Microsomal binding sites for antioestrogens in rat liver. Properties and detergent solubilization

The properties of an antioestrogen binding site (AEBS), which has high affinity and specificity for nonsteroidal antioestrogens and structurally related compounds, have been studied in rat liver microsomes. When subcellular organelles were separated on Percoll density gradients the distribution of the AEBS paralleled that of NADPH-cytochrome c reductase, indicating that the AEBS is associated with the endoplasmic reticulum. Saturation analysis showed that [3H]tamoxifen was bound to a single class of saturable binding sites in liver microsomes with a KD of 0.9 +/- 0.1 nM at 0 degrees C. The equilibrium KD was not significantly different at 22 degrees C. The KD calculated from the association and dissociation rate constants for [3H]tamoxifen binding at 0 degrees C and 22 degrees C was compatible with the KD measured at equilibrium. Ligand specificity studies using tamoxifen analogues showed qualitatively similar structure-affinity relationships for the AEBS from both rat liver and the MCF 7 breast cancer cell line. In general structural modifications caused correspondingly greater changes in affinity for rat liver AEBS than for MCF 7 AEBS. The AEBS was solubilized from microsomal membranes with sodium cholate. This was the only detergent of nine tested that solubilized the site in high yield without loss of activity. Solubilization using cholate was more effective in the presence of 1 M-NaCl. In the solubilized state there was an apparent loss of [3H]tamoxifen binding activity which could be restored by dilution of the detergent. Gel filtration indicated an Mr of 440,000-490,000 for the AEBS-cholate complex. These studies demonstrate that rat liver contains high concentrations of a microsomal AEBS which has similar properties and specificity to the AEBS previously described in human breast cancer cells. This site can be solubilized by sodium cholate to supply material suitable for further purification.