Iron binding, a new function for the reticulocyte endosome H(+)-ATPase.
The significance of the H(+)-ATPase in iron absorption by rabbit reticulocytes is explored using isolated endosomes, effects of inhibitors, and the purified proton pump. We have recently reported H(+)-ATPase-mediated iron transfer across a liposomal membrane (Li et al., 1994). In this report, the effect of H(+)-ATPase inhibitors on iron mobilization is investigated at pH 6.0 in the presence of 15 microM FCCP in order to dissociate 59Fe(III) from transferrin and eliminate the kinetic effects of acidification by the ATPase. Iron transport by isolated endosomes is decreased 50% by the cation pore inhibitor dicyclohexylcarbodiimide (DCCD) for ascorbate-mediated iron mobilization and increased by 40-50% when NADH and ferrocyanide are used as electron donors. In contrast, the ATPase hydrolysis inhibitors N-methylmaleimide (NEM) and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD) increase iron mobilization when NADH and ferrocyanide are used as reductants but have negligible effects for ascorbate. The differential inhibition or enhancement by DCCD, NEM, and NBD with respect to the reductants used for mobilization indicates that the H(+)-ATPase may be involved in the multiple pathways or iron transport found in isolated rabbit reticulocyte endosomes. Effects of inhibitors of ATP hydrolysis suggest significant structural interactions between the proton pump and sites for iron binding and/or reduction. The isolated H(+)-ATPase binds iron as revealed by using nondenaturing electrophoretic and chromatographic methods. One class of iron binding sites is suggested to be the 17.5 kDa proton pore subunits of the H(+)-ATPase which also covalently react with DCCD.(ABSTRACT TRUNCATED AT 250 WORDS)
Li
C Y
CY
Department of Medicine, Louisiana State University Medical Center, Shreveport 71130-3932, USA.
Watkins
J A
JA
Hamazaki
S
S
Altazan
J D
JD
Glass
J
J
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D011992:Endosomes / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D007501:Iron / Q000378:metabolism* / Q000494:pharmacology; D007504:Iron Radioisotopes / Q000378:metabolism; D011485:Protein Binding; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*
D007504:Iron Radioisotopes; D004024:Dicyclohexylcarbodiimide; D007501:Iron; D006180:Proton-Translocating ATPases; D009327:4-Chloro-7-nitrobenzofurazan; D005033:Ethylmaleimide