Characterization of erythropoietin receptor of murine erythroid cells.
Radioiodinated or biologically tritiated recombinant human erythropoietin was used to characterize receptors for this hormone on the surface of Friend erythroleukemic cells (745A and TSA8) and cells from mouse erythropoietic tissues (liver from fetus and spleen from animals made anemic by injection of Friend virus or phenylhydrazine). Specific binding of erythropoietin to these cells was time-dependent and dose-dependent. Binding studies at 37 degrees C showed that dissociation constants of erythropoietin-receptor complexes were in the range of 100-300 pM. The number of receptors on erythroleukemic cells increased after treatment with dimethylsulfoxide. Covalent binding of 125I-erythropoietin to its receptors with a cross-linking reagent, disuccinimidyl suberate or glutaraldehyde, resulted in the formation of two major radiolabeled products that migrated as 120-kDa and 140-kDa species on sodium dodecyl sulfate/polyacrylamide electrophoresis gels under reducing conditions. Under non-reducing conditions, both 120-kDa and 140-kDa species disappeared and two cross-linked products, a minor product with a molecular mass of 250 kDa and a major product of high molecular mass that kept it from migration into the separating gels, appeared. The relationship of the cross-linked products found under non-reducing conditions with those under reducing conditions remains to be clarified.
Sasaki
R
R
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Yanagawa
S
S
Hitomi
K
K
Chiba
H
H
D000818:Animals; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000032:analysis*; D004921:Erythropoietin / Q000378:metabolism*; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins / Q000378:metabolism; D013154:Spleen / Q000378:metabolism
D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin