Solubilization and purification of rat pituitary gonadotropin-releasing hormone receptor.
Gonadotropin-releasing hormone (GnRH) receptors were solubilized from rat pituitary membrane preparations in an active form by using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid). The solubilized receptor exhibits high affinity, saturability, and specificity. The soluble supernatant retained 100% of the original binding activity when stored at 4 or -20 degrees C in the presence of 10% glycerol. The receptors were resolved into two components on the basis of chromatography on wheat germ agglutinin-agarose. Homogeneous receptor preparation was obtained by two cycles of affinity chromatography on immobilized avidin column coupled to [biotinyl-D-Lys6]GnRH. The overall recovery of the purified receptor was 4-10% of the initial activity in the CHAPS extract, and the calculated purification -fold was approximately 10,000 to 15,000. Analysis of iodinated purified GnRH receptors by autoradiography indicated the presence of two bands, Mr = 59,000 and 57,000. This was confirmed by photoaffinity labeling of the partially purified receptors and suggests that both components can specifically bind the hormone.
D000818:Animals; D002064:Buserelin / Q000378:metabolism; D002793:Cholic Acids; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008722:Methods; D008970:Molecular Weight; D010902:Pituitary Gland / Q000032:analysis*; D051381:Rats; D011966:Receptors, LHRH / Q000302:isolation & purification*; D012995:Solubility
D002793:Cholic Acids; D011966:Receptors, LHRH; D002064:Buserelin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate