BRITTON CHANCE 



The application of these methods to the study of the re- 

 spiratory chains of phosphorylative* and nonphosphorylative 

 DPNH oxidase systems of mitochondria and of mitochondrial 

 fragments is outlined here. 



THE SUBSTRATE-INHIBITOR METHOD 



The examination of a respiratory chain by means of specific 

 substrates or inhibitors rests largely upon the generality: com- 

 ponents of the respiratory chain above the point of action of 

 the added reagent become more reduced in the case of a sub- 

 strate, t and more oxidized in the case of an inhibitor. If the 

 inhibitor is added to a system in which the substrate and oxygen 

 are already present, the components above the site of action of 

 the inhibitor will become more oxidized and those below will 

 become more reduced (see, for example. Figure 4 of Ref. 12). 

 The point in the respiratory chain at which the effect of an in- 

 hibitor upon the steady state shifts from an oxidation to a reduc- 

 tion, or vice versa, is called the crossover point. This crossover 

 point locates the site of action of the inhibitor. 



CYTOCHROME b AND SLATER' S FACTOR 



A clear example of the substrate-inhibitor technique is 

 afforded by the effect of antimycin-A upon the DPNH oxidase 

 system of phosphorylating liver mitochondria, as illustrated by 

 Figure 3. Since phosphate acceptor is lacking electron transfer 

 is in progress at a slow rate before the addition of the inhibi- 

 tor. Upon addition of the inhibitor, the spectroscopic record 

 shows an oxidation of reduced cytochrome c (the 550 m/x absorp- 

 tion peak of ferrocytochrome c disappears and leaves a trough 

 at 550 m/i) and cytochrome b becomes reduced (the peak of re- 

 duced cytochrome b appears at 563 m/x). Thus the crossover 

 point is between cytochromes b and c, and the theorem above 



* In collaboration with Dr. G. R. Williams. 



t "Above" refers to the oxygen side, "below" to the substrate side of 

 the point of action. 



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