Peschek G A, Wastyn M, Trnka M, Molitor V, Fry I V, Packer L
Institute of Physical Chemistry, University of Vienna, Austria.
Biochemistry. 1989 Apr 4;28(7):3057-63. doi: 10.1021/bi00433a048.
Functionally intact plasma membranes were isolated from the cyanobacterium (blue-green alga) Anacystis nidulans through French pressure cell extrusion of lysozyme/EDTA-treated cells, separated from thylakoid membranes by discontinuous sucrose density gradient centrifugation, and purified by repeated recentrifugation. Origin and identity of the chlorophyll-free plasma membrane fraction were confirmed by labeling of intact cells with impermeant protein markers, [35S]diazobenzenesulfonate and fluorescamine, prior to membrane isolation. Rates of oxidation of reduced horse heart cytochrome c by purified plasma and thylakoid membranes were 90 and 2 nmol min-1 (mg of protein)-1, respectively. The cytochrome oxidase in isolated plasma membranes was identified as a copper-containing aa3-type enzyme from the properties of its redox-active and EDTA-resistant Cu2+ ESR signal, the characteristic inhibition profile, reduced minus oxidized difference spectra, carbon monoxide difference spectra, photoaction and photodissociation spectra of the CO-inhibited enzyme, and immunological cross-reaction of two subunits of the enzyme with antibodies against subunits I and II, and the holoenzyme, of Paracoccus denitrificans aa3-type cytochrome oxidase. The data presented are the first comprehensive evidence for the occurrence of aa3-type cytochrome oxidase in the plasma membrane of a cyanobacterium similar to the corresponding mitochondrial enzyme (EC 1.9.3.1).
通过对溶菌酶/乙二胺四乙酸处理过的细胞进行法式压榨细胞挤出操作，从蓝细菌（蓝绿藻）集胞藻中分离出功能完整的质膜，通过不连续蔗糖密度梯度离心将其与类囊体膜分离，并通过反复再离心进行纯化。在膜分离之前，用非渗透性蛋白质标记物[35S]重氮苯磺酸盐和荧光胺对完整细胞进行标记，从而证实了无叶绿素质膜部分的来源和特性。纯化的质膜和类囊体膜氧化还原型马心脏细胞色素c的速率分别为90和2 nmol min-1（mg蛋白质）-1。从其氧化还原活性和抗乙二胺四乙酸的Cu2+电子自旋共振信号特性、特征性抑制谱、还原态减去氧化态差光谱、一氧化碳差光谱、一氧化碳抑制酶的光作用和光解离光谱，以及该酶的两个亚基与针对反硝化副球菌aa3型细胞色素氧化酶的亚基I和II以及全酶的抗体的免疫交叉反应，鉴定出分离的质膜中的细胞色素氧化酶为含铜的aa3型酶。所呈现的数据是关于蓝细菌质膜中存在与相应线粒体酶（EC 1.9.3.1）相似的aa3型细胞色素氧化酶的首个全面证据。
