Action of glycerol and sodium molybdate in stabilization of the progesterone receptor from rat trophoblast.
This study compares the stabilizing effects of glycerol and sodium molybdate on the progesterone receptor in vitro. Trophoblastic tissue from day 12 embryos was homogenized in TD buffer (10 mM Tris, 1 mM dithiothreitol at pH 7.8, 4 degrees C) with 30% glycerol (v/v) (TDG-30%) and/or 10 mM sodium molybdate (TDG-30% + Mo and TD + Mo, respectively). Receptor was partially purified from cytosol by ammonium sulfate precipitation and incubated overnight (4 degrees C) with [3H]P +/- unlabeled progesterone in appropriate buffer to measure binding properties of the receptor under various buffer conditions. The effects of glycerol and sodium molybdate on sedimentation behavior of receptor in 5-20% sucrose gradients was studied in other experiments. Binding parameters were indeterminate when the receptor was incubated in the basic TD buffer only. However, in the presence of molybdate and/or glycerol high affinity binding was maintained at 0 and 15 degrees C (Kd congruent to 2 nM). In TD + Mo media high affinity binding was lost (Kd = 14 and 35 nM at 0 and 15 degrees C, respectively). The receptor always sedimented as 4-5 S forms unless molybdate was present whereupon they were replaced by 7-8 S moieties. Under conditions of high salt (0.3 M KCl) high affinity was preserved by glycerol, as in low salt conditions, and a new 6-7 S moiety occurred; in addition to this form, the 7-8 S aggregate was retained in the presence of molybdate. Thermodynamic studies showed that the addition of molybdate to glycerol media did not alter the energy of activation for progesterone-receptor interaction measured at 0 and 15 degrees C; however, at 30 degrees C molybdate was necessary to prevent denaturation of receptor. Glycerol and molybdate must be present from the time of tissue extirpation for maximum stabilization. Denaturation begins immediately and is only partially reversible. It is concluded that glycerol interacts with the surface of the receptor molecule in such a way as to favor the folded native state which preserves high affinity interactions whereas molybdate interacts directly with receptor to maintain 7-8 S aggregates which increases the availability of binding sites.
D000818:Animals; D002499:Centrifugation, Density Gradient; D005260:Female; D005990:Glycerol / Q000494:pharmacology*; D006358:Hot Temperature; D008982:Molybdenum / Q000494:pharmacology*; D011247:Pregnancy; D051381:Rats; D011980:Receptors, Progesterone / Q000187:drug effects*; D014327:Trophoblasts / Q000032:analysis*
D011980:Receptors, Progesterone; D008982:Molybdenum; C024687:sodium molybdate(VI); D005990:Glycerol