Purification of α and β subunits and subunit pairs from rat liver mitochondrial F₁-ATPase

This chapter describes a procedure for the purification of α and β subunits and subunit pairs from rat liver mitochondrial F₁-ATPase. The procedure for separation based on cold treatment with subsequent purification is described. The protocols combining cold treatment for dissociation of the rat liver mitochondrial F₁-ATPase together with chromatographic separation allow the purification of the two major subunits, α and β, and two subunit complexes, αγ and βγ. The complexes are both stable to treatment and are not affected by the presence of dithiothreitol during cold treatment or purification. Neither the individual subunits (α and β) nor the subunit complexes (αγ and βγ) are capable of catalysis. However, under the appropriate conditions (the addition of Mg²⁺ and ATP), the supernatant and sediment fractions reconstitute to yield 100% of the control activity, indicating that they are still competent.