Figure 3.

GILT is an acid optimal thiol reductase. (A) Lysozyme (negative control), GILT (550 nM), or thioredoxin (1.04 μM) was incubated with ¹²⁵I-F(ab′)₂ (55 nM) under different pH conditions for 45 min at 37°C. (B) Lysozyme or GILT (550 nM) was incubated with ¹²⁵I-F(ab′)₂ (55 nM) at pH 4.0 and 37°C, and the reaction was stopped with excess iodoacetamide at different periods of time. (C) To quantitate the effect of substrate denaturation on GILT activity, 125 pM to 1 nM SDS-denatured ¹²⁵I-F(ab′)₂ (Left) and 14.5 nM to 116 nM native ¹²⁵I-F(ab′)₂ (Right) were incubated for 10 min at 37°C with 69 nM or 1.1 μM GILT, respectively, stopping the reaction by the addition of iodoacetamide. Samples were separated by nonreducing SDS/PAGE (12%) and visualized by autoradiography. In A and B, the gels are shown, and the positions of F(ab′)₂, Fab, H′ and light chains are indicated (Left). The intensity of specific bands (pixel density) was quantitated and plotted in A as percent reduced heavy and light chains after background subtraction and in B as percent of maximum. In C, the bands were converted to molarity of substrate. The results are presented as Lineweaver–Burke plots.