ADP‐Ribosylation in Permeable HeLa S3 Cells

Publisher Website
Google Scholar
Abstract
ADP‐ribosylation in permeabilized metaphase and interphase cells using [32P]NAD at pH 8.0 have been compared. Incorporation into trichloroacetic acid insoluble material was 4–5‐times greater in metaphase cells. 17–22% was in the soluble fraction which contained material released from the cells, 16–22% in the 0.2 M HCl extract (histones) of the cell ghosts and the remaining activity in the residual fraction. Fractions were analyzed using dodecylsulphate/polyacrylamide gel electrophoresis at pH 6.0. The soluble fractions from metaphase and interphase cells exhibited three common unidentified ADP‐ribosylated proteins corresponding to 78000, 54000 and 36000 Da. In addition metaphase cells contained several other ADP‐ribosylated proteins not present in interphase cells. The 0.2 M HCl extracts gave from metaphase cells radioactivity in the 32000–39000‐Da region suggesting ADP‐ribosylation of histone H1 with up to 10 residues of ADP‐ribose and in the 17000–20000‐Da region indicating ADP‐ribosylation of core histones. The pattern of ADP‐ribosylation of core histone in metaphase and interphase cells was qualitatively similar whereas the number of ADP‐ribose residues per H1 molecule was higher in metaphase cells. The residual fraction contained free poly(ADP‐ribose) and oligo(ADP‐ribose). The results do not lend support to a special function of ADP‐ribosylated histones in the mitotic event while certain ADP‐ribosylated non‐histone proteins may be specific for metaphase cells.