Redox, transferrin-independent, and receptor-mediated endocytosis iron uptake systems in cultured human fibroblasts.
Sepharose beads bound to 55Fe-transferrin (Tf) were used to evaluate Tf-dependent iron uptake not employing receptor-mediated endocytosis (RME). The iron of 55Fe2-Tf-Sepharose was reduced and taken up by cultured human fibroblasts in a time- and concentration-dependent fashion (Km 7 microM; Vmax 128 pmol/mg/min). This redox system resembled that for Tf-independent iron uptake (Tf-IU, evaluated using 55Fe-citrate) in several ways. 1) NH4Cl did not inhibit iron uptake from 55Fe-citrate and 55Fe2-Tf-Sepharose but did inhibit uptake from 55Fe2-Tf (RME system). 2) Iron uptake and reduction from 55Fe2-Tf-Sepharose and 55Fe-citrate increased with temperature hyperbolically, differing from the sigmoidal curve for RME uptake. 3) The subcellular distributions of iron from 55Fe-citrate and 55Fe2-Tf-Sepharose resembled each other and differed from that for 55Fe2-Tf. 4) The optimal pH for iron reduction and uptake using 55Fe2-Tf-Sepharose or 55Fe-citrate was less than pH 5.5, while that for iron uptake from 55Fe2-Tf was pH 7.4. 5) The uptake and reduction of iron from 55Fe2-Tf-Sepharose was inhibited by ferric citrate and by transition metals. We conclude that both Tf-independent and non-RME, Tf-dependent iron uptake proceed via a common redox system for iron. The mechanisms of cellular iron uptake can be separately evaluated in fibroblasts using 55Fe-citrate, 55Fe2-Tf, and 55Fe2-Tf-Sepharose beads.
Oshiro
S
S
Section on Human Biochemical Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
Nakajima
H
H
Markello
T
T
Krasnewich
D
D
Bernardini
I
I
Gahl
W A
WA
D002478:Cells, Cultured; D004705:Endocytosis; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D010084:Oxidation-Reduction; D011990:Receptors, Transferrin / Q000378:metabolism*; D012685:Sepharose; D013347:Subcellular Fractions / Q000378:metabolism; D013696:Temperature; D014168:Transferrin / Q000378:metabolism*
D011990:Receptors, Transferrin; D014168:Transferrin; D012685:Sepharose; D007501:Iron