Figure 4.

The catalytic subunit of p193 is a PARP that ADP-ribosylates itself. The catalytic domain of p193 (255–611) was expressed and purified from E. coli. In vitro assays containing p193 (255–611) were incubated in the presence of [³²P]NAD+ and the products separated by SDS-PAGE followed by Coomassie blue staining (left panel) and by autoradiography (right panel). Reactions contained 1 μg of p193 (255–611) and 1.3 μM [³²P]NAD+. Preincubation of p193 (255–611) at 65°C for 10 min before the addition of labeled NAD+ inactivated the activity (heat). Reactions were supplemented with either 1 mM unlabeled NAD+ (cold chase) or with 1 mM 3ABA, the PARP inhibitor (inhibitor).