The effects of sodium molybdate on the equilibrium and kinetics of [3H]estradiol binding, and on the differential ammonium sulfate precipitation and activation of the calf uterine estrogen receptor, were investigated. The magnitude of the fast component of the biphasic [3H]estradiol-receptor dissociation, which is proportional to the fraction of the receptor in the nonactivated state, was markedly increased by the presence of 10–20 mM molybdate, indicating an inhibition of receptor activation. A small fraction of the receptor (10-20%) was consistently found in the slower [3H]estradiol-dissociating (activated) form because a very slow rate of receptor activation still occurred in the presence of molybdate, or a small fraction of the receptor was initially present in the activated state, even in the absence of estradiol. The degree of inhibition of receptor activation by molybdate was proportionally reduced by the ionic strength of the buffer: in 0.4 M KCl, there was no inhibition of receptor activation by molybdate. Dialysis of the molybdate-treated cytosol reversed the inhibitory action of molybdate on receptor activation; however, once activation had occurred, the addition of molybdate did not shift the equilibrium between the two forms of the receptor toward the nonactivated state. The [3H]estradiol equilibrium binding analysis of the receptor in the presence of molybdate showed a decrease in the positive cooperative binding of estradiol. The Hill coefficient (nH) was reduced from 1.62 ± 0.08 to 1.40 ± 0.03. Pretreatment of the cytosols with DNA-cellulose prior to the [3H]estradiol equilibrium binding assay further reduced positive cooperativity: nh = 1.13 ± 0.06 in the presence and nH = 1.58 ± 0.05 in the absence of molybdate. DNA-cellulose pretreatment of the cytosol removed the small fraction of the receptor that was initially present in the activated state. Sedimentation analyses with sucrose gradients containing 0.4 M KCl showed that molybdate inhibited the transformation of the receptor from the 4S to the activated 5S form. A small fraction (10-20%) of the receptor in the presence of molybdate was seen consistently in the activated 5S form in agreement with the results of the [3H]estradiol-receptor dissociation assay. The sedimentaton coefficients of the estrogen receptor in low-salt buffers were not significantly affected by the molybdate. In the absence of molybdate, the receptor was precipitated at 30% ammonium sulfate and was shown to be transformed to the activated receptor, while in the presence of molybdate the receptor was precipitated at 30–50% ammonium sulfate saturation and shown to be in the nonactivated form. This differential ammonium sulfate precipitation can be used to provide approximately a 30-fold purification of the activated receptor. These data indicate that molybdate maintains and interacts exclusively with the nonactivated state of the receptor.
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