PYRIDINE NUCLEOTIDES IN MITOCHONDRIA 211 



latter two groups found unexpectedly high concentrations of TPNH. The 

 mean values obtained by these authors, and in later studies, are listed in 

 Table I (rat liver) and Table II (heart). 



In Table I are shown the values obtained in the Amsterdam laboratory 

 over the last 4 years by five different workers. DPN +, TPN + and DPNH, 

 TPNH w'ere determined in acid and alkali extracts, respectively, of the 

 freshly prepared mitochondria. Total DPX and TPN refer to the amounts 

 of oxidized nucleotides found when the mitochondria were treated in such 

 a way as to convert all the pyridine nucleotides into the oxidized form (see 

 below). The total DPN and TPN contents of our preparations have 

 remained almost constant during this period. Purvis has also obtained the 

 same values for mitochondria prepared by our procedure in Brandeis. 



The Amsterdam values for the total DPN content are quite similar to 

 those reported by Birt and Bartley [6] for DPN ^+ DPNH, but are con- 

 siderably higher than the others in Table I.* Our total TPN values are 

 rather higher than the (TPN "^ + TPNH) measured by other workers. It is 

 not known to what extent these differences represent differences in the 

 nutritional status of the rats used, or in the methods used to isolate the 

 mitochondria. All workers report considerable variation from preparation 

 to preparation. 



With respect to the DPNH/DPN+ and TPNH/TPN+ ratios our 

 preparations closely resemble those of Klingenberg and Slenczka [8]. 

 Birt and Bartley 's preparations contain much more of the oxidized pyridine 

 nucleotide. This difference is probably connected with the method of 

 preparation of the mitochondria. f 



Aliss Bailie has found essentiallv the same values by our enzymic 

 fluorimetric procedure, which is similar to that used by Jacobson and 

 Kaplan [14] and Purvis [11], and by a spectrophotometric method, which 

 differs somewhat from others described in respect to the determination of 

 the reduced pyridine nucleotides. The alkali extract is neutralized, treated 

 with a-ketoglutarate, NH4 and glutamate dehvdrogenase to oxidize the 

 DPNH and TPNH, and deproteinized with HCIO4. DPN + and TPN + 

 are determined spectrophotometrically on the completely deproteinized 

 solution by successive additions of ethanol + alcohol dehydrogenase (at 

 pH 10) and isocitrate and isocitrate dehydrogenase (at pH 7-4). The 

 spectrophotometric procedure is rather more reproducible but less 

 sensitive than the fiuorimetric. 



* Klingenberg et al. [9] have suggested that the method used by Holton et al. 

 [15] to determine the DPN + content of rat-liver mitochondria might also estimate 

 TPN ^, owing to traces of TPN ^-specific alcohol dehydrogenase in our prepara- 

 tions of this enzyme. This was not the case. TPN + is not estimated either in the pro- 

 cedure used by Holton et al. [15] or in our recent work (see Purvis [11], Table I). 



t A more recent paper {Biochem. J. 76, 328 (i960)) reports more of th^ 

 reduced nucleotides. 



