Poly(ADP-ribose) polymerase in HeLa cells--a high resolution two-dimensional gel analysis.
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).
Prasad
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Department of Radiation Medicine, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, Washington, D.C. 20007.
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D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D015153:Blotting, Western; D004563:Electrochemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D006367:HeLa Cells / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011506:Proteins / Q000378:metabolism
D011506:Proteins; D000246:Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases