116 PIERRE H. GONSE 



cinate Amytal-treated sample. Respiration is then inhibited while 

 terminal cytochromes are further reduced. Also shown in Fig. 8 is 

 the peculiar effect of a detergent, Triton, which without increasing 

 respiration brings about a significant increase in cytochrome oxidase 

 reduction. 



Azide appears to have a peculiar effect on the aerobic steady state 

 of lactate oxidation in bull spermatozoa. From a combination of 

 three spectra recorded on the same preparation at 25 °C, one can 

 calculate and obtain directly the percentage of reduction for pyridine 

 nucleotides, flavoproteins, and cytochromes (Fig. 9). Azide has a 

 multiple action; it increases the reduction of terminal cytochromes, 

 rt 3 , a, and c, as one would expect from that inhibitor, but has no 

 effect or rather a reoxidizing action on cytochrome b, while flavo- 

 proteins and pyridine nucleotides are more effectively reduced. The 

 phenomenon occurring at the level of cytochrome b is better seen 

 in low-temperature spectra recorded on "stabilized steady states" 

 (Fig. 10). That cytochrome oxidase is a primary site of action for 

 azide in bull spermatozoa is confirmed by the fact that the alpha ab- 

 sorption band of cytochrome a, normally at 600 m^, is shifted to 596 



Fig. 9. Difference spectra of bull spermatozoa at 25°C. Cells washed 

 and suspended in RP, pH 7.2. Sample H4726. Solid line: (lactate 20 mM, 

 aerobic) — (aerobic, endogenous substrates). Dotted line: (azide 10 mM, 

 lactate, aerobic) — (aerobic, endogenous substrates). Dashed line: (azide, 

 lactate, aerobic) — (lactate, aerobic). Reduction by azide in the aerobic 

 steady state in per cent: 10 (cyt a s ), 30 (a), 25 (r + c x ), about (b), 1 1 (FP), 

 19 (PN) calculated by difference between spectrum 2 and spectrum 1. 

 Experimental check of those figures is directly afforded by spectrum 3 

 where reduction by azide is (%): 12 (cyt a 3 ), 28 (a), 25 (c + c x ), about 

 (b), 9(FP), 14 (PN). 



