Insulin receptor isolation studies.

The observation that N alpha,B1-biotinylinsulin binds firmly to resins in which succinoylavidin is covalently attached to AH Sepharose 4B and can be retrieved by exposure of the resins to 20 mM biotin provided the basis for the present investigations. Solubilized, partially purified insulin receptor from human placenta binds to affinity resins in which N alpha,B1-biotinylinsulin is noncovalently attached to AH Sepharose 4B-immobilized-succinolylavidin. Exposure of the receptor loaded resin to 20 mM biotin results in liberation of a high molecular weight material containing bound 125I-biotinylinsulin, which precipitates with polyethyleneglycol and cross reacts with human insulin receptor antibodies. The technique is biospecific and appears to be applicable to the purification of insulin receptors on a preparative scale. Crude solubilized insulin receptor from human placenta is contaminated with "insulinase" which is inhibited by N-ethylmaleimide. HPLC provides a tool to assess "insulinase" activity that is more sensitive than the TCA precipitation method.