Nuclear binding of progesterone in hen oviduct. Binding to multiple sites in vitro

Abstract

Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1x10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1x10(-9)-1x10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei.

Nuclease resistance and the enrichment of native nuclear acceptor sites for the avian oviduct progesterone receptor.

“…Similar binding results were observed using the milder (DNase/exonuclease/Sl nuclease) digestion method. Thus, the saturable, high-affinity binding of PR to NAPf displayed similar properties as did the PR binding to intact NAP (8,9,11) or chromatin (7,36).…”

“…Nuclei and chromatin were isolated and purified using modifications of methods described (7,9,13,16). All steps were performed at 4°C.…”

“…DNA was isolated from hen spleen nuclei as described (8,13,16 7.4/10 mM MgCl2) at a concentration of 1 mg of DNA per ml at 4TC for 2 hr. The NAP was then centrifuged at 100,000 x g for 30 min in a Beckman TL-100 microultracentrifuge.…”

“…In contrast to the receptor binding to pure DNA sequences alone (5, 6), the binding of the oviduct PR to subfractions of chromatin containing nonhistone protein-DNA complexes, termed nucleoacidic protein or NAP, has been shown to be receptor dependent and saturable, of high affinity (7)(8)(9)(10)(11), to be receptor specific (12), and to generate patterns of bindings similar to that measured in vivo (13)(14)(15)(16). Further, the capacity of the PR to bind to nuclear acceptor sites reflects the ability of progesterone to alter RNA polymerase activity in nuclear run-off experiments (17, t) and to specifically induce the avidin gene (18).…”

The binding of [3H]oestradiol-receptor complexes to calf uterine chromatin

“…The most extensive work on nuclear acceptors has been performed by Spelsberg and co-workers (Spelsberg et al, 1975;Webster et al, 1976;Pikler et al, 1976;Spelsberg et al, 1979), using the chick progesterone receptor. Other steroid-hormone systems are currently under investigation (KlyzsejkoStefanowicz et al, 1976;Hamana & Iwai, 1978;Perry & Lopez, 1978;Weinberger & Veneziale, 1980; Taylor et al, 1980).…”