1 86 E. R. REDFEARN 



both cases, on a molar basis, ubiquinone is present in a considerable excess 

 over the cytochromes. On an electron-carrying basis the ubiquinone/cyto- 

 chrome ratio is, of course, increased further by a factor of two to give 

 ratios of 20-24. The reason for this large excess of ubiquinone is not clear 

 at the moment although it has important consequences when discussing its 

 possible function in the respiratory chain. It is interesting to note the 

 extraction experiments already described indicate that relatively large 

 amounts of ubiquinone may be removed from mitochondrial particles 

 without apparently having drastic effects on enzymic activities. Thus it is 

 possible that only the stoicheiometric amount is necessary for efficient 

 operation of the respiratory chain. 



The results of a study of the kinetics of ubiquinone reactions in heart- 

 muscle preparations, the action of inhibitors on these reactions and a 

 discussion of the possible function of ubiquinone in the non-phosphorylat- 

 ing respiratory chain have been presented recently [17, 15, 16]. To 

 summarize briefly, the rate of reduction of ubiquinone by DPNH or 

 succinate is less than the total electron flux as measured by the substrate 

 oxidase rates ; most of the endogenous ubiquinone appeared to be accessible 

 to both substrates; inhibitor studies indicate that its site of action is 

 between the flavoproteins and the antimycm-A-sensitive region. Three 

 possible schemes for the position of ubiquinone in the non-phosphorylating 

 chain can be put forward [16]. These are: (i) that ubiquinone is on the 

 main respiratory chain mediating the reaction between the flavoproteins 

 and the cytochromes, (ii) that it reacts only with the flavoproteins to form 

 a blind-alley pathway, and (iii) that it is on a branch pathway linking the 

 flavoproteins with cytochrome (\ via the antimycin-A-sensitive region. 



In order to try to elucidate the mode of action of ubiquinone in the 

 intact p»hosphorylating system we have begun experiments with rat-liver 

 mitochondria. The mitochondria were prepared by a modification of the 

 method of Schneider and Hogeboom [18] and ubiquinone determined by 

 the method of Pumphrey and Redfearn [i]. Respiratory control and P/0 

 ratios were determined with the oxygen electrode [19], and steady-state 

 oxidation-reduction levels of ubiquinone were measured in the different 

 metabolic states [20] of the mitochondria. Typical spectra are shown in 

 Fig. I. It can be seen that in the absence of added substrate or ADP 

 (State i) the ubiquinone is 45^0 reduced while on adding ADP (State 2) 

 it becomes 38°(, reduced. In the presence of added substrate (succinate) 

 but no ADP (State 4) the ubiquinone is 80",, reduced but on adding ADP 

 (State 3) falls to 72% reduction. The results of experiments on four 

 different mitochondrial preparations are shown in Table VI. When 

 succinate is the substrate ubiquinone is largely reduced (80-89°,,) in State 4 

 and becomes less reduced (70-86%) in State 3 while the corresponding 

 figures for ^-hydroxybutyrate are 53-72"^, (State 4) and 40 63",, (State 3). 



