Phosphorylation and protein kinase activities of chromosomal non histone proteins from chick embryo fibroblasts.

Confluent chick embryo fibroblasts were cultured in vitro in (i) medium which prevented the cells from dividing, (ii) medium which stimulated the cells to divide synchronously, (iii) medium without lysine in which the cells were blocked in G1. Chromosomal non histone proteins (NHP) were extracted from cells pulse labelled with 32P phosphate, and the radioactivity analyzed by acrylamide gel electrophoresis. Several radioactive peaks were found all along the gel in the NHP from confluent and stimulated cells. The highest phosphorylation was found in the fast moving proteins, but the stimulation of the cells increases the phosphorylation of the slower moving proteins. In the NHP from cells cultured in the medium without lysine only the slow migrating proteins were phosphorylated. NHP were extracted from unlabelled cell cultures in the three different media, incubated with [gamma-32P] ATP and analyzed by acrylamide gel electrophoresis. Highly labelled peaks were observed in the fast moving proteins from stimulated cells and from cells cultured in a medium deprived from lysine. By comparing in vivo and in vitro phosphorylation, it can be concluded that in confluent cells the turnover of bound phosphate is slow. In stimulated cells there is a fast turnover of the phosphate bound to fast turnover of the phosphate bound to a small group of fast migrating proteins and very little turnover of the phosphate bound to slow migrating proteins. The cells were incubated with labelled lysine and NHP analyzed by gel electrophoresis. The radioactivity of individual NHP varied with the culture conditions, but in all cases, there was little radioactivity in the fast moving proteins. The phosphate groups submitted to a fast turnover are bound to stable proteins. Phosvitin and casein kinase activities were measured in the NHP fractions. Nine-ten peaks of activities were observed with each substrate. Some variations were observed which apparently correlate with the culture conditions.