Partial resolution of the enzymes catalyzing oxidative phosphorylation. 13. Structure and function of submitochondrial particles completely resolved with respect to coupling factor.

Abstract 1. Passage of submitochondrial particles through a Sephadex column activated the adenosine triphosphatase activity 5- to 10-fold and subsequent treatment with urea removed virtually all ATPase (coupling factor 1). The resulting submitochondrial particles, exposed sequentially to Sephadex and urea (SU-particles), catalyzed the oxidation of succinate but phosphorylation was insignificant. P:O ratios exceeding 0.5 were obtained on addition of coupling factors 1, 2, and 3. 2. Without addition of coupling factor 1, low concentrations of oligomycin or rutamycin did not stimulate phosphorylation in SU-particles. However, in the presence of coupling factor 1 and optimal concentrations of rutamycin considerable phosphorylation rates were observed. Similar to rutamycin, dicyclohexylcarbodiimide stimulated oxidative phosphorylation at low concentrations and inhibited oxidative phosphorylation at high concentrations. 3. Reconstituted SU-particles which catalyzed oxidative phosphorylation with a P:O ratio of 0.5 catalyzed the energy-driven reversal of electron flow from succinate to diphosphopyridine nucleotide at a very slow rate. However, addition of a crude mitochondrial extract restored this activity to a rate about half of that observed in the original submitochondrial particles. 4. In electron micrographs SU-particles were found to be devoid of inner membrane spheres. On reconstitution with coupling factor 1, the inner membrane spheres reappeared. The reconstituted particles were morphologically indistinguishable from untreated submitochondrial particles. Calculations indicate that coupling factor 1 is responsible for most, if not all, of the inner membrane spheres.