NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP): Applications to enzyme and mitochondrial reaction kinetics, in vitro
NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP): Applications to enzyme and mitochondrial reaction kinetics, in vitro
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DOI：
10.1016/s0006-3495(04)74141-7
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发表时间：
2004-01-01
影响因子：
3.4
通讯作者：
Balaban, RS
中科院分区：
文献类型：
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作者：
Joubert, F;Fales, HM;Balaban, RS
采用光漂白后NADH酶依赖的荧光恢复法(ED-FRAP)研究线粒体的酶动力学。质量、光学和核磁共振数据与UV-NADH光解反应为NADH-->；NAD(圆)-->；H++e(-)相一致。总的净反应为O-2+2NADH+2H(+)-->；2NAD(+)+2H(2)O，或在其他竞争电子受体如细胞色素c，NADH+2Cyt(Ox)-->；NAD(+)+H++2Cyt(Red)的存在下。溶液pH可以区分这些自由基清除途径。这些净反应代表了NADH的光氧化为NAD(+)。建立了动力学模型和获取方案，通过改变NADH光解水平来改变[NADH]和[NAD]，以提取动力学参数。所使用的紫外线照射水平不会损害线粒体功能或酶活性。在线粒体中，[NADH]是一种高亲和力的产物抑制剂，显著降低NADH的再生率。基质NADH再生仅略高于NADH净消耗率，表明NADH再生过程远未达到平衡。与鱼藤酮处理的制剂相比，对活性线粒体中NADH再生的评估揭示了除基质[NADH]和[NAD]之外的其他调节元件，这些元件尚未完全表征。这些研究表明，NADH的快速紫外光分解为NAD是评价酶系统稳态动力学性质的有效工具。最初的数据支持这一观点，即线粒体中NADH的再生过程远未达到平衡，并可能受到NADH水平和其他几个基质因素的控制。
NADH enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) was evaluated for studying enzyme kinetics in vitro and in isolated mitochondria. Mass, optical, and nuclear magnetic resonance spectroscopy data were consistent with the UV NADH photolysis reaction being NADH-->NAD(circle)-->H+ + e(-). The overall net reaction was O-2+2NADH+2H(+)-->2NAD(+) + 2H(2)O, or in the presence of other competing electron acceptors such as cytochrome c, NADH+2Cyt(ox)-->NAD(+) + H+ + 2Cyt(red). Solution pH could differentiate between these free-radical scavenging pathways. These net reactions represent the photooxidation of NADH to NAD(+). Kinetic models and acquisition schemes were developed, varying [NADH] and [NAD] by altering NADH photolysis levels, for extracting kinetic parameters. UV irradiation levels used did not damage mitochondrial function or enzymatic activity. In mitochondria, [NADH] is a high affinity product inhibitor that significantly reduced the NADH regeneration rate. Matrix NADH regeneration only slightly exceeded the net rate of NADH consumption, suggesting that the NADH regeneration process is far from equilibrium. Evaluation of NADH regeneration in active mitochondria, in comparison to rotenone-treated preparations, revealed other regulatory elements in addition to matrix [NADH] and [NAD] that have yet to be fully characterized. These studies demonstrate that the rapid UV photolysis of NADH to NAD is an effective tool in evaluating the steady-state kinetic properties of enzyme systems. Initial data support the notion that the NADH regeneration process is far from equilibrium in mitochondria and is potentially controlled by NADH levels as well as several other matrix factors.