SOLUBILIZATION AND PROPERTIES OF THE DPNH DEHYDROGENASE IO7 



inhibitory to DPNH dehydrogenase, ahhough the situation is quite 

 different in liver [15]. Since at high ferricyanide concentrations the curve 

 is very steep, however, for accurate and rehable assays it is clearly desirable 

 to determine the activity at infinite ferricyanide concentration. 



The biphasic nature of the curve relating activity to ferricyanide con- 

 centrations at or above 6 x lo^* m DPXH (Fig. i) is not seen at low 

 (2 X 10"^ M or less) DPNH concentrations (Fig. 2). The break in the 

 curve also disappears on solubilization of the dehvdrogenase (Fig. 3), as 

 expected from the fact that this procedure separates the flavoprotein from 

 the respiratory chain and thus leaves only one reaction site for ferricvanide. 



8- 



6- 



2 4 6 8 10 



I/ml, Fe(CN)j^** 



Fig. 3. Ferricyanide assay of dehydrogenase after solubilization with phos- 

 pholipase A. 



One of the many reasons why assays of this enzyme conducted at fixed 

 ferricyanide concentrations tend to be unreliable is the narrow region of 

 DPNH concentrations in which apparently optimal activity is observed. 

 As shown in Fig. 4, both the DPNH oxidase and the DPNH-ferricyanide 

 assays are seriously inhibited by substrate concentrations in excess of i . 5 

 to 2 X iQ-^ M. "While inhibition of DPNH dehydrogenases and DPNH 

 cytochrome reductases by excess substrate is a fairly common finding, it 

 is interesting to note that the inhibition, at least with the enzyme under 

 discussion, is competitive with respect to the electron acceptor (Fig. ^). 

 Thus the inhibition by moderately high DPNH concentration disappears 

 at infinite concentration of ferricyanide. 



Returning for the moment to Fig. 3, it may be noted that the soluble 

 dehydrogenase employed here is completely insensitive to antimycin A 

 and to amytal. It may be remembered that ETP, the starting material 

 employed for the extraction of the enzyme, is 100" q inhibited by amytal 



