Radiolabeling of mammalian cells in tissue culture by use of acetic anhydride. Potential value for studying the dynamics of protein turnover in living cells.

When Chinese hamster ovary cells, growing in monolayer culture, were treated with trace quantities of radioactive acetic anhydride, label was very rapidly introduced into both the plasma membrane and intracellular components, all of which reached similar specific radioactivities. The cells themselves showed no measurable loss in viability. The incorporated label was recovered predominantly in the form of acetyl groups on proteins. Since tyrosyl hydroxyls were not extensively acetylated, it seems likely that substitution occurred mainly on amino groups. There was no indication that the acetyl groups once introduced were labile or that the modified proteins were discriminated against by the cells, since, in dividing cells, the acetylated proteins were as long-lived as those labeled with radioactive l-leucine. Nor was there any evidence for reutilization of labeled acetate following protein turnover in cells maintained in a nonproliferating state on a medium lacking the essential amino acid l-isoleucine. We suggest that acetylation could provide an ideal means for introducing a very short “pulse” of radioactivity into proteins of living cells. In addition, the method should be extremely useful for studying processes such as enzyme induction and protein turnover where the problem of amino acid reutilization has long been recognized as a possible basis for artifacts.