2-DIMENSIONAL DIFFUSION OF F1F0-ATP SYNTHASE AND ADP ATP TRANSLOCATOR - TESTING A HYPOTHESIS FOR ATP SYNTHESIS IN THE MITOCHONDRIAL INNER MEMBRANE
2-DIMENSIONAL DIFFUSION OF F1F0-ATP SYNTHASE AND ADP ATP TRANSLOCATOR - TESTING A HYPOTHESIS FOR ATP SYNTHESIS IN THE MITOCHONDRIAL INNER MEMBRANE
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DOI：
10.1016/0005-2736(91)90114-n
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发表时间：
1991-11-04
期刊：
影响因子：
--
通讯作者：
HACKENBROCK, CR
中科院分区：
文献类型：
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作者：
GUPTE, SS;CHAZOTTE, B;HACKENBROCK, CR
在这里，我们报告的第一个实验确定的横向扩散系数的F1 F0-ATP合酶和ADP/ATP转运蛋白在大鼠肝线粒体的分离内膜。针对从大鼠肝线粒体分离的F1 F0-ATP合酶开发的兔IgG被确定为对合酶亚基具有免疫特异性，特别是F1的α-β-双联体、γ和δ亚基以及F0的亚基2、3和4。该IgG与丽沙明-罗丹明缀合，用作荧光探针以监测合酶在膜中的扩散。IgG细胞色素bc 1复合物，制备和标记类似，被用作荧光探针扩散的氧化还原组分。曙红马来酰亚胺被确定为特异性标记的ADP/ATP转运蛋白在分离的内膜，并被用作特异性探针的扩散的转运蛋白。利用光漂白后的荧光恢复，F_1F_0-ATP合成酶的实验平均侧向扩散系数为8.4.10（-10）cm ~ 2/s，是细胞色素b_1复合物的2倍，而ADP/ATP转运体的平均侧向扩散系数为1.7.10（-9）cm ~ 2/s，是细胞色素b_1复合物的4倍，这表明三种组分都是独立的。维扩散剂使用这些扩散系数和应用一些基本假设，我们计算了理论上的二维扩散控制的碰撞频率和推导出的碰撞效率（质子转移每碰撞）之间的每三个质子转移的氧化还原复合物和F1 F0-ATP合酶和ADP/ATP转运蛋白通过处理的氧化还原组分作为质子供体和合酶和转运蛋白质子受体。这些碰撞效率支持的物理可能性的扩散为基础的，随机的碰撞过程中的质子转移和ATP的合成在线粒体内膜。
We report here the first experimentally determined lateral diffusion coefficients of the F1F0-ATP synthase and the ADP/ATP translocator in isolated inner membranes of rat liver mitochondria. Rabbit IgG developed against the F1F0-ATP synthase isolated from rat liver mitochondria was determined to be immunospecific for the synthase subunits, notably the alpha-beta-doublet, gamma and delta-subunits of F1 and subunits two, three and four of F0. This IgG, conjugated with lissamine-rhodamine, was used as a fluorescent probe to monitor the diffusion of the synthase in the membrane. IgG to cytochrome bc1 complex, prepared and labeled similarly, was used as a fluorescent probe for diffusion of this redox component. Eosin maleimide was determined to specifically label the ADP/ATP translocator in the isolated inner membrane and was used as a specific probe for the diffusion of the translocator. Using fluorescence recovery after photobleaching, the experimental average lateral diffusion coefficient of the F1F0-ATP synthase was determined to be 8.4.10(-10) cm2/s or twice that of cytochrome bc1 complex while the diffusion coefficient of the ADP/ATP translocator was 1.7.10(-9) cm2/s or four times that of cytochrome bc1 complex suggesting that all three components are independent two-dimensional diffusants. Using these diffusion coefficients and applying a number of basic assumptions, we calculated the theoretical two-dimensional diffusion-controlled collision frequencies and derived collision efficiencies (protons transferred per collision) between each of the three proton-transferring redox complexes and both the F1F0-ATP synthase and ADP/ATP translocator by treating the redox components as proton donors and the synthase and translocator as proton acceptors. These collision efficiencies support the physical possibility of a diffusion-based, random collision process of proton transfer and ATP synthesis in the mitochondrial inner membrane.