RESPIRATION AND OXIDATIVE PHOSPHORYLATION 121 



between pyridine nucleotides and cytochrome b. With succinate as 

 substrate, Amytal has no spectroscopic or respiratory effect. A cross- 

 over point for antimycin A is located between cytochromes b and r, , 

 and for DNP between cytochromes c and a. The location of these 

 points is, by and large, the same as that found in other cells or mito- 

 chondria. 



Parallel with the spectroscopic studies, experiments were con- 

 ducted on the effect of the same inhibitors on respiration and mo- 

 tility of Spisula and dog spermatozoa. Azide action on endogenous 

 respiration of Spisula spermatozoa is, as in bull sperm, a complex 

 one (Fig. 13). At concentrations from 1 to 3 mM, activation of res- 

 piration is noted, but some inhibition occurs at higher levels of 

 azide. In some samples the phenomena balance one another, and 

 there is no inhibition at 20 mM azide; in others, however, where ac- 

 tivation had not occurred at 3 mM azide, inhibition is brought about 

 by 20 mM azide, but at most this involves only a 50% inhibition of 

 respiration. Motility decreases progressively for azide concentrations 

 over 1 mM and is completely arrested by 20 mM azide. This inhibi- 

 tion of motility begins at concentrations which activate respiration, 

 and thus points to an uncoupling effect of azide as was suggested by 

 the data on bull spermatozoa. Here again, the high respiration ob- 

 tained by the addition of DNP is more sensitive to azide than is 

 endogenous respiration, and shows that inhibition is dependent on 

 turnover number. As in the bull, at the end point of titration (20 

 mM azide), the noninhibited respiration, expressed in volume of 

 oxygen consumed, is the same with or without DNP. The titration 

 curve with azide in the presence of DNP follows a sigmoid pattern 

 which could indicate that the secondary phenomenon, the activation 

 of respiration, is superimposed here, too, but occurs at lower azide 

 concentrations of 0.05 to 1 mM. 



As in Spisula, inhibition of respiration by azide is not complete in 

 dog spermatozoa; 30% of exogenous respiration is resistant to 10 mM 

 azide (Fig. 14). Motility is progressively inhibited in the range 0.2 to 

 5 mM and completely lost at 5 mM azide. It is important to note that 

 during the titration, the effect of azide on motility is not uniform. 

 Some spermatozoa remain fully motile, others are completely ar- 

 rested. As azide concentration increases, increasing numbers of im- 

 motile cells are found, a fact which indicates a threshold effect. This 



