GAPDH, but not the variant C152G or C156G, stimulates APE1 AP
endonuclease activity. A, increasing concentrations of either
purified GST (lanes 9–12) or GST-GAPDH (lanes
13–16) were incubated with purified His-APE1 (lanes
9–16) for 5 min at room temperature before assaying for AP
endonuclease activity. Shown are the controls, substrate (80 ng) alone
(lane 1), and the following purified proteins Endo IV (0.15 pmol,
lane 2), His-APE1 (lanes 3 and 4), GST (lanes
5 and 6), and GST-GAPDH (lanes 7 and 8). The
arrows indicate positions of the 42-mer substrate and the 20-mer
product. B, increasing amounts (0, 0.16, 0.64, and 0.8 pmol) of
either GST-GAPDH (lanes 2–5), or the variants C152G (lanes
6–9), C156G (lanes 10–13), and C247G (lanes
14–17) were incubated with a fixed amount of purified His-APE1 (0.4
pmol each, lanes 1–17) assayed for AP endonuclease activity.
Also shown are substrate alone (lane 18) and Endo IV (0.06 pmol,
lane 19).