Role of TPN in Control of Glycolysis 231 



The experimental evidence indicates that triphosphopyri- 

 dine nucleotide may inhibit glycolysis in a brain supernatant 

 system, under certain well defined conditions. 



Material and Methods 



The glycolytic system consisted of the supernatant of a 

 10 per cent homogenate of rat brain in 0-15M-KC1 [with 

 • 1 volume of • 1 M-tris(hydroxymethyl)aminomethane (Tris) 

 buffer, pH 7-4 in some experiments], obtained by centrifuga- 

 tion at 5,000 g for 10 minutes (83) or at 105,000 g during 60 

 minutes (S3); 0-5 ml. of any of these preparations was added 

 to a reaction mixture containing ATP, ADP, DPN, Mg, 

 inorganic phosphate and Tris or bicarbonate buffers. The 

 total volume was 1-6 ml. The exact composition of the 

 incubation medium varied somewhat in the course of the 

 experiments and will be indicated in each table or figure. The 

 system was incubated at 37° for 30 minutes, with air (or 

 oxygen with 5 per cent COg) as the gas phase. 



Occasionally, fresh or aged mitochondria were added to the 

 system. Mitochondria were prepared from a 10 per cent rat 

 liver homogenate in • 25 m sucrose, by centrifugation first at 

 600 g and then at 5,000 g (Schneider and Hogeboom, 1950). 

 Fresh mitochondria were washed twice with sucrose and 

 twice or three times with isotonic KCl before use. Aged 

 mitochondria were obtained by incubating the mitochondria, 

 after the second washing with sucrose, in glass-distilled water 

 during two hours at room temperature, with shaking (Reina- 

 farje and Potter, 1957). They were then washed with isotonic 

 KCl. The final suspension of the mitochondria was made in 

 KCl (or KCl-Tris), at a concentration of 50 or 100 mg.-equiva- 

 lents of liver per 0-1 ml. 



Materials — The nucleotides used were commercial products 

 obtained from Sigma Chemical Company, St. Louis, Missouri, 

 or Pabst Laboratories, Milwaukee, Wisconsin. TPN was 

 purified from metal contaminants by passing it through 

 Dowex-50. About 100 mg. of the commercial preparation was 



