Chronic treatment with azide in situ leads to an irreversible loss of cytochrome c oxidase activity via holoenzyme dissociation
Chronic treatment with azide in situ leads to an irreversible loss of cytochrome c oxidase activity via holoenzyme dissociation
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DOI：
10.1074/jbc.m112303200
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发表时间：
2002-03-29
影响因子：
4.8
通讯作者：
Moyes, CD
中科院分区：
文献类型：
--
作者：
Leary, SC;Hill, BC;Moyes, CD
用非常低水平的叠氮化物（I-50 < 10 μ（M））长期处理培养的细胞导致细胞色素c氧化酶（考克斯）活性的缓慢（t（1/2）= 6 h）、不可逆的损失。叠氮化物介导的考克斯损失并不伴随着其他线粒体酶的抑制，并不依赖于通过氧化磷酸化的电子通量。虽然叠氮化物治疗也降低了CuZn超氧化物歧化酶和过氧化氢酶的活性（但不是含量），但一系列促氧化剂（和抗氧化剂）未能模拟（或防止）叠氮化物的作用，认为考克斯活性的损失不是由于自由基清除的结果妥协。考克斯活性的丧失并不归因于线粒体蛋白质合成速率的降低或考克斯亚基mRNA或蛋白质水平的下降（考克斯I、II、IV）。使用铜（CuCl 2 Cu-His）和铜螯合剂（neocuproine，bathocuproine）的共孵育实验表明，叠氮化物效应不受与Cu-A或Cu-B相互作用的影响。与此相反，差光谱和高效液相色谱分析表明，叠氮诱导的细胞色素aa（3）含量的损失，虽然没有达到相同的程度，催化活性。使用精制的抑制时间过程结合蓝色天然电泳证实了相对于考克斯活性的考克斯含量的差异叠氮化物效应，并确定全酶解离发生在催化活性损失之后。总的来说，这些数据表明，考克斯缺陷可以通过增强全酶解离，可能通过与其血红素部分的结构或协调的相互作用而产生。
Chronic treatment of cultured cells with very low levels of azide (I-50 < 10 mu(M)) leads to slow (t(1/2) = 6 h), irreversible loss of cytochrome c oxidase (COX) activity. Azide-mediated COX losses were not accompanied by inhibition of other mitochondrial enzymes and were not dependent upon electron flux through oxidative phosphorylation. Although azide treatment also reduced activity (but not content) of both CuZn superoxide dismutase and catalase, a spectrum of pro-oxidants (and anti-oxidants) failed to mimic (or prevent) azide effects, arguing that losses in COX activity were not due to resultant compromises in free radical scavenging. Loss of COX activity was not attributable to reduced rates of mitochondrial protein synthesis or declines in either COX subunit mRNA or protein levels (COX I, II, IV). Co-incubation experiments using copper (CuCl2 Cu-His) and copper chelators (neocuproine, bathocuproine) indicated that azide effects were not mediated by interactions with either Cu-A or Cu-B. In contrast, difference spectroscopy and high performance liquid chromatography analyses demonstrated azide-induced losses in cytochrome aa(3) content although not to the same extent as catalytic activity. Differential azide effects on COX content relative to COX activity were confirmed using a refined inhibition time course in combination with blue native electrophoresis, and established that holoenzyme dissociation occurs subsequent to losses in catalytic activity. Collectively, these data suggest that COX deficiency can arise through enhanced holoenzyme dissociation, possibly through interactions with the structure or coordination of its heme moieties.