pH-dependent effects of sodium tungstate on the steroid-binding properties of the hen oviduct progesterone receptor.
ABSTRACT Effects of sodium tungstate on the steroid-binding properties of hen oviduct progesterone receptor were examined and were found to be pH-dependent. When freshly prepared hen oviduct cytosol containing progesterone receptor was heated at 37 degrees C for 20 min, its ability to bind [3H]progesterone decreased to 20% level of unheated samples. At pH 7, presence of 2-3 mM tungstate during the above incubation period reduced this loss of binding. At higher tungstate concentrations (greater than 5 mM), this stabilizing effect was gradually abolished. Similar results were obtained with preparations that contained [3H]progesterone-receptor complexes; 70-80% of which remained after a 20 min incubation at 37 degrees C in the presence of 2-3 mM tungstate at pH 7. At pH 8, presence of tungstate (1-10 mM) during the 37 degrees C incubation stabilized both the steroid-bound and the unoccupied progesterone receptor in a concentration-dependent manner. The extent of steroid binding by the receptor at 4 degrees C remained unchanged in the presence of up to 10 mM tungstate at both pH 7 and pH 8 assay conditions while presence of 20 mM tungstate lowered this binding capacity. These results indicate that tungstate effects may be mediated via its interaction with the progesterone receptor.
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ABSTRACT: The chick oviduct cytosol progesterone receptor, when complexed with ligand, can be exposed to urea concentrations as high as 3 M (at 0 degrees C) without loss of steroid binding capacity. The ligand dissociation rate is increased greater than or equal to 10 fold under these conditions. The "native" 8 S form of the receptor is progressively converted to a 4 S species by urea (greater than 2 M) as seen in ultracentrifugation analysis. This conversion is inhibited by Na2MoO4 (5-50 mM) suggesting that molybdate stabilizes the 8 S molecule by direct interaction. At urea concentrations above 2 M, the ligand-free receptor looses progressively its binding capacity. The "transformed", 4 S receptor was less stable than the 8 S species, and could not be protected by molybdate.Biochemical and Biophysical Research Communications 08/1983; 114(2):479-87. DOI:10.1016/0006-291X(83)90805-7 · 2.28 Impact Factor
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ABSTRACT: Recently we reported that adding molybdate to crude steroid-free cytosol at 0 degree C results in a dose-dependent reduction in the binding of [3H]aldosterone ([3H]ALDO) to Type I adrenocorticosteroid receptors. In the experiments outlined here, we found that addition of molybdate to steroid-free brain cytosol produces a 30-50% increase in the subsequently measured maximal specific binding capacity (BMAX) of [3H]ALDO-Type I receptors if the cytosol is subjected to Sephadex G-25 gel filtration prior to steroid addition. These manipulations were found to have no effect on the equilibrium dissociation constant (Kd) of the receptors. In contrast, when gel filtration of steroid-free cytosol was performed in the absence of molybdate, there was a 2-fold increase in the Kd and over a 50% reduction in the subsequently measured BMAX of [3H]ALDO-Type I receptors. When molybdate was added to this steroid-free cytosol immediately following gel filtration, there was no reduction (or increase) in Type I receptor [3H]ALDO binding capacity compared with non-gel-filtered controls. The addition of as little as 2 mM molybdate to crude steroid-free cytosol was found to stabilize the binding capacity of Type I receptors during exposure to 22 degrees C incubations; however, when gel-filtered steroid-free cytosol was exposed to these conditions at least 10 mM molybdate was required to stabilize Type I receptor binding capacity. Adding the sulfhydryl reducing reagent, dithiothreitol, to the various steroid-free cytosols had little effect on [3H]ALDO-Type I receptor binding.(ABSTRACT TRUNCATED AT 250 WORDS)Neurochemical Research 09/1988; 13(8):707-13. DOI:10.1007/BF00971592 · 2.55 Impact Factor