The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.

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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.

Plasma catecholamines, as determined by a new improved method, are increased in hypothyroidism and decreased in hyperthyroidism. In addition, plasma catecholamine values are inversely correlated with total thyroxine values in hyperthyroidism. (/ Clin Endocrinol Metab 36: 587, 1973) T HERE IS disagreement in the literature concerning the effect of abnormal thyroid function on levels of the catecholamines and their metabolites in the blood and urine (1-5). Some unconfirmed studies have suggested the blood and urine levels might be increased in hyperthyroidism and decreased in hypothyroidism (3,6). In contrast, Kuschke et al. (5) reported that urinary norepinephrine was low in 8 of 20 cases of hyperthyroidism and high in 3 cases of hypothyroidism. Wiswell et al. (7) reported slightly increased urinary norepinephrine in 20 hypothyroid patients. Levine et al. (8) reported that urinary norepinephrine and vanillylmandelic acid (VMA) appeared to be low in some cases of hyperthyroidism. Current popular thinking, a result of conflicting reports, is that abnormal thyroid function exerts little effect on the catecholamines and their metabolites in the blood and urine (1,7,(9)(10)(11).We recently began using a simple, reliable plasma catecholamine assay based on Renzini's two-column method for chromatographic purification. This assay is more sensitive and more specific than previous assays (12,23). On reviewing our initial clinical experience with this assay, we noted that several patients with hypothyroidism had increased plasma catecholamine values. Therefore, a prospective study was designed to evaluate plasma catecholamines in hypothyroidism and hyperthyroidism. The results indicate that plasma catecholamines are increased in hypothyroidism and decreased in hyperthyroidism. In addition, plasma catecholamine values are inversely correlated with total thyroxine values in hyperthyroidism. Materials and MethodsPatients with clinical hypothyroidism or hyperthyroidism and abnormal total thyroxine levels were studied. Mean ( ± SD) total thyroxine levels were 1.1 ± 0.7 jxg/100 ml (range, 0.1-2.4) in hypothyroid patients and 15.7 ±: 2.3 ng/ml (range, 12.0-19.0) in hyperthyroid patients; in our laboratory, the normal range for total thyroxine is 4-11 |xg/100 ml. Normal plasma catecholamine values were obtained from healthy normotensive (diastolic pressure < 90 mm Hg) subjects as determined by history and physical examination. The total thyroxine assay has been described (13). The plasma catecholamine assay was performed according to the method of Renzini et al. (12) with slight modification. The details of the method will be published elsewhere (23). Briefly the method involves the purification of catecholamines by alumnia and Amberlite CG-50 (Na+ form) columns. The boric acid eluate from the Amberlite CG-50 column was used for fluorimetry. Epinephrine and norepinephrine were measured collectively. Norepinephrine was used as the standard. The molar extinction coefficient of epinephrine and norepinephrine are almost identica...

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