Purification of follitropin receptor from bovine calf testes.
Follitropin (FSH) receptors were solubilized from pure light membranes of bovine calf testis, using an optimum detergent to protein ratio of 0.01. The soluble FSH receptor fraction was gel filtered through Sepharose 6B to isolate an active fraction (6B-Fr-1) which behaved as a complex of FSH receptor and Gs protein. The 6B-Fr-1 was concentrated by ultrafiltration and further purified by sequential Sepharose 4B gel filtration, DEAE-cellulose chromatography (to separate the receptor from Gs protein), and wheat germ lectin affinity chromatography. The purified receptor had an FSH-binding capacity of approximately 3.47 nmol/mg of protein with a Kd of 1.9 X 10(-10) M. Yield was 526 micrograms/11.5 kg tested. Radioiodinated, as well as unlabeled purified FSH receptor, migrated on sodium dodecyl sulfate-polyacrylamide gels as a single major band of Mr approximately 240,000. This band was not affected by 8 M urea treatment prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but treatment with dithiothreitol induced the loss of the 240-kDa band, with appearance of an Mr approximately 60,000 band. The availability of highly purified, stable FSH receptor should allow direct studies on its structure-function relationships.
Dattatreyamurty
B
B
Department of Biochemistry, Albany Medical College, New York 12208.
Zhang
S B
SB
Reichert
L E
LE
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005640:Follicle Stimulating Hormone / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D008970:Molecular Weight; D011962:Receptors, FSH / Q000302:isolation & purification* / Q000378:metabolism; D013737:Testis / Q000032:analysis*
D046911:Macromolecular Substances; D011962:Receptors, FSH; D006165:Guanylyl Imidodiphosphate; D005640:Follicle Stimulating Hormone; D004229:Dithiothreitol