Affinity chromatography and purification of the insulin receptor of liver cell membranes.

Relatively simple and rapid procedures are described for the large-scale preparation of liver membranes that contain virtually all of the high affinity insulin-binding activity of liver homogenates. The presumed insulin recepotr, which is extracted from these membranes in soluble form with Triton X-100, can be further purified by ammonium sulfate fractionation (3-fold purification) or by diethylaminoethyl-cellulose chromatography (60-fold purification). Several insulin-agarose derivatives have been synthesized that can efficiently extract the insulin-binding protein from the detergent extracts of the membranes. The receptor macro-molecule can be eluted from the affinity columns in high (50-80%) yield by use of urea-containing buffers of moderately low pH. The receptor, thus purified by small-scale affinity chromatography experiments, approaches theoretical purity on the basis of its specific activity. This protein is purified about 250,000-fold from the liver homogenate by detergent extraction and affinity chromatography.