Abstract
This chapter describes the purification and characterization of the estradiol receptor kinase from calf uterus cytosol, which is a member of a new class of protein kinases—namely, calmodulin-stimulated protein-tyrosine kinases. The physiological role of this enzyme is corroborated by the recent finding that the estradiol receptor is phosphorylated on tyrosine in whole uterus. Calf uterus estradiol receptor is a phosphoprotein and that phosphorylation–dephosphorylation of this receptor is controlled by a cytosol receptor-tyrosine kinase that activates the hormone binding, and by a nuclear receptor-phosphotyrosine phosphatase that inactivates this binding. The receptor is purified from calf uterus by affinity chromatography on heparin-Sepharose followed by affinity chromatography. (NH4)2SO4 is used to separate most of the receptor, which is precipitated at 0-25% saturation, from the kinase. Heparin-Sepharose separates the residual receptor and the enzyme coprecipitated by 25-50% (NH4)2SO4 saturation. High ionic strength and heparin drastically decrease the sedimentation coefficient of the cytosol estradiol receptor. Therefore, it is not possible to exclude that in intact cells, estradiol receptor kinase and estradiol receptor are loosely associated.
Published Version
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