Purification and characterization of poly(ADP-ribose) polymerase from <i>Sarcophaga peregrina</i>

Abstract

Poly(ADP-ribose) polymerase (PARP) was purified from embryonic NIH-Sape-4 cells of Sarcophaga peregrina almost to homogeneity. The purification was carried out using phosphocellulose and 3-aminobenzamide-Sepharose column chromatographies. The purified enzyme had an apparent molecular weight of 113kDa on SDS-polyacrylamide gel electrophoresis. The addition of DNA was essential for the activity of the purified enzyme, and the reaction was stimulated by the presence of histone and magnesium ion. The Km for NAD was 43μM and the optimum temperature for the PARP activity ranged from 20-25°C. MALDI (matrix-associated laser desorption ionisation)-TOF (time-of-flight) mass spectrometric analysis gave a signal at 112, 402. This value was close to 113, 033, the predicted mass from its cDNA sequence. This suggests that full length PARP is present as a major native form without extensive post-translational modification.