Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase

Duriez, P.J., Desnoyers, S., Hoflack, J.C., Shah, G.M., Morelle, B., Bourassa, S., Poirier, G.G. and Talbot, B. (1997) Characterization of anti-peptide antibodies directed towards the automodification domain and apoptotic fragment of poly (ADP-ribose) polymerase. Biochimica et Biophysica Acta (BBA) - Biomembranes, 1334 (1), 65-72. (doi:10.1016/S0304-4165(96)00077-3). (PMID:9042367)

Abstract

Poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30) is a highly conserved nuclear enzyme present in higher eukaryotes. PARP is activated following DNA damage, is implicated in DNA repair, and its proteolysis has been shown to be an early marker of programmed cell death or apoptosis. In order to better understand the role of PARP in apoptosis and DNA repair and also to study PARP automodification, we have developed anti-peptide sera directed against four peptides from the conserved automodification domain of PARP. Four peptides were synthesized according to the four branched Multiple Antigenic Peptide (MAP) system and injected into rabbits. Immune sera were titrated by ELISA and analysed in Western blotting experiments on cell lines. The sera were also analysed for their capacity to inhibit PARP activity in an in vitro assay. Of the eight sera developed (two for each peptide), a serum directed against a peptide localized at the C-terminal part of the automodification domain of PARP (#422) appeared to be the best antibody to detect PARP from different species. All antipeptide antibodies were efficient in detecting the apoptotic fragment of PARP during programmed cell death in HL-60 apoptotic cells. None of the serum alone was able to completely inhibit PARP activity but combinations of the sera could significantly reduce automodification of PARP consistent with the localization of half of the automodification sites on bovine PARP. Sera were also used to map proteolysed purified PARP and to immunoprecipitate purified bovine PARP.

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Contributors

Author: P.J. Duriez

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