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Comparison of two methods for assaying complex I activity in mitochondria isolated from rat liver, brain and heart.

机译:两种测定大鼠肝,脑和心脏线粒体中复杂I活性的方法的比较。

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摘要

AIMS: To establish a more sensitive, reliable, and convenient assay for complex I activity. MAIN METHODS: Two of the most widely used methods - the conventional NADH method and a newly developed 2, 6-dichlorophenolindophenol (DCIP)-coupled method - were compared and optimized. KEY FINDINGS: The DCIP method gave a higher enzyme sensitivity and comparable rotenone sensitivity in heart mitochondria while the NADH method gave higher rotenone sensitivity with a relatively lower enzyme activity in the brain and liver mitochondria. Addition of bovine serum albumin (BSA) to the reaction mixture greatly improved the accuracy of the NADH assay. The reaction conditions were optimized for use with a microplate reader that requires only small amounts of mitochondria or tissue. SIGNIFICANCE: Considering the important contribution of the non-specific rotenone-insensitive activity in the complex I assay, it is suggested that the NADH method with BSA addition should be adopted for assaying complex I activity in the brain or liver samples, while the DCIP method is the better choice for heart samples.
机译:目的:建立一种更灵敏,可靠和方便的复杂I活性测定方法。主要方法:比较和优化了两种最广泛使用的方法-常规的NADH方法和新开发的2,6-二氯苯酚吲哚酚(DCIP)偶联方法。主要发现:DCIP方法在心脏线粒体中具有较高的酶敏感性和相当的鱼藤酮敏感性,而NADH方法在小鼠的脑和肝线粒体中具有较高的鱼藤酮敏感性,而酶活性相对较低。向反应混合物中添加牛血清白蛋白(BSA)大大提高了NADH测定的准确性。反应条件已优化,可与仅需要少量线粒体或组织的酶标仪一起使用。意义:考虑到非特异性鱼藤酮不敏感活性在复合物I测定中的重要贡献,建议应采用添加BSA的NAD​​H方法测定脑或肝脏样品中的复合物I活性,而DCIP方法是心脏样本的更好选择。

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