DISCUSSION ON PHOSPHORYLATION 195 



Fritz Lipmann, Massachusetts General Hospital: 



On several occasions during this symposium acetylphosphate has 

 been mentioned as an intermediate in carbohydrate breakdown. 

 Since I have not yet published a complete account of my experi- 

 ments, I should like to take this opportunity to summarize the evi- 

 dence so far accumulated for the formation of acetylphosphate as an 

 intermediate in pyruvic acid oxidation. Early in my study of the 

 oxidation of pyruvic acid in lactic acid bacteria it was observed that 

 inorganic phosphate was an integral part of the pyruvic acid oxida- 

 tion system (1). In partial explanation of its necessity it was shown 

 that with oxidation of pyruvic acid, phosphate could be transferred 

 to adenylic acid to form adenosinepolyphosphate (2). In other words, 

 pyruvic acid oxidation generated energy-rich phosphate bonds 

 (terminology of ref. 3). In analogy to Negelein and Bromel's phos- 

 phoglycerylphosphate (4), acetylphosphate was then suspected to be 

 the phosphorylated intermediate between pyruvate and adenosine 

 polyphosphate. In confirmation it was found that synthetical acetyl- 

 phosphate enzymatically transferred phosphate to adenylic acid 

 with the formation of adenosinetriphosphate (5). This observation 

 led to a reinvestigation of the phosphate turnover in pyruvic acid 

 oxidation. In the bacterial metabolism pyruvate is fortunately not 

 oxidized farther than the acetate stage. Under favorable conditions, 

 therefore, one could hope to demonstrate the accumulation of a 

 phosphorylated precursor of acetic acid. 



Earlier attempts had always failed to disclose any disappearance 

 of inorganic phosphate during the oxidation of pyruvate. Now that 

 acetylphosphate was suspected as intermediate, its stability was 

 studied, and it was found that its formation would have been over- 

 looked because in the course of all the known procedures of phos- 

 phate assay it would have been decomposed to inorganic phosphate. 

 Therefore the problem arose of finding a method for the detennina- 

 tion of inorganic phosphate in the presence of a compound of the 

 stability of acetylphosphate, since this compound does not with- 

 stand the alkalinity of the magnesia mixture tolerated by creatine- 

 phosphate, nor the acidity of the molybdate reagent. It appeared 

 possible, however, to completely precipitate inorganic phosphate as 

 calcium phosphate in dilute alcohol at a pH of 8, where acetylphos- 

 phate is stable. The use of this more delicate precipitation procedure 

 showed indeed that large amounts of inorganic phosphate disap- 

 peared during pyruvate oxidation with or without fluoride (6). 

 Phosphorylation, oxygen consumption, and pyruvate disappearance 



