Department of Biochemistry, Chulalongkorn University, Bangkok, Thailand.
BMC Biochem. 2010 Aug 7;11:30. doi: 10.1186/1471-2091-11-30.
When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na+ from cells to maintain the cytoplasmic Na+ concentration. Until now, the ATPase involved in Na+ transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na+ transport of alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity conditions.
The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+ but not K+, Li+ and Ca2+. The apparent Km values for Na+ and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F1F0-ATPase based on the usual subunit pattern and the protection against N,N'-dicyclohexylcarbodiimide inhibition of ATPase activity by Na+ in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na+ upon the addition of ATP. The apparent Km values for this uptake were 3.3 and 0.5 mM for Na+ and ATP, respectively. The mechanism of Na+ transport mediated by Na+-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na+-stimulated ATPase was observed upon the addition of ATP with Na+ but not with K+, Li+ and Ca2+. The Na+- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP) but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na+ uptake activity. The uptake of Na+ was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na+-stimulated ATPase of the proteoliposomes.
The data demonstrate that Na+-stimulated ATPase from A. halophytica, a likely member of F-type ATPase, functions as an electrogenic Na+ pump which transports only Na+ upon hydrolysis of ATP. A secondary event, Na+- and ATP-dependent H+ efflux from proteoliposomes, is driven by the electric potential generated by Na+-stimulated ATPase.
当细胞暴露在高盐环境中时，它们会发展出一种机制，将多余的 Na+从细胞中排出，以维持细胞质中的 Na+浓度。到目前为止，尚未对蓝藻中参与 Na+运输的 ATP 酶进行鉴定。本研究旨在对嗜碱耐盐的鱼腥藻 Aphanothece halophytica 的 ATP 酶进行鉴定及其在 Na+运输中的作用进行研究，以了解 A. halophytica 在高盐条件下的生存机制。
纯化的酶在有 Na+存在的情况下催化 ATP 的水解，但不能催化 K+、Li+和 Ca2+的水解。Na+和 ATP 的表观 Km 值分别为 2.0 和 1.2 mM。该酶可能是 F1F0-ATP 酶，基于常见的亚基模式以及 Na+在依赖 pH 的方式下对 ATP 酶活性的 N,N'-二环己基碳二亚胺抑制的保护作用。用纯化的酶重建的脂质体在加入 ATP 时可以摄取 Na+。该摄取的表观 Km 值分别为 3.3 和 0.5 mM 用于 Na+和 ATP。揭示了 A. halophytica 中 Na+-刺激的 ATP 酶介导的 Na+运输的机制。用吖啶橙作为探针，在用 Na+和 ATP 而不是用 K+、Li+和 Ca2+处理时，观察到用 Na+-刺激的 ATP 酶重建的脂质体腔中的碱化。脂质体腔的 Na+-和 ATP 依赖性碱化被羰基氰化物 m-氯苯腙 (CCCP) 刺激，但被可渗透的阴离子硝酸盐抑制。脂质体显示出 ATP 酶活性和 ATP 依赖性 Na+摄取活性。CCCP 和硝酸盐均增强了 Na+的摄取。另一方面，CCCP 和硝酸盐都被证明可以耗散由脂质体中 Na+-刺激的 ATP 酶产生的预形成电势。
数据表明，鱼腥藻 A. halophytica 的 Na+-刺激的 ATP 酶是 F 型 ATP 酶的一个可能成员，作为一种电致性 Na+泵，在水解 ATP 时仅运输 Na+。脂质体中 Na+-和 ATP 依赖性 H+的外排是由 Na+-刺激的 ATP 酶产生的电势驱动的。
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