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N L Eberhardt

Publications and source records attributed to N L Eberhardt.

At least 73 records · Page 4Linked to original sources
PubMed1984

Interactions of the nuclear thyroid hormone receptor with core histones.

These studies concern the interactions of the rat liver thyroid hormone nuclear receptor with histones and factors influencing the receptor's assay and stability. Heating certain crude receptor preparations at 50 degrees C produces a selective loss of triiodothyronine (T3) but not thyroxine (T4) binding activity, whereas, with more purified preparations, such heating decreases both T3 and T4 binding. The selective T3-binding loss in crude preparations was found to be due to the simultaneous denaturation of the receptor's high-affinity hormone-binding activity for both T3 and T4 and generation of new low-affinity T4-binding sites. The fraction in which T4 binding can be activated could be separated from the receptors by Sephadex G-100 chromatography. Core histones stimulated both T3- and T4-binding activity of 6-fold-purified receptor preparations, and data from several different experimental approaches suggest that this stimulation is due to the capability of the core histones to prevent the receptor from binding to or being denatured by Sephadex G-25 assay columns. The core histones were also found to stabilize 500-fold-purified but not 6-fold-purified or crude receptor preparations. A number of other acidic or basic proteins had little or none of these stimulatory effects, whereas a few proteins (such as the insulin B chain and histone H1) did have activity, although it was less than that of the core histones. There were no significant differences between the purified core histone subfractions (H2A, H2B, H3, and H4). That core histones can interact with the thyroid hormone receptors was demonstrated more directly by the finding that the receptors bind to histone-Sepharose but not Sepharose or insulin- or ovalbumin-Sepharose columns and that this binding was blocked by core histones at concentrations suggestive of an affinity for the receptor-core histone interaction of around 3 microM at 0.15 M salt concentration. The results demonstrate the utility of the histones in the assay and stabilization of purified thyroid hormone receptors, but they fail to support our previous hypothesis of a receptor subunit where T3- but not T4-binding activity is regulated selectively by histones. However, the results indicate that histones may interact with the receptors with some degree of specificity, and they raise the possibility that the histones participate in the nuclear localization of the receptors.

Animals↗
PubMed1984

Mechanisms of glucocorticoid hormone action.

This work summarizes some of our studies of the mechanisms of glucocorticoid action, including aspects of steroid binding to receptors, the activation of glucocorticoid-receptor complexes and the regulation of expression of endogenous and transferred glucocorticoid-responsive genes. Studies of the receptor-steroid interaction support the notion that steroid entry is passive. A comparative analysis of binding in isolated cytosol and intact cells suggests that the initial receptor-steroid binding reaction and not subsequent steps such as activation and nuclear binding, is predominantly responsible for the high-affinity state that is generated. The binding is driven by entropy and enthalpy changes at low temperature; at higher temperatures it is driven by entropy changes, with enthalpy working against it. Studies of the activation of the receptor-glucocorticoid complex with the use of highly purified receptors suggest that this step is associated with a change in charge of the receptor-glucocorticoid complex (such as would occur with a dephosphorylation reaction), whereas the data do not support the notion that dissociation of a bound RNA or of receptor oligomers is responsible for generating the nuclear- and DNA-binding activity of the complex. Studies of the regulation by glucocorticoids of expression of the endogenous rat growth hormone (rGH) gene in cultured rat pituitary tumor (GC, GH3D6) cells suggest that glucocorticoids increase the expression of this gene by multiple mechanisms. First, there is a modest direct stimulation of transcription by a mechanism(s) that does not depend on protein synthesis; however, if the cells have been exposed to thyroid hormone for several hours, the steroid exerts a much greater increase in rGH pre-mRNA levels. Secondly, the steroid appears to stimulate some relatively stable function or functions that increase the ability of thyroid hormone to increase rGH levels. Thirdly, the steroid probably increases rGH mRNA stability, since the fold-increases in rGH mRNA exceed those of transcription. Finally, the steroid may, by unknown mechanisms, affect rGH mRNA polyadenylation. The gene transfer experiments utilized the rat and human (h) GH genes and hybrid genes containing either rGH and Herpes Simplex virus thymidine kinase (TK) gene sequences or the human metallothionein-IIA (hMT-IIA) and TK gene sequences. The steroid was found to regulate hMT-IIA gene expression in all glucocorticoid-responsive cell types tested by actions on its 5'-flanking DNA. By contrast, the glucocorticoid regulated GH gene expression in some but not all glucocorticoid-responsive cell types.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1984

Expression and hormonal regulation of the rat growth hormone gene in transfected mouse L cells.

Expression of the rat growth hormone (rGH) gene in the pituitary and in cultured pituitary tumor cells is regulated by glucocorticoid hormones. After co-transfer of cloned DNA containing the rGH gene with the herpes simplex virus (HSV) thymidine kinase (tk) gene into mouse Ltk- cells, rGH gene transcripts were detected in eight of fifteen tk+ cell lines. However, in all eight clones, the predominant rGH gene transcript was only about 0.75 kb, 0.3 kb shorter than pituitary rGH mRNA. The 0.75-kb transcripts, examined from one clone, L-rGH-4, lacked sequences derived from exons 1 and 2 of the rGH gene. Although transcripts larger than 0.75 kb were detected, the normal 2.2-kb rGH gene primary transcript was present only at very low levels. Nuclease mapping studies also failed to reveal transcripts initiated at the normal rGH gene promoter, but instead revealed transcripts with 5' termini arising within intron B of the gene. These data suggest either that transcripts arise from internal promoters within the rGH gene or that a transcript initiated upstream from the normal promoter was processed abnormally. Dexamethasone increased the levels of the 0.75-kb rGH gene transcripts about fourfold in all eight clones expressing rGH mRNA. These data suggest that structural elements important for glucocorticoid-mediated influences on regulation of GH gene expression are contained within the transferred rGH gene fragment and can function even when the normal rGH gene promoter is not used and the pattern of expression is grossly abnormal.

Animals↗
PubMed1984

Interaction of small nuclear ribonucleoproteins with simian virus 40 in CV-1 cells: is U2 snRNA involved in regulating replication?

The small nuclear RNAs (snRNAs) in African Green Monkey kidney cells (CV-1 cells) were examined by polyacrylamide gel electrophoresis. Methodology was developed to improve their extraction from enriched fractions. Cellular fractionation studies and subsequent analysis of these RNAs indicate that they are tightly associated with chromatin. Treatment of cells with alpha-amanitin totally suppressed transcription of U1, U2, U4, U5, and partially suppressed transcription of U6, suggesting that these snRNAs are transcribed by RNA polymerase II. Upon infection of the cells by simian virus 40 (SV40), overall transcription of these and other cellular RNAs was stimulated. Gel filtration and formaldehyde crosslinking studies indicated that the ribonucleoproteins (snRNPs) containing snRNAs are associated with the viral minichromosome. Nucleotide sequence comparisons show extensive sequence complementarity between the 5' end of U2 RNA, the replication origin of SV40, and a prokaryotic RNA (RNA I) that is involved in control of plasmid replication. The clustered homologies between these RNAs and the association of snRNAs with the SV40 chromosome suggest that snRNAs may be evolutionarily related to small RNAs from plasmids and are consistent with an hypothesis that U2 RNA may be involved in DNA replication.

Antigens, Viral, Tumor↗
PubMed1982

Photoaffinity labeling of human thyroxine-binding prealbumin with thyroxine and N-(ethyl-2-diazomalonyl)thyroxine.

To facilitate studies of thyroid hormone-binding proteins, we have synthesized and tested the photoaffinity analogues N-(ethyl-2-diazomalonyl)-3,5,3'-triiodothyronine (EDM-T3) and N-(ethyl-2-diazomalonyl)thyroxine (EDM-T4). The binding affinities of L-EDM-T4 and D-EDM-T4 to human thyroxine-binding prealbumin were 4% and 13.2%, respectively, that of L-thyroxine (L-T4). For comparison the affinities of L-EDM-T3 and D-EDM-T3 to crude rate liver nuclear receptor preparation were 0.1% and 0.7%, respectively, that of L-triiodothyronine (L-T3). Photolysis of prealbumin-[125I]-L-EDM-T4 complexes at 254 nm resulted in covalent linkage of [125I]-L-EDM-T4 to prealbumin as judged by sodium dodecyl sulfate gel electrophoresis. Virtually no labeling was observed in the absence of photolysis. Photolabeling of prealbumin was specific for the high-affinity hormone binding site since it was (a) completely blocked during photolysis in the presence of excess 3,5,3',5'-tetraiodothyroacetic acid, (b) saturated at high [125I]-L-EDM-T4 concentrations, (c) prevented when the hormone binding site had been previously blocked by dansylation of prealbumin, and (d) blocked competitively by T3 and T4 with inhibition constants (K1) similar to the dissociation constants (Kd) for these ligands. Analysis of prealbumin photolabeling by direct attachment of [125I]-L-EDM-T4 or attachment of unlabeled L-EDM-T4 followed by titration of the remaining sites with [125I]-L-T4 indicated a photolabeling efficiency of 54-61% at 63-67% site occupancy. After destruction of the diazo group by preirradiation, L-EDM-T4 was found to label prealbumin after further irradiation; 19-26% photolabeling efficiency could be achieved by using preirradiated reagent at 87-91% site occupancy. This carbene-independent photoattachment was also specific for a high-affinity hormone binding site. The mechanism of the carbene-independent process may involve attachment via radical formation following photoinduced loss of the thyronine ring iodine. Accordingly, specific covalent cross-linking of L-T4 to prealbumin was demonstrated; however, the photolabeling efficiency was much lower than that with preirradiated EDM-T4. Both EDM-T4 and T4 were employed to photolabel prealbumin, thyroxine binding globulin, and albumin in unfractionated human serum.

Affinity Labels↗
PubMed1982

Photoaffinity labelling of the rat liver nuclear thyroid hormone receptor with [125I]triiodothyronine.

[125I]Triiodothyronine (T3) was used as a photoreactive probe for the thyroid hormone nuclear receptor in photoaffinity labelling experiments. Autoradiograms of photolysis products electrophoresed on either one or two-dimensional gels showed that [125I]T3 covalently, but nonspecifically, labelled many proteins in the partially purified receptor preparations used. However, one of these proteins with an estimated molecular weight of 47,000 and an isoelectric point of approximately 6.2 +/- 0.5 pH units appears to be the thyroid hormone receptor, since, in contrast to the other proteins, its photoinduced labelling was blocked by concentrations of T3 and thyroxine (T4) similar to those that inhibit binding of [125I]T3 by the receptor in equilibrium binding assays. In addition, the isoelectric point of the photolabelled protein agrees with that determined in separate equilibrium isoelectric focusing studies. These results indicate that [125I]T3 can serve as a photoreactive probe for the thyroid hormone nuclear receptor, and they suggest that this receptor is a single polypeptide chain of molecular weight 47,000 with an isoelectric point of 6.2 +/- 0.5 pH units.

Affinity Labels↗
PubMed1981

Development of support matrices for affinity chromatography of thyroid hormone receptors.

Certain cellular responses to thyroid hormones appear to be mediated by non-histone chromatin protein receptors. Purification of these proteins is important for an investigation of the detailed mechanisms of their regulatory role. In the present studies, we report the development of an affinity chromatographic system that can be used to purify thyroid hormone receptors solubilized from nuclei. Amine-substituted hormone analogs were prepared with D and L isomers of T3; these bind to the receptor. This finding supports the hypothesis that thyroid hormones fit into the receptor with the amino groups accessible from outside the binding site. Although L-triiodothyronine (LT3) (the naturally occurring isomer) binds more tightly (relative Kd = 1.0 nM) to the nuclear receptor than D-triiodothyronine (DT3) (relative Kd = 2.0 nM), the amine-substituted analog of DT3 binds more tightly than the LT3 analog (DT3 analog, relative Kd = 40 nM; LT3 analog, relative Kd = 1500 nM). Agarose-based gels containing DT3 and LT3 covalently coupled by their amino groups were also prepared. Binding of receptor to these gels was biospecific in that it could be inhibited by prior incubation of the receptors with LT3. In addition, as predicted by the analog studies, the DT3 affinity gels were more effective than LT3 gels in adsorbing receptor. Elution of receptor from the LT3-derived gels was achieved in a predicted volume and concentration of counter-ligand in elution buffer. These results suggest that affinity chromatography can be applied to the purification of thyroid hormone receptors.

Animals↗
PubMed1979

Regulation of activity of chromatin receptors for thyroid hormone: possible involvement of histone-like proteins.

Thyroid hormone receptors lose their capability for high-affinity binding of the biologically active triiodothyronine after solubilization and separation from other chromatin proteins. The high-affinity triiodothyronine-binding capacity can be reconstituted by addition of a histone-containing extract of chromatin of purified core histones (H2A, H2B, H3, and H4); a number of other acidic or basic proteins tested were ineffective. The data support a model of the receptor in which a "core" receptor subunit that contains a thyroid hormone-binding site interacts with a regulatory subunit, which is possibly a histone or histone-like species. This interaction with the "core" subunit enables the resulting "holo" receptor to bind biologically active hormones. These data also suggest that histones or related proteins can modulate the activity of nonhistone chromosomal proteins that are involved in regulating the expression of specific genes.

Animals↗
PubMed1975

Malate dehydrogenase, circular dichroism difference spectra of porcine heart mitochondrial and supernatant enzymes, binary enzyme-coenzyme, and ternary enzyme-coenzyme-substrate analog complexes.

Circular dichroism spectra and circular dichroism difference spectra, generated when porcine heart mitochondrial and supernatant malate dehydrogenase bind coenzymes or when enzyme dihydroincotinamide nucleotide binary complexes bind substrate analogs, are presented. No significant changes are observed in protein chromophores in the 200- to 240-nm spectral range indicating that there is apparently little or no perturbation of the alpha helix or peptide backbone when binary or ternary complexes are formed. Quite different spectral perturbances occur in the two enzymes with reduced coenzyme binding as well as with substrate-analog binding by enzyme-reduced coenzyme binding. Comparison of spectral perturbations in both enzymes with oxidized or reduced coenzyme binding suggests that the dihydronicotinamide moiety of the coenzyme interacts with or perturbs indirectly the environment of aromatic amino acid residues. Reduced coenzyme binding apparently perturbs tyrosine residues in both mitochondrial malate dehydrogenase and lactic dehydrogenase. Reduced coenzyme binding perturbs tyrosine and tryptophan residues in supernatant malate dehydrogenase. The number of reduced coenzyme binding sites was determined to be two per 70,000 daltons in the mitochondrial enzyme, and the reduced coenzyme dissociation constants, determined through the change in ellipticity at 260 nm, with dihydronicotinamide adenine dinucleotide binding, were found to be good agreement with published values (Holbrook, J. J., and Wolfe, R. G. (1972) Biochemistry 11, 2499-2502) obtained through fluorescence-binding studies and indicate no apparent extra coenzyme binding sites. When D-malate forms a ternary complex with malate dehydrogenase-reduced coenzyme complexes, perturbation of both adenine and dihydronicotinamide chromophores is evident. L-Malate binding, however, apparently produces only a perturbation of the adenine chromophore in such complexes. Since the coenzyme has been found to bind in an open conformation on the surface of the enzyme and the substrate analogs bind at or very near the dihydronicotinamide moiety binding site, protein conformational changes are implicated during ternary complex formation with D-malate which can effect the adenine chromophore at some distance from the substrate binding site.

Animals↗