The H(+)-ATPase from reticulocyte endosomes reconstituted into liposomes acts as an iron transporter.
The H(+)-ATPase from reticulocyte endosomes was purified and reconstituted into liposomes, and protein-dependent iron transport was observed. Reconstitution of the H(+)-ATPase into liposomes was performed by sonicating a lipid mixture, with a composition similar to the reticulocyte plasma membrane, in a buffer containing ferric citrate. The nonencapsulated iron:citrate was removed by gel filtration and the proteoliposomes diluted into 1 mM FerroZine. Upon addition of ascorbate, an initial efflux of 2.9 +/- 0.3 x 10(-2) mumol of iron/mg of ATPase/min and 56 +/-7% of total internal Fe(II) was detected by formation of the Fe(II)-FerroZine complex with an absorbance at 562 nm or radioactivity of 59Fe(II)-FerroZine following separation using gel filtration. Both thiosulfate and ferrocyanide could substitute for ascorbate. Citrate or EGTA could substitute for FerroZine. The initial rate of Fe(II) efflux was decreased by 41 or 17% using 100 microM of the cation channel inhibitor N,N'-dicyclohexylcarbodiimide or 70 microM of the ATP hydrolysis inhibitor N-ethylmaleimide, respectively, but was unaffected by the presence of ATP. The amount of iron transported was decreased 51 or 39% by 100 microM N,N'-dicyclohexylcarbodiimide or 70 microM of the ATPase inhibitor 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. The amount of Fe(III) transport was 80% lower than Fe(II) when reductants were not present internally or externally although the apparent rate constants were identical when ascorbate was externally present. These results suggest that this vacuolar H(+)-ATPase may transport iron.
Li
C Y
CY
Department of Medicine, Louisiana State University Medical Center, Shreveport 71130.
Watkins
J A
JA
Glass
J
J
D000818:Animals; D001692:Biological Transport; D002850:Chromatography, Gel; D007501:Iron / Q000378:metabolism*; D008081:Liposomes; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*
D008081:Liposomes; D007501:Iron; D006180:Proton-Translocating ATPases