Fluorescence polarization measurement of the hormone-binding site interaction.
Fluorescence polarization methodology has been applied to the binding of fluorescent-labeled prolactin, growth hormone and estradiol to subcellular fractions prepared from rabbit mammary and uterine tissue. Equilibrium measurements treated by Scatchard plots have shown that there are high affinity sites (K approximately 10(9) 1 mol-1), as well as lower affinity sites (K approximately 10(8) 1 mol-1) for both hormones. The binding of the fluorescent labeled hormone to microsomal or cytosol fractions has been shown to be inhibited by the prior addition of native, unlabeled hormone. Kinetic results on the interaction of prolactin with the microsomal fraction are consistent with a bimolecular reaction involving significant structural rearrangements during the reaction (not diffusion controlled). The forward rate constant calculated from data on initial rates was found to be 1.7 X 10(5) 1 mol-1 sec-1. Stopped flow kinetic measurements on the reaction between fluorescent-labeled estradiol and cytosol binding sites show that at low temperatures, the reaction goes in two distinct steps separable in time. The second step may be the reaction found by others (utilizing sedimentation velocity methods) which precedes translocation of the hormone-binding site complex to the nucleus. Fluorescence polarization makes it possible to observe both the formation and dissociation of hormone-binding site complexes over a time scale down to a fraction of a second and at concentrations down to the nanogram per nl range.
D000818:Animals; D002021:Buffers; D006063:Chorionic Gonadotropin / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D007700:Kinetics; D008321:Mammary Glands, Animal / Q000378:metabolism; D008861:Microsomes / Q000378:metabolism; D009904:Optical Rotation; D011388:Prolactin / Q000378:metabolism*; D011817:Rabbits; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014599:Uterus / Q000378:metabolism
D002021:Buffers; D006063:Chorionic Gonadotropin; D011956:Receptors, Cell Surface; D011960:Receptors, Estrogen; D004958:Estradiol; D011388:Prolactin