Characterization of membrane action of steroids

Abstract

This neuroprotocol describes in detail a neuronal membrane radioreceptor assay for progesterone and testosterone. These steroids are linked through different C positions in the molecule to bovine serum albumin (BSA) to form a large steroid-BSA complex that can be radioiodinated through tyrosyl residues in the BSA molecule, generating a hydrophilic ligand of high specific activity. A crude synaptosomal membrane fraction (P2 pellet) prepared from different regions of the CNS of the male or female rat is used as the source of neuronal membranes, and the assay is carried out in Tris-binding buffer (pH 7.4) at 4°C for 30 min. With this assay, stereospecific binding sites with an affinity constant in the micromolar range were shown at several CNS target sites for these steroids. High capacity and high selectivity as well marked physiological changes indicate that these binding sites most likely correspond to a new group of steroid membrane receptors different from classical nuclear receptors.