Lê-Quôc D, Lê-Quôc K
Laboratoire de Biochimie, UA CNRS 531, UFR Sciences et Techniques, Besançon, France.
Arch Biochem Biophys. 1989 Sep;273(2):466-78. doi: 10.1016/0003-9861(89)90506-7.
Dysfunction of mitochondria after oxidation of endogenous NAD(P)H, especially after calcium accumulation, has been abundantly reported, but the causes of membrane perturbations did not receive a full explanation. In light of several additional observations reported in this study, we propose a general scheme which shows the sequential processes that are likely involved in the appearance of calcium-induced membrane leakiness. Addition of acetoacetate, oxaloacetate, or ketomalonate to rotenone-treated mitochondria led to a massive oxidation of both NADH and NADPH. Under these conditions, stimulation of fatty acid oxidation could be observed. This process was shown to be accompanied by a reduction of intramitochondrial NADP+. The reduction of NADP+ was inhibited by uncouplers, electron transfer inhibitors and N,N'-dicyclohexylcarbodiimide. It was thus probably catalyzed by the mitochondrial transhydrogenase. Oxidation of pyridine nucleotides in the presence of acetoacetate induced (i) a slight decrease in the number of sulfhydryl groups reactive with N-ethylmaleimide (but no change in the amount of intramitochondrial reduced glutathione) and (ii) modifications of the kinetics and the orientation of the ADP/ATP carrier. In the presence of calcium ions, acetoacetate-stimulated fatty acid oxidation promoted an extensive swelling of mitochondria. Uptake of calcium ions into the matrix was a critical factor for triggering the swelling. Thiols, if they were added at a sufficiently high concentration, suppressed the swelling. Also ligands of the ADP/ATP carrier which stabilized the m-state conformation of the protein, exerted an efficient protective action. Three essential interacting factors emerge from this study: (i) The crucial role of the ADP/ATP carrier orientation in promoting the calcium-induced membrane destabilization. More precisely, it has been shown that the ADP/ATP carrier adopts the c-state conformation (i.e., nucleotide binding site facing the cytoplasm) during fatty acid oxidation. (ii) The modification of a very small number of sulfhydryl groups of mitochondrial protein. These groups are probably in an oxidized state when the level of reduced pyridine nucleotides is low. (iii) The prevailing role of the transhydrogenase, the function of which is also intimately associated with fatty acid oxidation. After energization, transhydrogenase can hinder thiol oxidation and therefore partially protect the membrane structure.
内源性NAD(P)H氧化后，尤其是钙积累后线粒体功能障碍已有大量报道，但膜扰动的原因尚未得到充分解释。根据本研究报告的其他一些观察结果，我们提出了一个总体方案，该方案展示了钙诱导膜渗漏出现可能涉及的一系列过程。向经鱼藤酮处理的线粒体中添加乙酰乙酸、草酰乙酸或酮丙二酸会导致NADH和NADPH大量氧化。在这些条件下，可以观察到脂肪酸氧化受到刺激。该过程伴随着线粒体内NADP+的减少。NADP+的减少受到解偶联剂、电子传递抑制剂和N,N'-二环己基碳二亚胺的抑制。因此，它可能是由线粒体转氢酶催化的。在乙酰乙酸存在下吡啶核苷酸的氧化诱导了：(i)与N-乙基马来酰亚胺反应的巯基数量略有减少（但线粒体内还原型谷胱甘肽的量没有变化）以及(ii)ADP/ATP载体的动力学和方向发生改变。在钙离子存在下，乙酰乙酸刺激的脂肪酸氧化促进了线粒体的广泛肿胀。钙离子进入基质是引发肿胀的关键因素。巯基如果以足够高的浓度添加，则会抑制肿胀。同样，稳定蛋白质m态构象的ADP/ATP载体配体也发挥了有效的保护作用。这项研究揭示了三个重要的相互作用因素：(i)ADP/ATP载体方向在促进钙诱导的膜不稳定中的关键作用。更确切地说，已经表明在脂肪酸氧化过程中，ADP/ATP载体采取c态构象（即核苷酸结合位点面向细胞质）。(ii)线粒体蛋白质中极少数巯基发生修饰。当还原型吡啶核苷酸水平较低时，这些基团可能处于氧化状态。(iii)转氢酶的主导作用，其功能也与脂肪酸氧化密切相关。在供能后，转氢酶可以阻碍巯基氧化，从而部分保护膜结构。
