Hydrodynamic characterization of the photoaffinity-labeled insulin receptor solubilized in Triton X-100.
The insulin receptor in isolated rat liver plasma membranes was covalently labeled with the photoreactive insulin analogue NB-29-[(4-azido-2-nitrophenyl)acetyl]insulin and solubilized with the nondenaturing detergent Triton X-100. The resulting protein-detergent complex was characterized by gel filtration on Sepharose 6B, sedimentation rate determination in linear sucrose gradients, and equilibrium isopycnic centrifugation in NaBr and CsCl. The labeled insulin receptor was found in two forms. The Strokes radii and s20,w's of the two receptor-detergent complexes (R1 and R2) were (mean +/- SEM) 7.08 +/- 0.04 and 3.62 +/- 0.05 nm and 10.45 +/- 0.04 and 6.54 +/- 0.15 S, respectively. The two forms appeared to have the same buoyant density, 1.285 +/- 0.002 g cm-3. The dissociation of R2 from R1, or its reaggregation, either with itself or with other unlabeled proteins, to give R1 proceeded without chemical modification. Mild reduction of disulfide bonds (1 mM 1,4-dithiothreitol) increased the dissociation of R2 from R1. These results indicate that the solubilized receptor binds significant amounts of detergents, that the insulin binding component of the receptor binds to other receptor components by hydrophobic interactions, and that one or more components of the insulin receptor contain intrachain disulfide bonds.
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002462:Cell Membrane / Q000378:metabolism; D003902:Detergents; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D051381:Rats; D011972:Receptor, Insulin / Q000302:isolation & purification / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D003902:Detergents; D007328:Insulin; D046911:Macromolecular Substances; C021962:insulin, 4-azido-2-nitrophenyl-; D011092:Polyethylene Glycols; D017830:Octoxynol; D011972:Receptor, Insulin