Rapid purification of F₁-ATPase from Rat Liver Mitochondria Using a Modified Chloroform Extraction Procedure Coupled to High-Performance Liquid Chromatography

This chapter describes a procedure for rapid purification of F₁-ATPase from rat liver mitochondria using a modified chloroform extraction procedure coupled to high-performance liquid chromatography (HPLC). The most rapid method for isolation of F₁-ATPase from mammalian sources is employing chloroform for solubilization of the F₁ from the membrane. Purified inner membrane vesicles are extracted with chloroform in the presence of ATP and EDTA. The resulting F_l has a high specific activity, is reconstitutively active, catalyzing high rates of ATP synthesis, and can be crystallized. In addition, the reconstituted system is coupled and therefore capable of ATP synthesis, indicating that the F₁ is functionally intact. A one-step HPLC procedure is added to further purify the preparation if desired. Sucrose, present in the enzyme preparation after high-speed centrifugation, is a problem with the HPLC columns employed, so the desalting column is used to exchange the enzyme into a compatible buffer.