Purification of insulin-like growth factor I receptor from human placental membranes
| Autor: | T R, LeBon, S, Jacobs, P, Cuatrecasas, S, Kathuria, Y, Fujita-Yamaguchi |
|---|---|
| Rok vydání: | 1986 |
| Předmět: |
Octoxynol
Placenta Cell Membrane Detergents Antibodies Monoclonal Receptors Cell Surface Receptors Somatomedin Binding Competitive Chromatography Affinity Polyethylene Glycols Molecular Weight Kinetics Solubility Pregnancy Humans Insulin Electrophoresis Polyacrylamide Gel Female Insulin-Like Growth Factor I |
| Zdroj: | The Journal of biological chemistry. 261(17) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|