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ADP-ribosylation in permeable HeLa S3 cells
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 78 000, 54 000 and 36 000 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 32 000-39 000-Da region suggesting ADP-ribosylation of histone H1 with up to 10 residues of ADP-ribose and in the 17 000-20 000-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.
Insights
ADP-ribosylation is significantly higher in metaphase cells compared to interphase cells. While core histone modification is similar, histone H1 and non-histone proteins show distinct ADP-ribosylation patterns during mitosis.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- ADP-ribosylation is a post-translational modification involved in various cellular processes.
- Histones undergo ADP-ribosylation, particularly histone H1, which is implicated in chromatin condensation.
Purpose of the Study:
- To compare ADP-ribosylation patterns in permeabilized metaphase and interphase cells.
- To investigate the specific roles of ADP-ribosylated proteins, including histones, during mitosis.
Main Methods:
- Permeabilized metaphase and interphase cells were incubated with [32P]NAD.
- Cellular fractions (soluble, histone extract, residual) were analyzed.
- Dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate and identify ADP-ribosylated proteins.
Main Results:
- ADP-ribosylation was 4-5 times greater in metaphase cells.
- Metaphase cells showed unique ADP-ribosylated non-histone proteins not found in interphase cells.
- Histone H1 in metaphase cells incorporated more ADP-ribose residues than in interphase cells.
Conclusions:
- The study suggests that specific ADP-ribosylated non-histone proteins, rather than ADP-ribosylated histones, may play a role in mitotic events.
- The findings do not support a specialized function for ADP-ribosylated histones during mitosis.