The Significance of Respiratory Chain Oxidations 629 



These results are expressed in Table 2 (a) as //moles of pyridine nucleo- 

 tide/g of mitochondrial protein (assuming a conventional protein/N ratio 

 of 6/1). Table 2 (b) also includes measurements from Purvis (1958) on liver 

 mitochondrial coenzymes expressed on the same basis. Apart from somewhat 

 higher results for both DPNH and TPNH obtained by Purvis using the 

 fluorimetric methods of Kaplan, the agreement is again satisfactory. 



The last line of Table 2 (b) shows values obtained by Purvis after 5 min 

 aerobic incubation at 30°C of the mitochondrial suspension in presence of 

 inorganic phosphate (0-04 m), or with adenosine diphosphate (ADP, 0-002 m) 

 in presence of nicotinamide, or with dinitrophenol. Two effects occurred: 

 (1) the entire DPNH and TPNH became oxidized, and (2), a very remarkable 

 result, the total amount of both DPN and TPN detectable in the suspension 

 was approximately doubled. Neither the method of analysis used by Clock 

 and McLean, nor that of Purvis, seems capable of estimating this hidden form 

 of DPN and TPN, which Purvis suggests is the 'high energy' intermediate, 

 (DPN --^ I) or (TPN '--' I), often postulated as participating in oxidative 

 phosphorylation, and frequently discussed during this meeting. The nature 

 of compounds causing release of free coenzyme is consistent with this idea. 

 Direct synthesis of the extra coenzyme during incubation appears improbable 

 since ATP, unlike ADP, is ineffective. The mechanism suggested is : 



DPNH + fp + I ^ DPN ~ I + fpHg 



followed by release of DPN+ from the (DPN ---' I) complex by the second 

 intermediate, X, which becomes (X --^ I) and then reacts with inorganic 

 phosphate to give (X '~ P). If ADP is present as an acceptor, this complex 

 transfers its 'high energy' phosphate to ADP forming ATP; the presence 

 of dinitrophenol causes the breakdown by hydrolysis of the 'high energy' 

 intermediate. In either case the bound pyridine nucleotide is set free. The 

 estimated (DPN ~ I) at 1-67 /imoles/g mitochondrial protein (Purvis; Table 

 2 (b)) is considered to be consistent with the content (1-5 //.moles/g protein) 

 of 'high energy' compounds (judged by their rapid formation of ATP on 

 addition of ADP) in similar mitochondrial suspensions, obtained previously 

 by Eisenhardt and Schracliinger (1958). It seems to be assumed by Purvis 

 that TPN /^ I will not phosphorylate ADP. [Jacobson and Kaplan (1957, 

 Table V) incubated liver mitochondria for 5 min at 37°C in 0-02 m phosphate, 

 pH 7-5; they also obtained nearly complete oxidation of TPNH and DPNH 

 but no increase in the total pyridine nucleotide estimated (ATPNH — 1-25; 

 ADPNH — 0-12; A(DPN+ + TPN+) + 1-4 //moles/g mitochondrial protein; 

 calculated from these authors' data).] 



These recent and important experiments have been described at some 

 length, because if confirmed they may necessitate revision of all previous 

 analyses of pyridine nucleotides in mitochondria. Presumably their effect 

 on total cellular pyridine nucleotide is somewhat less serious; but even so. 



