Mikael Gillner

High-affinity binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin in cell nuclei from rat liver

The intranuclear binding of radioactive 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in rat liver has been studied both in vivo and in vitro. Following the intravenous administration of [1,6-3H]TCDD, a maximum uptake by cell nuclei could be observed at 2 h after injection with a concurrent decrease in the cytosolic uptake. Using linear sucrose density gradient centrifugation, dextran-coated charcoal adsorption assay, DEAE-Sepharose ion-exchange chromatography, competition, enzymatic and saturation studies, a high-affinity binding protein for TCDD in liver cell nuclei could be demonstrated both in vivo and after an exchange in vitro of intravenously administered unlabelled 2,3,7,8- tetrachlorodibenzofuran (TCDBF) for [3H]TCDD. Sucrose density gradient analysis showed a size of 4-5 S for both the cytosolic and nuclear TCDD binding entity. The specific binding of [3H]TCDD to nuclear components was heat labile and saturable and had an equilibrium dissociation constant of 1.05 nM. Based on a differential susceptibility to specific hydrolases, i.e. DNAase, RNAase, trypsin and pronase, the binding entity appears to be a 4-5 S salt-extractable protein.

The dioxin receptor: a comparison with the glucocorticoid receptor.

The physico-chemical properties of the dioxin and glucocorticoid receptors from rat liver and wild-type and mutant cell lines were investigated and compared. In rat liver, the receptors are virtually indistinguishable. Both are highly asymmetrical proteins with axial ratios of 12-15, have Stokes radii of 6 nm and sedimentation coefficients of approximately 4 S. This results in a calculated apparent mol. wt of approximately 100,000. The dioxin receptor from the mouse hepatoma cell line Hepa 1c1c7 represents an atypical form of the dioxin receptor with a pronounced tendency to aggregate to form Mr approximately equal to 300,000 complexes in high ionic strength and in the absence of sodium molybdate. In the presence of sulphydryl reducing agents, however, the Hepa 1c1c7 dioxin receptor dissociates to an Mr approximately 100,000 species. In analogy to the nt- mutant glucocorticoid receptor in mouse lymphoma cells, there is no gross change in the structure of the nt- dioxin mutant in mouse hepatoma cells compared with the wild-type receptor. The nt- dioxin receptor does, however, have a reduced affinity for DNA.

The TCDD receptor in rat intestinal mucosa and its possible dietary ligands.

Induction of aryl hydrocarbon hydroxylase (AHH) by polycyclic aromatic hydrocarbons and other inducers such as 2,3,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to occur following binding of the inducer to a soluble receptor protein similar to steroid hormone receptors. This receptor is usually called the TCDD receptor, since TCDD has the highest affinity of all known ligands for the receptor. In the present paper a receptor for TCDD in cytosol from rat intestinal mucosa has been studied, using isoelectric focusing in polyacrylamide gel. This receptors biochemical properties were found to be similar to those of the TCDD-receptor in rat liver cytosol. The dissociation constant (Kd) of the 3H-TCDD-receptor complex in rat intestinal mucosa was 0.7-3.1 nM, and it was present at a concentration of 70-80 fmol/mg protein. Starvation did not significantly increase the receptor level. The affinities of some potential dietary ligands for the TCDD receptor in rat intestinal mucosa were also studied. Indole-3-carbinol had 1/2,600 of the affinity of TCDD for the receptor protein. Butylated hydroxyanisole (BHA), transstilbene oxide and quercetinpentamethylether competed even more weakly with 3H-TCDD for binding to the receptor. The biological significance of the occurrence of low-affinity ligands of dietary origin for the TCDD receptor is uncertain at the present time.

A new structure-affinity relationship for TCDD receptor binding

We have investigated the capacity of various indoles to inhibit specific binding of [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gels. Of these indoles, indolo[3,2-b]carbazole was the most active. The IC50 value for TCDD receptor binding of indolo[3,2-b]carbazole as well as for 2,3,7,8-tetrachlorodibenzofuran was 3.6 nM. We have also studied the influence on binding exerted by introduction of some substituents on indolo[3,2-b]carbazole. Substitution with methyl groups at the 5 and 11 positions resulted in an increased affinity (IC50 1.2 nM) for the TCDD receptor as compared to the parent compound. Computer-supported molecular structure studies indicated that if the van der Waals radii of atoms are included, a rectangle of 6.8 × 13.7 A may account for the binding of high-affinity ligands to the TCDD receptor.

Molecular basis for human immunodeficiencies.

Major emphasis is currently being placed on unraveling the molecular basis of various forms of primary human immunodeficiencies. It is clear from recent studies that not only can different mutations give rise to different phenotypes but the same mutation may result in quite diverse clinical pictures. A correct diagnosis at the DNA level therefore becomes increasingly important in view of the possibility of gene therapy.

The dioxin receptor: Characterization of its DNA-binding properties☆

The binding of the rat hepatic dioxin and glucocorticoid receptors to the polyanionic matrices heparin-Sepharose and DNA-cellulose in vitro and to cell nuclei in vivo was studied under various conditions. In a non-liganded and non-activated state both receptors eluted from heparin-Sepharose at a low ionic strength and were not retained on DNA-cellulose. Following ligandation and activation in vitro both receptors showed an increased affinity for heparin-Sepharose and were retained on DNA-cellulose. In analogy to these in vitro data, it was found that a high salt concentration (0.4 M KCl) was required to extract in vivo liganded dioxin receptor from purified nuclear preparations in contrast to that previously reported for non-liganded nuclear receptors. Limited proteolysis of both dioxin and glucocorticoid receptors resulted in molecular species of similar binding properties with regard to DNA-cellulose and heparin-Sepharose. We conclude that, in addition to the dioxin and glucocorticoid receptors showing considerable similarities in their physicochemical properties, they may also share a similar structural organization with regard to functional domains.

Autoradiography of 2,3,7,8-tetrachloro [14C]-dibenzo-p-dioxin (TCDD): Accumulation in the nasal mucosa

Abstract Autoradiography of 2,3,7,8-tetrachloro [ 14 C] dibenzo-p-dioxin in three strains of mice showed a marked uptake and long-time retention of radioactivity in the nasal mucosa and the liver. Similar results were obtained in late gestational fetuses, although the placental passage was limited.

Detection of specific binding of [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) in human leukocytes using electrofocusing in polyacrylamide gel

The binding of [1,6-3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) in human leukocyte cytosol has been studied using electrofocusing in polyacrylamide gel. One single peak of bound [3H]TCDD is found after completed focusing with an isoelectric point of 6.0. The binding capacity for 1.5 nM [3H]TCDD in the leukocyte cytosol was completely saturated by incubation in the presence of 150 nM unlabelled 2,3,7,8-tetrachlorodibenzofuran. [3H]TCDD also binds to a single binding species in human serum and plasma which focuses at pH 5.2 and which is not saturated by the addition of a 100-fold excess of unlabelled 2,3,7,8-tetrachlorodibenzofuran to the incubation. The levels of specific binding of [3H]TCDD in leukocytes from healthy blood donors ranged from 0 to 50 fmol/mg cytosolic protein.

Polyacrylamide concentration gradient gel electrophoresis of the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver cytosol

An assay based on polyacrylamide concentration gradient gel electrophoresis for the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) receptor in rat liver cytosol was developed. [3H]TCDD-labeled cytosol was treated with dextran-coated charcoal and electrophoresed for 3000 Vh in polyacrylamide concentration gradient gels (T = 2.5-20%). A Tris-borate running buffer (pH 8.35) without salt and detergents was used. When the polyacrylamide concentration gradient was concave a linear calibration curve of log molecular weight vs. migration distance was obtained for the standard proteins used. Under these conditions the Mr value of the TCDD receptor from rat liver cytosol was estimated to be 316,000 +/- 27,000 before, and 246,000 +/- 12,000 after limited proteolysis. The Bmax for the specific binding of [3H]TCDD was between 35 and 43 fmol/mg cytosolic protein and the Kd of the binding ranged from 0.8 to 1.1 nM, as judged by saturation and Scatchard analysis.

THE DETECTION AND FUNCTION OF THE CYTOSOLIC RECEPTOR FOR 2,3,7,8-TETRACHLORODIBENZO-p-DIOXIN (TCDD) AND RELATED COCARCINOGENS

ABSTRACT A specific cytosolic receptor protein with high affinity for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related cocarcinogens can be demonstrated in rat liver cytosol by isoelectric focusing in polyacrylamide gel. This method can be used for the detection and quantitation of the receptor. The method can also be used for the detection of biologically active environmental pollutants in the particulate fraction of air samples by measuring the competition for binding of TCDD to the receptor. Using isoelectric focusing, it was possible to demonstrate the occurrence of a TCDD-binding protein in human lymphocyte cytosol. This protein has very similar characteristics to the TCDD receptor in rat liver cytosol. After the binding of TCDD to the receptor, the complex can bind to DNA. The receptor cannot bind to DNA in the absence of ligand (TCDD). Following the injection of radioactive TCDD, there is a peak in the uptake of radioactivity in rat liver cytosol after 30 min. After 2 hours there is a peak in the uptake of radioactivity in the nuclear fraction. Thus, the TCDD receptor shows many characteristics similar to the steroid hormone receptors.