High-resolution separation of molybdate-stabilized progestin receptors using high-performance liquid chromatography

Abstract

Molecular heterogeneity of the human uterine progestin receptor was investigated employing sucrose density gradient centrifugation and high-performance liquid chromatography (HPLC) in size-exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Synthetic progestomimetic ligands, [ 3H] R5020 and [ 3H] ORG-2058, were used to identify these receptors. Rapid centrifugation with a vertical tube rotor showed both 8–9 S and 4–5 S receptor species in the presence of 10 m M sodium molybdate with a 90–96% recovery. [ 3H] R5020 displayed greater non-specific binding than [ 3H] ORG-2058. When separated receptor preparations were labeled, each with a different ligand, mixed and separated on optimized gradients, at least two receptor isoforms were identified in the components sedimenting at 8–9 S. HPSEC confirmed the presence of receptor isoforms displaying different molecular size and shape dependent upon the progestin ligand used. When the surface charge properties were examined by HPIEC using AX-1000, two distinct species were observed irrespective of the radioactive ligand. The first peak appeared in the void volume similar to the position of free steroid, indicating the possibility of ligand stripping by the column. The second peak bound steroid specifically and eluted with 100 m M phosphate. If either 8-S or 4-S progestin receptors were first separated by gradient centrifugation then by HPIEC, both receptor isoforms eluted with 60 m M phosphate. Re-chromatography of these on HPIEC also gave the isoform eluting at 60 m M phosphate. HPCF of ligand-bound receptors on AX-500 columns also identified one isoform eluting at pH 5.6–6.1. Using a combination of HPLC techniques and sucrose gradient centrifugation, heterogeneity of the progestin receptor has been demonstrated.