Purification and characterization of the neu/erb B2 ligand-growth factor from bovine kidney.
A neu/erb B2 ligand growth factor (NEL-GF) was purified to homogeneity from bovine kidney by a procedure involving ammonium sulfate fractionation (35-70% saturation) followed by sequential column chromatography on DEAE-cellulose (DE52), Sulfadex (sulfated Sephadex G-50), heparin-Sepharose 4B, and Superdex 75 (fast protein liquid chromatography). NEL-GF was found to be a 25-kDa polypeptide according to the analysis by gel filtration on Superdex 75 and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. NEL-GF stimulated the tyrosine-specific autophosphorylation of the neu/erb B2 gene product purified by immunoabsorbent and tyrosine-specific phosphorylation of the neu/erb B2 gene product in intact dihydrofolate reductase (DHFR/G-8 cells (NIH 3T3 cells transfected with rat c-neu). NEL-GF also down-regulated the cell surface neu/erb B2 gene product in DHFR/G-8 cells. NEL-GF was mitogenic toward NIH 3T3 cells, DHFR/G-8 cells, A431 cells (human epidermoid carcinoma cells), and SK-BR-3 cells (human breast carcinoma cells) but inactive toward bovine aorta endothelial cells. NEL-GF was sensitive to 0.1% trifluoroacetic acid but resistant to 5% beta-mercaptoethanol and appeared to be distinct from a neu protein-specific activating factor (Davis, J. G., Hamuro, J., Shim, C. Y., Samanta, A., Greene, M. I., and Dobashi, K. (1991) Biochem. Biophys. Res. Commun. 179, 1536-1542) and a 30-kDa glycoprotein which competed with a monoclonal antibody for binding to the neu/erb B2 gene product (Lupu, R., Colomer, R., Zugmaier, G., Sarup, J., Shepard, M., Slamon, D., and Lippman, M. E. (1990) Science 249, 1552-1555).
Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, Missouri 63104.
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D016475:3T3 Cells; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D015536:Down-Regulation; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification* / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins; D018719:Erb-b2 Receptor Tyrosine Kinases; D013762:Tetrahydrofolate Dehydrogenase