Membrane Mg2+-(Ca2+)-Activated Adenosine Triphosphatase of Escherichia coli: Characterization in the Membrane-Bound and Solubilized States
Membrane Mg2+-(Ca2+)-Activated Adenosine Triphosphatase of Escherichia coli: Characterization in the Membrane-Bound and Solubilized States
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大肠杆菌膜 Mg2-(Ca2)-激活的三磷酸腺苷酶：膜结合和溶解状态的表征
DOI：
10.1128/jb.104.3.1203-1212.1970
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发表时间：
1970
影响因子：
3.2
通讯作者：
D. Evans
中科院分区：
文献类型：
--
作者：
D. Evans
进一步研究了膜结合的镁离子激活和钙离子激活的三磷酸腺苷酶(EC 3.6.1.3；ATPase)活性。一系列阴离子(即硝酸盐、碘化钠、氯化物和醋酸盐)对膜结合的镁-(钙)-三磷酸腺苷酶的抑制程度与这些阴离子的相对杂化或增溶效果有关。在pH 9.0和37℃条件下，用0.04%十二烷基硫酸钠从洗涤的膜影中溶解该酶。溶解的镁离子-(钙)-三磷酸腺苷酶的活性先是升高，然后是相当快的失活，这两种三磷酸腺苷酶的活性都特别不稳定。十二硫醇、0.01M tris(hydroxymethyl)amino-methane-hydrochloride缓冲液(pH 9.0)、0.2mM氯化镁和环境温度的联合稳定作用促进了酶的部分纯化，凝胶过滤技术估计其相对分子质量约为100,000。总的来说，大肠埃希菌的膜相关的镁-(钙)-ATPase既类似于线粒体膜ATPase，也类似于巨大芽孢杆菌和溶血微球菌的膜ATPase。特别值得注意的是，已知的线粒体ATPase、线粒体氧化磷酸化和粪链球菌膜结合型Mg2+-ATPase的抑制剂N，N‘-二环己基碳二亚胺(DCCD)既能抑制大肠杆菌的膜结合型，又能抑制其溶解形式。大肠埃希菌膜相关的镁-(Ca~(2+))-ATPase对阴阳离子的敏感性，它的异位行为，以及它对DCD抑制的敏感性，支持这种酶在膜的氧化磷酸化和离子转运等生物活性中起关键作用的观点，可能是多功能的。
The membrane-associated Mg2+-activated and Ca2+-activated adenosine 5′-triphosphatase (EC 3.6.1.3; ATPase) activities of Escherichia coli were further characterized. The degree of inhibition of membrane-bound Mg2+-(Ca2+)-ATPase by a series of anions (i.e., sodium salts of nitrate, iodide, chloride, and acetate) was found to correlate with the relative chaotropic, or solubilizing, effectiveness of these anions. The enzyme was solubilized from washed membrane ghosts by treatment with 0.04% sodium lauryl sulfate at pH 9.0 and 37 C. Solubilized Mg2+-(Ca2+)-ATPase exhibited an initial increase in activity, followed by fairly rapid inactivation, both ATPase activities being particularly cold-labile. The combined stabilizing effects of lauryl mercaptan (1-dodecanethiol), 0.01 m tris(hydroxymethyl)amino-methane-hydrochloride buffer (pH 9.0), 0.2 mm MgCl2, and ambient temperature facilitated partial purification of the enzyme, the molecular weight of which was estimated to be approximately 100,000 by the gel filtration technique. In general, the membrane-associated Mg2+-(Ca2+)-ATPase of E. coli resembles both mitochondrial membrane ATPase and the well-characterized membrane ATPases of Bacillus megaterium and Microcococcus lysodeikticus. It is of particular interest that N,N′-dicyclohexylcarbodiimide (DCCD), a known inhibitor of mitochondrial ATPase, of mitochondrial oxidative phosphorylation, and of the membrane-bound Mg2+-ATPase of Streptococcus faecalis was found to inhibit both the membrane-bound and the solubilized forms of E. coli Mg2+-(Ca2+)-ATPase. The sensitivity of the membrane-associated Mg2+-(Ca2+)-ATPase of E. coli to both anions and cations, its allotopic behavior, and its susceptibility to inhibition by DCCD favor the idea that this enzyme plays a key, probably polyfunctional, role in such biological activities of the membrane as oxidative phosphorylation and ion transport.