In this immunoradiometric assay (IRMA) of parathyrin (PTH) a polyclonal anti-amino-PTH(1-34) is the capture antibody and a radiolabeled monoclonal anti-hPTH(44-68) is the second antibody. Gel filtration of serum from a hyperparathyroid patient yielded only a single peak of PTH, corresponding to the elution position of synthetic PTH(1-84). Healthy elderly individuals (ages 78 +/- 5 y, mean +/- SD, n = 45) had PTH concentrations (21 +/- 13 ng/L) not significantly higher than those from healthy younger (38 +/- 11 y) adults (20 +/- 8 ng/L, n = 94). Assay results agreed well with those obtained with a carboxyl-terminal PTH assay both in normal subjects (r = 0.63, P less than 0.001) and in patients with primary hyperparathyroidism (r = 0.59, P less than 0.001). Both assays equally discriminated patients with surgically confirmed primary hyperparathyroidism from normal individuals, but the PTH(1-84) IRMA also allowed a nearly absolute discrimination between normal subjects and patients with primary hypoparathyroidism (undetectable serum PTH in 18 of 21 cases) and secondary hypoparathyroidism (caused by hypercalcemia that was caused by a malignant tumor, PTH 1.3 +/- 1.3 ng/L, n = 32). Moreover, the PTH(1-84) IRMA is more sensitive (detection limit in serum, 0.8 ng/L) and easier and quicker to perform than the carboxyl-terminal assay.

The thermodynamics of the interaction of glucocorticoids with their receptor were studied in cytosol from human lymphoblastoid cells. The rate and affinity constants of dexamethasone and cortisol between 0 degree and 25 degrees C were calculated by curve-fitting from time-course and equilibrium kinetics. The data were consistent with a simple reversible bimolecular interaction. Arrhenius and Van't Hoff plots were curvilinear for both steroids. At equilibrium, the solution for the equation delta G = delta H - T X delta S (eqn. 1) was (in kJ X mol-1) -47 = 36 - 83 (dexamethasone) and -42 = -9 - 33 (cortisol) at 0 degree C. Enthalpy and entropy changes decreased quasi-linearly with temperature such that, at 25 degrees C, the respective values were -50 = -75 + 25 and -43 = -48 + 5. Thus, for both steroids, the interaction was entropy-driven at low temperature and became entirely enthalpy-driven at 20 degrees C. Thermodynamic values for the transition state were calculated from the rate constants. For the forward reaction, eqn. (1) gave 45 = 84 - 39 (dexamethasone) and 46 = 60 - 14 (cortisol) at 0 degree C, and 44 = 24 + 20 (dexamethasone) and 46 = 28 + 18 (cortisol) at 25 degrees C. These data fit quite well with a two-step model [Ross & Subramanian (1981) Biochemistry 20, 3096-3102] proposed for ligand-protein interactions, which involves a partial immobilization of the reacting species governed by hydrophobic forces, followed by stabilization of the complex by short-range interactions. On the basis of this model, an analysis of the transition-state thermodynamics led to the conclusion that no more than half of the steroid molecular area is engaged in the binding process.

The intracellular receptor for thyroid hormone is a protein found in chromatin. Since thyroid hormone stimulates transcription of the growth hormone gene through an unknown mechanism, the hypothesis that the thyroid hormone-receptor complex interacts with defined regions of this gene has been investigated in a cell-free system. Nuclear extracts from human lymphoblastoid IM-9 cells containing thyroid hormone receptors were incubated with L-3,5,3'-tri[('I]iodothyronine and calf thymus DNA-cellulose. Restriction fragments of the human growth hormone gene were added to determine their ability to inhibit labeled receptor binding to DNA-cellulose. These fragments encompassed nucleotide sequences from about three kilobase pairs upstream to about four kilobase pairs downstream from the transcription initiation site. The thyroid hormone-receptor complex bound preferentially to the 5'-flanking sequences of the growth hormone gene in a region between nucleotide coordinates -290 and -129. The receptor also bound to an analogous promoter region in the human placental lactogen gene, which has 92% nucleotide sequence homology with the growth hormone gene. These binding regions appear to be distinct from those that are recognized by the receptor for glucocorticoids, which stimulate growth hormone gene expression synergistically with thyroid hormone. The presence of thyroid hormone was required for binding of its receptor to the growth hormone gene promoter, suggesting that thyroid hormone renders the receptor capable of recognizing specific gene regions. L-3,5,3'-Triiodothyronine (T3), the active form of thyroid hormone, modulates the expression of a number of genes (1, 2), and this is ascribed to the association of T3 with receptors localized in chromatin (3). In rat pituitary tumor cells the concentration of growth hormone (GH) mRNA increases in response to physiological concentrations ofT3 (refs. 4 and 5).Although additional mechanisms have not been ruled out, this response reflects an increased rate of transcription of the GH gene (6, 7), presumably through a direct effect of the receptor (8). Moreover, T3 acts synergistically with glucocorticoid hormones in stimulating GH gene transcription (6, 9). Binding regions for the rat (10, 11) and human (12) glucocorticoid receptors have been identified in the human GH gene upstream and downstream from the transcription initiation site (cap site). Homologous binding regions have also been detected (12) in the human placental lactogen (chorionic somatomammotropin, CS) gene, which has a 92% overall nucleotide sequence homology with the human GH gene (13). In contrast, the question of specific recognition sites for thyroid hormone receptors on any DNA has remained open. We have now searched for such sites in the human GH and CS genes (12) using the human thyroid hormone receptor in a cell-free system. This system contained [1251]T3-labeled nuclear extracts from cultured human lymphoblastoid cells of the IM-9 line and cloned fragments from the genes of interest. MATERIALS AND M...

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An in vitro competition assay was used to investigate whether binding sites for the human glucocorticoid receptor occur in the human genes for growth hormone (hGH) and placental lactogen (chorionic somatomammotropin, hCS). These genes display 95% sequence homology. Two receptor-binding regions were found in the hGH gene, one of which is located within 290 bp upstream, and one within 251 bp downstream from the transcription initiation site. Two binding regions homologous to those in the hGH gene were found in the hCS gene. The receptor-binding DNA fragment from the structural part of the genes, but not that from their promoter area, contained a sequence homologous to a 15-bp consensus sequence proposed earlier for the glucocorticoid receptor binding site. It is unlikely that the putative difference in glucocorticoid sensitivity between the hGH and hCS genes is accounted for by major differences in glucocorticoid receptor binding pattern.