Isolated washed Ehrlich ascites tumor cells when incubated in the presence of inorganic phosphate labeled with P32 incorporated this label into a phos-phoprotein fraction with a specific activity about 30 times higher than that of the other acid insoluble phosphate-containing components (128). A similar high rate of turnover of phosphoproteins in various tissues of the rat and in the Yoshida sarcoma had been previously reported by other workers. However, in all these studies the phosphoprotein fraction was not a pure chemical entity but was grossly contaminated with other phosphorus-containing substances, and considerable uncertainty existed as to the exact identity of the highly radioactive components of this fraction.

A detailed chemical identification of the radioactive component of the phosphoprotein fraction of the Ehrlich ascites tumor was therefore undertaken (128). New procedures were developed for the isolation of phos-phoserine by chromatography on ion exchange resin after partial acid hydrolysis of the phosphoproteins. This technique combined with paper chromatography and recrystallization of radioactive materials in the presence of carrier phosphoserine made possible the identification, isolation, and determination of the radioactivity of the phosphoserine derived from phosphoproteins.

Experiments were carried out in which Ehrlich ascites tumor cells were incubated in the presence of orthophosphate labeled with P32. After partial acid hydrolysis of the protein, the phosphoserine was isolated and re-crystallized to constant specific activity. It was found that the phosphate moiety of phosphoserine in the phosphoproteins of the Ehrlich ascites tumor undergoes a truly remarkable rate of turnover, since the specific activity of the pure phosphoserine was actually higher than the specific activity of the crude fraction from which it was derived. The possibility was suggested that this rate of turnover is the result of enzymatic activity on the part of enzymes which function by cyclic phosphorylation and dephos-phorylation of the serine hydroxyl group. Subsequent work in a number of laboratories, especially that of Sutherland and his collaborators, has shown that mechanisms of this kind do, in fact, play an important part in cell physiology.