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pH-dependent effect of C200S and C211S mutations on thiol reductase activity of precursor GILT. Precursor GILT was immunoisolated from the supernatants of COS-7 cells expressing wild-type, C200S, and C211S GILT. Eluates were immunoblotted for GILT expression (A) and 100, 50, and 25 μl of the eluates were assayed for reduction of F(ab′)2 at pH 5 (B) and pH 7 (D). (C and E) Subsequent quantitation. [Ctrl(−): buffer at pH 5; purified GILT: 1 μg.]
