Thyroid hormone binding in rat liver cytosol

Binding of 3,5,3'-tri-iodothyronine (T3) and thyroxine (T4) to components of perfused rat liver supernatant fraction and isolated liver cell cytosol was studied. Of the four binding fractions in supernatant (X, A, Y and Z) separable by gel chromatography, both T3 and T4 bound preferentially to the A-fraction, which was shown to contain albumin as the major binding protein. When cytosol prepared from isolated cells was examined, T4 was again bound mainly in the A-fraction; however, T3 was observed to bind predominantly in the Y-region. Hormone binding to soluble protein in the latter system is thought to reflect the pattern in vivo, better than does binding in supernatant, although the possibility exists that the concentration of albumin observed in cytosol may be artificially high due to transfer of membrane-bound albumin during cell disruption. Nevertheless, albumin (possibly derived from more than one intracellular source) is capable of binding T4 in vivo. The presence of this protein within the hepatocyte may thus contribute to the high T4 binding capacity of the liver compared to other tissues.     

Binding of cytosol receptor-glucocorticoid complexes by isolated nuclei of glucocorticoid-responsive and nonresponsive cultured cells.

Nuclear binding of the AtT-20 cytosol receptor-glucocorticoid complex was studied in a cell-free system using nuclei from steroid-responsive (AtT-20) and nonresponsive (EPO-G1) cell lines, both of which synthesize ACTH. The AtT-20 cell line was derived from a mouse pituitary adenocarcinoma, while the EPO cell line was established from a human malignant melanoma. The nonresponsive EPO cells lacked a cytosol receptor for glucocorticoids, and, when whole cells were incubated with labeled glucocorticoid, they were unable to concentrate the steroid in their nuclei. A cell-free system using AtT-20 cytosol preincubated with labeled glucocorticoid was used to study binding by isolated nuclei. Binding to isolated nuclei from both cell lines was indistinguishable, in terms of temperature sensitivity, binding capacity, and saturability. Sucrose density gradient analyses of KCl extracts of nuclei labeled under these cell-free conditions showed 3.2-3.6 S peaks. In contrast, a 4.0 S peak was observed consistently when unreacted cytosol was analyzed on high-salt gradients, suggesting that interaction with nuclei from both cell lines caused the receptor to alter its sedimentation characteristics. These findings suggest either that all cells contain nuclear acceptor sites and that target cell responsiveness is conferred solely by the presence or absence of the cytosol receptor, or that binding sites detected in isolated nuclei may be different from those observed in intact cells and may, in fact, obscure them.     

Interaction of rat liver glucocorticoid receptor with histones

The activated glucocorticoid-receptor complexes (GRC) from rat liver bind tightly to histone (from calf thymus)-agarose and cannot be eluted with 3 M KCl or 50% ethylene glycol, but can be eluted with 20 mM pyridoxal 5'-phosphate (PALP). Purified activated GRC were found to have much higher affinity for histones H3 and H4 (arginine-rich histones) than for histones H2A and H2B (slightly lysine-rich histones) and to have negligible affinity for histone H1 (lysine-rich histone). The GRC bound to immobilized histones could be eluted with PALP, but not with its related compounds, such as pyridoxamine, pyridoxamine 5'-phosphate, pyridoxal, and pyridoxine, suggesting a specific effect of PALP. Not only activated GRC but also unactivated GRC were found to bind to immobilized histones. However, from their profiles of elution from a histone-agarose column by 20 mM PALP, the activated GRC seemed to have higher affinity than unactivated GRC for histone. Our results suggest that the binding of GRC to histones could be associated with a mechanism for alteration of the chromosome structure by the hormonal signals, before the binding of the GRC to a specific sequence of DNA in regulatory elements of glucocorticoid-responsible genes.     

Sequence-specific binding of androgen-receptor complexes to prostatic binding protein genes

Prostatic binding protein is a complex glycoprotein comprising three components, C1, C2 and C3, organized into two different heterodimers (C1-C3 and C2-C3). The rat ventral prostate genes encoding all three constituent polypeptides are expressed under androgenic control. Analysis of genomic fragments containing the genes and flanking sequences revealed in each case one androgen receptor-binding region upstream of or within the promoter and another in the first intron. The effect of androgens on the expression of these genes may, therefore, be mediated by these direct receptor-DNA interactions. The genomic fragments which contain androgen receptor-binding regions all contain nucleotide sequences reminiscent of glucocorticoid response elements (GRE). Mutations in these sequences in restriction fragments and in synthetic oligonucleotides significantly decreased their affinity for androgen-receptor complexes and their introduction into nonspecific sequences conferred affinity for androgen-receptor complexes. Based on these data, a consensus sequence for putative androgen response elements (ARE) is proposed. However, despite the specific recognition of these sequences by the androgen receptor in vitro, only the C3(1) intronic fragment could confer significant androgen responsiveness on a heterologous promoter. While this could be due to the fact that the GRE-like sequences present in the other fragments are not strong AREs, alternative hypotheses are being investigated currently. Not least of these is that the similar localization of the binding sites in each gene might underlie a more complex androgen regulation mechanism.     

ATP-dependent association of nuclear proteins with isolated rat liver nuclei.

In vitro association of Xenopus nucleoplasmin and mammalian nonhistone chromosomal high mobility group 1 (HMG1) protein with nuclei isolated from rat liver was examined. Efficient association of nuclear proteins with isolated nuclei requires ATP, HCO3-, and Ca2+. Association occurred at 33 degrees C but not at 4 degrees C. ATP could be replaced by adenosine 5'-[alpha,beta-methylene]triphosphate (pp[CH2]pA), a nonhydrolyzable ATP analog. pp[CH2]pA associated with nuclei at 33 degrees C and nucleoplasmin and HMG1 rapidly associated with the pp[CH2]pA-bound nuclei at 4 degrees C. Competition studies showed that these associations at both 33 degrees C and 4 degrees C were specific. More than 80% of the bindings of nuclear proteins to the nuclear surface were blocked by wheat germ agglutinin.     

Identification of 3,5-diiodo-L-thyronine-binding proteins in rat liver cytosol by photoaffinity labeling

In this study, we obtained evidence for the presence of cytosolic-binding proteins for 3,5-diiodo-L-thyronine (3,5-T(2)). UV irradiation of rat liver cytosol with [(125)I]3,5-T(2) resulted in specific covalent attachment of (125)I to three polypeptides with apparent molecular masses of 86, 66, and 38 kDa. The photoaffinity labeling of all three proteins was strongly inhibited (by about 90%) when the reaction was carried out in the presence of a 10-fold excess of unlabeled 3,5-T(2) or T(3). However, whereas inhibition by 3,5-T(2) was nicotinamide adenine dinucleotide phosphate reduced (NADPH) independent, T(3) inhibited only in the presence of NADPH. The 38-kDa protein, which showed the greatest affinity for 3,5-T(2), was partially purified by preparative fast-performance liquid chromatography. Its binding activity was optimal at pH 7.4, stable between 0 and 37 C, and already maximal after 5-10 min of incubation. The finding that a 38-kDa cytosolic-binding protein binds 3,5-T(2) in the absence of NADPH, but T(3) only in a NADPH-dependent manner, suggests that it may serve to regulate intracellular T(3)/3,5-T(2) translocation in a way that depends on the nicotinamide adenine dinucleotide phosphate/NADPH ratio.     

Glucocorticoid-binding proteins of rat heart cytosol and their possible role in the transfer of glucocorticoids into nuclei

Binding of [³H]glucocorticoids by cytosol and nuclei isolated from rat heart has been studied. Two types of glucocorticoid-binding proteins (binder I and binder II) were found in the heart cytosol of adrenalectomized rats. Binder I is similar in its properties to serum transcortin. It does not bind dexamethasone, has fast kinetics of corticosterone binding, is insensitive to SH-blockers, thermostable (not inactivated at short exposure to 50°C), and precipitates in the range of 40 to 60% saturation by ammonium sulphate. Binder II corresponds in its properties to dexamethasone binding proteins of typical target tissues for glucocorticoids. It is characterized by relatively slow kinetics of dexamethasone binding, is sensitive to SH-blockers, highly thermolabile (inactivated completely at short exposure to 37°C when not bound to steroids) and precipitates at 0 to 25% saturation with ammonium sulphate. Corticosterone was found to exert strong competitive inhibitory effect on the binding of [³H]dexamethasone by binder II which demonstrates approximately equal affinity of this protein to both steroids. Nuclei isolated from hearts of normal rats are not able to accumulate [³H]dexamethasone and [³H]corticosterone in the absence of cytosol at 4°C and 20°C. [³H]Corticosterone in the complex with binder I, in the absence of binder II, is not accumulated by nuclei. Nuclei are able to accumulate [³H]dexamethasone, bound to binder II at 4°C. Accumulation is increased by 4-fold after 20 min incubation at 20°C. An increase in temperature of higher than 20°C or prolongation of incubation over 20 min decreases accumulation of protein-bound dexamethasone by nuclei. Our data demonstrate the role of binder II as a heart cytoreceptor which might participate directly in the transfer of glucocorticoids into cell nuclei.     

Binding of glucocorticoids and glucocorticoid-receptor complexes with rat liver nuclear matrix

Studies on binding glucocorticoids and cytosol glucocorticoid-receptor complexes with rat liver nuclear matrix were conducted. Zones of binding 3H-dexamethasone and 3H-dexamethasone-receptor complexes, characterized by saturability and high affinity, were found in nuclear matrix structures. A lower level of binding was noted during incubation of nuclear matrix and cytosol from the liver of rats aged 24 mos. than in the use of the same subcellular fractions isolated from the liver of rats aged 6 mos.     

Studies of a thyroid hormone and androgen dependent protein in rat liver cytosol.

Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of the liver cytosol of euthyroid male rats revealed a prominent band (molecular weight, 26 000 daltons), designated Protein II, which was virtually absent in the cytosol of hypothyroid animals. Injection of 500 mug triiodothyronine (T3) per 100 g body weight resulted in a maximal increase in the level of Protein II, reaching 90% of the euthyroidal level 3 days after hormone administration. Concomitant studies with the liver mitochondrial enzyme alpha-glycerophosphate dehydrogenase (alpha-GPD) indicated that this T3 dose also resulted in a maximal enzyme response in this time period. Since we have estimated that 500 mug of T3 will saturate nearly all nuclear T3 binding sites, these results support the concept that the synthesis of both proteins is limited by nuclear binding. Protein II was absent in the liver cytosol of female rats but could be induced in ovariectomized female rats by androgens. Treatment of male rats with oestradiol resulted in disappearance of Protein II. Since administration of testosterone to hypothyroid male rats caused only a minimal increase in the amount of Protein II, the absence of the protein in hypothyroid animals was not due to androgen deficiency. Similarities in the molecular weight and the response to hormonal manipulation of Protein II and of the urinary alpha2uglobulin, previously reported by Roy (1973) raise the possibility that these proteins are the same. The high concentration of Protein II in male rat cytosol and the relative ease in its identification by SDS polyacrylamide gel electrophoresis make it a potentially useful model protein for the study of thyroid hormone action at the cellular level.     

An unusual sex steroid-binding protein in mature male rat liver cytosol.

Mature male rat liver cytosol contains a moderate affinity and capacity estrogen-binding protein in at least a 200-fold higher level than mature female or immature male rat liver cytosol. Binding of estradiol to this protein is very rapid, is stabilized by EDTA, and is inhibited by divalent cations. This is the major binding protein for [3H]estradiol ([3H]E2) in mature male rat liver cytosol, and it has properties clearly distinguishing it from putative liver or uterine estrogen receptors. In addition to binding [3H]E2, this protein seems to rapidly bind a [3H]5alpha-dihydrotestosterone ([3H]DHT) metabolite at the same binding site. The binding of this androgen metabolite is stabilized by EDTA and is inhibited by divalent cations. The binding properties of the [3H]DHT metabolite suggest that these binding sites are not classical androgen receptors. Cytosol binding levels of both the [3H]E2 and the [3H]DHT metabolites change in a similar direction in resonse to endocrine manipulation. The putative liver estrogen receptor level, determined after partial purification (in a redissolved 30% ammonium sulfate-precipitated fraction), seems to change in an opposite direction in response to these same endocrine manipulations.     

Replication of total and mitochondrial DNA in rat liver with aging

Replication of total (t-DNA) and mitochondrial (mt-DNA) liver DNA of adult (6 months) and old (24 months) female rats labelled with i.v. ³H-thymidine has beeb studied. The most part of t-DNA radioactivity was shown to result from the replication of nuclear DNA (n-DNA). 10, 30, 70 and 120 min after labelling, intracellular acid-soluble ³H-radioactivity of old rats was significantly higher than that of adult ones. This may be due to the change in precursors concentration with aging. Thus, despite the lack of differences between the specific radioactivities of t-DNA and mt-DNA of adult and old animals during all labelling periods used the true replication rate, i.e., specific DNA radioactivity/volume of intracellular pool of label (relative DNA radioactivity), was significantly higher in adult animals during all labelling periods.Decrease of n-DNA and mt-DNA replication rate may lead to lowering of energetic potential and cell renewal in the liver with aging.     

Differences in the physicochemical characteristics of androgen-receptor complexes formed in vivo and in vitro

This study compares the physicochemical characteristics of the androgen-receptor hormone complexes formed in vitro by incubation of prostatic cytosol with tritiated 5 alpha-dihydrotestosterone (DHT) and methyltrienolone (R1881; 17 beta-hydroxy-17 alpha-methyl-4,9, 11-estra-trien-3-one) with those of hormone-receptor complexes formed in vivo upon hormone injection. [3H]DHT and [3H]R1881 had similar affinities for the androgen receptor in vitro (Kd = 0.3 nM). Dissociation of DHT at 0 C from the receptor complexes formed in vitro or in vivo was much slower than that of R1881. Furthermore, DHT and R1881 dissociated much more slowly from the cytoplasmic receptor labeled in vivo than in vitro. The sedimentation characteristics of the in vitro and in vivo formed hormone-receptor complexes were similar when analyzed on sucrose density gradients containing 400 mM KCl and 10 mM Na2MoO4. Higher concentrations (50 mM) of Na2MoO4, however, prevented the salt-induced disaggregation of the in vitro formed receptor complexes, which sedimented at 7-8S. In contrast, androgen-receptor complexes formed in vivo sedimented as 5.5S complexes, even in the presence of 50 mM molybdate. These differences were paralleled by the elution patterns from Sephacryl S-200. A further difference was found in the sensitivity of the hormone-receptor complex to the organic mercurial reagent mersalyl acid. This reagent, at 0.2 mM, induced ligand exchange of 80-90% of the in vitro formed hormone-receptor complexes, whereas it was nearly ineffective with complexes formed in vivo. Finally, the prostatic androgen receptor content 1 h after injection of radioactive steroid into castrated rats was 12-14 pmol/mg DNA, while incubation of tissue slices at 37 C yielded only 3-4 pmol receptor/mg DNA.     

Determination of the content of specific estrogen-binding protein in the rat liver cytosol

A method of quantitative determination of the areas of binding of a peculiar estrogen-binding protein (PEBP) in the cytosol of the rat liver by means of ion-exchange sorption on DEAE-cellulose is suggested. For differential determination of the PEBP contribution to the total estradiol (E2) binding with cytosol proteins a unique capacity of PEBP to form complexes with 3H-E2, which were rapidly decomposed on addition of an excess of unlabeled E2, was used. Theoretical prerequisites and experimental substantiation of the suggested method of the PEBP testing are considered. The content of the binding PEBP areas in the cytosol of the liver of male rats, measured by the mentioned method, constituted (5.8 +/- 0.7).10(-12) mol per 1 mg of protein (M +/- m, according to the data of 18 determinations). No analogous areas of E2 binding were revealed in the cytosol of the liver in female rats. It was also demonstrated that the cytosol of the liver of male rats contained specific areas of testosterone (T) binding, forming highly labile complexes with 3H-T, rapidly decomposing in the presence of an excess of T and E2.