2l8 



E. C. SLATER, M. J. BAILIE AND J. BOUMAN 



reported by Purvis and the bulk of our results, in that the DPNH content 

 increased, but not to the same extent as the decrease of DPN + (Table 

 VIB). This result suggests the possibility that, whenever we have a prepara- 

 tion of fresh rat-liver mitochondria containing some "extra DPN", we 

 can increase the amount by adding succinate. However, it must be empha- 

 sized that Table YIB describes a single result, which has not yet been 

 reproduced. 



At this stage, we must conclude that the tightly-coupled rat-liver 

 mitochondria which we normally prepare in Amsterdam rarely contain 

 "extra DPN", and that this substance cannot usually be induced by a 

 short-term incubation with succinate. Further progress clearly required a 

 reproducible method of inducing the "extra DPN". 



TABLE VII 



Effect of Incubation with Glutamate or Succinate in Pi-Deficient Med- 

 ium, AND of the Subsequent Addition of Dinitrophenol on DPN+ and 

 DPNH Contents of Rat-Liver Mitochondria 



Unpublished experiments of B. Winter and M. Bailie. Mean values (/xmoles/g. 

 protein.) 



Substrate : 



Glutamate 



Glutamate 



Succinate 



Analytical Method: 



Fluorimetric 



Spectro- 

 photometric 



Fluorimetric 



No. of expts. 



Changes on incubation zvith substrate 



Z1DPN+ -009 



JDPNH -063 



J(DPN+ + DPNH) -072 



Changes on subsequent addition of dinitrophenol 

 JDPN+ 1-88 



J DPNH -079 



J(DPN+ + DPNH) I 09 



* Means 2 08 and 023, respecti\ely, if one doubtful value is omitted. 



If the " extra DPN " is really an intermediate in oxidative phosphoryla- 

 tion, as we hope, we should expect that it would accumulate in the absence 

 of inorganic phosphate or ADP. For this reason, we tried incubation with 

 glutamate in the presence of ADP, hexokinase, glucose, and nicotinamide. 

 Under these conditions, any traces of inorganic phosphate are rapidly 

 esterified and accumulate as hexose monophosphate. The rate of respira- 

 tion is only about one-seventh that obtained on the addition of inorganic 

 phosphate [23, 24]. 



Figure 2 (A) and the second column of Table VII show the results of a 



