Biomedical subjects
Publications and source records attributed to J Mester.
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When Rat uterus was incubated at 37 degrees with estradiol-7 alpha-butyric acid (OII-7 alpha-bu), no interference was observed with the intracellular estradiol receptors. In addition, OII-7 alpha-bu did not display estrogenic effect such as in vivo inhibition of LH secretion in Rat and in vitro increased activity of the enzyme ornithine-decarboxylase in the chick oviduct. Contrary to these negative findings, we have observed preoptic and septal cells in the guinea pig where micro-iontophoresis of OII-7 alpha-bu triggers changes of the electric activity within a second. We submit therefore, that this latter response is due to an interaction between the acid estrogen and the neuron membrane.
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An alternative to affinity chromatography purification of proteins based on the use of a water-soluble biospecific polymer has been applied to the estrogen receptor from calf uterus. The receptor (4S-trypsin form) was bound to a dextran-estradiol conjugate (molecular weight approximately 500,000) and the complex was isolated by gel filtration. Highly purified receptor was subsequently released from the dextran-estradiol conjugate by exchange with [3H]estradiol.
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1. Exchange techniques were developed for measurement of total progesterone receptor binding sites concentration in the cytosol and nuclei of chicken oviduct. 2. The level of progesterone receptor in the cytosol was under both oestrogen and progesterone control. Primary stimulation by oestradiol benzoate increased the receptor concentration from approximately 10 000 sites/cell to approximately 40 000 sites/cell in the magnum cytosol; subsequent withdrawal from oestrogen treatment led to a decrease to approximately 14 000 sites/cell after 6 weeks. After progesterone administration (3 mg/kg) to oestrogen-stimulated, withdrawn chicken, the receptor concentration decreased to approximately 40% of the initial level within the first 4 h; afterwards the receptor level rose again and by 40 h exceeded slightly the initial one. 3. The nuclear levels of the receptor reached a maximum at 1 h after the progesterone injection; however, the gain of the binding sites by the nuclei (approximately 900/nucleus) did not account for their loss from the cytosol. The maximum nuclear receptor level was not influenced by the dose of progesterone within range of 1-10 mg/kg. 4. The extractibility of the nuclear progesterone receptor by 0.5 M NaCl was strongly influenced by preincubation of the nuclei at 30 degrees C; irrespective of this preincubation, approximately 12% of the nuclear receptor resisted extraction with 2 M NaCl/5 M urea.
Macromolecular components with properties of oestrogen receptors have been identified in the 0.5 M KCl nuclear soluble, the nuclear insoluble and the cytosol fractions of laying hen and immature (2--4 weeks, untreated by hormone) chicken oviduct. 7n the 0.5 M KCl extract of laying hen oviduct nuclei, a receptor, of protein nature according to the effects of enzymic treatments, has been identified. It exhibits high affinity for oestradiol with an apparent equilibrium association constant KA = 4 - 109 M-1 at 4 degrees C. The binding of [3H] oestradiol is abolished by 1 muM oestriol, oestrone and diethylstilboestrol, but not by the same concentration of progesterone, testosterone, and cortisol. Sucrose gradient ultracentrifugation studies in the presence of 0.5 M KCl indicate a sedimentation coefficient of 4.3 S, and there is partial aggregation in low-ionic-strength medium. The estimated number of binding sites per nucleus is about 5000, as calculated from DNA content of chick diploid genome. Most of the binding sites were found to be occupied by endogenous oestrogen(s). Oestradiol dissociates from the receptor according to an apparent two-step mechanism. The half-life time for the faster dissociation step is 18 h at 0 degrees C, 25 min at 20 degrees C and 10 min at 30 degrees C, and for the slower one is 180 h, 115 min and 60 min, respectively. In the 0.5 M KCl extract of immature chicken oviduct nuclei, there are approximately 500 receptor sites per nucleus; their affinity for oestradiol is the same as in the case of laying hen soluble nuclear receptor. After repeated extractions of nuclei with 0.5 M KCl medium, a substantial quantity of oestrogen binding sites remains in the residual fraction. Binding characteristics of this insoluble nuclear receptor resemble those of the soluble nuclear receptor: high affinity for oestradiol (KA = 7 - 10(8) M-1 at 37 degrees C) and specificity for oestrogens. The estimated number of binding sites are approximately 2000/cell for laying hen, and approximately 1000/cell for immature chicken. In the high-speed supernatant fraction of laying hen oviduct homogenates, an oestrogen receptor is also present, but its concentration is low (less than or equal to 100 sites/cell) and at the limits of sensitivity of the methods used. In the cytosol of immature chicken oviduct, there are approximately 2500 oestradiol receptor sites per cell.
1. The nuclear-myofibrilar (800g pellet) fraction of the uterus from immature (22-23 days old) rats not exposed to oestrogen exhibits saturable binding of oestradiol. The nuclear binding capacity represents approximately 10% of that of the cytosol fraction (approx. 3.5 fmol/mug of DNA). The predominant part (0.3.5 fmol/mug of DNA) of the nuclear binind sites are present in the residual pellet after extraction with 0.5 M-KC1. 2. By using an exchange technique in vitro, determinations of the nuclear binding sites have been carried out after administration of 1 mug of oestradiol in vivo. Within 0.5h after the hormone injection, the concentration of nuclear bindng sites increased to approx. 0.4 fmol/mug of DNA in the 0.5 M-KC1-extractable fraction, and to approx. 1.2 fmol/mug of DNA in the residual fraction. Meanwhile the cytosol oestrogen-receptor concentration decreased to approx. 10% of its initial value. In the following period from 0.5 h after the oestradiol injection onwards, the concentration of nuclear oestrogen receptors decreased with halflife values of approx. 140 and 200 min for the KC1(0.5 M)-extractable and residual form respectively. At the same time, the cytosol receptor concentration increased to reach approx. 50% of the initial value by the 6h. This increase could not be blocked by cycloheximide. The initial concentration of cytosol receptor was restored approx. 11h after the injection and the increase during the 6-11h period was sensitive to cycloheximide inhibition, suggesting protein-synthesis-dependence of the process. 3. With the (more) physiological dose of oestradiol (0.1 mug), the decrease the cytosol receptor was only 50% by 4h and this was followed by a period (up to 12h after injection) during which the initial concentration was restored. During this period the increase of the receptor can be blocked by cycloheximide.
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