Target size of components in oxidative phosphorylation. Studies with a linear accelerator.
Target size of components in oxidative phosphorylation. Studies with a linear accelerator.
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氧化磷酸化组分的目标大小。
DOI：
10.1016/0005-2728(67)90019-9
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发表时间：
1967
期刊：
影响因子：
--
通讯作者：
Y. Kagawa
中科院分区：
文献类型：
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作者：
Y. Kagawa
线粒体膜上氧化磷酸化的实际大小和排列在很大程度上仍不清楚，尽管有几种理论认为线粒体膜上存在形态和功能单位，如“呼吸组装”(Mol.WT.1.4。IO6)1或“基本粒子”(摩尔。WT.1.3。IoE)2，由化学计量的电子传输载体组成。另一方面，Fern/~ndez-Moran a发现了线粒体膜内层直径为87 2~的颗粒，这些颗粒最初被认为代表基本颗粒2，a，但后来发现它们与偶联因子I(F1)，即线粒体ATPase(ATP磷酸水合酶，EC 3.6)相同。4)4，5.然而，固定后观察到的内膜太薄，无法容纳这些单位，而没有固定的内膜可能会被伪影n变形。本工作的目的是通过用高能电子照射线粒体并应用靶理论v来推导各种线粒体酶的分子量来原位测量这些组分的大小。新鲜大鼠肝线粒体悬液(蛋白质：0.25M蔗糖中蛋白质：20 mg/ml)按S所述制成悬浮液，1ml注入直径2.2 cm、深度0.3 cm的玻璃盘中，置于照射野中。在某些情况下，线粒体悬浮液被冷冻干燥，并将相当于1ml(0.107g)的冻干粉放入盘子中。碟子上覆盖了帕拉菲膜，以改善剂量分布。未冷冻样品的温度保持在0℃，冷冻或冻干样品的温度保持在78.5℃。然后用从直线加速器(日本电气公司，东京，NELAC10O6型)均匀发射的6 MeV电子束以大约1mrad/min的剂量率照射I6-2O培养皿，直到总剂量达到所需的值。剂量计用FeSO4溶液进行校准，如文献9所述。照射后，对样品进行如表I所示的酶活性或如表1所述的电子传递活性的测定。根据这一统计超微技术，用下面的方程式计算各酶的相对分子质量。由于酶的密度未知，而辐射效应与密度成正比，本报告中目标尺寸的单位是g/摩尔或g/靶NAvogaaro(即分子或目标重量)：不可能将尺寸表示为厘米3/摩尔。这个方程有两个条件：(I)活性随着剂量的增加而指数下降，而与剂量率无关(单次打击不可逆失活)；(2)间接影响，即由辐照H-CO产生的自由基的失活可以忽略不计(直接击中失活)。方程式是：
The actual size and arrangement of tile components of oxidative phosphorylation in the mitochondrial membrane in situ is still largely unknown, despite several theories on the presence of morphological and functional units in the membrane such as" respiratory assembly"(mol. wt. 1.4. 1o6) 1 or" elementary particle"(mol. wt. 1.3. IOe) 2, which are composed of stoichiometric amounts of electron-transport carriers. On the other hand, particles of 87 2~ diameter lining the inner mitochondrial membrane were discovered by FERN/~ NDEz-MoRAN a. These were initially suggested to represent elementary particles2, a, but were then found to be identical to coupling factor I (F1), ie mitochondrial ATPase (ATP phosphohydrase, EC 3.6. 1.4) 4, 5. However, the inner membrane observed after fixation is too thin to accommodate these latter units, whereas one observed without fixation might be deformed by artifacts n. The aim of this work was to measure the size of these components in situ by irradiating mitochondria with high-energy electrons and applying the target theory v to deduce the molecular weight of the various mitochondrial enzymes. A suspension of fresh rat-liver mitochondria (protein: 2o mg/ml in o. 25 M sucrose) was prepared as described s, and i ml of the suspension was pipetted into a glass dish (diameter 2.2 cm, depth o. 3 cm) which was placed into the field of irradiation. In some cases the mitochondrial suspension was lyophilized and an amount equivalent to i ml (o. lO7 g) of lyophilized powder was put into the dish. The dishes were covered with parafilm to improve dose distribution. The temperature was kept constant at o for unfrozen samples or at 78.5 for frozen or lyophilized ones. I6-2o dishes were then irradiated with a 6-MeV electron beam emitted uniformly from a linear accelerator (Nippon Electric Co., Tokyo, Model NELAC lOO6) at a dose rate of about I Mrad/min, until the total dose reached the desired value. The dosimeter was calibrated with FeSO 4 solution as described elsewhere 9. After the irradiation, samples were assayed for enzyme activity as indicated in Table I or for electrontransport activities as described 1.The molecular weight of each enzyme was calculated from this statistical ultramicrometry with the equation below. As the density of enzymes is unknown, while the radiation effect is proportional to density, the unit of the target size in this report is g/mole or g/target of NAvogaaro (ie molecular or target weight): it is not possible to express the size as cm3/mole. There are two conditions applying to this equation:(I) activity diminishes exponentially with increased dose irrespective of dose rate (single-hit irreversible inactivation);(2) indirect effects, ie inactivation by radicals produced from irradiated H cO, are negligible (direct-hit inactivation). The equation is: