The receptor for transferrin on murine myeloma cells: one-step purification based on its physiology, and partial amino acid sequence.
The receptor for transferrin is one of the major surface proteins of proliferating lymphocytes and other cells. It binds ferrotransferrin from serum and endocytoses it into an acidic nonlysosomal intracellular compartment where iron is released, but in which apotransferrin remains tightly bound to its receptor. Recycling of the apotransferrin-receptor complex to the cell surface is associated with a return to neutral pH and concomitant loss of affinity of apotransferrin for its receptor. Apotransferrin is then free to leave the cell and initiate a new cycle. We have exploited this cycle in a novel method for the purification of the receptor for transferrin. Murine myeloma cells were lysed in nonionic detergent, and the lysate passed over a column of ferrotransferrin-agarose at pH 7.4. After washing with sodium acetate at pH 5.0, iron was removed with sodium citrate pH 5.0 and desferrioxamine. Upon returning the pH to neutrality, the receptor was eluted and found to be homogeneous by SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. The degree of purification was estimated to be at least 3,000-fold, and the calculated yield was 10 to 20%. The purified receptor was capable of binding to transferrin. The receptor was digested with trypsin, and the resulting peptides were separated by reversed-phase high performance liquid chromatography in NH4HCO3. Selected peptides were rechromatographed in 0.1% trifluoroacetic acid, and their amino acid sequences were determined.
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins; D002460:Cell Line; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D051379:Mice; D009101:Multiple Myeloma / Q000032:analysis / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis; D011956:Receptors, Cell Surface / Q000032:analysis / Q000302:isolation & purification* / Q000502:physiology; D011990:Receptors, Transferrin; D014168:Transferrin / Q000378:metabolism
D001059:Apoproteins; D010446:Peptide Fragments; D011956:Receptors, Cell Surface; D011990:Receptors, Transferrin; D014168:Transferrin; C028284:apotransferrin