使用分离的线粒体进行 BAK/BAX 激活和细胞色素 c 释放测定。
BAK/BAX activation and cytochrome c release assays using isolated mitochondria.
机构信息
Icahn School of Medicine at Mount Sinai, Department of Oncological Sciences, One Gustave L. Levy Place, Box 1130, New York, NY 10029, USA.
出版信息
Methods. 2013 Jun 1;61(2):146-55. doi: 10.1016/j.ymeth.2013.03.030. Epub 2013 Apr 6.
Abstract
The mitochondrial pathway of apoptosis proceeds when the outer mitochondrial membrane (OMM) is compromised by the pro-apoptotic BCL-2 family members, BAK and BAX. Once activated, BAK and BAX form proteolipid pores in the OMM leading to mitochondrial outer membrane permeabilization (MOMP), and the release of inner membrane space proteins, such as cytochrome c, which promotes caspase activation. The use of isolated mitochondria has been instrumental to understanding the key interactions necessary to engage BAK and BAX activation, MOMP, and apoptosis. Furthermore, it is possible to biochemically define the relationships between BCL-2 family function and mitochondrial physiology using isolated systems. Our laboratory uses freshly isolated mitochondria from numerous sources to better understand BCL-2 family function and requirements for BAK and BAX activation. Here, we will discuss commonly used in vitro techniques to perform MOMP and cytochrome c release assays; and provide several key methodologies to implicate BAK and BAX activity in these processes.
摘要
线粒体凋亡途径是在外膜（OMM）被促凋亡的 BCL-2 家族成员 BAK 和 BAX 破坏时进行的。一旦被激活，BAK 和 BAX 在 OMM 中形成亲脂性孔，导致线粒体外膜通透性（MOMP）和内膜空间蛋白（如细胞色素 c）的释放，从而促进半胱天冬酶的激活。使用分离的线粒体对于理解使 BAK 和 BAX 激活、MOMP 和凋亡发生所必需的关键相互作用非常重要。此外，使用分离的系统可以在生化上定义 BCL-2 家族功能和线粒体生理学之间的关系。我们的实验室使用从多种来源分离的新鲜线粒体，以更好地理解 BCL-2 家族的功能以及 BAK 和 BAX 激活的要求。在这里，我们将讨论常用的体外技术来进行 MOMP 和细胞色素 c 释放测定；并提供几种关键方法学来表明 BAK 和 BAX 在这些过程中的活性。
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