Solubilization of pituitary GnRH binding sites by means of a zwitterionic detergent.
Solubilization in an active form of the pituitary plasma membrane protein carrying binding sites for gonadotropin-releasing hormone (GnRH) was possible by using as detergent CHAPS (3[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate), which is a zwitterionic derivative of cholic acid. In contrast, the use of the non-ionic detergent Triton X-100 resulted in the loss of GnRH binding sites. CHAPS at a concentration of 5-10 mM allowed the solubilization of up to 80% of available sites, and remaining non-solubilized sites retained unaltered binding properties. Characterization of solubilized binding sites of GnRH was achieved by using as radioactive ligand and standard a stable GnRH analog. [D-Trp6,(NEt)Pro9,desGly10]-GnRH. Maximum binding of GnRH to solubilized binding sites was possible in the presence of a very low amount of CHAPS in the incubation medium. Scatchard analysis of classical saturation experiments indicated the presence of a single class of high affinity, low capacity binding sites. The mean value for the affinity constant KA was 0.36 +/- 0.07 X 10(10) M -1 (n = 5) for solubilized GnRH binding sites, when analysed in the presence of 2 mM CHAPS; when solubilized GnRH binding sites were analysed in the absence of CHAPS, significantly higher KA values were observed, ranging from 1.2 to 5.9 X 10(10) M -1. Parallel analysis of the plasma membranes prior to solubilization indicated a mean KA value of 0.53 +/- 0.13 X 10(10) M -1 (n = 5). The binding specificity of the pituitary GnRH binding sites as seen by evaluating cross-reactions with a panel of agonists and antagonists of GnRH was not altered by solubilization. Gel filtration on Sephadex G-25 of solubilized binding sites preincubated with radioiodinated GnRH analog demonstrated the presence of a large molecular weight complex. In conclusion, CHAPS appears to be quite suitable for the solubilization of the binding site moiety of the pituitary GnRH receptor with good retention of the binding characteristics, thus offering a new possibility for the purification and characterization of the GnRH receptor protein.
D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002793:Cholic Acids; D002850:Chromatography, Gel; D003902:Detergents; D008565:Membrane Proteins / Q000302:isolation & purification*; D010902:Pituitary Gland / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D011966:Receptors, LHRH; D012995:Solubility; D013501:Surface-Active Agents
D002793:Cholic Acids; D003902:Detergents; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011966:Receptors, LHRH; D013501:Surface-Active Agents; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate