242 MAYNARD E. PULLMAN, HARVEY S. PENEFSKY AND E. RACKER 



on respiration and appears, therefore, to be primarily concerned in the 

 phosphorylation mechanism. Phosphorylation in the reconstituted system 

 is uncoupled by 2,4-dinitrophenol as well as by a number of other recog- 

 nized uncouplers including dicoumarol, chlorpromazine and triiodo-L- 

 thyronine. The maximal P:0 ratios obtained in this particular experiment 

 are somewhat less than those generally observed. The average maximal 

 P : O ratio was o • 6, with a range of o • 4 to o • 8. During the early phases of 

 this work, different preparations of the particulate fraction exhibited 

 residual and variable phosphorylation activity. Nevertheless, the addition 

 of the supernatant fraction never failed to result in a marked increase in 



TABLE I 



Effect of the Supernatant Solution on Phosphorylation Accompanying 



Succinate Oxidation 



Each Warburg vessel contained 0-05 m succinate, pH 7-4, 0-004 M MgCl2, 

 0'002 M ATP, 0-0I2 M potassium phosphate buffer, pH 7-4, 0-032 M glucose, 

 0-005 M tris, pH 7-4, o-ooi M EDTA, 0-06 mg. yeast hexokinase (25 to 40 units/ 

 mg.), I -6 mg. of the particulate fraction and the indicated amounts of the super- 

 natant solution (105 000 X g) in a final volume of 0-5 ml. Where added, 2,4- 

 dinitrophenol (DNP) was 0-0005 J^i- Incubations were carried out for 36 min. 

 at 30°. 



the P:0 ratio. In subsequent work, disruption of the mitochondria was 

 carried out in the presence of EDTA, which in confirmation of the results 

 reported by Linnane [7], consistently yielded particles in which phosphory- 

 lation was either low or absent. For reconstitution, these particles were 

 preincubated with the soluble protein and Mg + + and an aliquot of the 

 mixture was then added to a Warburg vessel containing the otherwise 

 complete reaction mixture (cf. Table I). 



Table II illustrates the dependency on the coupling factor for phos- 

 phorylation associated with the oxidation of various substrates. It may be 

 seen that isocitrate was oxidized by the submitochondrial particles at about 

 one-half the rate of succinate and that no esterification of Pj occurred in 

 the absence of the purified coupling factor. The oxidation of ^-hydroxy- 

 butyrate required the addition of DPN while glutamate oxidation occurred 



