SOLUBILIZATION AND PROPERTIES OF THE DPNH DEHYDROGENASE 



115 



Before leaving the subject it may be worth mentioning that the absolute 

 position and the height of the peak in the near visible region are strongly 

 dependent on pH (Fig. 14). 



The partly purified enzyme (specific activity = 130) was found to be 

 relatively insensitive to inhibition by /)-chloromercuribenzoate, completely 

 insensitive to dicoumarol (Fig. 3); it did not catalyze the reduction of 

 coenzyme Q^, significantly (with or without added mitochondrial lipid 

 and Triton X-ioo), and, under the assay conditions recommended by 

 Wosilait [22], the rate of reduction of menadione at V^^^ was less than 1% 

 of the rate of reduction of ferricyanide. These observations clearly dis- 



Lipoyl dehydrogenase assay 



04 0'8 1-2 



I///1. Iipoamide 



Fig. 15. Lipoyl dehydrogenase assay of DPXH dehydrogenase. Conditions 

 were as recommended by Massey [6]. The abscissa denotes the reciprocal volumes 

 (in jA.) of 0-058 M Iipoamide present in i ml. reaction mixture. 



tinguish the enzyme from DT diaphorase [23, 24] and from menadione 

 reductase [22]. 



Under the conditions of the lipoyl dehydrogenase assay employed by 

 Massey [6] the soluble extract obtained on treatment of FTP with phos- 

 pholipase A shows only a trace of activity on Iipoamide (Fig. 15): ratio of 

 activities on ferricyanide and Iipoamide, respectively, differ bv a factor of 

 about 500 between this preparation and diaphorase (Table II). This trace 

 of lipoyl dehydrogenase activity may well be due to a slight contamination 

 with diaphorase which would be probably removed in the purification pro- 

 cedure. The dehydrogenase may be readily distinguished from DPNH 

 cytochrome c reductase [2] by its much greater stability and bv the 

 extremely low rate of cytochrome c reduction even when assayed under 

 optimal conditions for Mahler's enzyme (Table III). That the residual 



