46 Discussion 



Rocker: Yeast hexokinase is not very susceptible to product inhibi- 

 tion. 



Siekevitz : We know that we obtained a breakdown of glucose-6-phos- 

 phate simultaneously with, and proportionally to, the increase in 

 activity of the hexokinase which was bound to the mitochondria. 



Lynen : How did you measure hexokinase activity? 



Siekevitz: By the changes in the nucleotide concentrations. 



Lipmann : Crane and I did some experiments where we added radio- 

 active phosphate to a dense suspension of mitochondria, and it was 

 found that in the very early part, within the first minutes, the specific 

 activity of the ATP proved to be greater than that of inorganic phos- 

 phate. This was very paradoxical. We then found that this was 

 caused by a dilution of the inorganic phosphate by intramitochondrial 

 phosphate which had not yet equilibrated (Crane, R. K., and Lipmann, 

 F. (1953). J. bioL Chem., 201, 245). I have some difficulty in under- 

 standing how such an observation compares with yours. 



Siekevitz : Our explanation is based firstly on the finding that we can 

 only get this effect by adding ADP, not AMP. The ADP would be 

 reacted upon by myokinase to give ATP on the surface and AMP inside. 

 The AMP on the inside would eventually go to AT3 2p so that this AT^^p 

 inside the mitochondria would be much higher in specific radioactivity 

 than the mixture of AT^ip and AT^ap on the outside. 



Lipmann: It seems, then, that the two sets of experiments cannot be 

 directly compared since we used AMP, and we assayed early when the 

 internal phosphate had very low specific activity, in other words, when 

 it was very far from being in equilibrium with the external phosphate. 



Siekevitz: Yes, we also noticed that the intramitochondrial inorganic 

 phosphate is lower in specific radioactivity, but only by 30 per cent, 

 than the extramitochondrial inorganic phosphate, and the inner phos- 

 phate has the same specific activity as the intramitochondrial terminal 

 ADP phosphate. Therefore, the newly synthesized inner ATP must have 

 come from the pool of mitochondrial inorganic phosphate ; and the 

 differences in specific activity between intra- and extramitochondrial 

 ATP could not have been due to differences in inorganic phosphate 

 radioactivity. 



Bartley: To return to the structure of the nuclear membrane. J. D. 

 Robertson (1959, Biochem. Soc. Symp., in press) showed electron micro- 

 graphs which indicated that the nuclear membrane was not attached to 

 the endoplasmic reticulum. The perinuclear pool was connected with 

 the exterior of the cell by the channels between the endoplasmic 

 reticulum. This view fits in better with studies on isolated nuclei which 

 apparently have their own bounding membrane. In Robertson's view 

 the fluid bathing the nucleus is connected with the extracellular fluid. 

 In your view the nucleus is in contact with the intracellular fluid. 



Siekevitz : According to the theory held in our laboratory, the nucleus 

 itself has no membrane. It is enclosed by the double membrane system 

 of the endoplasmic reticulum. Perhaps, when you isolate the nucleus 

 you also get part of this endoplasmic reticulum membrane. 



Bartley : In electron microscope pictures of isolated nuclei there is no 



