REDOX, TRANSFERRIN-INDEPENDENT, AND RECEPTOR-MEDIATED ENDOCYTOSIS IRON UPTAKE SYSTEMS IN CULTURED HUMAN FIBROBLASTS

Sepharose beads bound to Fe-55-transferrin (Tf) were used to evaluate Tf-dependent iron uptake not employing receptor-mediated endocytosis ( RME). The iron of Fe-55(2)-Tf-Sepharose was reduced and taken up by cu ltured human fibroblasts in a time- and concentration-dependent fashio n (K(m) 7 muM; V(max) 128 pmol/mg/min). This redox system resembled th at for Tf-independent iron uptake (Tf-IU, evaluated using Fe-55-citrat e) in several ways. 1) NH4Cl did not inhibit iron uptake from Fe-55-ci trate and Fe-55(2)-Tf-Sepharose but did inhibit uptake from Fe-55(2)-T f (RME system). 2) Iron uptake and reduction from Fe-55(2)-Tf-Sepharos e and Fe-55-citrate increased with temperature hyperbolically, differi ng from the sigmoidal curve for RME uptake. 3) The subcellular distrib utions of iron from Fe-55-citrate and Fe-55(2)-Tf-Sepharose resembled each other and differed from that for Fe-55(2)-Tf. 4) The optimal pH f or iron reduction and uptake using Fe-55(2)-Tf-Sepharose or Fe-55-citr ate was less than pH 5.5, while that for iron uptake from Fe-55(2)-Tf was pH 7.4. 5) The uptake and reduction of iron from Fe-55(2)-Tf-Sepha rose was inhibited by ferric citrate and by transition metals. We conc lude that both Tf-independent and non-RME, Tf-dependent iron uptake pr oceed via a common redox system for iron. The mechanisms of cellular i ron uptake can be separately evaluated in fibroblasts using Fe-55-citr ate, Fe-55(2)-Tf, and Fe-55(2)-Tf-Sepharose beads.