Purification of insulin receptor with full binding activity.
Insulin receptor was purified 2400-fold with an overall yield of 40% from human placental membranes by affinity chromatography on wheat germ agglutinin-Sepharose and insulin-Sepharose. The receptor was eluted from insulin-Sepharose using mild conditions, eliminating urea, so that it was stable and retained full insulin-binding activity. Chromatofocusing and gel filtration analysis indicated that the receptor preparation was apparently pure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three high molecular weight protein bands with Mr = 320,000, 300,000, and 270,000 under nonreducing conditions and two major protein bands with Mr = 135,000 and 90,000 under reducing conditions. The purified receptor showed a curvilinear Scatchard plot with maximum insulin binding of 28.5 micrograms per mg of protein. In comparison, the receptor eluted from insulin-Sepharose with previously used conditions in the presence of urea resulted in maximum insulin binding of only 6 micrograms per mg of protein. This indicates that a 4-to 5-fold increase in specific activity can be obtained by using the new elution conditions.
D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008970:Molecular Weight; D010920:Placenta / Q000032:analysis; D011247:Pregnancy; D011972:Receptor, Insulin / Q000302:isolation & purification* / Q000378:metabolism
D011972:Receptor, Insulin