METHOD FOR THE LOCALIZATION OF SITES FOR OXIDATIVE PHOSPHORYLATION
METHOD FOR THE LOCALIZATION OF SITES FOR OXIDATIVE PHOSPHORYLATION
复制标题
DOI：
10.1038/176250a0
复制
发表时间：
1955-01-01
期刊：
影响因子：
64.8
通讯作者：
WILLIAMS, GR
中科院分区：
文献类型：
--
作者：
CHANCE, B;WILLIAMS, GR
参与氧化磷酸化的呼吸链的部分通过阻断链的一部分的抑制剂或通过仅激活链的一部分的特定底物的分离显示磷酸化发生在还原的二磷酸吡啶核苷酸和氧之间，以及细胞色素c和氧之间。更精确的位点定位受到呼吸链所有部分的特异性底物和抑制剂的可用性的限制。我们的方法是基于氧化磷酸化起始对完整线粒体呼吸链组分稳态的影响的直接光谱观察。当向提供底物和磷酸盐的线粒体中加入腺苷二磷酸后呼吸突然增加时，我们观察到稳定状态下沿着呼吸链从脱氢酶部分的氧化到氧化酶部分的还原的变化的一致梯度”。根据这些观察结果，我们可以鉴定完整线粒体系统中氧化磷酸化的位点。根据Lardy和Wellman修改的Schneider方法制备线粒体。在通常的“等渗”介质中研究反应。以3-羟基丁酸酯为底物，制备物的磷/氧值约为3”。为了记录光谱变化的动力学，我们使用双光束差示分光光度计，其中两个单色器设置在由待测量的吸收带的半宽度不同的波长处。由于仅测量吸收的差异，因此记录对线粒体中发生的光透射的非特异性变化相对不敏感。磷酸化活性和呼吸通过基于振动铂微电极的技术同时监测。在记录中使用的波长对的组成部分，
SOLATION of portions of the respiratory chain involved in oxidative phosphorylation either by inhibitors that block a portion of the chain or by specific substrates that activate only a portion of the chain show that phosphorylation occurs between reduced diphosphopyridine nucleotide and oxygen", and cytochrome c and oxygen"*. A more exact localization of sites is limited by the availability of specific substrates and inhibitors for all parts of the respiratory chain. Our approach is based upon direct spectroscopic observations of the effect of the initiation of oxidative phosphorylation upon the steady state of the components of the respiratory chain of intact mitochondria". When respiration abruptly increases upon addition of adenosine diphosphate to mitochondria supplied with substrate and phosphate, we observe a consistent gradation of the changes in the steady state along the respiratory chain from an oxidation at the dehydrogenase portion to a reduction of the oxidase portion". On the basis of these observations, we can identify sites for oxidative phosphorylation in the intact mitochondrial system.The mitochondria were prepared according to the method of Schneiderº as modified by Lardy and Wellman". The reactions were studied in the usual “isotonic” medium. The preparations had values of phosphorus/oxygen of about 3 with 3-hydroxybutyrate as a substrate". For recording the kinetics of spectroscopic changes we use a double-beam differential spectrophotometer with the two monochromators set at wave-lengths differing by the half-width of the absorption band to be measured. Since only the difference of absorption is measured, the recording is relatively insensitive to non-specific changes of light transmission that occur in the mitochondria". The phosphorylative activity and the respiration are monitored simultaneously by a technique based upon the vibrating platinum microelectrode**. The pairs of wave-lengths that were used in the recording of the components of the