The proton-driven dissociation of oestradiol-receptor dimers as a preparative tool. Isolation of a 32 kDa fragment from porcine uteri and assignment of C-terminal origin by partial sequencing

Abstract

Homodimers of the porcine oestradiol receptor dissociated at pH 6.4. The monomers reassociate after neutralization. This property is retained in a 32 kDa receptor fragment generated by co-adsorbed endopeptidases from cytosolic receptor bound to heparin-Sepharose. The fragment was purified by successive gel filtrations in the dimer and monomer states. Precipitations with ethanol and (NH4)2SO4 respectively served as concentrating steps. In all, 10-15 nmol of the homogeneous fragment were recovered from 8 kg batches of porcine uteri with a approximately 10(5)-fold enrichment and in approximately 20% yields. Its oestradiol-binding capacity was identical with that of the intact receptor. The N-terminus was blocked. Two decapeptides from a tryptic digest were sequenced. One of them corresponded to amino acids 353-362 of the human receptor, a sequence fully conserved in all species investigated. The second peptide differed in positions 553, 554 and 557 from the 549-558 sequence of the human protein.

Characterization of Five Monoclonal Antibodies Raised against Domain E of the Porcine Estradiol Receptor

“…The estradiol receptor fragment of 32 kDa was purified to homogeneity from the cytosol of prepubertal porcine uteri as described (Thole et al, 1991).…”

“…We have recently published the purification of a steroid-binding estradiol receptor fragment in large amounts (Thole et al, 1991). This fragment from porcine uterus has a mass of 32 kDa and spans most of domain E. We used the fragment for the generation of monoclonal antibodies.…”

Surface Mapping of the Ligand‐Filled C‐Terminal Half of the Porcine Estradiol Receptor by Restricted Proteolysis

“…The effects of endogenous enzymes and endopeptidases artificially admixed to the soluble phase of homogenates or adsorbed to subcellular structures, have been repeatedly described by our group and others 1231. We exploited the release of a 32-kDa Cterminal fragment of the estradiol receptor from heparin-Sepharose by plasmin [15] for receptor purification by successive gel filtrations in the monomer and dimer states [9] and by immunoadsorption to carrier-linked mAb 13H2 111, 141.…”

“…We have previously shown, that the C-terminal 32-kDa half of the porcine estradiol receptor [9] extending from His267 to Ile595 [lo, 111 with a mass of 37.3 kDa can be tailored to a minimal steroid-binding core of two consecutive peptides of 17 kDa (Asn304-Lys467) and (Ser468-Lys531) 7 kDa by endopeptidase Lys-C. The Achromobacter lyticus protease used in that study [lo] is a very specific and aggressive enzyme.…”

“…alp barrels [6], parallel p strands flanked by a helices, as in serine proteases of the subtilisin type [7], and antiparallel sheets resembling the structures found in proteins (e.g. al-antitrypsin and ovalbumin) of the serine protease-inhibitor family [8] have been discussed.We have previously shown, that the C-terminal 32-kDa half of the porcine estradiol receptor [9] extending from His267 to Ile595 [lo, 111 with a mass of 37.3 kDa can be tailored to a minimal steroid-binding core of two consecutive peptides of 17 kDa (Asn304-Lys467) and (Ser468-Lys531) 7 kDa by endopeptidase Lys-C. The Achromobacter lyticus protease used in that study [lo] is a very specific and aggressive enzyme.…”