Studies on the mechanism of oxidative phosphorylation

1. A subunit (ETPH) having nearly theoretical P/O ratios may be prepared from heavy mitochondria from beef heart when both Mg2+ and Mn2+ are included in the medium during the isolation procedure. 2. Optimal concentration of the ions required during the preparation are 5 mM for Mg2+ and 10 mM for Mn2+. 3. The addition of succinate (1 mM) improved the reliability of the preparative procedure, and minimized seasonal difficulties in the preparation of ETPH during summer months. 4. Certain preparations of ETPH show increased lability of the phosphorylation mechanism, and deteriorate during the temperature equilibrium period of the assay, when shaken in the assay medium in the absence of substrate. This can be overcome by including substrate in the preliminary incubation and starting the assay reaction with ADP and the hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) trapping system.