2--hydroxy-5-9-dimethyl-2-allyl-6-7-benzomorphan and 3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate

Rat liver membranes (crude P2 membranes) were solubilized in 10 mM Tris-HCl, pH 7.4 containing 7 mM 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). The soluble fraction was designated the Extract 1. The 105,000 x g pellet was washed once, and then extracted a second time (Extract 2). The various resulting fractions were assayed for sigma (sigma) binding characteristics, using [3H](+)-pentazocine to label sigma 1 sites and [3H]1,3-di-o-tolylguanidine (DTG) in the presence of 1 microM dextrallorphan to label sigma 2 sites. Both of the extracts and resultant pellets (Pellet 1 and Pellet 2) contained sigma 1 and sigma 2 receptors, as indicated by the pharmacological profiles upon competition studies. The Kd and Bmax values for sigma 1 activity in the original P2 membranes were 8.3 +/- 0.73 nM and 5333 +/- 572 fmol/mg protein; Kd and Bmax for sigma 2 activity was 19 +/- 0.17 nM and 9190 +/- 800 fmol/mg protein. There were no changes in the radioligand Kd values of the two sites in the subsequent soluble and particulate fractions. However, while the sigma 1 and sigma 2 Bmax values in extracts and pellets were generally on the same order as those of P2 membranes, the actual sigma 2 to sigma 1 Bmax ratio varied markedly across the fractions. The ratio of sigma 2/ sigma 1 binding in Extract 1 and Extract 2 was 0.86 and 0.68, respectively, compared to a ratio of 1.7 in the original P2. However, the ratio in Pellet 2 was 3.8, twice that of the original P2 membranes. Furthermore, the Bmax value for sigma 1 sites in Pellet 2 did not change, whereas the sigma 2 Bmax increased 1.8 fold relative to the original P2 membranes. The changes in sigma 2/ sigma 1 binding ratio in extracts were observed using two different assay methods for soluble receptors (retention on polyethyleneimine-coated filters and polyethylene glycol precipitation) and is therefore not an artifact of assay procedure. These data suggest that, relative to sigma 1 receptors, sigma 2 receptors are more resistant to solubilization and become somewhat enriched in the particulate fractions. This supports the notion that sigma 1 and sigma 2 receptors are distinct macromolecules and may indicate different modes of association with the cell membrane.

Topics: Animals; Binding, Competitive; Cell Fractionation; Cell Membrane; Cholic Acids; Detergents; Guanidines; Haloperidol; In Vitro Techniques; Liver; Male; Pentazocine; Phenazocine; Protein Binding; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, sigma; Solubility; Subcellular Fractions

The zwitterionic detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS) produced optimal solubilization of (+)-[3H]SKF-10,047 binding sites from rat liver membranes at a concentration of 0.2%, well below the critical micellular concentration of the detergent. The pharmacological selectivity of the liver (+)-[3H]SKF-10,047 binding sites corresponds to that of sigma sites from rat and guinea pig brain. When the affinities of 18 different drugs at (+)-[3H]SKF-10,047 binding sites in membranes and solubilized preparations were compared, a correlation coefficient of 0.99 and a slope of 1.03 were obtained, indicating that the pharmacological selectivity of rat liver sigma sites is retained after solubilization. In addition, the binding of 20 nM [3H]progesterone to solubilized rat liver preparations was found to exhibit a pharmacological selectivity appropriate for sigma sites. A stimulatory effect of phenytoin on (+)-[3H]SKF-10,047 binding to sigma sites persisted after solubilization. When the solubilized preparation was subjected to molecular sizing chromatography, a single peak exhibiting specific (+)-[3H]SKF-10,047 binding was obtained. The binding activity of this peak was stimulated symmetrically when assays were performed in the presence of 300 microM phenytoin. The molecular weight of the CHAPS-solubilized sigma site complex was estimated to be 450,000 daltons. After solubilization with CHAPS, rat liver sigma sites were enriched to 12 pmol/mg of protein. The present results demonstrate a successful solubilization of sigma sites from rat liver membranes and provide direct evidence that the gonadal steroid progesterone binds to sigma sites. The results also suggest that the anticonvulsant phenytoin binds to an associated allosteric site on the sigma site complex.

Topics: Animals; Binding Sites; Cell Membrane; Cholic Acids; Culture Techniques; Haloperidol; Liver; Male; Phenazocine; Rats; Rats, Inbred F344; Receptors, Opioid; Receptors, sigma; Tritium