Assessment of progestin receptor polymorphism by various synthetic ligands using HPLC.
Tritiated R5020 and [3H]ORG-2058 were utilized to investigate apparent polymorphism of progestin receptors by vertical-tube gradient centrifugation and HPLC in size exclusion (HPSEC), ion-exchange (HPIEC) and chromatofocusing (HPCF) modes. Rapid centrifugation (3 h) following molybdate stabilization (1 h) showed mainly 8-9S receptor species with 90-96% recovery. [3H]R5020 appeared to associate with a receptor isoform sedimenting faster than that bound to [3H]ORG-2058. Excess unlabeled R5020 did not eliminate all of the [3H]R5020 binding by the 8-9S component suggesting some nonspecific association while excess unlabeled ORG-2058 suppressed this binding by either ligand. Separate labeling of cytosol with each ligand and mixing prior to gradient separation showed at least two receptor species isoforms sedimenting in the 8-9S region with mol. wt of 190,000 and 173,000 D. Sephacryl S-300 chromatography revealed two radioactive peaks with either ligand but with slight molecular weight differences. HPSEC confirmed the presence of isoforms with different molecular size and shape as a function of the radioactive ligand employed. HPIEC showed the presence of two labeled receptor species irrespective of the ligand used. The first peak appeared at the void volume of the column (10 mM), co-eluted with free ligand, indicating the possibility of ligand stripping by the column. The second peak bound both steroids specifically and eluted with 100 mM phosphate. HPCF identified a single specific receptor eluting at a pH of 5.6-6.1, but with free steroid in the void volume irrespective of the ligand used. [3H]ORG-2058 appeared to be less prone to the stripping phenomenon than was [3H]R5020. These data suggest these ligands either bind to different progestin receptor species or they modify receptor characteristics in a fashion that allows separation based upon size and shape.
van der Walt
L A
LA
Wittliff
J L
JL
D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005260:Female; D006801:Humans; D008024:Ligands; D011110:Polymorphism, Genetic; D011282:Pregnenediones / Q000378:metabolism; D011397:Promegestone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000032:analysis*; D014316:Tritium
D008024:Ligands; D011282:Pregnenediones; D011980:Receptors, Progesterone; D014316:Tritium; C020175:16 alpha-ethyl-21-hydroxy-19-nor-4-pregnene-3,20-dione; D011397:Promegestone