A method for determining oligo- and poly(ADP-ribosy)ated enzymes and proteins in vitro

Evidence for regulation of NF-κB by poly(ADP-ribose) polymerase

“…The nuclear extracts or purified recombinant NF-κB proteins were incubated in a purified poly(ADP-ribosyl)ating enzyme system containing the indicated concentrations of purified PARP, NAD + and activated DNA, 25 mM Tris\HCl, pH 8n0, 10 mM MgCl # , 1 mM DTT and 0n05 % Triton X100 (TMDT). In some experiments, either MgCl # was omitted from the reaction mixture (TDT) to limit the chain elongation of pADPR [41] or unlabelled NAD + was replaced by [$#P]NAD + , as indicated in the respective experiment. The reaction was carried out at 25 mC for 30 min and terminated by the addition of a final concentration of 2n5 mM 3-AB.…”

“…As shown in Figure 6(A), both rp50 (lanes 1-3) and rMBP-p65 (lanes 5-7) were found to be poly(ADP-ribosyl)ated, whereas rGST-IκB (lane 12) and rMBP-lacZ (lane 9, included as a negative control) were not modified : p53(rMBP-p53), included as a positive control, was poly(ADPribosyl)ated also [47]. In this experiment, PARP reaction was carried out at a limited concentration of NAD + (10 µM) and in the absence of Mg# + (except for lane 16) to avoid excessive and heterogeneous chain elongation of the protein-bound polymer, which is known to cause large and heterogeneous shifts of the modified proteins from the position of the unmodified one upon analysis by SDS\PAGE [41]. Thus, under the reaction condition, all of the ADP-ribosylated proteins were located close to the position of the unmodified one, except that modified PARP showed a large shift due to an extensive automodification.…”

Poly(ADP‐ribosyl)ation of terminal deoxynucleotidyl transferase in vitor

“…As described in our previous reports [7, 311, when Mg2' was depleted and the appropriate ratio of added acceptor proteins to active DNA was used, the automodification of poly(ADP-ribose) polymerase was minimized and a preferential ADP-ribosylation of the acceptor proteins such as histones could be observed. Recently, we have demonstrated that this reaction system can be applied to a rapid screening of various acceptors of ADP-ribose [32].…”

“…Therefore, we used a reaction condition whereby exogenous acceptors of ADPribose are preferentially labeled with [3H]oligo(ADP-ribose), as we successfully employed this system in the analyses of other acceptors of ADP-ribose [32]. This method was as follows: 24 pg of terminal transferase were incubated at 25°C for 40 min in 0.6 ml of reaction mixture containing 5 mM Tris/HCl buffer, pH 8.0, 1 mM dithiothreitol, 5 pM…”

“…Samples were dissolved in an SDS-containing buffer and analyzed as described in a previous report [32] by using a 7.5% polyacrylamide gel (140 wide x 120 height x 2mm thickness). The electrophoresis was carried out at 30 mA for about 15 h. After termination of the electrophoresis, the gel was stained by Coomassie blue and destained according to the method of Weber and Osborn [33].…”