In situ F1 ATPase is involved in the coupling of substrate oxidation by mitochondria to the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate (Pi). Intact rat liver mitochondria have little ATPase activity. Disruption of the mitochondria causes loss of the normally tight coupling between oxidation and ATP synthesis and activates an ATPase that is sensitive to inhibitors of oxidative phosphorylation. The chapter describes two different procedures for purification of this enzyme from disrupted rat liver mitochondria using measurements of its ATPase activity as an assay procedure. It discusses analytical and purification procedures. Analytical procedure involves the measurement of ATPase activity and measurement of protein. The purification procedures involves the isolation of mitochondria, preparation of washed mitochondrial membranes, solubilization of F1 ATPase, chromatography on diethylaminoethyl (DEAE)-cellulose, chromatography on Sephadex G-200, and stability and storage of purified F1 ATPase.
ASJC Scopus subject areas
- Molecular Biology