Isolation and characterization of normal rat kidney cell membrane proteins with affinity for transferrin.
Studies were performed to identify membrane receptors for transferrin in cultured normal rat kidney (NRK) cells. Cells were surface iodinated or metabolically labeled with radioactive glycoprotein precursors. Membrane receptors for transferrin were solubilized with the nonionic detergent Triton X-100. The soluble transferrin receptor has been purified approximately 1500-fold by affinity chromatography using transferrin coupled to Sepharose. Experiments demonstrated that the receptor can be adsorbed to a transferrin-Sepharose gel and be eluted specifically with transferrin. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the receptor preparations obtained by one cycle of affinity chromatography display, in addition to components of Mr lower than 20 000, a major glycoprotein component of approximately 170 000. Solubilized receptor preparations subjected to two cycles of affinity chromatography revealed a single polypeptide of approximately 20 000 daltons. Further studies indicated that the 20 000-dalton polypeptide is a degradation product of the 170 000 glycoprotein. Immunological studies showed that antitransferrin antibodies specifically precipitate a transferrin-170 000 complex and that a specific antibody against 170 000 glycoprotein precipitates the same complex. These results suggest that the 170 000 glycoprotein associates with transferrin in specific fashion and that this protein may be a subunit of the transferrin receptor of NRK cells.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D051381:Rats; D011955:Receptors, Drug / Q000302:isolation & purification* / Q000378:metabolism; D014168:Transferrin / Q000378:metabolism*
D008565:Membrane Proteins; D011955:Receptors, Drug; D014168:Transferrin; D011092:Polyethylene Glycols