Oxidative phosphorylation in fractionated bacterial systems XLIII. Coupling factors associated with the NAD +-linked electron transport pathway

Abstract

Oxidative phosphorylation with the cell-free system from Mycobacterium phlei is dependent on the presence of both the particulate and soluble fractions. While the particles contain the bulk of the respiratory components, the soluble fraction contains a number of dehydrogenases which are necessary to initiate electron transport and protein components which are necessary for the coupling process. The supernatant components necessary for restoration of phosphorylation were resolved into two different coupling factors which appear to operate at different phosphorylative sites. The purified coupling factors appear to be protein in nature. The phosphorylation restored on addition of the purified coupling factors to the electron transport particles was found to be sensitive to uncoupling agents. The purified coupling factors were found to contain some ATPase, adenylate kinase, 14C-ADP-ATP and 32P i-ATP exchange activities. Antibodies prepared to either of the coupling factors was found to be specific for the coupling protein to which it was prepared. In addition, the antibodies were found to inhibit the phosphorylation elicited by that coupling factor. An additional coupling factor was found to be bound to the electron transport particles. The particulate bound factor was released from the particles by treatment with 1.0 m urea. The level of phosphorylation was increased by a combination of the different coupling factors. The coupling factors had no significant effect on the level of oxidation.