Differential solubilization of rat liver sigma 1 and sigma 2 receptors: retention of sigma 2 sites in particulate fractions.
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.
Torrence-Campbell
C
C
Unit on Receptor Biochemistry and Pharmacology, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892, USA.
Bowen
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WD
D000818:Animals; D001667:Binding, Competitive; D002458:Cell Fractionation; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D006146:Guanidines / Q000494:pharmacology; D006220:Haloperidol / Q000494:pharmacology; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D008297:Male; D010423:Pentazocine / Q000494:pharmacology; D010620:Phenazocine / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D017480:Receptors, sigma / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D012995:Solubility; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
D002793:Cholic Acids; D003902:Detergents; D006146:Guanidines; D017480:Receptors, sigma; C012134:SK&F 10047; D010620:Phenazocine; D006220:Haloperidol; C050232:1,3-ditolylguanidine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D010423:Pentazocine