Fig 1: PDI reductase activities of NusA-Nox2(357–570) compared to those of the NusA-Nox2(357-570) C369R, C371R mutant and to PDIA1 and PDIA3. (A) PDI reductase activity of recombinant NusA-Nox2(357–570) was assayed in a concentration range of 1–4 μM and plotted as non-linear regression (one site binding equation). (B) PDI reductase activity of recombinant NusA-Nox2(357–570) C369R, C371R mutant was assayed in a concentration range of 1–4 μM and plotted as a cubic spline curve. (C) PDI reductase activity of recombinant PDIA1 was assayed in a concentration range of 10–80 nM and plotted as linear regression. (D) PDI reductase activity of recombinant PDIA3 was assayed in a concentration range of 1–10 nM and plotted as linear regression. The assays were performed as described by Raturi and Mutus (2007) and detailed in the Materials and Methods Section. Results represent means ± SEM of 9 (NusA-Nox2(357–570), 4 (NusA-Npx2(357–570) C369R, C371R), 4 (PDIA1), and 3 (PDIA3) experiments.
Fig 2: PDI reductase activity of NusA-Nox2(357–570). (A) Recombinant NusA-Nox2(357–570) (2 μM) was assayed for disulfide reductase activity on dieosin glutathione disulfide (DE-GSSG), in the presence of DTT, as described by Raturi and Mutus (2007) and detailed in the Materials and Methods Section. Briefly, the reaction mixtures contained 800 nM DE-GSSG and 12.5 μM DTT and the kinetics of the increase in fluorescence were followed for 30 min, using an excitation wavelength of 519 nm and an emission wavelength of 545 nm. Results are expressed as Vmax (milli relative fluorescence units per min). (B) The dose dependence of the PDI reductase activity of NusA-Nox2(357–570) was assayed on DE-GSSG in the presence of DTT, as described in (A). The concentration of NusA-Nox2(357–570) was varied from 1 to 4 μM. The results of one characteristic experiment are illustrated. (C) The absence of the 369CysGlyCys371 triad in NusA-Nox2(372–570) or mutating Cys 369 and Cys 371 to Arg in NusA-Nox2(357–570) eliminates PDI reductase activity. NusA-Nox2(357–570), NusA-Nox2(372–570), and NusA-Nox2(357–570) C369R, C371R (all, at a concentration of 2 μM) were assayed for disulfide reductase activity on DE-GSSG in the presence of DTT, as described in (A). The results of one characteristic experiment are illustrated. (D) The PDI inhibitor phenylarsine oxide (PAO) interferes with the PDI activity of NusA-Nox2(357–570). Recombinant NusA-Nox2(357–570) (2 μM) was assayed for disulfide reductase activity on DE-GSSG in the presence of DTT, in the absence and presence of 50 μM PAO, as described in (A). The results of one characteristic experiment are illustrated.
Fig 3: Confocal laser analysis of PDIA3 (red) and SMILE (green) proteins in cultured human odontoblasts.(A) PDIA3 labeling is localized in the endoplasmic reticulum. (B) SMILE labeling is mainly present in vesicles and in some cells in the reticulum area. (C) Merged picture showing the colocalization of PDIA3 and SMILE in the endoplasmic reticulum. (D) A higher magnification of (C) showing yellow dots in the endoplasmic reticulum. Bar in A is 40 µm. Bar in D is 10 µm.
Fig 4: A polyclonal anti-PDIA3 antibody but not a polyclonal anti-PDIA1 antibody reacts with Nox2 by immunoblotting. (A) Polyclonal anti-PDIA3 antibody H-220 reacts with recombinant NusA-Nox2(357–570) but not with NusA-Nox2(372–570) and NusA. It reacts strongly with recombinant PDIA3 but not with recombinant PDIA1. It also recognizes a protein in the guinea pig macrophage membrane (sharp single band). (B) Polyclonal anti-PDIA1 antibody H-160 does not react with recombinant NusA-Nox2(357–570) and NusA-Nox2(372–570). It reacts with recombinant PDIA1 and weakly, with recombinant PDIA3. It also recognizes a protein in the guinea pig macrophage membrane (sharp double band). Nox2(357–570) numbered 1, 2, and 3 represent three batches of NusA-Nox2(357–570).
Fig 5: Effect of PDI inhibitors on NADPH oxidase activation in a cell-free system. Selected PDI inhibitors were tested for their ability to interfere with NADPH oxidase activation in an amphiphile- and p47phox-independent system. The reaction mixtures contained macrophage membrane liposomes (5 nM cytochrome b558 heme), p67phox(1–212) (300 nM), and prenylated Rac1 Q61L (300 nM). The membrane liposomes were preincubated with the PDI inhibitors for 5 min, followed by the addition of p67phox and Rac1, incubation for 5 min in the absence of an amphiphilic activator, and the addition of 240 μM NADPH, to initiate O·−2 production. The effect of the following inhibitors is described in the figure: (A) phenylarsine oxide (1.56–200 μM); (B) bacitracin (62.5–2000 μM); (C) gliotoxin (1.56–200 μM), and (D) scrambled RNAse (0.114–14.6 μM). Results represent means ± SEM of 3 experiments, for each of the inhibitors tested. IC50 values are indicated for phenylarsine oxide and bacitracin but could not be determined for gliotoxin and scrambled RNAse, due to the absence of curves amenable to kinetic analysis.
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