PYRIDINE NUCLEOTIDES IN MITOCHONDRIA 217 



this amount of " extra DPN ". Although we are under the impression that 

 some of our preparations of Uver mitochondria have the properties 

 ascribed to them by Purvis, thev turn up so rarelv that we could not be 

 absolutely certain that they were not due to analytical errors. 



Further evidence for the presence in liver mitochondria of a form of 

 diphosphopyridine nucleotide other than DPN and DPNH was obtained 

 by Pur\'is by studying the effect of adding substrates. Table V shows that 

 the addition of a-ketoglutarate, glutamate or succinate for 90 sec. at o^ 

 (without added substrate there is no change in the amount of DPN + and 

 DPNH during this period) caused a decline in the amount of DPN +, 

 without any increase in the amount of DPNH, i.e. some pyridine nucleo- 

 tide disappeared. Malate and fumarate, on the other hand, caused little 

 change in the DPN + or DPNH level. 



TABLE VI 



Effect of Added Succinate (90 sec. at o ) ox Forms of Diphosphopyridine 

 Nucleotide in Rat-Liver Mitochondria 



Unpublished experiments of J. Bouman, B. Winter and M. Bailie. Mito- 

 chondria suspended in o- 18-0-25 ^ sucrose. Value in /(moles/g. protein. 



Method No. ofexpts. JDPN^ J DPNH 



A. Mean results 



Fluorimetric 11 — o-6i +0-66 



Spectrophotometric 4 —0-94 +o-6o 



B. Single experiment (spectrophotometric method) 



DPN ^ DPNH " Extra DPN "* 



Fresh mitochondria 2-34 i'54 0"57 



+ Succinate (40 niM) o-(Si 2-28 i'36 



* Total DPN (determined by incubation in absence of substrate with 0-05 

 M P, for 10 min.) minus (DPN^ + DPNH). 



The results with succinate appeared to be in conflict with those of 

 Chance and Hollunger [22], who reported extensive reduction of DPN + 

 by this substrate. 



When we repeated these experiments with our preparations of liver 

 mitochondria, which contained little "extra DPN", the DPN + which 

 disappeared nearly always appeared as DPNH (see Table VL4). These 

 results support Chance and Hollunger [22], whose findings have in the 

 meantime been confirmed by Klingenberg et al. [9]. As a whole, they give 

 no support to the existence of another form of DPN. When, however, 

 succinate was added to one of our rare preparations which appeared to 

 contain "extra DPN", a result was obtained intermediate between that 



