Partial Resolution of the Enzymes Catalyzing Oxidative Phosphorylation

1. Amorphous membrane fragments depleted in P-lipids and cytochrome oxidase were isolated from bovine heart mitochondria and were reconstituted with P-lipids and coupling factors to yield vesicular structures. These vesicles catalyzed a 32Pi—ATP exchange and showed an induced enhancement of anilinonaphthalene sulfonate fluorescence on addition of ATP 2. 32Pi—ATP exchange and fluorescence enhancement were abolished by uncouplers of oxidative phosphorylation and by energy transfer inhibitors. The ATPase activity was inhibited by energy transfer inhibitors, but stimulated by uncouplers or by the combined action of nigericin and valinomycin in the presence of K+. Both ATPase activity and 32Pi—ATP exchange were inhibited by a specific antibody against coupling factor 1. 3. It was shown that the reconstitution of vesicular structures with functional activity required several hours. Rapid reconstitution resulted in inactive vesicles. Evidence for the formation of new vesicles from solubilized P-lipids was obtained by demonstrating inclusion of macromolecules such as 14C-labeled inulin or ferritin which could not be removed by washing.

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