Properties of solubilized nuclear triiodothyronine binding proteins.

Rat liver nuclear proteins bind 3,5,3'-triiodo-L-thyronine (T3) to essentially one class of sites (Ka approximately 1 X 10(10) M-1). Gel filtration and sucrose gradient centrifugation studies show a main T3 binding component with a Stokes radius of 33 A and a sedimentation coefficient of 3.5S, and variable amounts of high molecular weight binding components, most of them being reversible aggregates of the main component formed during storage. But the uniqueness of the nuclean T3 binding proteins (NTBP) cannot be ascertained from polyacrylamide gel electrophoresis data. During storage in the absence of reducing agents, NTBP aggregate and rapidly lose their ability to bind T3; T3--NTBP complexes also aggregate and progressively dissociate. This can be reversed by dithiothreitol. Bound T3 could temporarily stabilize the binding site but cannot protect NTBP against general conformational changes which follow the oxidation of their essential --SH group(s). NTBP are DNA binding proteins with probably a relative independence of their DNA and T3 binding sites: they bind T3 to the same class of high affinity sites whether complexed or not with DNA; bound T3 is not a prerequisite for DNA binding.     

Triiodothyronine binding to isolated liver cell nuclei.

Nuclei of euthyroid rat liver have been prepared from homogenates by sedimentation through 2.3M sucrose with or without a 0.25% Triton wash. Triiodothyronine is accumulated by these nuclei during incubation in vitro in solutions containing 0.32M sucrose, 1mM MgCl2 and 0.02M Tris-Cl buffer at pH 7.4 or 7.85. Specific T3 binding sites occupied at 10-1,000 pM T3 are saturated by excess unlabeled T3 (0.15 muM). Specific T3 binding at 20 C is maximal at 203 hr nad is proportional to amount of nuclei. Calcium ion enhances nuclear integrity by reduces T3 accumulation. EDTA and phosphate ion cause nuclear damage but increase T3 accumulation. Binding is unaffected by inhibition of energy dependent reactions or of RNA synthesis. It is markedly increased under certain conditions by addition of dithiothreitol (DDT). Binding does not require mediation of a cytosol protein. T3 binding is not prevented by RNAse or DNAse, but is obliterated by pronase. Te binds to a nuclear iodothyronine binding protein (NTBP) to form an NTBP-T3 complex similar to that form-d after in vivo administration of the hormone. The complex can be extracted from the nuclei by 0.4M KC-. T3 present in the NTBP-T3 complex resists accumulation by anion exchange resin at 0-2C, but is bound by resin after 20 min at 37C or after addition of 0.1 mM p-hydroxymercuribenzoate. At pH 7.85 and with 5 mM DTT, the apparent Ka for isolated nuclei is 0.2 times 10-10M-1 and the capacity is 508 pg T3/g wet tissue or 53 times 10-15 moles T3/100 mug DNA. The data may not represent total capacity, but rather the amount of T3 dissociated during the period of incubation so that NTBP-T3 can be exchanged with labeled T3. Among analogues tested, triiodothyroacetic acid appears to bind with four times the affinity of L-TX, D-T3 binds with equal affinity, and L-T4 with one-fourth the affinity of T3.     

Triiodothyronine nuclear binding in fetal and adult rabbit lung and cultured lung cells.

To investigate the possible mechanism of thyroid hormone action in the lung, we examined fetal and adult rabbit lung, and cell lines derived from lung, for specific nuclear binding sites for T3. Using incubation of isolated nuclei with L-[125I]T3 at 37 C, we found approximately 2400 specific binding sites/cell in fetal lung and 1120 sites/cell in adult lung, with a similar dissociation constant (approximately 500 pm) for both tissues. The L-2 and A549 cell lines, which may have originated from pulmonary type II alveolar cells, contained 2280 and 1580 nuclear sites/cell, and the dissociation constants were 280 and 200 pm, respectively. In fetal lung, the ability of various analogs to compete for L-T3 (100%) binding was: 3,5-diiodo-3'-isopropylthyronine, 81%; D-T3, 73%; L-T4, 6.7%; 3,3'-diiodothyronine, 0.19%; 3,5-dimethyl-3'-isopropyl-L-thyronine, 0.15%; and rT3, 0.08%. These findings indicate that both fetal and adult lung, and cultured lung cells, contain specific nuclear binding sites for T3, suggesting that these tissues and their type II alveolar cells may be directly influenced by thyroid hormones.     

GTP-binding proteins in rat liver nuclear envelopes.

Nuclear transport as well as reassembly of the nuclear envelope (NE) after completion of mitosis are processes that have been shown to require GTP and ATP. To study the presence and localization of GTP-binding proteins in the NE, we have combined complementary techniques of [alpha-32P]GTP binding to Western-blotted proteins and UV crosslinking of [alpha-32P]GTP with well-established procedures for NE subfractionation. GTP binding to blotted NE proteins revealed five low molecular mass GTP-binding proteins of 26, 25, 24.5, 24, and 23 kDa, and [alpha-32P]GTP photoaffinity labeling revealed major proteins with apparent molecular masses of 140, 53, 47, 33, and 31 kDa. All GTP-binding proteins appear to localize preferentially to the inner nuclear membrane, possibly to the interface between inner nuclear membrane and lamina. Despite the evolutionary conservation between the NE and the rough endoplasmic reticulum, the GTP-binding proteins identified differed between these two compartments. Most notably, the 68- and 30-kDa GTP-binding subunits of the signal recognition particle receptor, which photolabeled with [alpha-32P]GTP in the rough endoplasmic reticulum fraction, were totally excluded from the NE fraction. Conversely, a major 53-kDa photolabeled protein in the NE was absent from rough endoplasmic reticulum. Whereas Western-blotted NE proteins bound GTP specifically, all [alpha-32P]GTP photolabeled proteins could be blocked by competition with ATP, although with a competition profile that differed from that obtained with GTP. In comparative crosslinking studies with [alpha-32P]ATP, we have identified three specific ATP-binding proteins with molecular masses of 160, 78, and 74 kDa. The localization of GTP- and ATP-binding proteins within the NE appears appropriate for their involvement in nuclear transport and in the GTP-dependent fusion of nuclear membrane vesicles required for reassembly of the nucleus after mitosis.     

Roentgenographic contrast agents inhibit triiodothyronine binding to nuclear receptors in vitro.

The ability of roentgenographic contrast agents to inhibit binding of [125I]T3 to nuclear receptors was studied during incubation of rat liver nuclei or nuclear extracts in vitro and after ip administration of the agents in vivo. Ipodate, iodipamide, iopanoic acid, and diatrizoate inhibited binding of [125I]T3 in vitro. The most potent inhibitor was ipodate, which produced 50% inhibition of binding at 1.2 X 10(-4) M. When given orally in acute in vivo experiments, ipodate did not diminish binding to liver nuclear receptors. Ipodate appeared to inhibit in vivo metabolism of [125I]T3.     

Specific binding of solubilized adenylate cyclase to the erythrocyte cytoskeleton.

Concepts and criteria that have been developed for the study of the molecular organization of membrane-associated proteins are employed here to investigate the interaction of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] with other membrane components. Detergent-solubilized adenylate cyclase can be shown to bind to erythrocyte-derived Triton X-100 shells containing cytoskeletal elements. This binding appears to be saturable with respect to adenylate cyclase concentration, and it is enhanced by the presence of divalent cations. Preactivation of the enzyme with 5'-guanylyl imidodiphosphate and isoproterenol, or with NaF, is a prerequisite for effective binding. Two exceptions to this general observation are noted: rat brain adenylate cyclase, which binds without prestimulation, and rat testicular cytosolic adenylate cyclase, which fails to bind under any of the conditions tried. The binding sites of the Triton X-100 shells are inactivated or released by treatment with various concentrations of trypsin or KCl. Moreover, exposure of the Triton X-100 shells to increasing temperatures results in a progressive loss of the adenylate cyclase binding capacity. On the basis of these and other findings, it is suggested that the adenylate cyclase complex possesses two principal domains that allow it to interact with both cytoskeletal elements and the lipid bilayer. The specific modulation of these interactions may be involved in the hormonal regulation of adenylate cyclase activity.     

Hypothyroidism-induced changes in triiodothyronine binding to nuclei and cytosol-binding proteins in rat liver.

A tracer dose of [125I]T3 was given iv to normal, thyroidectomized, and propylthiouracil-fed rats and the distribution of radioactivity in serum and liver fractions was studied over 1 h. Total liver homogenate and serum 125I were higher at all times in hypothyroid rats and, in all groups, showed a continuous fall over the period studied. Hepatic nuclear 125I was maximal at 20 min in all and was greater in hypothyroid rats; there was more 125I in the hepatic cytosol of normal rats than in that from either thyroidectomized or propylthiouracil-fed animals. Binding studies with [125I]T3 and purified hepatic neclear preparations in vitro indicated that both the association constant, Ka (1.08-9.0 x 10(9) M-1) and the capacity (500-600 pg/mg DNA) in thyroidectomized and goitrogen-treated rats were similar to those obtained with normal animals. Cytosol, on the other hand, showed a decrease in binding capacity without change in affinity in livers of hypothyroid rats. Analysis of binding data by Hill plots indicated the presence of both positive and negative cooperativity in binding of T3 by rat liver cytosol proteins. In the in vitro experiments, higher serum radioactivity alone could not account for increases in the hepatic nuclear 125I in the hypothyroid rats because cytosol 125I (presumably in dynamic exchange with both blood and nuclei) was less. Consequently, cytosol T3-binding proteins may regulate the free T3 concentration in the cell and, thus influence the distribution of the hormone in other cellular compartments.     

A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.     

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.     

Cell cycle-regulated binding of nuclear proteins to elements within a mouse H3.2 histone gene.

The histone gene family in mammals consists of 15-20 genes for each class of nucleosomal histone protein. These genes are classified as either replication-dependent or -independent in regard to their expression in the cell cycle. The expression of the replication-dependent histone genes increases dramatically as the cell prepares to enter S phase. Using mouse histone genes, we previously identified a coding region activating sequence (CRAS) involved in the upregulation of at least two (H2a and H3) and possibly all nucleosomal replication-dependent histone genes. Mutation of two seven-nucleotide elements, alpha and omega, within the H3 CRAS causes a decrease in expression in stably transfected Chinese hamster ovary cells comparable with the effect seen upon deletion of the entire CRAS. Further, nuclear proteins interact in a highly specific manner with nucleotides within these sequences. Mutation of these elements abolishes DNA/protein interactions in vitro. Here we report that the interactions of nuclear factors with these elements are differentially regulated in the cell cycle and that protein interactions with these elements are dependent on the phosphorylation/dephosphorylation state of the nuclear factors.     

Studies on cytosol thyroid hormone binding proteins in the rat liver: Part I. Stability and binding characteristics of thyroid hormone binding proteins

Many studies demonstrated the presence of cytosolic thyroid hormone binding proteins (CTHBPs) in various tissues, but the physiologic significance of these CTHBPs is not clear, partially because of the lack of information about the physicochemical properties of CTHBPs as purified forms. Since the difficulty in isolating these CTHBPs is considered to be due to instability during the various procedures for their isolation, studies on the stability of CTHBPs of rat liver were performed using a charcoal binding method to separate bound and free hormones. Binding characteristics of CTHBPs of rat liver were also determined. Specific triiodothyronine (T3) binding sites of cytosolic T3 binding protein (CT3BP) of rat liver were destroyed as the time progressed in homogenate at 0 degrees C, and Aprotinin (500 U/ml) had little effect in protecting these binding sites. T3 binding sites were stable in the form of cytosol at -20 degrees C up to 10 weeks. Dithiothreitol (DTT) had no effect on T3 binding to cytosol. T3 binding to CT3BP was pH-dependent with maximum specific binding at pH 7.4. T3 binding to CT3BP was stable at 4 degrees C overnight but was destroyed rapidly at 37 degrees C. Interestingly, specific T3 binding sites of CT3BP were completely abolished by dialysis, and Ca2+ or Mg2+ had no effect on retaining the specific binding sites. Thus, CT3BP was supposed to require some dialysable small molecule(s) to maintain specific T3 binding sites. Scatchard plot of T3 binding to crude cytosol revealed a high affinity, limited capacity T3 binding site with affinity constant (Ka) of 5.9 X 10(7) M-1 and maximum binding capacity (MBC) of 118 ng/g. liver. Relative affinities of T3 analogues for CT3BP were determined by comparing the molar concentrations of T3 analogues required for 50% inhibition of tracer 125I-T3 binding. If the affinity of L-T3 was assigned 100, D-T3 would have a value of 66.1; L-T4, 22.3; D-T4, 16.5; Triac, 6.2; and both Tetrac and reverse T3 were less than 1. Thus, the binding characteristics of CT3BP were fundamentally different from those of nuclear T3 receptor. Cytosolic thyroxine (T4) binding protein (CT4BP) of rat liver was relatively stable compared with CT3BP in homogenate at 0 degrees C. CT4BP was also stable in the form of cytosol at -20 degrees C for 10 weeks. CT4BP was pH-dependent with maximum specific binding at pH 7.4. It was stable at 4 degrees C overnight but destroyed rapidly at 37 degrees C. Specific T4 binding was decreased by dialysis but not abolished completely.(ABSTRACT TRUNCATED AT 400 WORDS)     

DNA binding specificity of two homeodomain proteins in vitro and in Drosophila embryos.

In previous experiments, the homeodomain proteins even-skipped and fushi-tarazu were found to UV cross-link to a surprisingly wide array of DNA sites in living Drosophila embryos. We now show that UV cross-linking gives a highly accurate measure of DNA binding by these proteins. In addition, the binding of even-skipped and fushi-tarazu proteins has been measured in vitro to the same DNA fragments that were examined in vivo. This analysis shows that these proteins have broad DNA recognition properties in vitro that are likely to be important determinants of their distribution on DNA in vivo, but it also shows that in vitro DNA binding specificity alone is not sufficient to explain the distribution of these proteins in embryos.     

Insulin binding to solubilized material from fat cell membranes: Evidence for two binding species

The components of fat cell membranes responsible for the binding of insulin were solubilized by treatment with the nonionic detergent Triton X-100. By using a polyethylene glycol precipitation method to assay specific insulin binding, the soluble preparation was shown to have insulin-binding characteristics similar to those of intact fat cells. Further studies of this preparation by polyacrylamide gel electrophoresis in the presence of (125)I-labeled insulin demonstrated two distinct insulin binding activities, designated species I and II. The two species were separated by electrophoresis in the absence of iodo-labeled hormone and eluted from the gel. Scatchard analysis of the insulin binding data for species I showed a curvilinear plot with the initial portion having a K(d) of 1.3 x 10(-10) M. The Scatchard plot for species II was linear with a K(d) of 6.0 x 10(-9) M. Desoctapeptide insulin and glucagon failed to compete for the insulin-binding sites in both species whereas desalanine insulin was an effective competitor. High concentrations of proinsulin competed with the iodo-labeled hormone for binding to species I but not to species II. In the presence of a low concentration of (125)I-labeled insulin (0.3 nM) some species I activity appeared to be converted to species II activity; there was no evidence of interconversion between the two species in the absence of insulin. Neither species degraded insulin as measured by trichloroacetic acid precipitation or rebinding to intact fat cells. These findings indicate the existence in the adipocyte plasma membrane of two insulin-binding species that have distinct physicochemical properties.     

Effect of fatty acids on rat liver nuclear T3-receptor binding.

The effects of selected fatty acids (linoleic, oleic, and palmitic) on triiodothyronine (T3)-receptor binding were compared in isolated rat hepatocytes, rat liver nuclei, and receptor protein. Scatchard analysis indicated that the inhibition of T3-receptor binding by fatty acids was characterized by an increase in Kd and no change in maximum binding capacity (MBC). In isolated receptors, the rank order of potency for inhibition was linoleic acid greater than oleic acid greater than palmitic acid. The Ki for oleic acid in isolated receptors was the same as that for whole nuclei (15.4 +/- 1.3 v 16.3 +/- 1.9 mumol/L, respectively), indicating that the inhibition of nuclear T3 binding is probably at the level of the receptor protein itself. In isolated hepatocytes, linoleic acid was more potent than oleic acid in inhibiting T3 binding to nuclear receptors. Cell-associated T3 was not affected by the presence of fatty acids, implying that cellular uptake of T3 was not inhibited. High concentrations of fatty acids were necessary for inhibition of T3-receptor binding in isolated hepatocytes, with linoleic acid being one to two orders of magnitude less potent in isolated hepatocytes compared with isolated receptors (Ki, 179 +/- 12 v 4.4 +/- 0.5 mumol/L, respectively). It is concluded that the inhibitory effect of fatty acids on T3-receptor binding in isolated rat hepatocytes probably occurs at the level of the nuclear receptor, and does not involve an inhibition of the access of T3 to the receptor. However, in vivo it seems unlikely that fatty acids will have access to the nuclear receptors in sufficiently high concentrations to affect T3-receptor binding in liver cells.     

Identification of potato nuclear proteins binding to the distal promoter region of the proteinase inhibitor II gene.

Potato nuclear proteins specifically bind to a DNA sequence at the most 5' distal region of the promoter of a potato proteinase inhibitor II gene. Binding studies using the electrophoretic mobility-shift assay showed the appearance of two protein-DNA complexes in the presence of both tuber and leaf nuclear protein extracts. Mechanical wounding of the leaves had no effect on the amount of specific protein-DNA complexes formed. DNase I protection analysis and binding to synthetic oligonucleotides identified the sequence 5'-GAGGGTATTTTCGTAA-3' as the target for the noncooperative binding of two potato nuclear proteins to the upstream element. Methylation interference experiments showed that guanine nucleotides separated by one turn of the DNA helix were in close contact with the proteins. The binding ability of a series of mutated synthetic oligonucleotides further defined the sequence requirements for protein binding, which appeared to contact one side of the DNA helix.     

Progesterone-binding proteins: in vitro binding and biological activity of different steroidal ligands.

Progesterone-binding proteins from human, rabbit, sheep and guinea pig myometrial cytosol, all induced with oestradiol, as well as from pregnant guinea pig myometrium and plasma were investigated. The physico-chemical properties of the oestradiol-induced binding proteins were very similar in all the species studied. In all, 63 steroids were tested for their ability to compete with tritiated progesterone for the binding sites on these six proteins and their relative affinities were determined. The studies reveal that the ligand specificities of oestrogen-induced myometrial binding proteins from human, rabbit and sheep are rather similar, whereas that from guinea pig myometrium has different binding characteristics. The properties of the binding proteins from pregnant guinea pig uterus and plasma differ substantially from all of the induced proteins. It is clear from the different physico-chemical characteristics and binding specificities that the oestrogen-induced myometrial protein of the guinea pig is not the same as that which appears in the myometrium and plasma during pregnancy. The binding energies of the well bound progestational compounds were of the order of -12 Kcal/mole, half of which stems from the interaction of the steroid nucleus with the protein. The specific interaction of the protein with the two functional groups, the 3-keto-4-ene system and the acetyl sid chain each contributed-3 Kcal/mole. In the case of the rabbit, sheep and human proteins a 17alpha-ethynyl-17beta-hydroxyl function could replace the acetyl side chain. For a large number of steroids reasonable agreement existed between the degree of binding to the rabbit myometrial protein and in vivo biological activity (Clauberg-McPhail test) in the same species. The data suggest that as far asthe binding aspect is concerned, the rabbit is an appropriate model for assessing the biological activity of compounds under development for human application. The in vitro binding system is also a useful tool to assess whether steroids need to be bio-activated before eliciting a biological response.