Insulin and epidermal growth factor-urogastrone: affinity crosslinking to specific binding sites in rat liver membranes.
Both insulin and human epidermal growth factor-urogastrone (EGF/URO) can be covalently linked to specific rat liver membrane binding sites by glutaraldehyde coupling followed by sodium borohydride reduction to yield affinity-labeled membrane constituents sufficiently stable for solubilization and further analysis by various techniques. Solubilization of membranes covalently labeled with (125)I-labeled insulin yields a component with chromatographic properties identical to those of a soluble insulin receptor characterized in previous studies. A second soluble insulin-binding component that is not revealed by the affinity-labeling method and that has not yet been reported can also be detected. Membranes similarly labeled with (125)I-labeled EGF/URO yield one major and two minor ligand-specific soluble (Triton X-100) affinity-labeled components, as detected by chromatography on Sepharose 6B. Further analysis of the EGF/URO-labeled components by affinity chromatography on concanavalin A-Sepharose, by disc gel electrophoresis, and by enzymatic digestion suggests that the major specific binding component for EGF/URO in liver membranes is a glycoprotein subunit of approximately 100,000 daltons that possesses a 20,000-dalton portion inaccessible to proteolytic cleavage when the subunit is anchored in the membrane. The affinity labeling approach described should prove of use for the study of other polypeptide receptors that, like the EGF/URO receptor, lose their ligand recognition property subsequent to membrane solubilization.
D000345:Affinity Labels; D000818:Animals; D001894:Borohydrides; D002462:Cell Membrane / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism*; D005768:Gastrointestinal Hormones / Q000378:metabolism*; D005976:Glutaral; D007328:Insulin / Q000378:metabolism*; D008099:Liver / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011092:Polyethylene Glycols; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001894:Borohydrides; D005768:Gastrointestinal Hormones; D007328:Insulin; D010455:Peptides; D011956:Receptors, Cell Surface; D011092:Polyethylene Glycols; D004815:Epidermal Growth Factor; D011972:Receptor, Insulin; D005976:Glutaral