Substrate specificity of (A) PaExoY and (B) VnExoY. (A) Reactions containing 5 ng or 10 ng (for GTP) or 50 or 100 ng PaExoY (for ATP, UTP, CTP) that was activated by Mg-ATP-actin (3 μM skeletal α-actin) were started by the addition of NTP substrate (2 mM) and allowed to proceed for 15 or 30 min. Mg2+ or Mn2+ was present at 15 mM. (B) Reactions were performed in reaction buffer containing 50 mM KCl and contained 5 ng or 10 ng (for ATP), 5 μg or 10 μg (for GTP or UTP), 0.25, or 0.5 μg (for CTP) of VnExoY that was activated by Mg-ATP-actin (3 μM skeletal α-actin) or in the absence of actin (ATP bkg) were started by the addition of NTP substrate (2 mM) and allowed to proceed for 10 or 20 min (ATP), 60 or 120 min (GTP), or 30 or 60 min (UTP, CTP). Values were extracted from the linear slope of conversion to cNMP or correspond to averages of specific activities measured in reactions containing different amounts of enzyme. Error bars correspond to s.d. of at least two independent experiments.