Purification of insulin-like growth factor I receptor from human placental membranes.
Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.
D000911:Antibodies, Monoclonal; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D007334:Insulin-Like Growth Factor I / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D017830:Octoxynol; D010920:Placenta / Q000378:metabolism*; D011092:Polyethylene Glycols; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D017451:Receptors, Somatomedin; D012995:Solubility
D000911:Antibodies, Monoclonal; D003902:Detergents; D007328:Insulin; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011092:Polyethylene Glycols; D007334:Insulin-Like Growth Factor I; D017830:Octoxynol