124 BRIXTON CHANCE 



suggesting that only ATP is required for pyridine-nucleotide reduction. 

 These considerations eliminate hypotheses which postulate that DPN- 

 linked substrates are acting directly or through an ATP-activated step, 

 since succinate or ATP alone should cause the observed effects. 



A remarkable feature of the reaction and one which seems to differ 

 considerably from the results so far obtained by Klingenberg and Azzone 

 (this meeting) is the low concentration of ATP required for pyridine- 

 nucleotide reduction. Under favourable conditions it has been observed 



Fluorescence excitation 365nn/i 

 Fluorescence measurement 450m/i 



lOmM succinate 

 PH. M + ==jf_' 



State 1 



Fig. 6. 



Fig. 6. Illustrating separation of 

 electron and energy-transfer require- 

 ments for succinate-linked pyridine- 

 nucleotide reduction. Addition of 

 succinate alone causes no fluores- 

 cence change, while loo jum ATP 

 causes a large fluorescence increase 

 corresponding to pyridine-nucleo- 

 tide reduction. Pigeon-heart mito- 

 chondria suspended in sucrose- 

 " tris "-mannitol medium, pH 7-4, 

 temperature 26° (Expt. 133d). 

 (Reproduced with permission of 

 The Journal of Biological Chemistry.) 



Fluorescence excitation 365m/i 

 Fluorescence measurement 450myu 



TS/jM ATP 



4mM succinate 



P.H Mv»_>: 



(state 1) 



Fig. 7. 



Fig. 7. Illustrating separate 

 energy and electron-transfer require- 

 ments for succinate-linked pyridine- 

 nucleotide reduction. Pigeon-heart 

 mitochondria suspended in mannitol- 

 sucrose-"tris" medium, pH 7 '4, 

 temperature 25°. Downward deflec- 

 tion of fluorescence trace indicates 

 increase of pyridine-nucleotide re- 

 duction (Expt. 133d). (Reproduced 

 with permission of The Journal of 

 Biological Chemistry). 



that as little as two ATPs per DPNH are required [15]. This result is 

 obtained under relatively poor conditions for optimal efficiency of the 

 reaction since the mitochondria are capable of breaking down ATP with- 

 out expending its energy in the reduction of pyridine nucleotide. The small 

 ATP requirement for DPN reduction also sets a value for any other sub- 

 stances that might react with ATP. First, the conditions of the experi- 

 ment are such that about 20 /xM DPN is reduced on addition of about 60 

 /xM ATP. Thus readily detectable chemical changes are caused by the 

 ATP reaction which can easily be confirmed by extraction of the mito- 

 chondria and subsequent analysis. 



