Solubilization and purification of the gonadotropin (GTH II) receptor from rainbow trout (Oncorhynchus mykiss) ovaries.
The rainbow trout (Oncorhynchus mykiss) ovarian gonadotropin (GTH II) receptor was solubilized by extraction with the nonionic detergent 1% Triton X-100 in the presence of 20% glycerol. The hormone-binding characteristics of the soluble receptors were similar to those of membrane-bound receptors: the Scatchard plot of the equilibrium binding data produced a straight line, suggesting that the solubilized GTH II receptors, like membrane-bound receptors, contained a single class of high affinity 125I-sGTH II binding sites with an association constant of 2-5 x 10(10) M-1 (Ka = 1.4-2 x 10(10) M-1 for membrane-bound receptor). The maximal binding capacity was very low and varied from 7 to 17 fmol/mg proteins (about 5 fmol/mg ovarian membrane protein). The soluble receptor was purified by a simple and rapid immunoaffinity chromatography. The sGTH II-solubilized receptor complex was adsorbed to anti-sGTH II beta-subunit gammaglobulins covalently linked to Sepharose 4B and then eluted with an acidic buffer. About 50% of the binding activity present in the Triton X-100 extract was recovered in the pH 4 eluate. The other binding sites were eluted as a hormone-receptor complex and/or a damaged form. The free purified receptor presented a Ka of 1.3 x 10(10) M-1 in agreement with those found in membrane preparation and solubilized extract.
Quesnel
H
H
Laboratoire de Physiologie des Poissons, INRA, Campus de Beaulieu, Rennes, France.
Breton
B
B
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D007457:Iodine Radioisotopes; D007700:Kinetics; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010053:Ovary / Q000378:metabolism*; D011967:Receptors, Gonadotropin / Q000302:isolation & purification / Q000378:metabolism*
D007457:Iodine Radioisotopes; D011967:Receptors, Gonadotropin