The porcine LH/hCG receptor. Characterization and purification.
Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.
Wimalasena
J
J
Moore
P
P
Wiebe
J P
JP
Abel
J
J
Chen
T T
TT
D000818:Animals; D001667:Binding, Competitive; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003338:Corpus Luteum / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D007986:Luteinizing Hormone / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011974:Receptors, LH; D013552:Swine
D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D011974:Receptors, LH; D007986:Luteinizing Hormone