Iron binding, a new function for the reticulocyte endosome H(+)-ATPase

Area of Science:

• Cell Biology
• Biochemistry
• Hematology

Background:

• Iron absorption is crucial for red blood cell formation.
• The proton pump (H(+)-ATPase) is involved in cellular transport processes.
• Previous research indicated H(+)-ATPase-mediated iron transfer across liposomal membranes.

Purpose of the Study:

• To investigate the role of H(+)-ATPase in iron absorption by rabbit reticulocytes.
• To elucidate the mechanisms of iron mobilization and transport in isolated endosomes.
• To identify potential iron binding sites on the H(+)-ATPase.

Main Methods:

• Utilized isolated endosomes from rabbit reticulocytes.
• Employed specific inhibitors of H(+)-ATPase (DCCD, NEM, NBD) and electron donors (ascorbate, NADH, ferrocyanide).
• Investigated iron mobilization at pH 6.0 with FCCP to isolate ATPase effects.
• Used nondenaturing electrophoretic and chromatographic methods to assess protein-ligand interactions.

Main Results:

• Dicyclohexylcarbodiimide (DCCD) inhibited ascorbate-mediated iron mobilization but enhanced NADH/ferrocyanide-driven transport.
• N-methylmaleimide (NEM) and NBD increased iron mobilization with NADH/ferrocyanide but had minimal effect with ascorbate.
• Isolated H(+)-ATPase demonstrated direct iron binding, with potential sites on 17.5 kDa proton pore subunits.

Conclusions:

• H(+)-ATPase is implicated in multiple iron transport pathways within reticulocyte endosomes.
• Inhibitor responses suggest structural interactions between the proton pump and iron binding/reduction sites.
• The H(+)-ATPase itself directly binds iron, potentially via its proton pore subunits.