The Role of ATPase in Oxidative Phosphorylation* 



Maynard E. Pullman, Harvey S. Pexefsky and E. Racker 



Division of Xutrition and Physiology, 

 The Public Health Research Institute of the City of Xew York, Inc., 



X.Y., U.S.A. 



In previous communications we reported the resolution of mechanically 

 fragmented beef heart mitochondria into a particulate and a soluble 

 protein component, both of which were required for oxidative phosphory- 

 lation [i, 2]. The particulate fraction catalyzed the oxidation of a number 

 of substrates with little or no concomitant phosphorvlation. The addition 

 of the soluble component to the particulate fraction recoupled the respira- 

 tion to phosphorylation. A summary of the properties of the reconstituted 

 system and of the soluble factor, as well as some of the more recent 

 developments with this system will form the subject of this discussion. 

 Since a detailed description of the experimental procedures was presented 

 elsewhere [3, 4] only the salient features will be considered here. 



Beef heart mitochondria, prepared according to the method of Green 

 et al. [5], were disrupted in vacuo in the presence of glass beads bv means 

 of a high-speed reciprocal Xossal shaker [6]. The suspension was centri- 

 fuged for 20 min. at 26 000 x g yielding a brown, gelatinous residue which 

 was discarded and a yellow, turbid supernatant fluid. The supernatant 

 solution was recentrifuged at 105 000 x g for 30 min. A red-brown gela- 

 tinous residue (residue i) and a faintly turbid, yellow supernatant fluid 

 were obtained. The supernatant fluid was decanted and clarified by 

 centrifugation for an additional 30 min. at 105 000 x g, vielding a clear 

 yellow solution. Residue i was washed by homogenizing in 0-2^ m 

 sucrose containing 0-002 M EDTA and centrifuged at lo^oooxg for 

 30 min. The washing procedure was repeated with 0-2^ m sucrose and 

 the final residue i was suspended in 0-25 M sucrose. 



As shown in Table I residue i catalyzed the oxidation of succinate with 

 little or no accompanying phosphorylation. Addition of increasing amounts 

 of the supernatant solution (105 000 xg) resulted in almost a tenfold 

 increase in phosphate uptake. The coupling factor had no significant effect 



* This work was supported by Grants Xos. A- 12 19 and C-3463 from the 

 National Institutes of Health, United States Public Health Service, Bethesda, 

 Maryland. 



VOL. II. — R 



