MOTIONAL DYNAMICS OF FUNCTIONAL CYTOCHROME-C DELIVERED BY LOW PH FUSION INTO THE INTERMEMBRANE SPACE OF INTACT MITOCHONDRIA
MOTIONAL DYNAMICS OF FUNCTIONAL CYTOCHROME-C DELIVERED BY LOW PH FUSION INTO THE INTERMEMBRANE SPACE OF INTACT MITOCHONDRIA
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DOI：
10.1016/0005-2728(93)90102-l
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发表时间：
1993-04-05
期刊：
影响因子：
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通讯作者：
HACKENBROCK, CR
中科院分区：
文献类型：
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作者：
CORTESE, JD;HACKENBROCK, CR
我们研究了细胞色素c在完整的功能性大鼠肝细胞中的运动动力学。为此，功能性的FITC-细胞色素c（异硫氰酸荧光素单衍生的细胞色素c）通过将细胞色素c封装到asolectin脂质体中，然后通过低pH诱导的脂质体与线粒体外膜融合，被掺入完整线粒体的膜间隙（IMS）中。获得了15%-50%之间的细胞色素c控制富集（每个分子掺入1800-7200个分子）。所有的细胞色素c纳入，无论数量，参与电子传递的功能，指示功能，独立的随机扩散。共振能量转移测定从IMS截留的功能性FITC-细胞色素c的十八烷基罗丹明B纳入线粒体膜。共振能量转移从FITC-细胞色素c到十八烷基罗丹明B在分离的线粒体内膜或外膜（IMM和OMM，分别）也进行了测量。我们发现离子强度（I）对细胞色素c与孤立的IMM和OMM的接近度的影响存在实质性差异。与孤立IMM的相互作用非常动态，即，非常依赖于I，细胞色素c与IMM的结合仅在非常低的I下才显著。I依赖的相互作用的细胞色素c与孤立的OMM的I依赖性比IMM。然而，FITC-细胞色素c基本上是从IMM和OMM在生理I释放。在不同的外部体积I下，使用以下方法估计了FITC-细胞色素c在以凝聚构型掺入完整线粒体的IMS中后与每个线粒体膜的接近程度：（a）从IMS-截留的FITC-细胞色素c到均匀分布在两个线粒体膜中的十八烷基罗丹明B-标记的共振能量转移;和（B）从IMS包埋的FITC-细胞色素c到浓缩在OMM中的十八烷基罗丹明B-标记的共振能量转移。共振能量转移表明，细胞色素c和两个IMS-膜表面之间的平均距离随着IMS-I的增加而增加，在150 mM I下接近85埃的最大可测量距离。该结果与FITC-细胞色素c和完整线粒体的两个膜在生理1，即，当细胞色素C在电子传递中的活性最高时。我们的研究结果揭示了一个主要的三维扩散模式IMS-细胞色素c在其功能的电子传递在完整的线粒体在生理I，并提供了进一步的证据，线粒体电子传递是一个过程，其独立扩散的电子传递，氧化还原组分之间的随机碰撞驱动。
We have investigated the motional dynamics of cytochrome c in the intact, functional rat liver mitochondrion. To do this, functional, FITC-cytochrome c (fluorescein isothiocyanate monoderivatized cytochrome c) was incorporated into the intermembrane space (IMS) of intact mitochondria through encapsulation of cytochrome c into asolectin liposomes followed by low pH-induced fusion of the liposomes with the outer membranes of the mitochondria. A cytochrome c controlled enrichment of between 15%-50% (1800-7200 molecules incorporated per mitochondrion) was obtained. All cytochrome c incorporated, regardless of the quantity, participated in the function of electron transport, indicative of a functional, independent random diffusant. Resonance energy transfer was determined from the IMS-entrapped functional FITC-cytochrome c to octadecylrhodamine B incorporated into the mitochondrial membranes. Resonance energy transfer from FITC-cytochrome c to octadecylrhodamine B in isolated inner or outer mitochondrial membranes (IMM and OMM, respectively) was also measured. We found substantial differences in the effects of ionic strength (I) on the proximity of cytochrome c to isolated IMM and OMM. Interactions with isolated IMM were very dynamic, i.e., very I-dependent, and cytochrome c binding to IMM was significant only at very low I. I-dependent interactions of cytochrome c with isolated OMM were less I-dependent than those for the IMM. However, FITC-cytochrome c was essentially released from IMM and OMM at physiological I. The proximity of FITC-cytochrome c to each mitochondrial membrane after its incorporation into the IMS of intact mitochondria in the condensed configuration was estimated at different external, bulk I using: (a) resonance energy transfer from IMS-entrapped FITC-cytochrome c to octadecylrhodamine B-label evenly distributed in both mitochondrial membranes; and (b) resonance energy transfer from IMS-entrapped FITC-cytochrome c to octadecylrhodamine B-label concentrated in the OMM. Resonance energy transfer showed that the average distance between cytochrome c and the two IMS-membrane surfaces increased with increasing IMS-I, approaching a maximal measurable distance of 85 angstrom at 150 mM I. This result is consistent with a dissociation of FITC-cytochrome c and both membranes of intact mitochondria at physiological 1, i.e., when the activity of cytochrome c in electron transport is highest. Our findings reveal a primarily three-dimensional diffusion mode for IMS-cytochrome c during its function in electron transport in intact mitochondria at physiological I, and offer further evidence that mitochondrial electron transport is a process driven by random collisions between its independently diffusing electron transferring, redox components.