FIGURE 7.

Nonreducing SDS-PAGE after blocking of free thiols. A, S- and U-forms of replicase were precipitated with 5% TCA and treated with the oxidative agent, AMS, to modify free thiols. This procedure is known to inhibit disulfide bond exchange. The replicases were then subjected to SDS-PAGE (5%-15%) without any reducing agent. In the AMS− lane, the TCA-precipitated replicase before AMS treatment was applied. In the DTT+ lanes, the applied replicase was treated with DTT (10 mm) for 5 min at 37 °C after modification with AMS. The black arrowhead indicates the high molecular weight proteins. As AMS has a molecular weight of 0.5 kDa, the bands showed a shift in migration following AMS treatment. B, wild-type and Cys533(β) mutant replicase purified by standard methods were treated with AMS and subjected to nonreducing SDS-PAGE. The S-form replicase was applied again for comparison. C, Western blotting analysis. S-form replicase subjected to nonreducing SDS-PAGE was analyzed by Western blotting using monoclonal antibodies specific for β, EF-Tu, and EF-Ts subunits.