RESPIRATION AND OXIDATIVE PHOSPHORYLATION 119 



state 2, uncoupling by DNP without increase of respiration (no 

 exogenous substrate); state 3, high respiration in the presence of 

 exogenous substrate and uncoupling agent (DNP); state 4, inter- 

 mediary respiration with substrate alone. In the preparations of 

 spermatozoa the addition of azide gives inhibition which is de- 

 pendent upon the rate of respiration or the turnover number as 

 shown in Fig. 1 1 where results are computed for a given number of 

 cells and therefore at constant cytochrome concentration. All titra- 

 tions end along a line of azide sensitivity from 0.1 to 20 mM con- 

 centration. Samples with low respiration thus would appear not to 

 be inhibited although spermatozoa do not carry any special azide- 

 insensitive oxidative mechanism. 



These observations are summarized in Fig. 12. The average level 

 of reduction in the steady state of substrate oxidation (succinate or 

 lactate) is shown in the upper section of the figure. The highest rate 

 of reduction is observed with cytochrome b, which here plays, as in 

 other types of cells, the role of bottleneck in electron transfer 

 (Chance, 1955). However, it should be noted that pigments are not 

 in equal amounts (i.e., cyt b is one-third of cyt a) and therefore, in 

 absolute values, there is more flavine reduced, for instance, than 

 cytochrome b in the steady state (see Table I). 



Results from a series of experiments on azide action are grouped 

 at the lower left of Fig. 12. The higher reduction of terminal com- 

 ponents, cytochromes a 3 , a, and c, is shown, as well as that of the 

 initial ones, pyridine nucleotides and flavoproteins. In between, a 

 phenomenon resembling uncoupling occurs at the level of cyto- 

 chrome b. At this site there is a minimal reducing effect which at 

 low turnover is in fact a reoxidation. This could be interpreted as 

 due to the superimposition of two actions; first, the general increase 

 of reduction following action of azide on cytochrome oxidase which 

 is dependent upon turnover number, and second, a specific un- 

 coupling exerted on cytochrome b. Other results are also in favor of 

 an uncoupling action of azide, such as the data obtained on Spisula 

 spermatozoa where respiration can be activated by azide. 



Crossover points for antimycin A, Amytal, and DNP are given at 

 the lower right of Fig. 12. Crossover points as defined by Chance and 

 Williams (1956) are sites along the electron transfer chain where 

 blocking occurs. Below such a point, on the substrate side, reducing 



