Protein kinase activity of the partially purified insulin receptor from human adipocytes.

Insulin receptors were partially purified by wheatgerm agglutinin chromatography from adipocytes of fasted healthy female subjects. The partially purified receptors showed binding characteristics similar to those of intact calls with an apparent affinity for insulin (half maximal binding) of 1.6 X 10(-9) mol/l. Insulin receptor alpha- and beta-subunits were identified by affinity labelling and phosphorylation with (gamma-32p)ATP, respectively. The electrophoretic mobility was 135 K for the alpha-subunit, and 97.5 K for the beta-subunit. The intrinsic tyrosine kinase activity of the insulin receptor was demonstrated by autophosphorylation of receptors purified by immunoprecipitation, and by phosphorylation of a synthetic substrate: poly(Glu, Tyr (4:1]. The kinase was activated by insulin in a dose-dependent manner with half maximal stimulation at 8 X 10(-10) mol/l. The Km value for ATP was 50 mumol/l. The dose-response relationship between percentage maximal kinase activation and fractional receptor occupancy by insulin was sigmoidal with half maximal effect when 35% of receptors are occupied. It is suggested that positively cooperation interactions between the receptor monomers are involved in stimulation of kinase activity and receptor autophosphorylation by insulin.