Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form

Journal Article · Mon Dec 31 23:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00216a004· OSTI ID:5736323

Hosoi, Takayuki; Sawyer, S T; Krantz, S B ^[1]

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1. Vanderbilt Univ. School of Medicine, Nashville, TN (USA)

Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-((3-((4-((p-azido-m-({sup 125}I)iodophenyl)azo)benzoyl)amino)propanoyl)oxy)-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to {sup 125}I-EP. Recently, D'Andrea and co-workers cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands cross-linked with {sup 125}I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.

• OSTI ID: 5736323
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 30:2; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACYLATION
AUTORADIOGRAPHY
AZO COMPOUNDS
AZO DYES
BETA DECAY RADIOISOTOPES
CHEMICAL REACTIONS
CHROMATOGRAPHY
CROSS-LINKING
DAYS LIVING RADIOISOTOPES
DNA
DYES
ELECTRON CAPTURE RADIOISOTOPES
ELECTROPHORESIS
ERYTHROPOIETIN
GLOBULINS
GLOBULINS-BETA
GLYCOPROTEINS
HORMONES
INTERMEDIATE MASS NUCLEI
INTERNAL CONVERSION RADIOISOTOPES
IODINE 125
IODINE ISOTOPES
ISOTOPES
LIGANDS
LIQUID COLUMN CHROMATOGRAPHY
MEMBRANE PROTEINS
METALLOPROTEINS
MITOGENS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PEPTIDE HORMONES
POLYMERIZATION
PROTEINS
RADIOISOTOPES
RECEPTORS
RECOMBINANT DNA
SEPARATION PROCESSES
TRANSFERRIN