Properties of LH/hCG receptors in porcine corpus luteum homogenates and subcellular fractions, and factors influencing the recovery of membrane-bound hormone.
An evaluation of several techniques commonly used to separate bound from free hormone was made for the binding of radiolabelled human chorionic gonadotropin to mid-luteal pig corpus luteum homogenates and subcellular fractions. Compared to millipore filtration with 0.22 or 0.45-mu HAWP filters, centrifugation of homogenates at 2 500 x gav or 1 000 x gav for 15 min recovered only 50 and 33% respectively of the total bound hormone present. This discrepancy was accentuated further when hormone binding to post-nuclear subcellular fractions was studied. Centrifugation of a nuclear fraction at 2 500 x gav for 15 min recovered 70% of the membrane-bound receptors present (as measured by filtration), whereas centrifugation of mitochondrial, lysosomal and microsomal fractions under these conditions recovered only 46, 16 and 4% respectively. Divalent metal ions influenced hormone binding in 2 ways: (i) Specific bonding of hCG was greatest in the absence of metal ions and in the presence of low levels of chelating agents. Increasing concentrations of magnesium and calcium ions appeared to interfere with the interaction of hormone and receptor. (ii) Divalent metal ions reduced the recovery of particle-bound hormone by centrifugation, at both high and low speeds. The results illustrate the pitfalls of using low-speed centrifugation to recover membrane-bound hormone-receptor complexes when small membrane vesicles are present.
D000818:Animals; D006063:Chorionic Gonadotropin / Q000378:metabolism; D003338:Corpus Luteum / Q000032:analysis*; D004247:DNA / Q000032:analysis; D005260:Female; D007986:Luteinizing Hormone / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis* / Q000378:metabolism; D013347:Subcellular Fractions / Q000032:analysis; D013552:Swine
D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D007986:Luteinizing Hormone; D004247:DNA