FIGURE 5.

Dug2p-Dug3p functions as a complex to cleave GSH. A, gel filtration profile of the Dug2p-Dug3p complex. Recombinant purified Dug2p and Dug3p were mixed in 1:1 stoichiometry, dialyzed in 300 mm NaCl, 50 mm Tris-HCl, pH 8.0, 1 mm GSH buffer for 1 h, and loaded on a Superdex S200 gel filtration column. Elution profile was monitored by absorbance at 280 nm, and four peaks were seen. Peak 1, high molecular mass fraction; peak 2, 346 kDa; peak 3, 156 kDa; peak 4, 53 kDa. B, relative activity of different Dug2p-Dug3p fractions obtained from gel filtration. GSH degradation activity of different fractions is measured by using a coupled assay using Dug1p and monitoring the cysteine formed. 1, unfractionated Dug2p-Dug3p complex; 2, high molecular mass fraction; 3, 346-kDa fraction; 4, 156-kDa fraction; 5, 53-kDa fraction. Values are plotted as mean ± S.E.