Fluorescence Spectroscopic Studies of Pressure Effects on Na+,K+-ATPase Reconstituted into Phospholipid Bilayers and Model Raft Mixtures

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• 作者：Ewa Powalska ; Sascha Janosch ; Evamaria Kinne-Saffran ; Rolf K. H. Kinne ; C. F. L. Fontes ; Julio A. Mignaco ; Roland Winter
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：February 13, 2007
• 年：2007
• 卷：46
• 期：6
• 页码：1672 - 1683
• 全文大小：190K
• 年卷期：v.46,no.6(February 13, 2007)
• ISSN：1520-4995

文摘

To contribute to the understanding of membrane protein function upon application of pressureas relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymologicalbiotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na⁺,K⁺-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay thatcouples ATP hydrolysis to NADH oxidation. The data show that the activity of Na⁺,K⁺-ATPase isreversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated.To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstitutedinto various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneityincluding model raft mixtures. To yield additional information on the conformation and phase state of thelipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique weredetermined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order.Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures leadto a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in mostcases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, asignificant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers.Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zeroaround 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect ofpressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressuresensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasinghydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventuallysubunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation,probably because of the dissociation and partial unfolding of the subunits.