COUPLING OF REDUCED PYRIDINE NUCLEOTIDE OXIDATION 1 77 



enzyme has a very high turnover number [9] it requires rather high 

 levels of substrate in order to function efficiently- 



This effect may be illustrated by comparing the activity of two types of 

 diaphorase enzvmes, the purified DT diaphorase and the D diaphorase of 

 the mitochondrial respiratory chain prepared by extraction of mitochondria 

 with Lubrol W. The diaphorase activity of these two enzymes was 

 compared in the oxidation of both added DPNH and of DPNH generated 

 with a system containing alcohol dehydrogenase and ethanol, which at the 

 pH used has an equilibrium unfavourable to the production of DPNH. 

 As may be seen in Table IV the two diaphorase activities were chosen so 

 as to react at similar rates with added DPNH as substrate. In the alcohol 

 dehydrogenase system D diaphorase could still function efficiently; how- 

 ever, the reaction rate with DT diaphorase was greatly reduced as com- 

 pared with the activity with added DPNH. 



TABLE IV 



Comparison of the Activities of DT Diaphorase axu DPNH Diaphorase of 

 Mitochondria with Added DPNH and with a DPNH -Generating System 



Diaphorase activity 



Purified DT diaphorase Mitochondrial D diaphorase 

 (/Ltmoles DCPIP reduced/niin.) 



DPNH 0020 0017 



Ethanol, DPN, alcohol 



dehydrogenase 00024 00165 



The assay system contained 004 m.M DCPIP, o-i niM DPNH or DPN, 33 

 mM ethanol, excess alcohol dehydrogenase, o 33 niM KCN, oi",, albumin, and 

 0-05 mM orthophosphate (pH 75). Reaction followed hy JE^on- [Ernster, Daniel- 

 son, and Ljunggren, unpublished]. 



It seems then that DT diaphorase would function in the cell only when 

 the levels of reduced pyridine nucleotide are high. As Glock and AIcLean 

 [10] and others [12] have shown this is generally found only in the case of 

 TPNH. It is assumed, therefore, that the function of DT diaphorase is 

 primarily with regard to the TPNH of the cell. 



Although the bulk of this enzyme is located in the cytoplasm it can also 

 be extracted from mitochondria [5]. Here the function of the enzyme is 

 perhaps even more obscure than it is in the cytoplasm. Numerous trans- 

 hydrogenases have been reported which would presumably allow the 

 oxidation of TPNH through the active DPNH oxidase of mitochondria. 



As has been reported pre\"iously [4, 8] DT diaphorase can be demon- 

 strated in mitochondria by b} passing the site of Amytal inhibition in the 



VOL. II. — N 



