212 



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



Oxidation and reduction of mitochondrial pyridine nucleotide 



The first study of the oxidation and reduction of pyridine nucleotides 

 in mitochondria was carried out by Chance and WilHams [7], who made 

 the important discovery that, in the presence of substrate, the pyridine 

 nucleotides were largely reduced when the respiration of the mitochondria 

 was " inhibited " owing to lack of ADP, and were oxidized by the additions 

 of ADP. These measurements were made by a sensitive spectrophoto- 

 metric technique, which enabled the determination of the absorbancy 



o 4 



"T I I I I I I I T 



/30H 15 mM 



STATE 3 /state 3^* 



\STATE 



^ ADP 63mM 



> PO4 4 OmM 



ADP 7mM 



PO4 4 OmM 



J — I I I I \ L 



~i — I — r 



/JOH l5mM 



• DPN 

 oTPN 



ADP 063mM 

 PO4 4 OmM 



ADP 7mM ^'*^^ ' 

 PO4 4 OmM 



I I I 1 I 



12 3 4 5 6 7 8 1 2 3 4 5 6 7 



Time (min) 



Fig. I. DPN + and TPN + contents of rat-liver mitochondria in different 

 "States" [7]. Rat-liver mitochondria (final concn. 2-8 mg. protein/ml.) were 

 suspended in o- 21 M sucrose, 33 mM nicotinamide, 2-5 mM MgCU final volume 

 2-7 ml. The following additions were made: at zero time, 07 mM ADP and 

 4-0 mM Pj; at 2 min., 15 mM j8-hydroxybutyrate ; at 4-5 min., 0-63 mM ADP 

 and 4-0 mM Pj. DPN + and TPN + were determined on aliquots of the same sus- 

 pension. Abbreviations: PO4, Pj; j80H, /3-hydroxybutyrate. Temperature, 0°. 

 Unpublished experiment of Dr. J. L. Purvis. 



changes at 340 m/^i in a mitochondrial suspension. Chance and Williams 

 [16] interpreted these absorbancy changes as reflecting changes in the 

 oxido-reduction state of DPN. This became uncertain when Clock and 

 McLean [13] and Jacobson and Kaplan [14], by specific enzymic tests on 

 deproteinized extracts, showed that rat-liver mitochondria contained much 

 more TPNH than DPNH. However, a recent study by Klingenberg and 

 Slenczka [8], who used both Chance's method of direct spectrophoto- 

 metric observation of the mitochondrial suspension and the less sensitive 

 but more specific enzymic assays on deproteinized extracts, have confirmed 

 the most important findings of Chance and Williams. In particular, they 

 showed that the rapid absorbancy changes w^hich follow exhaustion or 



