingolipids ; sodium potassium ATPase

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