Summary
A59Fe assay was designed to detect an Fe(III) binding capacity in NP-40 solubilized proteins from rabbit reticulocyte endocytic vesicles. The iron binding capacity had an apparent molecular weight as determined by gel exclusion chromatography of 450,000 daltons. The iron binding moiety coincided with the major nontransferrin iron-containing material of endocytic vesicles labeled in vivo by incubation of cells with59Fe,125I-labeled transferrin. The material solubilized from vesicles with NP-40 exhibited two classes of saturable binding sites, one with an association constant for59Fe-citrate of 3.63×109 m −1 and with 6.6×10−12 moles of iron bound per mg protein and the other with a constant of 3.96×108 m −1 and 1.0×10−12 moles of iron bound per mg protein. These affinities are sufficient to satisfy the sobulility characteristics of Fe(III) at pH 5.0. Most of the59Fe bound both in vivo and in vitro to the iron binding moiety could be displaced with56Fe and an equivalent amount of59Fe could subsequently be rebound in vitro. The iron binding assay was adopted to vesicle proteins separated by SDS-polyacrylamide gel electrophoresis with subsequent transfer to nitrocellulose and revealed an iron binding activity of molecular weight approximately 95,000 daltons.
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Aisen, P., Leibman, A., Zweier, J. 1978. Stoichiometric and site characteristics of the binding of iron to human transferrin.J. Biol. Chem. 253:1930–1937
Armstrong, N.J., Morgan, E.H. 1983. The effect of lysosomotrophic bases and inhibitors of transglutaminase on iron uptake by immature erythroid cells.Biochim. Biophys. Acta 762:175–186
Bakkeren, D.L., Jeu-Jaspers, C.M.H. de, Kroos, M.J., Eijk, H.G. van 1987. Release of iron from endosomes is an early step in the transferrin cycle.Int. J. Biochem. 19:179–186
Choe, H.-R., Moseley, S.T., Glass, J., Nunez, M.T. 1987. Rabbit reticulocyte coated vesicles carrying the transferrin-transferrin receptor complex: I. Purification and partial characterization.Blood 70:1035–1039
Enns, C.A., Larrick, J.W., Suomalainen, H., Schroder, J., Sussman, H. 1983. Co-migration and internalization of transferrin and its receptor on K562 cells.J. Cell Biol. 97:579–585
Glass, J., Nunez, M.T., Robinson, S.H. 1980. Transferrin-binding and iron-binding proteins of rabbit reticulocyte plasma membranes. Three distinct moieties.Biochim. Biophys. Acta 598:293–304
Iacopetta, B.J., Morgan, E.H. 1983. The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes.J. Biol. Chem. 258:9108–9115
Jandl, J.H., Katz, J. 1963. The plasma-to-cell cycle of transferrin.J. Clin. Invest. 42:314–336
Karin, M., Mintz, B. 1981. Receptor-mediated endocytosis of transferrin in developmentally totipotent mouse teratocarcinoma stem cells.J. Biol. Chem. 256:3245–3252
Laemmeli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685
Martinez-Medellin, J., Schulman, H.M. 1972. The kinetics of iron and transferrin incorporation into rabbit erythroid cells and the nature of stromal-bound iron.Biochim. Biophys. Acta 264(2):272–274
Nunez, M.T., Cole, E.S., Glass, J. 1983. The reticulocyte plasma membrane pathway of iron uptake as determined by the mechanism of α,α′-dipyridyl inhibition.J. Biol. Chem. 258:1146–1151
Nunez, M.T., Glass, J. 1983. The transferrin cycle and iron uptake in rabbit reticulocytes. Pulse studies using59Fe,125I-labeled transferrin.J. Biol. Chem. 258:9676–9680
Nunez, M.T., Glass, J. 1985. Iron uptake in reticulocytes. Inhibition mediated by the ionophores monensin and nigerisin.J. Biol. Chem. 260:14707–14711
Octave, J.-N., Schneider, Y.-J., Crichton, R.R., Trouet, A., 1982. Transferrin protein and iron uptake by isolated rat erythroblasts.FEBS Lett. 137(1):119–123
Paterson, S., Armstrong, N.J., Iacopetta, B.J., McArdle, H.J., Morgan, E.H. 1984. Intravesicular pH and iron uptake by immature erythroid cells.J. Cell Biol. 120:225–232
Renswounde, J. van, Bridges, K.R., Harford, J.B., Klausner, R. 1982. Receptor-mediated endocytosis of transferrin and the uptake of Fe in K562 cells: Indentification of nonlysosomal acidic compartment.Proc. Natl. Acad. Sci. USA 79:6186–6190
Speyer, B.E., Fielding, J. 1974. Chromatographic fractionation of human reticulocytes after uptake of double labeled59Fe,125I transferrin.Biochim. Biophys. Acta 332:192–200
Spiro, T.G., Bates, G., Saltman, P. 1967. The hydrolytic polymerization of ferric citrate: II. The influence of excess citrate.J. Am. Chem. Soc. 89:5559–5562
Young, S.P., Aisen, P. 1980. The interaction of transferrin with isolated hepatocytes.Biochim. Biophys. Acta 663:145–153
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Nunez, M.T., Pinto, I. & Glass, J. Assay and characteristics of the iron binding moiety of reticulocyte endocytic vesicles. J. Membrain Biol. 107, 129–135 (1989). https://doi.org/10.1007/BF01871718
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DOI: https://doi.org/10.1007/BF01871718