FIGURE 5.

NEM sensitivity of the Nfs1p·Isd11p complex versus Nfs1p by itself. A, A. vinelandii NifS (50 ng) was incubated with increasing concentrations of NEM for 10 min at 30 °C. Following addition of excess DTT (10 mm) to neutralize free NEM, NifS was precipitated with ammonium sulfate as in Fig. 4A. The protein precipitates were solubilized with buffer A and incubated with PLP (150 μm) and [³⁵S]cysteine (5 μCi) for 10 min at 30 °C. Following TCA precipitation, samples were analyzed by non-reducing SDS-PAGE and autoradiography. B, the preformed Nfs1p·Isd11p complex (100 ng) or Nfs1p by itself (100 ng) was treated with increasing concentrations of NEM as in A. Free NEM was neutralized with DTT, and the protein pellets obtained after ammonium sulfate precipitation were solubilized with buffer A. Isd11p (100 ng) was then added only to Nfs1p alone samples. All reaction mixtures were supplemented with PLP (150 μm) and [³⁵S]cysteine (5 μCi), incubated at 30 °C for 10 min, and analyzed. The intensity of Nfs1p-S-³⁵SH for NEM-untreated Nfs1p·Isd11p (lane 1) was considered 100%.