Tipton Peter, Su Tong, Hannink Mark
University of Missouri, Columbia, MO, United States.
University of Missouri, Columbia, MO, United States.
Methods Enzymol. 2018;607:353-372. doi: 10.1016/bs.mie.2018.05.017. Epub 2018 Jun 23.
Phosphoglycerate mutase family member 5 (PGAM5) is a serine/threonine phosphatase that has been localized to both inner and outer mitochondrial membranes. PGAM5 has been suggested to regulate multiple aspects of mitochondrial dynamics, including fission/fusion and mitophagy, through phosphatase-dependent and phosphatase-independent mechanisms. Understanding how the phosphatase activity of PGAM5 is regulated will provide new insight into signaling mechanisms that link changes in cell physiology with mitochondrial function. In this chapter, we describe methods for obtaining both multimeric and dimeric complexes of PGAM5 and for characterizing their kinetic properties. The ability to purify different PGAM5 complexes and to characterize their kinetic properties will enable detailed biophysical studies of the quaternary structures of the various PGAM5-containing complexes. The phosphatase activity of different PGAM5 complexes varies over three orders of magnitude. We suggest that the ability to generate PGAM5 complexes that have a wide range of phosphatase activities will facilitate screens to identify small molecules that modulate the phosphatase activity of PGAM5.
磷酸甘油酸变位酶家族成员5（PGAM5）是一种丝氨酸/苏氨酸磷酸酶，定位于线粒体内外膜。有人提出，PGAM5通过磷酸酶依赖性和非磷酸酶依赖性机制调节线粒体动力学的多个方面，包括裂变/融合和线粒体自噬。了解PGAM5的磷酸酶活性如何被调节将为连接细胞生理变化与线粒体功能的信号传导机制提供新的见解。在本章中，我们描述了获得PGAM5多聚体和二聚体复合物以及表征其动力学特性的方法。纯化不同PGAM5复合物并表征其动力学特性的能力将使我们能够对各种含PGAM5复合物的四级结构进行详细的生物物理研究。不同PGAM5复合物的磷酸酶活性在三个数量级上变化。我们认为，生成具有广泛磷酸酶活性的PGAM5复合物的能力将有助于筛选出调节PGAM5磷酸酶活性的小分子。
