2o8 E. C. SLATER, M. J. BAILIE AND J. BOUMAN 



by intact liver mitochondria, and by Christie and Judah's [4] similar 

 finding with respect to reduction of DPN.* 



The intramitochondrial pyridine nucleotides are also inaccessible to 

 DPN +-destroying enzymesf which are present in the microsome fraction 

 [5]. The fact that the pyridine nucleotides are retained within the mito- 

 chondria, even when the mitochondrial suspension is diluted, provides the 

 opportunity of studying the oxidation and reduction of the mitochondrial 

 pyridine nucleotides during the operation of oxidative phosphorylation, 

 an opportunity availed of with great success by Chance and Williams [7], 

 and by Klingenberg and his co-workers [8, 9]. 



For some time, we, like others, have been interested in the possibility 

 that a compound of DPN with some substance, variously known as C, I 

 or X, might be an intermediate in chemical reactions which link intra- 

 cellular respiration with the synthesis of ATP, About 2 years ago. Dr. 

 Purvis announced from our laboratory that he had evidence that rat-liver 

 mitochondria contained, besides DPN + and DPNH, a third form of 

 diphosphopyridine nucleotide, termed "extra DPN", which accumulated 

 in and disappeared from the mitochondria in a manner that might be 

 expected for an intermediate of oxidative phosphorylation [10, 11]. This 

 conclusion has been criticized by others, whose experiments gave no 

 evidence of a diphosphopyridine nucleotide compound other than DPN + 

 and DPNH. 



During the last 2 years. Dr. Purvis has been continuing this investi- 

 gation in Dr. Kaplan's laboratory in Brandeis, while we have been following 

 up other aspects in Amsterdam. Although our studies are not yet com- 

 pleted, I thought it only fair to other workers in the field that we bring out 

 an interim report of our experiments. 



Pyridine nucleotide content of isolated mitochondria 



Huennekens and Green's [2] measurements of the pyridine nucleotide 

 contents of cyclophorase preparations were followed by determinations by 

 Holton [12] of the DPN + content of isolated rat-heart sarcosomes, and by 

 Glock and McLean [13] and Jacobson and Kaplan [14] of the DPN +, 

 DPNH, TPN + and TPNH content of isolated rat-liver mitochondria. The 



* Birt and Bartley [6] have recently confirmed by direct analysis that mito- 

 chondria can exclude added DPN + and DPNH from participation in intramito- 

 chondrial processes of oxidation and reduction. TPN + and TPNH, on the other 

 hand, can enter the mitochondria readily. 



t Abbreviations : DPN +, DPNH, oxidized and reduced diphosphopyridine 

 nucleotide ; DPN, all forms of diphosphopyridine nucleotide ; TPN +, TPNH, 

 TPN, corresponding symbols for compounds of triphosphopyridine nucleotide ; 

 ADP, ATP, adenosine di- and triphosphate; P,, inorganic phosphate; EDTA, 

 ethylenediaminetetraacetate. 



