TURNOVER OF THE ACYL PHOSPHATES OF HUMAN AND MURINE PROTHYMOSIN-ALPHAIN-VIVO

Prothymosin alpha is a small, highly acidic, abundant, nuclear, mammal ian protein which is essential for cell growth, Our laboratory has rec ently shown that primate prothymosin alpha contains stoichiometric amo unts of phosphate on the glutamyl groups of the protein and that in vi tro the phosphate undergoes rapid hydrolysis or transfer to a nearby s erine residue, Here an assay for the presence of acyl phosphates in vi vo has been developed by measuring stable phosphoserine and phosphothr eonine in. vitro, The assay was used to determine the half-life of the acyl phosphates on prothymosin alpha in vivo by pulse-labeling HeLa c ells with [P-32]orthophosphate and chasing using three different techn iques: permeabilization with digitonin to allow extracellular ATP to e quilibrate with the intracellular pool; electroporation in the presenc e of ATP to reduce the specific activity of [P-32]ATP by expansion of the pool; and incubation with inorganic phosphate, Regardless of the m ethod, the phosphate turned over with a half-life of 75-90 min. The ab ility of cells to phosphorylate old prothymosin alpha molecules was es tablished by demonstrating equivalent labeling of the protein with [P- 32]orthophosphate in the presence and absence of cycloheximide. The ha lf-life of the acyl phosphates was also studied in resting and growing NIH3T3 cells, with measured values of 30-35 and 70 min, respectively. Our data suggest that the ''activity'' of prothymosin alpha involves the turnover of its acyl phosphates and that it participates in a func tion common to all nucleated mammalian cells regardless of whether the y are quiescent or undergoing rapid proliferation, This is the first m easurement of the stability of protein-bound acyl phosphates in vivo.