Figure 1.
GSNO and GSNO(ox) inhibit IDE.
The effect of 10−4 M GSNO or GSNO(ox) on 125I-Insulin degradation by partially purified liver IDE is shown in the absence (open bars) or presence (grey bars) of ascorbate [10−3 M]. Inhibition by GSNO (NO donor) is prevented by ascorbate, while GSNO(ox) inhibition is not. All graphs represent the mean ± SEM of at least three independent experiments. *P<0.01compared to no addition. #P<0.01 compared to GSNO without ascorbate.
Figure 2.
The nitrosylation of purified recombinant IDE by GSNO, but not GSNO(ox) is demonstrated using the biotin switch method. IDE was either not treated (lanes 2 and 6), treated with 10−4 M GSNO(ox) (lanes 3 and 7) or 10−4 M GSNO (lanes 4 and 8). Lanes 2 through 4 show controls without the biotin reagent. Lanes 6 through 8 show enzyme subjected to biotin labeling. Lanes 1 and 5 are molecular weight markers with indicated MW. IDE at approximately 110 K MW is indicated.
Figure 3.
Proteasome chymotrypsin-like activity with GSNO and GSNO(ox) treatment.
Partially-purified rat IDE enzyme with proteasome was treated with increasing concentrations of NO donor and assessed for chymotrypsin-like activity using a fluorogenic proteasome substrate. (A) Proteasome activity with GSNO (○), GSNO+Ascorbate [10−3 M] (•). (B) Proteasome activity with GSNO(ox) (□), GSNO(ox)+Ascorbate [10–3 M] (▪). Inhibition by GSNO (NO donor) is prevented by ascorbate, while GSNO(ox) inhibition is not. Mean ± SEM of at least three independent experiments; **P<0.01 compared to no addition.
Figure 4.
Effect of GSNO or GSNO(ox) on purified proteasome.
Purified proteasome (no IDE) was treated with increasing concentrations of NO donor and assessed for chymotrypsin-like activity using a fluorogenic proteasome substrate; GSNO (○), GSNO(ox) (□). Purified proteasome is not susceptible to inhibition by NO. Mean ± SEM of three experiments; a is P<0.05 compared to no addition; b is P<0.05 GSNO(ox) compared to GSNO at 10−5 M; c is P = 0.058 GSNO(ox) compared to GSNO at 10−4 M.
Figure 5.
Glutathionylation of IDE by GSNO(ox).
Post-translational modification of IDE by glutathionylation was measured by Western blotting with an anti-glutathione antibody (right panel). Anti-IDE blot of the same gel is shown in the left panel. Partially-purified IDE was left untreated (lane 1) or treated with GSNO [10−6, 10−5, or 10−4 M, lanes 2 to 4 respectively] or GSNO(ox) [10−6, 10−5, or 10−4 M, lanes 6–8, respectively]. Molecular weight markers, with their sizes indicated, are in lane 5. Lane 8 (right panel) shows increased glutathione staining with GSNO(ox), while GSNO had no effect. Blot is representative of 4 similar experiments.
Figure 6.
Glutathionylation of purified IDE by GSSG.
Post-translational modification of IDE by glutathionylation was measured by Western blotting with an anti-glutathione antibody (right panel). Anti-IDE blot is shown on the left. Partially-purified IDE was left untreated (lane 1) or treated with GSH [10−4, 10−3, or 10−2 M, lanes 2 to 4 respectively] or GSSG [10−4, 10−3, or 10−2 M, lanes 7 to 9, respectively]. Molecular weight markers, with their sizes indicated, are in lane 5. Lane 6 is a blank. GSSG dose-dependently increased glutathionylation of IDE, while GSH had no effect. Blot is representative of 4 similar experiments.
Figure 7.
Effect of GSH and GSSG on insulin degradation.
125I-Insulin degradation by IDE is shown after treatment with GSH and GSSG in the presence and absence of ascorbate [10−3 M]. (A) Insulin degradation curve fit with increasing concentrations of GSH (○) and GSSG (□). (B) Insulin degradation curve fit with GSH+Ascorbate [10−3 M] (•) and GSSG+Ascorbate [10−3 M] (▪). GSSG inhibits IDE, while GSH appears to increase activity. Mean ± SEM of at least 3 independent experiments; *P<0.05, **P<0.01 compared to no addition.
Figure 8.
Inhibition of IDE by GSSG is reversible with DTT.
IDE was incubated without or with GSSG (10−4 M) and then dialyzed in the absence (open bars) or presence (shaded bars) of DTT (10−2 M) before assay of insulin degrading activity. GSSG inhibits IDE, while DTT reverses the effect and reveals latent degrading activity. Mean ± SEM of three replicates. The graph is representative of 4 independent experiments with varying levels of insulin degradation (TCA solubility).
Figure 9.
Direct effect of GSH on the partially-degraded 125I-insulin and 125I-amyloid-β.
Substrates were incubated with IDE in the presence of GSH or GSSG [10−2 M]; left side of each graph. Alternatively, IDE was incubated with substrate and GSH or GSSG, then IDE was heat-inactivated. An additional 10−2 M GSH was added after enzyme inactivation and incubated for another 15 min before TCA precipitation (right side of each graph). Gray bars are Untreated Enzyme; white bars are Enzyme + GSH; black bars are Enzyme + GSSG. (A) Insulin degradation. (B) Amyloid β degradation. Background TCA solubility in the absence of enzyme was subtracted. Treatment with GSH after heat inactivation of the enzyme increases TCA solubility of insulin products by breaking disulfides. Amyloid-β, not having any disulfide bonds, is unaffected either before or after enzyme inactivation. Mean ± SEM of three independent experiments. *P<0.05, **P<0.01 compared to control.
Figure 10.
HPLC analysis of insulin degradation products.
Insulin degradation products were qualitatively measured after incubation with (A) partially-purified IDE, (B) partially-purified IDE + GSH [10−2 M], and (C) partially-purified IDE + GSSG [10−2 M]. The peaks that changed in size with GSH treatment are identified with arrows (1, 2, and 3). GSH shifted the product pattern by decreasing peaks 2 and 3, and increasing peak 1. GSSG inhibits IDE and reduced all products.