Isolation of dissimilar components of the 8.5S nonactivated uterine progestin receptor

Abstract

Sucrose gradient analysis of the binding of our monoclonal antibody (secreted by cell line KN 382/ECl) to [17 α-methyl-3H] R5020-labeled rabbit uterine progestin receptors revealed that the antibody bound only to the 8.5S form (Kd = 0.86 nM) and not to the 7S and smaller complexes. 125I-labeled antibody, on the other hand, bound to both the 8.5S complex and a component of dissociated receptor. Calculation of the relative mass (Mr) of the 125I-labeled immunoglobulin G1 (lgG1) – protein complex indicated the addition of a 60 000 Mr peptide. Electrophoretic analysis of immunoaffinity purified receptor substantiated this by revealing two protein bands [Formula: see text]. Sequential washing of adsorbed receptor was accompanied by dissociation of the bound steroid and the Mr 92 000 peptide. The [Formula: see text] peptide could only be completely eluted from the immunoadsorbent under denaturing conditions. Autofluorography of receptor complexes covalently bound with [17α-methyl-3H]R5020 revealed two bands, one with a [Formula: see text] and the second with a [Formula: see text]. Upon immunoprecipitation both peptides precipitated with the [Formula: see text] and [Formula: see text] peptides. Gel electrophoresis demonstrated that the [Formula: see text] peptide and the [Formula: see text] did not comigrate. We have concluded that the nondissociated 8.5S rabbit uterine progestin receptor complex contains [Formula: see text] and [Formula: see text] nonsteroid-binding components, in addition to the [Formula: see text] and [Formula: see text] steroid binders and that the antibody preferentially recognizes the [Formula: see text] nonsteroid-binding peptide.