Biosynthesis of bacterial glycogen: purification and characterization of ADPglucose pyrophosphorylase with modified regulatory properties from Escherichia coli B mutant CL1136-504

The l-thyroxine binding site in human serum thyroxine-binding globulin was investigated by affinity labeling with N-bromoacetyl-l-thyroxine (BrAcT₄). Competitive binding studies showed that, in the presence of 100 molar excess of BrAcT₄, binding of thyroxine to thyroxine-binding globulin was nearly totally abolished. The reaction of BrAcT₄ to form covalent binding was inhibited in the presence of thyroxine and the affinity-labeled thyroxinebinding globulin lost its ability to bind thyroxine. These results indicate BrAcT₄ and thyroxine competed for the same binding site. Affinity labeling with 2 mol of BrAcT₄/mol of thyroxine-binding globulin resulted in the covalent attachment of 0.7 mol of ligand. By amino acid analysis and high voltage paper electrophoresis, methionine was identified as the major residue labeled (75%). Lysine, tyrosine, and histidine were also found to be labeled to the extent of 8, 8, and 5%, respectively.     

Partial amino acid sequence of human thyroxine-binding globulin. Further evidence for a single polypeptide chain.

The NH₂-terminal amino acid of highly purified thyroxine-binding globulin has been identified by dansyl chloride, cyanate and Edman degradation methods. All three gave alanine as the only amino terminal residue. Carbamylation and Edman degradation of the denatured protein yielded 0.86 and 0.98 – 1.05 mole of alanine per mole of protein, respectively. These data further indicate that thyroxine-binding globulin is composed of a single polypeptide chain. Automated Edman degradation gave the partial sequence as: Ala-Ser-Pro-Glu-Gly-Lys-Val-Thr-Ala-Asp-Ser-Ser-Ser-Gln-(Pro)-X-Ala-(Ser)-Leu-Tyr- A computer search revealed no homology of the NH₂-terminal segment of thyroxine-binding globulin with human prealbumin. The NH₂-terminal portion of prealbumin contains part of the thyroxine binding site.     

Allosteric modulation of hormone release from thyroxine and corticosteroid-binding globulins

The release of hormones from thyroxine-binding globulin (TBG) and corticosteroid-binding globulin (CBG) is regulated by movement of the reactive center loop in and out of the β-sheet A of the molecule. To investigate how these changes are transmitted to the hormone-binding site, we developed a sensitive assay using a synthesized thyroxine fluorophore and solved the crystal structures of reactive loop cleaved TBG together with its complexes with thyroxine, the thyroxine fluorophores, furosemide, and mefenamic acid. Cleavage of the reactive loop results in its complete insertion into the β-sheet A and a substantial but incomplete decrease in binding affinity in both TBG and CBG. We show here that the direct interaction between residue Thr(342) of the reactive loop and Tyr(241) of the hormone binding site contributes to thyroxine binding and release following reactive loop insertion. However, a much larger effect occurs allosterically due to stretching of the connecting loop to the top of the D helix (hD), as confirmed in TBG with shortening of the loop by three residues, making it insensitive to the S-to-R transition. The transmission of the changes in the hD loop to the binding pocket is seen to involve coherent movements in the s2/3B loop linked to the hD loop by Lys(243), which is, in turn, linked to the s4/5B loop, flanking the thyroxine-binding site, by Arg(378). Overall, the coordinated movements of the reactive loop, hD, and the hormone binding site allow the allosteric regulation of hormone release, as with the modulation demonstrated here in response to changes in temperature.     

Comparison of the modelled thyroxine binding site in TBG with the experimentally determined site in transthyretin.

The structure of cleaved thyroxine-binding globulin (TBG) has been modelled on the crystal structure of cleaved alpha 1-antitrypsin (a member of the serine proteinase inhibitor, serpin, superfamily) based on the high sequence homology exhibited by the two proteins. Particular attention was paid to the identification and modelled characteristics of the thyroxine binding site. The primary aim of the study was to compare the site qualitatively with the crystallographically determined binding site of transthyretin, the other major transporter of thyroxine, in an attempt to explain the higher binding affinity of the site compared with the known thyroxine binding site in transthyretin (10(10) versus 10(8) M-1). The proposed binding site shares some similar characteristics with the transthyretin binding site but also includes a cluster of aromatic residues which are entirely absent in transthyretin. It is proposed that this might account for the substantial difference in binding affinities.     

Study of the binding of thyroxine by thyroxine-binding prealbumin by a dialysis method using a fixed free concentration of ligand (author's transl)]

In order to study ligand-protein binding in solution, a dialysis method was used in which the free concentration of ligand can be controlled. The method has certain advantages and was applied to the binding of thyroxine by thyroxine-binding prealbumin, a system about which the results found in the literature are not in good agreement. From the isotherm drawn according to the Scatchard plot, it was found that thyroxine-binding prealbumin only presents a single binding site for thyroxine per molecule, the association constant being 1.7 . 10(8) M-1.     

Rat liver iodothyronine monodeiodinase. Evaluation of the iodothyronine ligand-binding site

Ligand binding characteristics of rat liver microsomal type I iodothyronine deiodinase were evaluated by measuring dose-response inhibition and apparent Michaelis-Menten or inhibitor constants of iodothyronine analogues to compete as substrates or inhibitors for the natural substrate L-thyroxine. These data show strong correlations with the binding requirements of hormone analogues to serum thyroxine-binding prealbumin since iodothyronine analogues with a negatively charged side chain, a negative charge or hydrogen bonding function in the 4'-position, tetraiodo ring substitution, and a skewed hormone conformation are structural features shared in common which markedly affect enzyme activity and protein binding affinity. 3,3',5'-Triiodo-L-thyronine is the most potent natural substrate (IC50 = 0.3 microM) and tetraiodothyroacetic acid is the most potent inhibitor (IC50 = 0.2 microM). Both thyroxine (T4)-5'- and T4-5-deiodination pathways are inhibited by these potent analogues, providing further evidence for a single enzyme catalyzing the rat liver microsomal deiodination reactions. These data also show that L-hormone analogues are preferentially deiodinated via the T4-5'-deiodination pathway, whereas D-analogues produce products via the T4-5-deiodination pathway. The thyroxine-binding prealbumin complex was used to model the interaction of thyroid hormones with the deiodinase active site. Computer graphic modeling of the prealbumin complex showed that only those analogues which are potent deiodinase inhibitors or substrates can be accommodated in the hormone binding site. This model suggests the design of functionally specific ligands which can modulate peripheral thyroid hormone metabolism and act as antithyroidal drugs.     

A thyroxine binding globulin (TBG)-like protein in the sera of developing and adult rats

We report evidence based on equilibrium binding, electrophoretic, autoradiographic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). We show that in the sera of postnatal developing animals, the thyroxine and the triiodothyronine binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as thyroxine carrier to the thyroid binding prealbumin, but retains the major role as binder of triiodothyronine i.e. of the biologically active thyroid hormone.     

Vitamin A and thyroxine carrier proteins in chicken plasma: steady-state control of the plasma level of free retinol-binding protein and free thyroxine.

1. The binding parameters of prealbumin-2 with retinol-binding protein and thyroxine (T4) revealed the existence of distinct and multiple sites for both retinol-binding protein and T4. 2. From the analysis of binding parameters of retinol-binding protein with prealbumin-2 it is clear that under steady-state conditions about 99% of the holo-retinol-binding protein remains bound to prealbumin-2. 3. Equilibrium dialysis studies on binding properties of thyroid hormones with prealbumin-2 revealed that it has a single high affinity site and three low affinity sites. 4. The occurrence of three carrier proteins for thyroid hormones, thyroxine-binding globulin, prealbumin-2 and albumin has been demonstrated. However, the chicken thyroxine-binding globulin differs from human thyroxine-binding globulin by being relatively less acidic and occurring at a two-fold lower concentration. But the thyroid hormone binding parameters are comparable. 5. Highly sensitive methods were developed for determination of T4 binding capacities of the various proteins and plasma level of total T4 by fractionation of carrier proteins and further quantitatively employing in electrophoresis and equilibrium dialysis. 6. The thyroxine-binding proteins were found to be of two types, one (viz., thyroxine-binding globulin) of great affinity but of low binding capacity, which mainly acts as reservoir of T4, and another (viz., prealbumin-2) of low affinity but of high binding capacity, which can participate predominantly in the control of the free T4 pool.     

Thyroxine-protein interactions. Binding constants for interaction of thyroxine analogues with the thyroxine binding site on human thyroxine-binding globulin.

The binding constants for interaction of various thryoxine analogues with the thyroxine binding site on human thyroxine-binding globulin have been determined. Equilibrium dialysis, at pH 7.4 and 37 degrees C, was used to measure the competitive effects of different iodothyronine compounds on the binding of 125I-labeled thyroxine to highly purified thyroxine-binding globulin. Relative to L-thyroxine, K = 6 . 10(9) M-1, the association constants of some important analogues were D-thyroxine, 1.04 . 10(9) M-1, 3,5-diiodo-3'-isopropyl-L-thyronine, 4.9 . 10(8) M-1; L-triiodothyronine, 3.3 . 10(8) M-1, 3,3',5'-DL-triiodothyronine (reverse triiodothyronine), 3.1. 10(8) M-1; tetraiodothyropropionic acid, 2.7 . 10(8) M-1; tetraiodothyroacetic acid, 2.6 . 10(8) M-1; 3', 5'- diiodo-DL-thyronine, 8.3 . 10(7) M-1; and 3,5-diiodo-DL-thyronine, 7.1 . 10(7) M-1. Calculation of the deltaG0 values for binding of the analogues indicates that a major contribution to the free energy favoring binding is made by the alanine side chain of thyroxine. A change in configuration of the alpha-amino group from the L to D form causes an unfavorable change of 1 kcal/mol in the free energy of binding. Removal of the alpha-amino group as in tetraiodothyropropionic acid causes an unfavorable change of 1.9 kcal/mol in the free energy of binding. With regard to ring substituents, the results indicate that the two inner 3,5-iodines make about the same contribution to binding as the two outer 3', 5'-iodines.     

Effect of long-chain fatty acids on the binding of thyroxine and triiodothyronine to human thyroxine-binding globulin

The effect of long-chain fatty acids on the binding of thyroxine to highly purified human thyroxine-binding globulin has been studied by equilibrium dialysis performed at pH 7.4 and 37 degrees C. At a fixed molar ratio of 2000:1 of fatty acid to thyroxine-binding globulin, the degree of binding inhibition based on the percent change in nK value relative to the control as determined from Scatchard plots was: palmitic, 0%; stearic, 0%; oleic, 76%; linoleic, 69%; and linolenic, 61%. At a 500:1 molar ratio of oleic acid to thyroxine-binding globulin, equivalent to 0.125 mM free fatty acid in serum, thyroxine binding was inhibited by 18%, increasing to 93% at a 4500:1 molar ratio. At molar ratios of oleic acid to thyroxine-binding globulin of 1000:1, 2000:1 and 4000:1, the degree of inhibition of triiodothyronine binding was 24%, 41% and 76%, respectively. The results indicate that the unsaturated long-chain fatty acids are potent inhibitors of thyroxine binding to thyroxine-binding globulin, whereas the saturated fatty acids have little or no effect on thyroxine binding.     

Thyroxine-binding proteins in liver and tail of Rana catebeiana undergoing metamorphosis.

Presence of a thyroxine-binding protein was demonstrated in vivo in cell sap of tail and liver of metamorphosing Rana catesbeiana tadpoles. Thyroxine-binding protein was not present in tail of prematamorphic tadpoles while it appeared during progressing metamorphosis roughly coinciding with the beginning of tail resorption. Susceptibility to pronase indicates that this thyroxine-binding macromolecule is protein in nature. Thyroxine-binding in liver was already present during premetamorphic stages and increased further during metamorphosis. A further difference between tail and liver thyroxine-binding protein was evidenced by molecular sieve chromatography on Sephadex G-200 indicating a molecular weight of thyroxine-binding protein in the tail of 60 000 as opposed to 42 000 for liver. Scatchard analysis of tail cell sap of tadpoles in metamorphic climax revealed a high affinity thyroxing binding site (Kd of 2 - 10(-10) M) of low capacity (1.7 pmol per mg protein) while tadpoles in premetamorphic stage had a thyroxine-binding site of lower affinity (9 - 10(-10) M) and higher capacity (4.8 pmol per mg protein). Thus affinity of thyroxine binding is 4-fold in metamorphic climax and appears to reflect the appearance of thyroxine binding observed in vivo.     

New aspects of isolation and purification of thyroxine-binding globulin from human biological fluids

Based on the new data concerning the multicomponent system of thyroxine-binding proteins in human plasma, some methodological aspects of isolation and purification of thyroxine-binding globulin (TBG) were examined, and a simple two-step procedure for TBG purification was developed. Normal human blood serum, retroplacental serum and amniotic fluid were used as TBG sources. The procedure includes affinity chromatography and adsorption chromatography on a hydroxyapatite column. A biospecific adsorbent was synthesized by stepwise binding of epichlorohydrin and thyroxine to Sepharose. The yield of pure TBG varied from 60 to 80%, depending on the TBG source used. The properties of TBG preparations from retroplacental serum and amniotic fluid were identical; both preparations contained a pregnancy-associated molecular variant, TBG-1. Two novel serum thyroxine-binding proteins were detected, isolated and partly characterized.     

Specific labeling of the thyroxine binding site in thyroxine-binding globulin: determination of the amino acid composition of a labeled peptide fragment isolated from a proteolytic digest of the derivatized protein

[125I] Thyroxine has been covalently bound to the thyroxine binding site in thyroxine-binding globulin by reaction with the bifunctional reagent, 1,5-difluoro-2,4-dinitrobenzene. An average of 0.47 mol of [125I] thyroxine was incorporated per mol protein; nonspecific binding amounted to 8%. A labeled peptide fragment was isolated from a proteolytic digest of the derivatized protein by HPLC and its amino acid composition was determined. Comparison with the amino acid sequence of thyroxine-binding globulin indicated partial correspondence of the labeled peptide with two possible regions in the protein. These regions also coincide with part of the barrel structure present in the closely homologous protein, alpha 1-antitrypsin.     

Demonstration and ontogenesis in the rat of a serum protein analogous to human thyroxine binding globulin

It has been reported evidence based on equilibrium binding, electrophoretic, immunoelectrophoretic studies, that the rat possesses a major high affinity thyroid hormone binding protein, with an electrophoretic mobility and binding properties similar to those of the human thyroxine binding globulin (TBG). It is shown that in the sera of postnatal developing animals, between 3 and 21 days, the thyroxine (T4) and the triiodothyronine (T3) binding activities increase up to 10 times over adult or foetal levels, due to a high transient post-natal surge of the rat TBG. In the adult serum, the TBG persists in decreased amounts: it then yields the predominant role as T4 carrier to the thyroid binding prealbumin (TBPA), but retains the major role as binder of T3, i.e. of the biologically active thyroid hormone.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and the prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool.Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the ‘buffering’ action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintainance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

Homeostatic regulation of free retinol-binding protein and free thyroxine pools of plasma by their plasma carrier proteins in chicken.

The mechanism underlying homeostatic regulation of the plasma levels of free retinol-binding protein and free thyroxine, the systemic distribution of which is of great importance, has been investigated. A simple method has been developed to determine the rate of dissociation of a ligand from the binding protein. Analysis of the dissociation process of retinol-binding protein from prealbumin-2 reveals that the free retinol-binding protein pool undergoes massive flux, and that prealbumin-2 participates in homeostatic regulation of the free retinol-binding protein pool. Studies on the dissociation process of thyroxine from its plasma carrier proteins show that the various plasma carrier proteins share two roles. Of the two types of protein, the thyroxine-binding globulin (the high affinity binding protein) contributes only 27% of the free thyroxine in a rapid transition process, despite its being the major binding protein. But prealbumin-2, which has lower affinity towards thyroxine, participates mainly in a rapid flux of the free thyroxine pool. Thus thyroxine-binding globulin acts predominantly as a plasma reservoir of thyroxine, and also probably in the 'buffering' action on plasma free thyroxine level, in the long term, while prealbumin-2 participates mainly in the maintenance of constancy of free thyroxine levels even in the short term. The existence of these two types of binding protein facilitates compensation for the metabolic flux of the free ligand and maintenance of the thyroxine pool within a very narrow range.     

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study

Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuous-wave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D₂O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.     

Comparison of human thyroxine-binding globulin purification by affinity chromatography procedures

Three procedures for the isolation of thyroxine-binding globulin from human serum, using affinity chromatography on triiodothyronine (T3) linked to Sepharose (A), thyroxine (T4) linked to Sepharose (B) or T3 linked to epoxy-Sepharose (C) as the first purification step, were compared. With the use of additional purification steps, the three procedures yielded pure thyroxine-binding globulin without desialylation. With procedure A, the initial binding of T4-binding globulin to T3-Sepharose was very low, yielding a poor final recovery (17%). Procedure B gave the highest yield (35%) after a three-step purification, with a low T4 content (0.15-0.30 mol/mol). Procedure C also gave a high yield (28%) after only two purification steps, with a T4 content greater than 0.7 mol/mol. The microheterogeneity of T4-binding globulin obtained with these three procedures was demonstrated by isoelectric focusing: five major bands were observed between pH 4.1 and 4.6, and intermediate faint bands (often doublets) in the same pH range. However, with procedures A and C, the most acidic bands (pH 4.10-4.20) were always absent. Thyroxine-binding globulin was preincubated with radioactively labelled T3 or T4 and the hormone-protein complex was analyzed by isoelectric focusing. The binding of T3--compared to that of T4--was reduced in the most acidic protein subspecies. These results suggest differences in the thyroid hormone binding properties of the various subspecies of human T4-binding globulin.     

Progesterone-binding globulin interaction with its steroid ligands: study of the protein binding site topography using spin labeled steroids and electron spin resonance spectroscopy

The binding site topography of progesterone-binding globulin (PBG) purified from pregnant guinea pig serum was examined using synthesized spin-labeled ligands and electron spin resonance (ESR) spectroscopy. A series of deoxycorticosterone-nitroxide (DOC-NO) derivatives were prepared, bearing the free radical on the side chain at increasing distance (d) from the steroid nucleus. The ability of the spin-labeled steroids to specifically bind to PBG was assessed by measurement of their relative binding affinity as compared to progesterone. ESR spectra of the bound steroid nitroxide radical were used to calculate the rotational correlation times tau c for the nitroxides as a function of their distance d to the protein-bound steroid nucleus. The data showed that the side chain nitroxide exhibited an unrestrained rotation in a water-like environment when d reached about 18 A. This would correspond to a PBG steroid binding site depth of about 28 A and suggests that the bound steroid in the PBG site is oriented with the side chain at C-17 directed toward the outside of the protein binding crevice.     

Thyroxine-protein interactions. Interaction of thyroxine and triiodothyronine with human thyroxine-binding globulin.

The effect of temperature on the binding of thyroxine and triiodothyronine to thyroxine-binding globulin has been studied by equilibrium dialysis. Inclusion of ovalbumin in the dialysis mixture stabilized thyroxine-binding globulin against losses in binding activity which had been found to occur during equilibrium dialysis. Ovalbumin by itself bound the thyroid hormones very weakly and its binding could be neglected when analyzing the experimental results. At pH 7.4 and 37 degrees in 0.06 M potassium phosphate/0.7 mM EDTA buffer, thyroxine was bound to thyroxine-binding globulin at a single binding site with apparent association constants: at 5 degrees, K = 4.73 +/- 0.38 X 10(10) M-1; at 25 degrees, K = 1.55 +/- 0.17 X 10(10) M-1; and at 37 degrees, K = 9.08 +/- 0.62 X 10(9) M-1. Scatchard plots of the binding data for triiodothyronine indicated that the binding of this compound to thyroxine-binding globulin was more complex than that found for thyroxine. The data for triiodothyronine binding could be fitted by asuming the existence of two different classes of binding sites. At 5 degrees and pH 7.4 nonlinear regression analysis of the data yielded the values n1 = 1.04 +/- 0.10, K1 = 3.35 +/- 0.63 X 10(9) M-1 and n2 = 1.40 +/- 0.08, K2 = 0.69 +/- 0.20 X 10(8) M-1. At 25 degrees, the values for the binding constants were n1 = 1.04 +/- 0.38, K1 = 6.5 +/- 2.8 X 10(8) M-1 and n2 = 0.77 +/- 0.22, K2 = 0.43 +/- 0.62 X 10(8) M-1. At 37 degrees where less curvature was observed, the estimated binding constants were n1 = 1.02 +/- 0.06, K1 = 4.32 +/- 0.59 X 10(8) M-1 and n2K2 = 0.056 +/- 0.012 X 10(8) M-1. When n1 was fixed at 1, the resulting values obtained for the other three binding constants were at 25 degrees, K1 = 6.12 +/- 0.35 X 10(8) M-1, n2 = 0.72 +/- 0.18, K2 = 0.73 +/- 0.36 X 10(8) M-1; and at 37 degrees K1 = 3.80 +/- 0.22 X 10(8) M-1, n2 = 0.44 +/- 0.22, and K2 = 0.43 +/- 0.38 X 10(8) M-1. The thermodynamic values for thyroxine binding to thyroxine-binding globulin at 37 degrees and pH 7.4 were deltaG0 = -14.1 kcal/mole, deltaH0 = -8.96 kcal/mole, and deltaS0 = +16.7 cal degree-1 mole-1. For triiodothyronine at 37 degrees, the thermodynamic values for binding at the primary binding site were deltaG0 = -12.3 kcal/mole, deltaH0 = -11.9 kcal/mole, and deltaS0 = +1.4 cal degree-1 mole-1. Measurement of the pH dependence of binding indicated that both thyroxine and triiodothyronine were bound maximally in the region of physiological pH, pH 6.8 to 7.7.