Receptor affinity chromatography.
Insulin receptor was purified in high yield from cultured 3T3-L1 mouse adipocytes using the bifunctional ligand N alpha B1-(biotinyl-epsilon-aminocaproyl)insulin in conjunction with avidin-Sepharose CL-4B. This ligand is 100% competent as insulin and 60% competent as biotin, as measured by competitive binding assays. The procedure requires preliminary removal of biotin-containing proteins on "native" avidin-Sepharose CL-4B. This matrix shows nearly the same biotin-binding characteristics as uncoupled avidin and can be regenerated by washing with 0.02 N HCl, causing only a minor loss of nonexchangeable biotin-binding sites. Insulin receptor is isolated by formation of a complex between the bifunctional ligand and the receptor, and then adsorption to "monomeric" avidin-Sepharose via the biotin moiety. This affinity matrix binds [14C]biotin with a Kd approximately equal to 0.2 microM and has exchangeable/nonexchangeable biotin binding sites in the ratio 9:1. Displacement of homogeneous insulin receptor is achieved by the addition of biotin; the elution is time-dependent, suggesting that it is accomplished by the prevention of rebinding.
D000273:Adipose Tissue / Q000032:analysis; D000818:Animals; D001360:Avidin; D001710:Biotin; D002846:Chromatography, Affinity / Q000379:methods*; D066298:In Vitro Techniques; D051379:Mice; D010047:Ovalbumin / Q000031:analogs & derivatives*; D011972:Receptor, Insulin / Q000302:isolation & purification*; D012685:Sepharose
D001360:Avidin; D001710:Biotin; D010047:Ovalbumin; D012685:Sepharose; D011972:Receptor, Insulin