Specificity and neutralizing capacity of antibodies elicited by a synthetic peptide of scorpion toxin

Polyclonal antibodies raised against a synthetic peptide (sequence 50-59) of Androctonus australis Hector toxin II can neutralize the effects of toxin II in vivo. The antigenic specificities of anti-peptide and anti-toxin antibodies were compared by competitive aqueous phase radioimmunoassay by using 125I-toxin II, chemically modified or homologous toxins, and the synthetic peptide 50-59, either free or bound to bovine serum albumin (BSA). The antipeptide and anti-toxin antibodies had a comparable high affinity for the native toxin, but anti-peptide antibodies exhibited a lower binding capacity. Anti-peptide antibodies had a higher affinity for native toxin than for the peptide 50-59 bound to BSA, used as immunogen, and were unable to recognize the free peptide. These results suggest that it is necessary to restrict the conformational freedom of the immunizing peptide in order to obtain anti-peptide antibodies with a high affinity for the toxin. The lysine residue at position 58 of toxin II, essential for toxicity, appears to be immunogenic when immunization is with peptide 50-59 bound to BSA and not with the native toxin. This residue is antigenic in the native toxin, however, as shown by the anti-peptide antibodies.     

Antipeptide antibodies of predetermined specificity recognize and neutralize the bioactivity of the pan-specific hemopoietin interleukin 3

Ten peptides that corresponded to portions of the T cell lymphokine pan-specific hemopoietin interleukin 3 (IL 3) were synthesized, coupled to keyhole limpet hemocyanin, and used to raise antipeptide antibodies in rabbits. These antisera reacted to varying degrees with native biologically active IL 3. Antibodies directed against peptides corresponding to residues 1-29 at the NH2 terminus, 123-140 at the COOH terminus, and to residues 64-82 and 91-112 were affinity-purified on peptide columns. Immunoabsorbent columns produced from affinity-purified antibodies to the 1-29, 91-112, and 123-140 although not the 64-82 peptide were effective in depleting biologically active IL 3 from conditioned medium. However, the antibodies specific for peptides 91-112 and 123-140 had only a low affinity for native IL 3 and it was only in the case of the anti-1-29 antibodies that a significant amount of IL 3 remained bound after extensive washing and could be recovered from the column by acid elution. The affinity-purified antibodies directed to peptides 1-29, 91-112, and 123-140 significantly inhibited the biological activity of IL 3, although with different dose-response characteristics. Anti-1-29 antibodies inhibited bioactivity over a wide range of concentrations (down to 20 ng/ml) although the inhibition was never complete. In contrast, the anti-91-112 antibodies, although effective only at high concentrations, produced complete inhibition of biological activity. These experiments demonstrated that antibodies to defined peptides can be used to generate antibodies to native IL 3 and should form useful tools in analyzing the structure and function of the native molecules.     

A human autoantibody specific for a unique conserved region of 28 S ribosomal RNA inhibits the interaction of elongation factors 1 alpha and 2 with ribosomes

An autoantibody reactive with a conserved sequence of 28 S rRNA (anti-28 S) was identified in serum from a patient with systemic lupus erythematosus. Anti-28 S protected a unique 59-nucleotide fragment synthesized in vitro against RNase T1 digestion. RNA sequence analysis revealed that it corresponded to residues 1944-2002 in human 28 S rRNA and 1767-1825 in mouse 28 S rRNA. These sequences are identical and highly conserved throughout all known eukaryotic 28 S rRNAs. In addition, this fragment is homologous to residues 1052-1110 of Escherichia coli 23 S rRNA that lies within the GTP hydrolysis center of the 50 S ribosomal subunit. Anti-28 S and its Fab fragments strongly inhibited poly(U)-directed polyphenylalanine synthesis, but had no effect on ribosomal peptidyltransferase activity. This effect resulted from inhibition of the binding of elongation factors EF-1 alpha and EF-2 to ribosomes and of the associated GTP hydrolysis. The inhibitory effect was almost completely suppressed by preincubation of anti-28 S with 28 S rRNA or in vitro synthesized RNA fragments containing the immunoreactive region. These results show that the immunoreactive conserved region of 28 S rRNA participates in the interaction of ribosomes with the two elongation factors in protein synthesis.     

Use of antibodies specific to defined regions of scorpion alpha-toxin to study its interaction with its receptor site on the sodium channel

Five antibody populations selected by immunoaffinity chromatography for their specificity toward various regions of toxin II of the scorpion Androctonus australis Hector were used to probe the interaction of this protein with its receptor site on the sodium channel. These studies indicate that two antigenic sites, one located around the disulfide bridge 12-63 and one encompassing residues 50-59, are involved in the molecular mechanisms of toxicity neutralization. Fab fragments specific to the region around disulfide bridge 12-63 inhibit binding of the 125I-labeled toxin to its receptor site. Also, these two antigenic regions are inaccessible to their antibodies when the toxin is bound to its receptor site. In contrast, the two other antigenic sites encompassing the only alpha-helix region (residues 23-32) and a beta-turn structure (residues 32-35) are accessible to their respective antibodies when the toxin is bound to its receptor. Together, these data support the recent proposal that a region made of residues that are conserved in the scorpion toxin family is involved in the binding of the toxin to the receptor.     

Synthesis of an antigenic site of native acetylcholine receptor peptide 159-169 of Torpedo acetylcholine receptor alpha-chain.

A region of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of the Torpedo electric organ, containing residues 161-166, has been proposed to be a major antigenic site in the native AChR protein. We report the synthesis of a peptide corresponding to residues 159-169, which contains the proposed antigenic region. In quantitative radiometric titrations, radiolabelled anti-(native AChR) antibodies from three different species, rabbit, rat and dog, exhibited considerable binding (approx. 15% relative to native AChR) to Sepharose-immobilized peptide 159-169, but did not bind significantly to Sepharose-immobilized unrelated proteins or peptides. Specificity was further confirmed by the finding that no rabbit anti-AChR antibodies bound to the peptide after absorption with native AChR. These data indicate that the region 159-169 contains an antigenic site that is readily accessible in solubilized native Torpedo AChR.     

Solvent effects on coupling yields during rapid solid-phase synthesis of CGRP(8-37) employing in situ neutralization.

The success of solid-phase peptide synthesis is often dependent upon solvation of the resin and the growing resin-bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide-resin in order to obtain satisfactory coupling yields for the rapid solid-phase peptide synthesis, using butyloxycarbonyl-(Boc)-amino acid derivatives, of human-alpha-calcitonin gene-related peptide(8-37) (CGRP(8-37)). Solvation of (p-methylbenzhydrylamine)copoly(styrene-1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8-37) (peptide-resin) was correlated to solvent Hildebrand solubility (delta) and hydrogen-bonding (delta(h)) parameters. Contour solvation plots of delta(h) vs. delta revealed maximum solvation regions of resin and peptide-resin. Maximum resin solvation occurred with N-methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide-resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide-resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide-resin solvation shifts to solvents with higher delta(h) values. DMF provided the most effective peptide-resin solvation and was the only solvent from which CGRP(8-37) was obtained as a single major product in the crude cleaved material.     

Solution structure of micelle-bound H5 peptide (427-452): a primary structure corresponding to the pore forming region of the voltage dependent potassium channel

A 26-mer peptide with the sequence of the pore forming region (residues 427-452) of the Shaker K(+) channel (H5 region) was chemically synthesized. Analyses by CD and two-dimensional 1H NMR spectroscopy were used to investigate the structure of the peptide bound to SDS micelles in solution, which are commonly used in biophysical studies. The tertiary structure of the peptide as a monomer was composed of an alpha-helix (431-438), a turn (439-442), and random coils (427-430, 443-452), and was very similar to that of the pore forming region of the native K(+) channel from Streptomyces lividans determined by X-ray analysis. This result suggests that even an isolated peptide forms a native-like conformation for residues from 431 to 442, depending on its intrinsic amino acid sequence and the surrounding environment.     

Redistribution of intermediate filament subunits during skeletal myogenesis and maturation in vitro

The distribution of intermediate filament (IF) subunits during maturation of skeletal myotubes in vitro was examined by immunofluorescence, using antibodies against two different types of chick IF subunits: (a) 58-kdalton subunits of fibroblasts (anti-58K), and (b) 55-kdalton subunits of smooth muscle (anti-55K). Anti-58K bound to a filament network in replicating presumptive myoblasts and fibroblasts, as well as in immature myotubes. The distribution in immature myotubes was in longitudinal filaments throughout the cytoplasm. With maturation, staining of myotubes by anti-58K diminished and eventually disappeared. Anti-55K selectively stained myotubes, and the fluorescence localization underwent a drastic change in distribution with maturation--from dense, longitudinal filaments in immature myotubes to a cross-striated distribution in mature myotubes that was associated with the I--Z region of myofibrils. However, the emergence of a cross-striated anti-55K pattern did not coincide temperally with the emergence of striated myofibrils, but occurred over a period of days thereafter.     

Detailed analysis of the region 9-30 of rat follicle stimulating hormone receptor: identification of peptide 20-30 as a potential hormone binding inhibitor

The extracellular domain (ECD) of the follicle stimulating hormone receptor (FSHR) has been shown to be a major determinant of hormone selectivity. The N-terminal 9-30 region, the sequence of which is unique to FSHR, has been extensively studied earlier and has been proposed to be an FSHR neutralizing epitope. In this study antipeptide antibodies specific to the peptide 9-30 were generated and used for identifying a specific immunodominant region within it. Overlapping peptides corresponding to the regions 9-19, 15-25 and 20-30 were synthesized. The ability of the antipeptide antibodies to 9-30 of FSHR to bind to different peptides was checked. The results indicated that the antibodies mainly recognized the peptide 20-30 and not the other two overlapping peptides. Further, the effect of the peptide 20-30 on the binding of radiolabeled FSH to its receptor was monitored. This peptide showed FSH-binding inhibitory activity with an IC(50) value of 0.598 x 10(-4)M and was more effective than the peptide 9-30 itself. Binding kinetics revealed that the observed effect of the peptide 20-30 is due to mixed type of inhibitory mechanism. This is the smallest peptide from the rat FSHR sequence having ability to inhibit FSH binding to its receptor by more than 90%.     

Differing accessibility in chromatin of the antigenic sites of regions 1-58 and 63-125 of histone H2B

Experiments with antibodies induced by separated fragments 1-58 and 63- 125 of H2B histone indicated that the 1-58 portion of the molecule is much more accessible in chromatin than is the 63-125 region. In immunoabsorption and immunoelectron microscopic assays with bovine and chicken chromatins, anti-1-58 antibodies reacted with sheared or unsheared chromatin both at low ionic strength (1 mM Tris-HCl) and in 0.14 M NaCl. Anti-63-125 antibodies were bound only weakly by chromatin at low ionic strength and not at all in 0.14 M NaCl. Antibodies to whole H2B showed intermediate reactivity with chromatin in both assays. In tests of immunofluorescence with unfixed calf liver nuclei in suspension, anti-1-58 caused nucleolar as well as nucleoplasmic fluorescence, whereas anti-63-125 did not lead to detectable fluorescence; anti-H2B showed intermediate staining intensity. In control experiments, anti-H1 antibody was bound by chromatin at low ionic strength but not in 0.14 M NaCl; anti-H3 antibody was bound poorly under either condition.     

Production and characteristics of human tissue plasminogen-activator antibodies with region-oriented synthetic peptides.

We selected six peptide sequences as belonging to potential epitopes of tissue plasminogen activator (tPA) using, as the main criterion for their choice, the location of the peptide sequences on the surface of the protein molecule. The six peptides (corresponding to amino acids 4-8, 11-16, 96-101, 272-277, 371-376 and 514-519) were synthesized, coupled to carrier proteins and injected into rabbits. All of these peptides elicited antibodies and 15-75% binding of the corresponding iodinated peptide was obtained with a 1:100 dilution of antiserum. Only two anti-(peptide) sera [anti-(tPA96-101) and anti-(tPA272-277)] reacted with intact tPA and its heavy chain in Western immunoblotting analysis. These two peptides sequences and fragment tPA11-16 appear to be involved in the structure of native antigenic epitopes of tPA, since they were recognized and antibodies present in antisera raised against native tPA. There was no interaction between anti-(tPA4-8) and anti-(tPA371-376) sera with intact one-chain or two-chain tPA. In the case of anti-(tPA4-8) cleavage of one-chain tPA to two-chain tPA and reduction of disulfide bonds exposed this epitope.     

Functional mapping of SPARC: peptides from two distinct Ca+(+)-binding sites modulate cell shape

Using synthetic peptides, we have identified two distinct regions of the glycoprotein SPARC (Secreted Protein Acidic and Rich in Cysteine) (osteonectin/BM-40) that inhibit cell spreading. One of these sites also contributes to the affinity of SPARC for extracellular matrix components. Peptides representing subregions of SPARC were synthesized and antipeptide antibodies were produced. Immunoglobulin fractions of sera recognizing an NH2-terminal peptide (designated 1.1) blocked SPARC- mediated anti-spreading activity. Furthermore, when peptides were added to newly plated endothelial cells or fibroblasts, peptide 1.1 and a peptide corresponding to the COOH terminal EF-hand domain (designated 4.2) inhibited cell spreading in a dose-dependent manner. These peptides exhibited anti-spreading activity at concentrations from 0.1 to 1 mM. The ability of peptides 1.1 and 4.2 to modulate cell shape was augmented by an inhibitor of protein synthesis and was blocked by specific antipeptide immunoglobulins. In addition to blocking cell spreading, peptide 4.2 competed for binding of [125I]SPARC and exhibited differential affinity for extracellular matrix molecules in solid-phase binding assays. The binding of peptide 4.2 to matrix components was Ca+(+)-dependent and displayed specificities similar to those of native SPARC. These studies demonstrate that both anti- spreading activity and affinity for collagens are functions of unique regions within the SPARC amino acid sequence. The finding that two separate regions of the SPARC protein contribute to its anti-spreading activity lead us to propose that multiple regions of the protein act in concert to regulate the interactions of cells with their extracellular matrix.     

Capping protein binding to S100B: implications for the tentacle model for capping the actin filament barbed end

S100B binds tightly to a 12-amino acid peptide derived from heterodimeric capping protein. In native intact capping protein, this sequence is in the C terminus of the alpha-subunit, which is important for capping the actin filament. This C-terminal region is proposed to act as a flexible "tentacle," extending away from the body of capping protein in order to bind actin. To this hypothesis, we analyzed the interaction between S100B and capping protein in solution. The C-terminal 28 amino acids of the alpha-subunit, the proposed tentacle, bound to S100B as a free synthetic peptide or a glutathione S-transferase fusion (K(d) approximately 0.4-1 microm). In contrast, S100B did not bind to whole native capping protein or functionally affect its capping activity. S100B does not bind, with any significant affinity, to the proposed alpha-tentacle sequence of whole native capping protein in solution. In the NMR structure of S100B complexed with the alpha-subunit-derived 12-amino acid peptide, the hydrophobic side of a short alpha-helix in the peptide, containing an important tryptophan residue, contacts S100B. In the x-ray structure of native capping protein, the corresponding sequence of the alpha-subunit C terminus, including Trp(271), interacts closely with the body of the protein. Therefore, our results suggest the alpha-subunit C terminus is not mobile as predicted by the tentacle model. Addition of non-ionic detergent allowed whole capping protein to bind weakly to S100B, indicating that the alpha-subunit C terminus can be mobilized from the surface of the capping protein molecule, presumably by weakening the hydrophobic binding at the contact site.     

Panning of a Phage Display Library against a Synthetic Capsule for Peptide Ligands That Bind to the Native Capsule of Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax with the ability to not only produce a tripartite toxin, but also an enveloping capsule comprised primarily of γ-D-glutamic acid residues. The purpose of this study was to isolate peptide ligands capable of binding to the native capsule of B. anthracis from a commercial phage display peptide library using a synthetic form of the capsule consisting of 12 γ-D-glutamic acid residues. Following four rounds of selection, 80 clones were selected randomly and analysed by DNA sequencing. Four clones, each containing a unique consensus sequence, were identified by sequence alignment analysis. Phage particles were prepared and their derived 12-mer peptides were also chemically synthesized and conjugated to BSA. Both the phage particles and free peptide-BSA conjugates were evaluated by ELISA for binding to encapsulated cells of B. anthracis as well as a B. anthracis capsule extract. All the phage particles tested except one were able to bind to both the encapsulated cells and the capsule extract. However, the peptide-BSA conjugates could only bind to the encapsulated cells. One of the peptide-BSA conjugates, with the sequence DSSRIPMQWHPQ (termed G1), was fluorescently labelled and its binding to the encapsulated cells was further confirmed by confocal microscopy. The results demonstrated that the synthetic capsule was effective in isolating phage-displayed peptides with binding affinity for the native capsule of B. anthracis.     

Identification of a linear peptide recognized by monoclonal antibody 2D7 capable of generating CCR5-specific antibodies with human immunodeficiency virus-neutralizing activity

CCR5 is the major coreceptor for human immunodeficiency virus (HIV) infection. The murine monoclonal antibody (MAb) 2D7, which recognizes a conformation-dependent epitope in the second extracellular loop of CCR5, is one of the most potent inhibitors of R5 virus cell entry. However, attempts to humanize 2D7 for in vivo human use have been unsuccessful so far. A filamentous phage library expressing random peptides was used to identify a peptide mimitope that is recognized by MAb 2D7. A synthetic peptide containing this sequence (2D7-2SK) bound to MAb 2D7 with high affinity and reversed its HIV type 1 (HIV-1) fusion inhibitory activity. The peptide contains sequence homologies to two distal regions of the second extracellular loop of human CCR5, both of which are required for MAb 2D7 binding. Rabbit anti-2D7-mimitope antibodies competed with MAb 2D7 for binding to the 2D7-2SK peptide in Biacore biosensor testing. Importantly, the rabbit anti-2D7-2SK antibodies bound to CCR5 on cells and specifically inhibited R5 (but not X4) envelope-mediated syncytium formation. These antibodies also neutralized infection of human peripheral blood mononuclear cells with R5 HIV isolates comparably to MAb 2D7. In summary, we have identified a novel peptide that closely mimics the MAb 2D7 epitope on CCR5. This peptide could be included as a potential vaccine candidate or to isolate 2D7-like human antibodies as entry inhibitors for R5 viruses.     

Solution structure of a K+-channel blocker from the scorpion Tityus cambridgei

A new K(+)-channel blocking peptide identified from the scorpion venom of Tityus cambridgei (Tc1) is composed of 23 amino acid residues linked with three disulfide bridges. Tc1 is the shortest known toxin from scorpion venom that recognizes the Shaker B K(+) channels and the voltage-dependent K(+) channels in the brain. Synthetic Tc1 was produced using solid-phase synthesis, and its activity was found to be the same as that of native Tc1. The pairings of three disulfide bridges in the synthetic Tc1 were identified by NMR experiments. The NMR solution structures of Tc1 were determined by simulated annealing and energy-minimization calculations using the X-PLOR program. The results showed that Tc1 contains an alpha-helix and a 3(10)-helix at N-terminal Gly(4)-Lys(10) and a double-stranded beta-sheet at Gly(13)-Ile(16) and Arg(19)-Tyr(23), with a type I' beta-turn at Asn(17)-Gly(18). Superposition of each structure with the best structure yielded an average root mean square deviation of 0.26 +/- 0.05 A for the backbone atoms and of 1.40 +/- 0.23 A for heavy atoms in residues 2 to 23. The three-dimensional structure of Tc1 was compared with two structurally and functionally related scorpion toxins, charybdotoxin (ChTx) and noxiustoxin (NTx). We concluded that the C-terminal structure is the most important region for the blocking activity of voltage-gated (Kv-type) channels for scorpion K(+)-channel blockers. We also found that some of the residues in the larger scorpion K(+)-channel blockers (31 to 40 amino acids) are not involved in K(+)-channel blocking activity.     

Radioimmunoassay of atrial natriuretic factor (ANF) in rat atria

We describe a solid phase radioimmunoassay for atrial natriuretic factor (ANF) and its application for measurement of this peptide in homogenates of rat atria. The method uses a synthetic 26 amino-acid fragment (8-33 ANF) of the native peptide. Sample (or standard) are incubated with the rabbit anti-8-33 ANF antiserum in peptide (8-33 ANF)-coated wells. Then an excess of I125 goat anti-rabbit IgG is added. The radioactivity bound is directly proportional to the amount of ANF present. The concentration of immunoreactive ANF has been found to be about 4 times higher in the right atrium than in the left atrium of the rat.     

Immunization with a peptide having both T cell and conformationally restricted B cell epitopes elicits neutralizing antisera against a snake neurotoxin.

We have synthesized a free peptide capable of eliciting antibodies that neutralize toxin alpha from Naja nigricollis, a protein that binds specifically to the acetylcholine nicotinic receptor. Of the five tested fragments that encompassed the whole toxin sequence, only fragment 24-41 stimulated T cells from BALB/c mice primed with the whole toxin and conversely, only T cells from mice primed with fragment 24-41 could be stimulated by both the toxin and priming peptide. No other peptides had such properties, indicating that only fragment 24-41 possessed T determinant(s) in BALB/c mice (H-2d haplotype). In agreement with the current view that B cell proliferation requires specific T cell stimulation, only fragment 24-41 elicited an antibody response. However, the antipeptide antisera failed to bind to the native toxin and thereby to neutralize it. Instead, it recognized an unfolded form of the toxin. The peptide 24-41 was then made cyclic. A circular dichroism analysis revealed that, in organic solvent, this peptide had a tendency to adopt a beta-sheet structure, as in the folded toxin, whereas the linear peptide adopted an helical structure. The cyclic peptide not only remained T stimulating but elicited antisera that recognized and neutralized the native toxin. Furthermore, the antisera cross-reacted with several toxin variants. Our data show, therefore, that it is possible to give an appropriate B cell specificity directly to a T cell-stimulating peptide, an approach that may be of value for the design of synthetic vaccines.     

Targeting of specific domains of diphtheria toxin by site-directed antibodies.

Antibodies highly selective for two functionally distinct regions of diphtheria toxin (DTx) were prepared using synthetic peptide conjugates as immunogens. Three peptides were selected for synthesis: sequence DTx141-157 on fragment A, which contains the putative protein elongation factor (EF-2) ADP-ribosyltransferase site; DTx224-237 on fragment B, selected on the basis of forming a predicted surface loop; and DTx513-526 on fragment B, forming a part of the region containing the putative receptor binding domain. All of the anti-peptide antibodies recognized the corresponding peptide, and also reacted with the toxin, specifically with the fragment containing the sequence against which they were raised, confirming the utility of this approach in generating fragment-specific antibodies. The anti-peptide antibody with the highest binding titre both to the peptide and to the native toxin was the one prepared against the sequence with the highest surface and loop likelihood indices of the three peptides selected. The similarity of the reactivity profiles with peptide and native and denatured toxin is consistent with the prediction that the region selected occurs in a surface loop and that the structure of the peptide is similar to the conformation of this region in the native protein. The epitopes for two of the anti-peptide antibodies were mapped. The results indicated that even though the antisera were raised to peptides containing 14 amino acids (aa) they were directed predominantly against a narrow region within the peptide, consisting of only 5-6 aa residues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antibodies directed to restricted sequences of the c-Erb a α hinge domain interfere with hormone or dna binding to recombinant α-type triiodothyronine receptor (c-Erb a α1) and detect structural changes

Abstract

In a previous study we showed that antibodies directed to restricted sequences in the hinge domain of α-type triiodothyronine (T3) receptor (TR) (aminoacids 150-166, 172-191, 144-162) selectively recognized and immunoprecipitated α-type TR in tissues. Furthermore, antibodies to peptide 172-191 (anti-α 172) strongly impaired T3 binding to natural TR α. To get more precise informations on the ability of these antibodies to recognize the TR, alter TR properties and/or detect conformational changes in TR, TR αl was produced in E. coli as a non-mutated, non-fusion protein from a rat c-erb A α1 cDNA inserted into pTrc99A vector. The recombinant c-Erb A αl protein, after solubilization with 5 M guanidine and progressive refolding, presented the main characteristics of TR α: unique or largely dominant band of 46 KDa in Western blots with the different anti-c-Erb A α antibodies; binding to DNA and to T3. Binding to DNA was markedly attenuated by anti-α 144 but not by anti-α 150 and anti-α 172. Binding to T3 was modified by anti-α 150 and -α 172 with different characteristics whether recombinant TR was previously bound to T3 or not, and with marked differences in comparative studies with natural TR. When liganded to T3, recombinant and natural TR αl present the same pattern of interaction with both antibodies: immunoprecipitation without any dissociation of T3 by anti-α 150; marked dissociation of bound T3 by anti-α 172. By contrast unliganded recombinant and natural TR are oppositely altered by these antibodies in their ability to bind T3: strong impairment restricted to anti-α 172 for natural TR, and to anti-α 150 for recombinant TR. Anti-α 144 did not interfere. These results lay emphasis on: 1/ the existence and biological relevance of different conformational states of TR α hinge domain, particularly whether TR is liganded or not to T3 and whether it is in a nuclear environment or bacterially-produced; 2/ an important role of the C-terminal part of hinge domain for efficient hormone binding, this involving a region that overlaps the α 150 and α 172 sequences.