engleski

The effect of membrane integrity disruption by freeze-thaw cycles on Na+/K+-ATPase activity and submembrane localization

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 are highly dependent on the interactions with neighboring membrane lipids. In this study we initially aimed to optimize conditions for spectrophotometric enzyme activity determination in brain tissue homogenates of wild type (wt) mice. More specifically, we analyzed the decrease in NKA activity after repeated freeze and thaw cycles. Dissected cortical tissue from wt mice was snap frozen in liquid nitrogen and stored at -80˚C until use and repeated spectrophotometric measurements showed changes of enzyme activity that corresponded to the number of freeze-thaw cycles. However, we detected an unexpected pattern of decreased followed by increased NKA enzyme activity after several freeze-thaw cycles. We hypothesized that this surprising rise in NKA activity is a consequence of NKA liberation from the inactive pool due to partial membrane integrity disruption caused by local interruption of intermolecular forces between NKA and lipids membrane’s components. To test this hypothesis we performed Western blotting to detect total NKA quantity after every freeze-thaw cycle. Western blotting showed that the total immunoreactivity of NKA stays the same, even though the NKA activity is significantly different after a specific number of freeze-thaw cycles. To further investigate the potential submembrane redistribution of NKA, we performed lipid rafts isolation after specific number of freeze-thaw cycles. The isolation of lipid rafts was performed from wt mice cortical homogenate using the nonionic detergent Brij O20 and sucrose density gradients ultracentrifugation. Successful lipid raft isolation was confirmed by Western blotting for known lipid raft markers, flotilin and ganglioside GM1, in addition to NKA. The results of lipid raft analysis showed that the submembrane localization of NKA undeniably changes after a specific number of freeze-thaw cycles, which corresponds to changes in NKA activity. In conclusion, this preliminary study revealed that modulation of membrane integrity causes the disruption of fine intermolecular interactions leading to changes in NKA localization to distinct membrane microregions and consequent change in NKA activity. Keeping in mind that NKA not only serves as an electrogenic pump, but has multiple roles in signaling pathways, and the consequences of impaired NKA function are extensive and severe, the possibility to modulate NKA pumping and signaling properties is attractive and gives a new prospect in the research of NKA linked disorders.

lipid rafts ; ATPase

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