Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells

Journal Article · Fri May 01 04:00:00 EDT 1987 · Blood; (United States)

OSTI ID:6477358

Mufson, R A; Gesner, T G

Erythropoietin (EPO) biosynthetically labelled with (/sup 35/S)cysteine was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant (/sup 35/S)EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The (/sup 35/S)-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 to 200 binding sites per cell at 37 degrees C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with /sup 125/I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation at 37 degrees C, most of the bound (/sup 35/S)EPO was internalized, whereas most of the (/sup 125/I)EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.

• Research Organization: Genetics Institute, Cambridge, MA
• OSTI ID: 6477358
• Journal Information: Blood; (United States), Journal Name: Blood; (United States) Vol. 5; ISSN BLOOA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ANIMAL CELLS
ANIMALS
AZIDES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BLOOD FORMATION
CHO CELLS
DAYS LIVING RADIOISOTOPES
DNA
ELECTRON CAPTURE RADIOISOTOPES
ERYTHROPOIESIS
ERYTHROPOIETIN
EVEN-ODD NUCLEI
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
MICE
MITOGENS
NITROGEN COMPOUNDS
NUCLEI
NUCLEIC ACIDS
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
PROTEINS
RADIOISOTOPES
REACTION KINETICS
RECOMBINANT DNA
RODENTS
SULFUR 35
SULFUR ISOTOPES
TRACER TECHNIQUES
VERTEBRATES