Solubilization and hydrodynamic characteristics of the erythropoietin receptor. Evidence for a multimeric complex.
In order to study the erythropoietin receptor in its native state, we solubilized erythropoietin-receptor complexes from spleen cell membranes of mice infected with the anemia strain of Friend virus using mild detergents. Among 11 tested detergents, Triton X-100 and Lubrol PX were the most effective. Triton X-100 was therefore selected for this study. The solubilized complexes appeared to be well representative of the total membrane receptor population as indicated by cross-linking experiments and affinity measurements. The hydrodynamic characteristics of the complexes were determined by gel filtration chromatography and ultracentrifugation through sucrose gradients prepared with H2O or D2O. Although erythropoietin-receptor-detergent complexes exhibited some heterogeneity, we determined the following minimal hydrodynamic values: sedimentation coefficient (s20,w): 11.7 +/- 0.8 S, Stokes radius: 7.7 +/- 0.2 nm, partial specific volume: 0.774 +/- 0.017 ml/g, giving a molecular mass of 458 +/- 66 kDa. The contribution of the detergent was estimated to be 28% from the measured partial specific volume, giving an estimated molecular mass of 330 +/- 48 kDa for the erythropoietin-receptor complex. The minimal molecular mass value was significantly greater than those obtained by polyacrylamide gel electrophoresis under denaturing conditions, strongly suggesting that the erythropoietin receptors were present as multimeric complexes. The nature of these complexes is discussed. Beside this major component our results revealed the presence of higher-molecular-mass erythropoietin binding components. We also demonstrated that erythropoietin-receptor complexes could be precipitated with anti-erythropoietin antibodies. This property should greatly improve the purification of erythropoietin receptors.
Mayeux
P
P
Unite INSERM 152, Institut Cochin de Génétique Moléculaire, Paris, France.
Casadevall
N
N
Lacombe
C
C
Muller
O
O
Tambourin
P
P
D000818:Animals; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004921:Erythropoietin / Q000378:metabolism; D005622:Friend murine leukemia virus; D046911:Macromolecular Substances; D051379:Mice; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D017467:Receptors, Erythropoietin; D012995:Solubility
D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D011092:Polyethylene Glycols; D017830:Octoxynol