Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures.
Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures.
复制标题
荧光光谱研究压力对重构为磷脂双层和模型筏混合物的 Na ,K( )-ATP 酶的影响。
作者：
Ewa Powalska;S. Janosch;E. Kinne;R. Kinne;Carlos Frederico Leite Fontes;J. Mignaco;R. Winter
为了有助于理解施加压力时膜蛋白的功能，例如深海生物的生理学或压力酶生物技术过程，我们使用将 ATP 水解与 NADH 氧化耦合的动力学测定法，研究了静水压对兔肾外髓质膜中富集的 Na+,K+-ATP 酶活性的影响。数据显示，Na+,K+-ATP酶的活性被低于2kbar的压力可逆地抑制。在较高压力下，酶不可逆地失活。为了能够探索脂质基质对酶活性的影响，酶也被重构为不同链长、构象、相态和异质性的各种脂质双层系统，包括模型筏混合物。为了获得有关脂质双层系统的构象和相状态的更多信息，还通过劳丹荧光技术确定了广义偏振值。酶的掺入导致脂质链顺序显着增加。一般来说，与天然质膜中的酶活性类似，高静水压会导致重组到各种脂质双层系统中的酶活性下降，并且在大多数情况下，观察到多相行为。有趣的是，在约 100 bar 的低压区域，观察到重组为 DMPC 和 DOPC 双层的酶的活性显着增加。高于 100-200 bar，活性增强，随后活性急剧下降直至约 800 bar，此时达到或多或少宽阔的平台值。对于所有测量的重构系统，酶活性在 2 kbar 左右降至零。观察到压力对模型筏混合物中酶活性的影响的不同情况。液体有序域和液体无序域的共存以及脂质混合物中脂质分选的可能性导致中压范围内的压力敏感性降低。 ATP酶活性的降低可能是由于疏水性错配的增加而引起的，从而导致蛋白质构象动力学的降低并最终导致亚基重排。高于约 2.2 kbar 的高压会不可逆地改变蛋白质构象，这可能是由于亚基的解离和部分展开所致。
To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical 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 that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly 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 reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined 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 lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant 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 about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure 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 pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.