Figure 1. ARIP4 is an active DNA-dependent ATPase.

ATPase activity was measured in a fluorimetric ATPase assay. (a, b) 500 ng of ARIP4wt (or 800 ng of ARIP4-K310A) and 250 μM ATP were incubated with different amounts of supercoiled plasmid DNA (pGL3Basic) or ssDNA (ΦX174). Estimated K_m (app) for DNA (at 250 μM ATP) equals 40±10 nM. (c) 500 ng of ARIP4, 15 nM supercoiled plasmid DNA (pGL3Basic) were incubated with different amounts of ATP. Estimated K_m for ATP (at 15 nM dsDNA) equals 25±10 μM. (d) For catalytic activity estimation, 250 μM ATP and 600 ng/ml salmon sperm DNA were incubated with different ARIP4 amounts. ARIP4 activity is estimated from the resulting line (R=0.97) and equals 600±100 nmol of ADP released·min⁻¹·(mg of ARIP4)⁻¹. Assuming that ARIP4 has a molecular mass of approx. 200 kDa, the catalytic activity is 120±20 ATP molecules hydrolysed per min.