Poly(ADP-ribose) polymerase in HeLa cells--a high resolution two-dimensional gel analysis.

Abstract

PADPRP is an eukaryotic enzyme responsible for poly(ADP-ribosyl)ation of several chromatin-associated proteins. Consequently, the modified proteins acquire various lengths of negatively charged, covalently bound oligo or poly(ADP-ribosyl) homopolymers. The present study was undertaken to get an insight into the charge and size heterogeneity of the various auto-modified species of PADPRP and other protein acceptors of (ADP-ribose) polymers. Toward this end, we analyzed HeLa cells using high resolution two-dimensional gel electrophoresis (2D-PAGE). Resolution of HeLa total cellular lysates in the basic pH range combined with immunoblots and activity-blots revealed extensive modification and processing of the enzyme. In addition to the native enzyme, a protein of approximately 116 kDa, we observed several proteins that exhibit immunoreactivity to an antibody prepared against a peptide encompassing the N-terminal 20 amino acids of the polymerase. Several protein species showed auto-modifying potential in an in situ activity blot assay. We have also localized the ADP-ribosylated proteins in permeabilized HeLa cells to demonstrate protein acceptors of poly(ADP-ribose).