Figure 7
Figure 7. Characterization of α2-macroglobulin interaction with hepcidin and confirmation of the biologic activity of the α2-macroglobulin-hepcidin complex. (A) Saturation binding curve of 125I-human hepcidin with nonactivated α2-M demonstrating a single class of noninteracting binding sites. (B) Increasing concentrations of unlabeled hepcidin competes with 125I-hepcidin preventing binding of the label to α2-M. (C) Migration of methylamine-activated human macroglobulin (α2-M-MA) differs from nonactivated α2-M. (D) Saturation binding curve of 125I-human hepcidin with α2-M-MA demonstrating sigmoidal binding. This indicated allosteric cooperativity and the identification of higher order hepcidin-binding to α2-M-MA. In contrast, nonactivated α2-M demonstrated a hyperbolic function that corresponded to 2 independent binding sites with the same affinity. (E) Western analysis demonstrating that the α2-M–hepcidin complex, but not the α2-M–albumin complex reduces ferroportin expression in J774 cells. (A,D) Samples of 100 μg α2-M or α2-M-MA were incubated for 1 hour at 37°C with increasing concentrations of 125I-human hepcidin. The samples were separated using native gradient (3%-12%) PAGE. After electrophoresis, the gel was scanned on a phosphorimager and analyzed using Aida and GraphPad software. (B) α2-M (143 nM) was incubated for 1 hour at 37°C with 125I-human hepcidin (717 nM) and increasing concentrations of unlabeled hepcidin from 150 to 1200 nM. The samples were separated using native gradient (3%-12%) PAGE. After electrophoresis, the gel was scanned and analyzed as in panels A and D. (C) α2-M and α2-M-MA were resolved on a native gel as described for panels A and C and then stained with Coomassie blue protein stain. (E) J774 cells were incubated for 6 hours at 37°C in culture media without FCS containing either hepcidin (0.7 μM), α2-M (2.8 μM), hepcidin (0.7 μM) + α2-M (2.8 μM), albumin (2.8 μM), or hepcidin (0.7 μM) + albumin (2.8 μM). Results in panels A, B, and D are mean ± SD from 3 separate experiments, while panel C shows a typical gel from 3 experiments performed. The Western analysis shown in panel E is from a typical blot, while the densitometric scan is mean ± SD from 5 or 6 experiments. *P < .05; **P < .01.

Characterization of α2-macroglobulin interaction with hepcidin and confirmation of the biologic activity of the α2-macroglobulin-hepcidin complex. (A) Saturation binding curve of 125I-human hepcidin with nonactivated α2-M demonstrating a single class of noninteracting binding sites. (B) Increasing concentrations of unlabeled hepcidin competes with 125I-hepcidin preventing binding of the label to α2-M. (C) Migration of methylamine-activated human macroglobulin (α2-M-MA) differs from nonactivated α2-M. (D) Saturation binding curve of 125I-human hepcidin with α2-M-MA demonstrating sigmoidal binding. This indicated allosteric cooperativity and the identification of higher order hepcidin-binding to α2-M-MA. In contrast, nonactivated α2-M demonstrated a hyperbolic function that corresponded to 2 independent binding sites with the same affinity. (E) Western analysis demonstrating that the α2-M–hepcidin complex, but not the α2-M–albumin complex reduces ferroportin expression in J774 cells. (A,D) Samples of 100 μg α2-M or α2-M-MA were incubated for 1 hour at 37°C with increasing concentrations of 125I-human hepcidin. The samples were separated using native gradient (3%-12%) PAGE. After electrophoresis, the gel was scanned on a phosphorimager and analyzed using Aida and GraphPad software. (B) α2-M (143 nM) was incubated for 1 hour at 37°C with 125I-human hepcidin (717 nM) and increasing concentrations of unlabeled hepcidin from 150 to 1200 nM. The samples were separated using native gradient (3%-12%) PAGE. After electrophoresis, the gel was scanned and analyzed as in panels A and D. (C) α2-M and α2-M-MA were resolved on a native gel as described for panels A and C and then stained with Coomassie blue protein stain. (E) J774 cells were incubated for 6 hours at 37°C in culture media without FCS containing either hepcidin (0.7 μM), α2-M (2.8 μM), hepcidin (0.7 μM) + α2-M (2.8 μM), albumin (2.8 μM), or hepcidin (0.7 μM) + albumin (2.8 μM). Results in panels A, B, and D are mean ± SD from 3 separate experiments, while panel C shows a typical gel from 3 experiments performed. The Western analysis shown in panel E is from a typical blot, while the densitometric scan is mean ± SD from 5 or 6 experiments. *P < .05; **P < .01.

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