Partial purification and characterization of erythropoietin receptors from erythroid progenitor cells.
Arch Biochem Biophys
; 278(2): 486-91, 1990 May 01.
Article
em En
| MEDLINE
| ID: mdl-2158283
ABSTRACT
We have partially purified and characterized erythropoietin (Epo) receptors of erythroid progenitor cells which were obtained from the spleens of anemia-inducing Friend virus infected mice. Membrane proteins of splenic erythroid progenitor cells were solubilized with 1% Triton X-102. Upon chromatography on DEAE-Sephacel anion-exchange columns, two distinct Epo receptor peak fractions referred to as Peak I and Peak II were identified by 125I-Epo binding assays using the polyethylene glycol precipitation method. The Peak I and Peak II samples were then individually chromatographed on an S-Sepharose column. The S-Sepharose-purified Peak I and Peak II samples were crosslinked with 125I-Epo in the presence and absence of excess unlabeled Epo by disuccinimidyl suberate treatment, and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography. Both Peak I and Peak II samples showed a radiolabeled peptide with a Mr 135K and the labeling was blocked by excess unlabeled Epo. Since the Mr of Epo is about 35K, Epo receptor peptide has a Mr approximately 100K. To determine whether Epo stimulates autophosphorylation of the receptors, the S-Sepharose-purified Peak I and Peak II samples were incubated with or without Epo, and then briefly incubated in the presence of [gamma-32P]ATP and Mn2+. The tyrosine residue phosphorylated protein was isolated by an immunochemical technique, and then analyzed by SDS-PAGE and autoradiography. The result showed that Epo stimulates phosphorylation of a 100-kDa peptide.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Células Precursoras Eritroides
/
Receptores de Superfície Celular
/
Reagentes de Ligações Cruzadas
Limite:
Animals
Idioma:
En
Revista:
Arch Biochem Biophys
Ano de publicação:
1990
Tipo de documento:
Article