50] Isolation and characterization of an inactivated complex of F1F0-ATPase and its inhibitory factors from yeast

Abstract

Publisher Summary This chapter discusses the procedure for the isolation and characterization of an inactivated complex of F 1 F 0 -ATPase and its inhibitory factors from Yeast. An intrinsic F 1 -ATPase inhibitor protein found in mitochondria regulates oxidative phosphorylation. ATPase inhibitors are obtained in purified forms from various sources, including beef heart, rat liver, skeletal muscle, and yeast. No inhibitor protein is present in purified F 1 -ATPase or F 1 F 0 -ATPase, but it is found in mitochondria and submitochondrial particles in an equimolar ratio to F 1 -ATPase, indicating that it is an integral subunit of the enzyme, but is readily released from the enzyme during the processes of isolation of the enzyme. In contrast to inhibition of F 1 -ATPase activity, F 1 F 0 -ATPase in mitochondria is inhibited even in the absence of ATP and Mg 2+ . This suggests the existence in mitochondria of factors that stabilize the inactivated complex between the enzyme and the inhibitor. The stabilizing factors, 9K and 15K proteins, are isolated from yeast mitochondria and purified by high-performance liquid chromatography. The factors also exert their action on isolated F 1 F 0 -ATPase, but not on F 1 -ATPase.