32 Philip Siekevitz 



of an experiment in which some answer was attempted. We 

 used a much larger amount of mitochondria in the oxidative 

 phosphorylation medium so that we could measure the con- 

 centrations of ADP and ATP in the mitochondria as well as 

 in the medium after spinning down the mitochondria. At the 

 same time, by employing radioactive inorganic phosphate, 

 we could obtain the specific radioactivities of these compounds 

 and compare them with the radioactivity of the glucose-6- 

 phosphate formed as a result of hexokinase activity. By 

 choosing certain conditions we could also obtain different 

 specific radioactivities for the ATP in the medium and the 

 ATP in the mitochondria, so that we could determine the 

 source of the phosphate of glucose-6-phosphate. It can be 

 seen that the large amounts of added hexokinase make for a 

 lower concentration of ATP in the medium but not for the 

 ATP within the mitochondria. However, when we examine 

 the specific activities of the glucose-6-phosphate, we can see 

 that the phosphate of this compound could only have come 

 from the terminal phosphate of the intramitochondrial ATP. 

 We thus postulated that the added hexokinase was acting not 

 on the ATP in the medium nor even on the ATP within the 

 mitochondria but on the ATP as it was leaving the mito- 

 chondria, i.e. at the mitochondrial membrane. We then tried 

 to verify the exactness of this site of action and Fig. 12 gives 

 the results. We added a hexokinase solution of a known 

 activity to mitochondria, and then, after spinning down the 

 mitochondria, we tested the hexokinase activity of the 

 supernatant solution remaining on top of the mitochondrial 

 pellet. The difference between the two activities should be 

 the amount of hexokinase which was brought down with (or 

 bound to) the mitochondria. We thus expected 10 per cent 

 of the activity to be brought down, but, as Fig. 12 shows, we 

 obtained activities of from three to ten times this amount. 

 In other words, we apparently increased our initial hexokinase 

 activity by having it bound to mitochondria. Ernster and 

 Lindberg (1952) have done much the same experiments and 

 have obtained the same results. The possible reasons for this 



