Separation of a Factor Indispensable for Coupled Phosphorylation from the Particulate Fraction of Mycobacterium phlei

The respiratory apparatus of bacteria appears to be associated with the cytoplasmic membrane. The particulate structures obtained by differential centrifugation of cell-free extracts contained the bulk of the respiratory activity of the cell and are referred to as the electron transport particles. This material, however, was not homogenous and was further separated into a number of distinct particulate fractions by density gradient centrifugation. The heaviest fraction appeared as a red pellet, comprised about 28% of the total particulate protein, and was found to contain the complete electron transport pathways for succinate and NAD+-linked oxidations. However, this fraction failed to couple phosphorylation to oxidation even in the presence of soluble coupling factors. Restoration of phosphorylation to the electron transport particles was found to require one of the light particulate fractions and soluble coupling factors. The light particulate fraction had little or no oxidative activity and was active in restoring phosphorylation even after prolonged exposure to sonic oscillation. Reassociation of the light and heavy particles required the presence of Mg++ ions. The light particulate fraction was required for restoration of phosphorylation with different electron donors as well as for the third phosphorylative site.