232 Van R. Potter and Hermann Niemeyer 



diluted in 2 ml. of water and passed in the cold through a small 

 column (2 cm. in length, 0-8 cm. in diameter) of Dowex-50 in 

 the hydrogen form. The nucleotide was retained, possibly not 

 as the consequence of an ionic exchange, but merely retained 

 in solution in the intergranular water. The elution from the 

 column was done with small portions of water (0-5 ml.). 

 The fractions were assayed enzymically. The recovery was 

 practically complete. TPNH was also purified from possible 

 metal contaminants by passing it through Dowex-50 in the 

 sodium form. The spectrophotometric studies showed no 

 alteration of the nucleotide with this procedure. Glucose- 

 6-phosphate (G-6-P) was the crystalline potassium salt from 

 Sigma. Fructose-1 : 6-diphosphate (F-di-P) and pyruvic acid 

 were purified preparations provided by Dr. G. A. LePage. 

 6-Phosphogluconate (6-PG) was either a Sigma product or one 

 prepared by Dr. C. Scholtissek by the procedure of Robison 

 and King (1931). 



Analytical procedures — Glucose was determined in barium 

 hydroxide-zinc sulphate filtrate (Somogyi, 19456) with the 

 reagents of Somogyi (1945a) and Nelson (1944). In some 

 experiments, glucose oxidase was used for this purpose. 

 Lactic acid was determined by the procedure of Barker and 

 Summerson (1941), with some of the modifications introduced 

 by HuUin and Noble (1954), and inorganic phosphate by the 

 method of Fiske and Subbarow (1925) as used by Lohmann 

 and Jendrassik (1926). TPN was determined in an aliquot 

 that was boiled in acid to destroy TPNH (Glock and McLean, 

 1955). After neutralization, i^ocitric dehydrogenase and iso- 

 citric acid were added and readings at 340 mpi were taken in 

 the Beckman-DU spectrophotometer until a plateau was 

 reached. The difference between the plateau and the initial 

 reading was converted into (jimole of TPN using the absorption 

 coefficient of 6 • 22 X 10^ (Horecker and Kornberg, 1948). An 

 approximate estimation of TPNH was considered to be the 

 reading at 340 m(ji, corrected by the appropriate control, 

 accepting that all the diphosphopyridine nucleotide (DPN) of 

 the system was in the oxidized form (Aisenberg and Potter, 



