Homogeneous functional insulin receptor from 3T3-L1 adipocytes. Purification using N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin and avidin-sepharose.
Insulin receptor was purified 10,000-fold from cultured mouse 3T3-L1 adipocytes in 35% overall yield. The specific activities of 125I-insulin binding and autophosphorylation increased in parallel, following the initial Triton X-100 extraction of membranes. The isolation protocol, performed entirely at pH 8.45, entailed adsorption by avidin-Sepharose CL-4B of a complex formed between Triton X-100-solubilized insulin receptor and N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin, and the specific elution of the complex with biotin. The avidin-Sepharose CL-4B was a partially denatured preparation, showing estimated dissociation constants of 0.2 microM for biotin and approximately 1 microM for the bifunctional ligand at, pH 7, 4 degrees C. The bifunctional ligand was characterized by 70% competency in binding to avidin, 100% competency in binding to solubilized insulin receptor, full stimulation of autophosphorylation of the isolated receptor, and maximal stimulation of hexose uptake by intact 3T3-L1 adipocytes. The insulin binding properties of the insulin receptor were uniform throughout this purification procedure. At pH 8.45, 4 degrees C, an average Kd = 0.72 nM was determined for a single class of noninteracting insulin binding sites. The apparent autophosphorylation of the beta-subunit was also unchanged following affinity chromatography. A single oligomeric structure was established for the purified receptor, composed only of 135,000- and 95,000-Da subunits, whose association was lost by denaturation in the presence of reducing agent. This single structure occurred in the initial Triton X-100 extract. The purified insulin receptor was capable of autophosphorylating the beta-subunit and catalyzed phosphorylation of protein substrates.
D000273:Adipose Tissue / Q000032:analysis*; D000818:Animals; D001360:Avidin; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives* / Q000378:metabolism; D051379:Mice; D011494:Protein Kinases / Q000378:metabolism; D011972:Receptor, Insulin / Q000302:isolation & purification*; D012685:Sepharose
D007328:Insulin; D001360:Avidin; D001710:Biotin; C045095:insulin, N(alpha)(B1), biotinyl-epsilon-aminocaproyl-; D012685:Sepharose; D011494:Protein Kinases; D011972:Receptor, Insulin