Large-scale column-free purification of bovine F-ATP synthase

Abstract

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at sufficient quantity, stability and purity. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favouring IF1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the GDN to LMNG ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.