Akira Murayama

Protection of steroid hormone receptors by protease inhibitors

Abstract Steroid hormone receptors are proteolyzed by different types of enzymes present in target tissues. Effective protease inhibitors protecting steroid hormone receptors in various target tissues were investigated. Progesterone receptor (PR) in hen oviduct and estrogen receptor (ER) in cow uterus were specifically protected by relatively low concentrations (0.5 mM) of leupeptin or antipain (inhibitors of serine and thiol proteases). It was indicated that two different types of enzymes which modify native glucocorticoid receptor (GR) are present in rat liver. One was inhibited by 1 mM leupeptin or 1 mM antipain, while the other was inhibited by 1 mM phosphoramidon (inhibitor of thermolysin like proteases) or 10 mM sodium molybdate. Native PR, ER, and GR were shown to have similar Stokes radii (44 A).

The strong hydrophobic domain of the activated estrogen receptor of porcine uterus

Basic estrogen receptor (ER) molecule (vero‐ER) of porcine uterus, which was previously shown to be the activated ER necessary to translocate from the cytoplasm into the nucleus, possesses a strongly hydrophobic nature. The strong hydrophobicity of vero‐ER was concealed through binding with ER‐binding factors (ERBFs). Vero‐ER lost its strong hydrophobicity and its capability to bind with ERBFs after limited proteolysis by endogenous protease. The strong hydrophobic domain of vero‐ER, indispensable for the nuclear translocation, was assumed to be located near the binding site with ERBFs.

A new assay of estrogen receptor by thin-layer gel filtration.

Abstract Application of a thin-layer gel filtration (TLG) method using Sephadex G-200 superfine for the assay of estrogen receptor (ER) is described. The method is based on the effective separation of specific receptor protein from nonspecific binding proteins and free hormone by TLG and subsequent determination of the radioactivity bound to specific ER. By the aid of fluorescein-labeled human γ-globulin, the position of specific ER can be visualized. The assay excludes completely the undesired fluctuation depending on the concentration of coexisting proteins and permits reliable and rapid quantitation of specific ER. Association constant and concentration of specific ER can be determined with 100 mg of mammary tumor tissue. The method has the additional advantage of not requiring expensive equipment.