Figure 5.

Role of sulfhydryl groups as targets in the inhibition of the ATPase activity of MSH2–MSH6 by cadmium. (a) Comparison of the inhibition of ATPase activity by NEM and Cd²⁺. MSH2–MSH6 (40 nM) was pre-incubated with 0.5 mM Cd²⁺ (2/6+Cd), 0.5 mM NEM (2/6+NEM) or a combination of both (2/6+Cd+NEM) at 4°C for 10 min and assayed for ATPase activity. To remove excess NEM and Cd²⁺, the pre-incubation mixture was extensively dialyzed (denoted as D in the Figure) and assayed for ATPase activity.(2/6+Cd)D indicates that MSH2–MSH6 was treated with Cd²⁺ followed by dialysis; (2/6+NEM)D indicates that MSH2–MSH6 was treated with NEM followed by dialysis; and (2/6+Cd)D+NEM indicates that MSH2–MSH6 was treated with Cd²⁺ followed by dialysis, and the treated with NEM. The comparison of the ATPase activities is shown here. The average of three experiments is presented, bars include the standard deviation. (b) Effect of DTT (2 mM) on the inhibition of the MSH2–MSH6 ATPase activity by Cd²⁺ (0.5 mM) and NEM (5 mM). MSH2–MSH6 (160 nM) was pre-incubated with Cd²⁺ for 15 min (2/6+Cd), Cd²⁺ for 15 min followed by DTT for 15 min(2/6+Cd+DTT), DTT for 15 min followed by Cd²⁺ for 15 min (2/6+DTT+Cd), NEM for 15 min (2/6+NEM), NEM for 15 min followed by DTT for 15 min (2/6+NEM+DTT), and DTT for 15 min followed by NEM for 15 min(2/6+DTT+NEM). The mixtures were then assayed for ATPase activity as described in Materials and Methods. The average of three experiments is presented, including the standard deviation.