A melatonin-based fluorescence method for the measurement of mitochondrial complex III function in intact cells.
A melatonin-based fluorescence method for the measurement of mitochondrial complex III function in intact cells.
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DOI：
10.1111/jpi.12079
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发表时间：
2013-11
影响因子：
10.3
通讯作者：
Zhang BX
中科院分区：
文献类型：
--
作者：
Fu JL;Zhang HM;Zhang H;Kamat A;Yeh CK;Zhang BX
线粒体复合物III（MC-3）在电子传递和氧化磷酸化中起着关键作用。受损的MC-3功能可能通过中断正常的生物能量学和增加活性氧产生和氧化应激而导致多种疾病。目前，MC-3的功能是通过在分离的线粒体或MC-3中以电化学方法测量细胞色素c还原酶活性来评估的。在这些分离过程中，线粒体复合体功能的细胞质微环境可能会被耗尽。非常需要开发一种可靠的方法来测量完整细胞或组织中的MC-3活性。本报告描述了一种新的基于荧光的方法来评估MC-3功能，即Qi网站电子转移，在完整的细胞。用人肾小球系膜细胞和畸胎癌细胞NT 2证明褪黑素诱导的2′，7 ′-二氯二氢荧光素（H2 DCF）氧化可被MC-3 Qi位点特异性抑制剂抗霉素A抑制，但不被MC-3 Qo位点特异性抑制剂Myxothiazol抑制，也不被线粒体复合物I抑制剂鱼藤酮抑制。这些结果表明，褪黑素诱导的H2 DCF氧化反应反映了MC-3 Qi位点的电子转移活性。在褪黑激素的吲哚环的R3和R5位置处的侧基的修饰结构降低了其诱导H2 DCF氧化的功效，表明褪黑激素与MC-3 Qi位点的特异性相互作用。这些结果表明，褪黑激素诱导的H2 DCF氧化的荧光性质提供了完整细胞中MC-3 Qi位点电子转移特异性测量。有趣的是，通过使用这种方法，发现转化或永生化细胞中的Qi位点电子转移活性显著高于非转化细胞。
Mitochondrial complex III (MC-3) plays a pivotal role in electron transfer and oxidative phosphorylation. Impaired MC-3 functions may contribute to a variety of diseases by interrupting normal bioenergetics and increasing reactive oxygen production and oxidative stress. Currently, MC-3 function is assessed by measuring the cytochrome c reductase activity spectrophotometrically in isolated mitochondria or MC-3. The cytoplasmic microenvironment critical for mitochondrial complex functions may be depleted during these isolation processes. The development of a reliable method to measure MC-3 activities in intact cells or tissues is highly desirable. This report describes a novel fluorescence-based method to assess MC-3 functions, i.e. Qi site electron transfer, in the intact cells. Human mesangial and teratocarcinoma NT2 cells were used to demonstrate that melatonin induced oxidation of 2′,7′-dichlorodihydrofluorescein (H2DCF) was inhibited by antimycin A, the MC-3 Qi site specific inhibitor; but not by myxothiazol, the MC-3 Qo site specific inhibitor, nor rotenone, the mitochondrial complex I inhibitor. These results indicate that melatonin induced oxidation of H2DCF is reflecting MC-3 Qi site electron transfer activities. Modifying structures of the side groups at the R3 and R5 positions of the indole ring of melatonin diminished its efficacy for inducing H2DCF oxidation, suggesting a specific interaction of melatonin with the MC-3 Qi site. These results suggest that the fluorogenic property of melatonin-induced H2DCF oxidation provides a MC-3 Qi site electron transfer specific measurement in intact cells. Interestingly, by using this method, the Qi site electron transfer activity in transformed or immortalized cells was found to be significantly higher than the non-transformed cells.
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DOI：
10.1006/bmmb.1994.1004
发表时间：
1994-02-01
期刊：
BIOCHEMICAL MEDICINE AND METABOLIC BIOLOGY
影响因子：
--
作者：
BIRCHMACHIN, MA;BRIGGS, HL;TURNBULL, DM
通讯作者：
TURNBULL, DM
DOI：
10.1152/ajpcell.1998.275.2.c367
发表时间：
1998-08-01
影响因子：
5.5
作者：
Turner, JT;Redman, RS;Quissell, DO
通讯作者：
Quissell, DO
影响因子：
3.5
作者：
Possel, H;Noack, H;Wolf, G
通讯作者：
Wolf, G
DOI：
10.1056/nejmoa1214091
发表时间：
2013-03-14
期刊：
The New England journal of medicine
影响因子：
--
作者：
Wang PY;Ma W;Park JY;Celi FS;Arena R;Choi JW;Ali QA;Tripodi DJ;Zhuang J;Lago CU;Strong LC;Talagala SL;Balaban RS;Kang JG;Hwang PM
通讯作者：
Hwang PM
影响因子：
4.7
作者：
Ren, Jun;Pulakat, Lakshmi;Whaley-Connell, Adam;Sowers, James R.
通讯作者：
Sowers, James R.