Intracellular modification of 125I-labeled epidermal growth factor by normal human foreskin fibroblasts.
Intracellular processing of 125I-labeled epidermal growth factor (EGF) in normal human foreskin fibroblasts was examined after incubation with saturating concentrations of [125I]EGF. This report describes the column chromatographic separation of multiple processed forms of EGF generated by human foreskin fibroblasts and their structural characterization. More than 95% of the cell-bound [125I]EGF was converted into multiple forms, which were separated into four distinct peaks of radioactivity using columns of Bio-Gel P-150 equilibrated with 0.2% sodium dodecyl sulfate. These were designated peaks 1-4. Cellular generation of these four peaks was dependent on culture conditions. Differences in absolute and relative amounts of peaks 1-4 were observed as a function of time of incubation at 37 C. In addition, chromatographic profiles of cell-associated 125I varied in relation to cell density. The radioactivity in peak 1 comigrated with 125I-labeled native EGF on nondenaturing polyacrylamide gels (pH 9.5), whereas peaks 2 and 3 exhibited more rapid electrophoretic mobilities. Electrophoretic mobilities of the radioactivity in peaks 2 and 3 were indistinguishable from those of chemically prepared derivatives of [125I]EGF which were lacking either one or six amino acid residues from the carboxyterminus, respectively. The EGF receptor bound the radioactive material in peak 2 with an affinity equal to or greater than that of EGF; however, the radioactivity in peak 3 was bound to a much lesser extent. The radiolabel in both peaks 2 and 3 was greater than 95% precipitable by antiserum to native EGF. The labeled material in peak 4 was composed of [125I]monoiodotyrosine, 125I-, and an unidentified peptide. None of the radiolabeled compounds in peak 4 interacted with the EGF receptor or with antiserum to native EGF.
Schaudies
R P
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Savage
C R
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D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism; D002452:Cell Count; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D007163:Immunosorbent Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D008247:Lysosomes / Q000378:metabolism; D008297:Male; D051379:Mice; D010446:Peptide Fragments / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D004815:Epidermal Growth Factor; D002268:Carboxypeptidases; D043424:Carboxypeptidase B; D014357:Trypsin