Ligand binding kinetics of a soluble full-length murine erythropoietin receptor.
The full-length murine erythropoietin receptor was expressed in Sf9 cells using a baculovirus vector. Erythropoietin receptors in solubilized Sf9 cell lysates bound erythropoietin with high affinity (92 pM). Erythropoietin receptor-125I-labeled erythropoietin association and dissociation kinetics using solubilized Sf9 cell lysates revealed a ka of 0.16 nM-1 min-1 and a kd of 0.00055 min-1 giving an observed KD of 3.45 pM. The erythropoietin receptors was partially purified from Sf9 cell lysates by chromatography on Con A Sepharose. When erythropoietin receptors were crosslinked to 125I-labeled erythropoietin and analyzed by SDS-7.5% PAGE protein complexes of 90 and 125 kDa were observed with receptors in solubilized lysate, and 170 and 190 kDa with the partially purified receptors.
D000818:Animals; D001345:Autoradiography; D002460:Cell Line; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004921:Erythropoietin / Q000302:isolation & purification / Q000378:metabolism; D007457:Iodine Radioisotopes; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008970:Molecular Weight; D017467:Receptors, Erythropoietin / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility; D018411:Spodoptera; D014162:Transfection
D007457:Iodine Radioisotopes; D008024:Ligands; D017467:Receptors, Erythropoietin; D011994:Recombinant Proteins; D004921:Erythropoietin