title: Solubilization of functional and stable follitropin receptors from light membranes of bovine calf testis.
abstract: Rapid destabilization of FSH receptor after solubilization by detergents is a serious problem complicating its purification and further study. We have developed a procedure for the solubilization of stable and functional FSH receptors with Triton X-100. The new protocol selectively utilizes pure lighter membranes isolated from bovine calf testes by preparative sucrose density gradient centrifugation as the source of receptor. The conditions of detergent solubilization were optimized to reduce the required ratio of Triton X-100 to membrane protein to a minimum. In addition, during detergent extraction the membranes were treated with petroleum ether to remove interfering neutral lipids, thus facilitating solubilization of FSH receptors by the detergent. FSH receptors so obtained appeared to be soluble by criteria such as failure to sediment at 145,000 X g after 90 min, passage through 0.22-micron Millipore filters, and retardation upon chromatography on Sepharose 6B column. Approximately 86% of receptors originally present in the light membranes were recovered after solubilization, with a 24-fold increase in specific activity. The detergent-soluble fraction has several interesting properties not previously reported. It contains only high affinity receptors for FSH (Ka = 1.02 X 10(10) M-1), which are stable in the absence of glycerol for 4 days at 1 degree C or 6 months at -80 degrees C. Luteinizing hormone and human chorionic gonadotropin receptor activity usually associated with detergent-solubilized extracts of testes is low due to incomplete solubility of these receptors under the conditions utilized for solubilization of FSH receptors. Of particular interest is the ability of the receptor in the detergent extract to respond to added FSH with stimulation of adenylate cyclase activity. Adenylate cyclase activity also responds to F- stimulation and the detergent extract retains full guanosine 5'-imidotriphosphate-binding activity. This suggests that under the extraction conditions employed, a high proportion of soluble receptors are associated with related components of the adenylate cyclase system. Our data are consistent with the notion that the solubilized hormone-binding sites represent the physiologically relevant and functional receptors originally present in the light membrane fraction of calf testis. The availability of this detergent-soluble, stable and functional receptor fraction in larger amounts (2.2 g of protein from each batch of 11.5 kg bovine calf testes) than heretofore possible should facilitate further studies on FSH receptor purification and its mechanism of action.
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002499:Centrifugation, Density Gradient; D005459:Fluorides / Q000494:pharmacology; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008722:Methods; D017830:Octoxynol; D011092:Polyethylene Glycols; D011962:Receptors, FSH / Q000302:isolation & purification*; D011974:Receptors, LH / Q000302:isolation & purification; D012995:Solubility; D013737:Testis / Q000032:analysis / Q000166:cytology*; D014461:Ultracentrifugation
chemical_list: D011962:Receptors, FSH; D011974:Receptors, LH; D006165:Guanylyl Imidodiphosphate; D011092:Polyethylene Glycols; D017830:Octoxynol; D000262:Adenylyl Cyclases; D005459:Fluorides