Abstract
Commercially available 125I-labeled erythropoietin, obtained by genetic engineering from a human gene, was used to characterize receptors for this hormone on the cell surface of rat erythroid progenitor cells. A low number of high affinity binding sites (487 +/- 32 sites/cell, Kd = 167 +/- 14 pm) were found. Nonerythroid cells and erythrocytes did not exhibit specific binding. The high affinity binding was reversible and displaced by unlabeled erythropoietin, but not by other hormones and growth factors. After incubation at 37 degrees C, nearly 35% of the specifically bound erythropoietin seemed to be internalized, as judged by resistance to acidic buffer treatment. Thus, binding showed characteristics of a hormone-receptor association. 125I-Erythropoietin-labeled cells were treated with the bifunctional reagent dissucinimidyl suberate. Analysis of the cellular extracts by polyacrylamide gel electrophoresis under denaturing and reducing conditions revealed that erythropoietin can be cross-linked to two molecules of 94 and 78 kDa, respectively. Both labeled bands disappeared when the cells were labeled in the presence of an excess of unlabeled erythropoietin. Under nonreducing conditions, a cross-linked band of 230-255 kDa was observed. The relationships between these bands are discussed.
Highlights
Obtained by genetic engineering from a human gene, Recently, methods have been described for the preparation was used to characterize receptors ftohris hormone on of erythroid progenitors from hemopoietic populations, either the cell surface of rat erythroid progenitocrells
Binding showed characteristics of a hormone-receptor association. lZ6I-Erythropoietin-lawith a high binding affinity ( K d = 5.2nM).We describe the preparation from rat fetal livers of cell suspensions essentially made of erythroid progenitors which depend on erythropoietin for proliferation and differentiation [13, 16]
Thesecells were used in this work to test thebinding of radioiodinated erythropoietin; we demonstrate the existence of specific and rebeled cells were treated with the bifunctional reagent versible high affinity binding and performed cross-linkage dissucinimidyl suberate
Summary
From the Laboratoire de Physwlogie AnimaleF, aculte des Sciences, F 51062 Reims Ceder,France. Binding of Radioactive Erythropoietin-Rat erythroid progenitor cells (1-10 X lo6) were incubated in 100 pl RPMI 1640medium containing 10% heat-inactivated bovine fetal serum and increasing concentrations (at least X10, and generally x50) of radioactive erythropoietin in the presence or absence of an excess of the non-labeled hormone (durations and temperatures of incubations are given under "Results"). Determination oCfell Surface-bound lz61-Erythropoietin-Rat erythroid progenitor cells, labeled and washed as described above, were resuspended in 1ml ice-cold acidic buffer (50 mM glycine/acetic acid, pH 3.0) containing 150mM NaC1. Affinity Cross-linkage--Rat erythroid progenitors were incubated for 3 h at 20"C in the presence of400800 PM '251-erythropoietin They were washed and resuspended in ice-cold phosphatebuffered saline at a density of 5-10 X lo6cells/ml.
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