Protease substrates inhibit binding of 3H-R5020 to the G-fragment in chick oviduct cytosol

Abstract

Our previous studies have shown that the binding of progestins to the chick oviduct progesterone receptor is inhibited by the protease substrates tryptophan methyl ester and tosyl arginine methyl ester. The K d of these substrates for the chick oviduct progesterone receptor is about 5 mM. Now we report the novel finding that incubation of chick oviduct cytosol for 18 to 24 hours at pH 7.8, 23° increases the affinity of tryptophan methyl ester and tosyl arginine methyl ester for the chick oviduct progesterone receptor by 5000 fold ( K d ∼ 1 μM). Our experiments also show that the K d of R5020, a synthetic progestin, is about 0.2 nM for the native form of the progesterone receptor and about 1.0 nM for the treated receptor. It is known from studies in several laboratories, including our own, that our incubation conditions cause the progesterone receptor to be cleaved to a fragment of about 20,000 molecular weight. It may be that limited proteolysis activafes the protease substrate binding site on the chick oviduct progesterone receptor.