io8 



THOMAS P. SINGER 



and antimvcin A in the DPNH oxidase assay and that the reduction of 

 ferricyanide by ETP is also inhibited by these reagents as far as the cyto- 

 chrome c site is concerned (Fig. i). That the sohible flavoprotein is 

 antimycin-insensitive is not surprising, since this inhibitor is thought to 



4- 



DPNH oxidase 



Fe(CN)^ assay 



DPNH (x10''m) 



Fig. 4. Inhibition of DPNH oxidase and of DPNH dehydrogenase activities 

 by excess substrate. Left: DPNH oxidase assay at 30°; 120 /Limoles phosphate, 

 pH 7-4, o-o6 mg. protein (DPNH oxidase, Crane, et al. [11]), and DPNH as 

 indicated in 3 ml. volume. Right: Ferricyanide assay at fixed acceptor concentra- 

 tion. Same conditions except that 0-09 mg. protein and 2-5 /xmoles K3Fe(CN)6 

 were present in each cuvette. Reaction time in both experiments about 30 sec. 



2 4 6 



l/ml. Fe(CN)g'" 



Fig. 5. Ferricyanide assay of soluble DPNH dehydrogenase at various con- 

 centrations of DPNH. For assay conditions see Fig. i. The DPNH concentrations 

 were: 1-5 x ic* m, • — •;3-o x 10"* m, o — O; and6-o x 10 * m, X — X. 



act between cytochromes h and c, but the fact that it is also amytal- 

 insensitive was contrary to expectations, since amytal had been thought 

 to interrupt the flow of electrons from DPNH to flavoprotein [16]. The 

 insensitivity of the isolated dehydrogenase to amytal and the insensitivity 



