DEMONSTRATION OF MEMBRANE ESTROGEN-BINDING PROTEINS IN RAT-BRAIN BY LIGAND BLOTTING USING A 17-BETA-ESTRADIOL-[I-125]BOVINE SERUM-ALBUMIN CONJUGATE

This paper describes a ligand blotting procedure to visualize membrane estrogen receptors/binding proteins immobilized on nitrocellulose mem branes. Using 17 beta-estradiol covalently Linked with [I-125]bovine s erum albumin (BSA) at the C-6 position (17 beta-E-6-[I-125]BSA) as a l igand, three major binding proteins with molecular masses of approxima tely 23, 28, and 32 kDa were identified from crude synaptosomal fracti ons (P2) of female rat brains. The binding of 17 beta-E-6-[I-125]BSA t o these proteins is selective for 17 beta-estradiol because BSA had no effect, and 17 alpha-E-6-BSA was at least two orders of magnitude les s potent than 17 beta-E-6-BSA in displacing the binding. In addition, [I-125]BSA and 17 alpha-E-6-[I-125]BSA at similar concentrations did n ot bind to these proteins. Competition and saturation assays indicate that the binding affinity of 17 beta-E-6-[I-125]BSA for these proteins was in the range of 1-10 nM. These proteins are not contaminants from cytosolic or serum estrogen binding proteins since no corresponding p rotein bands were found in cytosolic fractions. Three additional prote in bands with molecular masses of approximately 18, 40, and 130 kDa we re also detected, although inconsistently. The 23 and 40 kDa proteins seem to be concentrated in mitochondrial fractions (mP2), whereas the 28 and 32 kDa proteins are enriched in microsomal fractions (P3). Appl ication of digitonin-solubilized P2 fractions to 17 beta-estradiol-cou pled affinity columns resulted in significant purification of the 23 k Da protein as shown by Ligand blotting. This protein was later identif ied as oligomycin-sensitivity conferring protein (OSCP), as reported p reviously. These data indicate that specific estrogen binding proteins different from classical nuclear estrogen receptor (66 kDa) are prese nt in the cellular membranes of the female rat brain. The ligand blott ing technique described here would also be applicable for the identifi cation of other membrane steroid binding proteins/receptors using simi lar radiolabelled steroid-BSA conjugates. (C) 1997 Elsevier Science Lt d.