1974) Studies on Cortisosterone-Recepasr Complexes from Mouse Placenta. Can. b. Bisckem. 52, The ia vitm binding of radioactive stemids to components of mouse placental nuclei and cytoplasm was investigated using Sephadex or charcoal to remove unbound steroid. Specificity was indicated in csmpetitiom experiments using excess unlabelled competing steroids. Only the active glucocortisoids formed complexes that could be isolated from the nucleus. The binding properties of the cytoplasmic steroid-receptor complex were studied. From the time course of binding the complex was shown to be more stable at 0" than at 37", and the distribution of receptors i na the cytosol appeared to be homogeneous. The complex was labile to heat and to proteolytic digestion but did not appear to be aEected by nucleases or suBfiydry1 reagents. Kinetic analysis revealed the presence of high affinity specific binding sites with a dissociation constant sf 17.5 nil4 and a receptor site concentration of 0.26pmol/mg protein. The cortisosterone isolated from nuclear complexes and dexamethasone from cytoplasmic complexes were identified by chromatography and by cwrystallization as the unchanged steroid in each case. Wong, M. D. & Burton, A. SF. (1974) Studies on Corticosterone-Receptor Complexes from Mouse Placenta. Can. B. Bischern. 52, 190-1195Nous avom 6tudiC B a liaison in vitro des stCrsYdes radioactifs aux mnstituants des noyaux et du cytoplasrne placentaires de la souris utilisant le SCphadex ou H e sharbon de bois pour enlever les stCroYdes libres. Des expkriences de comlpttieisn, par addition en exc$s de stCroTdes comp6eiteurs non rnarquCs, ont permis de dkterminer la spkcificitC. Seuls Bes glucocorticoYdes forment des complexes qu'il est possible d'isoler des noyaux. Nous avons 6tudi6 les propri6tCs de liaison du cornplexe stkro?dsr6cepteur dans le cytoplasme. D'aprbs la liaison en fsmction du temps, le complexe est plus stable B 0" qu'8 39" et la dktributiom des rbcepteurs dans le cgrtosol serait homog&ne. Le complexe est instable B la chaleur et & la digestion protColytiqase mais il we semble pas affect6 par les nucl6ases et les r6actifs sulfhydryles. L'analyse cinktiqlare rkvble la prhence de sites de liaison sp$cifiques possCdant unae &nit& Blev6e; la constante dde dissociation est Be 17.5 nM et la concentration du site rhcepteur de 0.26 pmol/rng de protgine. La corticostCrone, isolCe des complexes nuclCaires, et la dexam6thasone, des compkxes cytoplasmiques, ssnt identifikes par c2arornatopraphie et cocristallisation cornme Ctamt le m&me st6rsYde dans chaque cas.[Traduit par le journal]

Mouse fibroblasts contain a macromolecular binding component (receptor) which binds glucocorticoids specifically and with high affinity. This study shows that there are three different cellular forms of bound receptor and that it is experimentally possible to markedly alter the subcellular distribution of these three forms. Cells incubated with (3H)triamcinolone acetonide were broken after hypotonic shock and a 7000g hypotonic supernatant was obtained; the pellet was extracted with 0.3 M KCl, yielding a nuclear extract; the remaining pellet was resuspended in water, sonicated, and assayed for "nuclear residual" (i.e., nonextractable) radioactivity. If whole cells are incubated at 0 degrees in a growth medium, almost all of the bound steroid is located in the hypotonic supernatant fraction. Incubation at 37 degrees produces a shift of the steroid-bound macromolecule into the nuclear extractable form, while omission of glucose and addition of KCN at 37 degrees markedly increase the nuclear residual form at the expense of both the nuclear-extractable and supernatant forms. Since DNase treatment of chromatin liberates a soluble steroid-receptor complex, we believe that the nuclear residual form may be steroid-receptor complex tightly bound to chromatin. We propose a model suggesting that an energy-requiring process is required to generate free receptor from the chromatin complex to complete the normal cellular recycling system.

¹⁴C-glucose uptake by mouse fetuses was reduced by doses of dexamethasone, 200 μg or more, which over 2 days caused fetal death. Uptake by strain C57B1/6J > A/J > SWV. Corticosterone or cortisol, 4 mg, caused neither reduced uptake nor fetal death. These differences occurred despite similar maternal hyperglycemia in all cases. Recovery of ³H-steroid after 15 min, total and unchanged steroid per gram fetal tissue was: corticosterone 1.7 and 0.3 %; dexamethasone 0.12 and 0.06 % of the dose. Rapid metabolism of corticosterone apparently prevents accumulation in fetal tissue sufficient to evoke a response under these conditions.

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