title: Synthesis of the transferrin receptor by cultures of embryonic chicken spinal neurons.
abstract: We have purified a glycoprotein from chicken sciatic nerves, sciatin, which has pronounced trophic effects on avian skeletal muscle cells in culture. Recent studies have shown that sciatin is identical to the iron-transport protein, transferrin, in terms of its physicochemical structure, immunological reactivity, and biological activity. To determine whether transferrin is synthesized and released by neuronal tissue, we incubated cultures of dissociated chicken spinal neurons in a medium free of L-leucine containing either L-3H-amino acids or L-[14C]leucine and immunoprecipitated transferrin with highly specific antibodies. The radiolabeled protein precipitated by rabbit heteroclonal, goat heteroclonal, or mouse monoclonal antitransferrin antibodies increased in specific activity in a linear manner for at least 30 min. Synthesis of this protein was abolished by the presence of puromycin (20 micrograms/ml) or cycloheximide (10(-5) M). The disappearance of the radiolabeled protein from cells was linear with a half-life (t 1/2) of 8-10 h. When immunoprecipitates were separated by SDS gel electrophoresis, a prominent band corresponding to transferrin (Mr 84,000) was visualized by staining with Coomassie Blue. However, when such gels were fluorographed, no radioactivity was apparent in the transferrin region of the gel although a prominent radioactive band was visualized at an Mr of 56,000. The protein of Mr 56,000 was not simply a degradation product of transferrin because this particular protein band was not generated by incubating radiolabeled transferrin with unlabeled neuronal homogenates. The protein of Mr 56,000 was purified from embryonic chicken brain and spinal cord by immunoabsorption chromatography on mouse monoclonal antitransferrin IgG conjugated to Sepharose 4B followed by affinity chromatography on immobilized transferrin. The purified protein bound radioiodinated transferrin and was precipitated by rabbit anti-chicken transferrin-receptor antibodies. Furthermore, this receptor protein was found to be localized on the plasma membrane of dorsal root ganglion neurons by immunocytochemistry using the peroxidase-antiperoxidase technique, and by blocking experiments, which showed that antitransferrin receptor IgG could inhibit the binding of fluorescein-conjugated transferrin at 4 degrees C to cultured neurons in vitro. From these data, we conclude that transferrin is not synthesized by cultures of chicken spinal cord neurons, but that the receptor for transferrin is synthesized by these cultures and is precipitated by antitransferrin antibodies as an antigen-receptor complex.
authors.forename: G J
authors.initials: GJ
authors.lastname: Markelonis
authors.forename: T H
authors.initials: TH
authors.lastname: Oh
authors.forename: L P
authors.initials: LP
authors.lastname: Park
authors.forename: C Y
authors.initials: CY
authors.lastname: Cha
authors.forename: C A
authors.initials: CA
authors.lastname: Sofia
authors.forename: J W
authors.initials: JW
authors.lastname: Kim
authors.forename: P
authors.initials: P
authors.lastname: Azari
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002478:Cells, Cultured; D002642:Chick Embryo; D005779:Immunodiffusion; D008970:Molecular Weight; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011990:Receptors, Transferrin; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D014168:Transferrin / Q000378:metabolism*; D014316:Tritium
chemical_list: D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; D014316:Tritium