100 



Britton Chance 



In the other two tests, the larger amounts of ADP cause 

 greater reduction of cytochrome in the steady state and a 

 longer duration of the cycle corresponding to the longer time 

 required to expend the added ADP. By plotting the amplitude 

 of these cycles against the added ADP concentration, we 

 obtain a saturation curve, indicated in Fig. 5. Half -maximal 



-O.IOi 



§ 



ro 

 I 



O 

 ro 

 ro 



I-O.OI5J 



10 

 [POj mM 



E 

 o 



.- 1^ 



w ro 



0) > 



Q O 



■^ ro 



Q. 



o 



Fig. 7. Comparison of the effects of phosphate and ADP 

 upon the steady state oxidation-reduction level of intra- 

 mitochondrial reduced pyridine nucleotide. Addition of 

 one of these substances to a mitochondrial suspension con- 

 taining the other causes an oxidation of the reduced pyri- 

 dine nucleotide. The relationship between the ADP and 

 phosphate concentrations and the extent of oxidation of 

 the pyridine nucleotide is indicated by the graph. The con- 

 centrations giving half-maximal effect are also indicated. 

 (Expt. no. 299b, 463b) [From Chance and Connelly, 1957. 

 Reproduced by permission of the Editors, Nature (Lond.).] 



effect is caused by 60 (jlm ADP under these conditions. A 

 similar experiment may be carried out with phosphate, 

 provided the substrate- and ADP-treated mitochondria are 

 suspended in a phosphate-free medium. As illustrated by 

 Fig. 6, such mitochondria show a negligible response to the 

 addition of an excess of ADP. The respiratory carrier (in this 

 case, reduced pyridine nucleotide) shows a slow increase of 

 oxidation in response to the addition of 6 • 8 mM phosphate. 



