ESTIMATION OF TOTAL PROLACTIN-BINDING SITES AFTER INVITRO DESATURATION

[125I]Iodoovine PRL ([125I]oPRL) binds with high affinity and specificity to crude membrane fractions prepared from either rabbit mammary gland or female rat liver. After completion of [125I]iodo-PRL binding, a short (5-min) exposure to magnesium chloride (4—5 M) resulted in a 91—97% dissociation of the bound [125I]iodo-PRL. Upon reincubation with fresh [125I]iodo-PRL in the absence or presence of 1 μg unlabeled oPRL, the membrane preparation specifically bound 89—105% of the label bound by control (H2O-treated) membranes. Similar results were observed with 4 M MnCl2. Efficient dissociation of bound PRL could also be obtained with 1.6—4 M ammonium thiocyanate and 4 M sodium trifluoroacetate, although MgCl2 was far superior in the rebinding studies, indicating some irreversible damage to the receptor by some of the dissociating agents. Similar positive results were obtained by first saturating he binding sites with unlabeled PRL, removing the PRL by MgCl2 treatment, and reincubating with labeled PRL. In either case, MgCl2 treatment did not alter the binding affinity (Ka, ∼ nM-1) but increased the number of available binding sites. In vivo desaturation of PRL-binding sites by previous treatment with CB-154 was also effective in increasing the amount of receptors measurable in rat liver. Ovariectomized rats were treated with estradiol (2.5 μg) twice a day for 7 days. Injection of 1 mg CB-154 6 h before sacrifice at 1700 h reduced the peak of plasma PRL from 1259 ± 214 to 64 ± 40 ng/ml, while PRL binding was increased from 18.7 ± 0.9% to 28.7 ± 1.2%. In vitro dissociation by MgCb was capable of removing endogenously bound lactogenic hormones circulating during pregnancy. During periods when placental lactogen levels were highest (days 11—13 and 17—20), prior treatment of membrane fractions from mid- and late pregnancy with MgCb increased binding to 219-228% of that observed in control membranes. The positive effect of PRL on PRL binding was also enhanced by MgCl2 treatment. Ovariectomized rats treated for 1 week with oPRL (1 mg twice a day) showed no significant change in available hepatic PRL-binding sites. However, prior treatment of liver membranes with MgCl2 increased PRL binding in the control group and also resulted in a 2.4-fold increase in binding in the animals injected with PRL. These data suggest that PRL-binding sites retain endogenously bound hormone even during preparation of membranes and that techniques such as the one described should be applied for the estimation of the total number of PRL-binding sites. © 1979 by The Endocrine Society.