Nonoxidative cadmium-dependent dimerization of Cd7-metallothionein from rabbit liver.

The effect of free Cd(II) ions on monomeric Cd7-metallothionein-2 (MT) from rabbit liver has been studied. Slow, concentration-dependent dimerization of this protein was observed by gel filtration chromatographic studies. The dimeric MT form, isolated by gel filtration, contains approximately two additional and more weakly bound Cd(II) ions per monomer. The incubation of MT dimers with complexing agents EDTA and 2-mercaptoethanol leads to the dissociation of dimers to monomers. The results of circular dichroism (CD) and electronic absorption studies indicate that the slow dimerization process is preceded by an initial rapid Cd-induced rearrangement of the monomeric Cd7-MT structure. The 113Cd NMR spectrum of the MT dimer revealed only four 113Cd resonances at chemical shift positions similar to those observed for the Cd4 cluster of the well-characterized monomeric 113Cd7-MT. This result suggests that on dimer formation major structural changes occur in the original three-metal cluster domain of Cd7-MT.     

Subunit structure and activity of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system solubilized in detergent

The original proposal of Saier stating that P-enolpyruvate-dependent mannitol phosphorylation is catalyzed by the monomeric form of the bacterial phosphotransferase enzyme IImtl, which would be the form predominantly existing in the phospholipid bilayer, whereas mannitol/mannitol-P exchange would depend on the transient formation of functional dimers, is refuted [Saier, M.H. (1980) J. Supramol. Struct. 14, 281-294]. The correct interpretation of the proportional relation between the rate of mannitol phosphorylation in the overall reaction and the enzyme concentration is that enzyme IImtl is dimeric under the conditions employed. Differences measured in the enzyme concentration dependency of the overall and exchange reactions were caused by different assay conditions. The dimer is favored over the monomer at high ionic strength and basic pH. Mg2+ ions bind specifically to enzyme IImtl, inducing dimerization. A complex formed by mixing inorganic phosphate, F-, and Mg2+ at sufficiently high concentrations inhibits enzyme IImtl, in part, by dissociation of the dimer. Enzyme IImtl was dimeric in 25 mM Tris, pH 7.6, and 5 mM Mg2+ over a large enzyme concentration range and under many different turnover conditions. The association/dissociation equilibrium was demonstrated in phosphate bufers, pH 6.3. The dimer was the most active form both in the overall and in the exchange reaction under the conditions assayed. The monomer was virtually inactive in mannitol/mannitol-P exchange but retained 25% of the activity in the overall reaction.     

Binding properties and stoichiometries of a palladium(II) complex to metallothioneins in vivo and in vitro

This paper will be the first to discuss the in vivo and in vitro properties of a Pd(II) complex, K2PdCl4, interacting with metallothioneins (MTs). In vivo experiments revealed that intraperitoneal injections of K2PdCl4 into rabbits led to the simultaneous synthesis of Pd-MT in the kidney and Zn7MT in the liver. The renal Pd-MT complex contains 3.6 +/- 0.3 Pd, 2.1 +/- 0.2 Zn, and 1.0 +/- 0.1 Cu per mole protein. It was found that pre-treatment with Zn(NO3)2 before K2PdCl4 injections significantly enhanced renal Pd-MT level. The same pre-treatment also increases hepatic Zn-MT levels. These results strongly suggest that Pd(II) ions can be bound in vivo by MT existing in the rabbit kidneys to form Pd-MT. Gel-filtration chromatographic studies after the incubation of either native Cd5Zn2MT2 or Zn7MT2 with K2PdCl4 in vitro demonstrate that Pd(II) ions promote the non-oxidative oligomerization of native MTs. Increasing the level of Pd(II) relative to MT led to a concomitant increase in the apparent yield of MT oligomers. At relatively low Pd-MT ratio, Pd(II) is found predominantly in the oligomers while the monomeric products are chiefly composed of the reactants, Cd5Zn2MT2 or Zn7MT2. Based on our experimental data, the mechanisms of the reactions between Pd(II) and MTs in vivo and in vitro are discussed.     

High-yield trapping of EGF-induced receptor dimers by chemical cross-linking

The binding of epidermal growth factor (EGF) to its plasma membrane receptor results in the stimulation of a tyrosyl residue-specific protein kinase, which has been shown to be part of the receptor. The mechanism by which EGF binding give rise to the stimulation of kinase activity is not understood in detail; however, a number of recent studies have implicated receptor dimerization or oligomerization in this process. We prepared Triton X-100 extracts of A431 cells in which the concentration of EGF receptors was on the order of 10(-7) M. When samples of the extracts were incubated with or without EGF and then treated with the high-yield cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3), covalent receptor dimers could be detected in high yield in samples that had been treated with both EGF and BS3, whereas only monomeric receptor was detected in untreated samples or in samples that had been treated with either EGF or BS3. The yield of receptor dimers trapped by cross-linking correlated with the stimulation of autophosphorylation by EGF and with the concentration of EGF present. EGF-induced receptor dimers were also efficiently cross-linked in highly purified receptor preparations, suggesting that EGF-induced dimerization is a process intrinsic to the receptor, requiring no additional accessory proteins.     

Epidermal growth factor induces rapid, reversible aggregation of the purified epidermal growth factor receptor

Epidermal growth factor (EGF) receptor from A-431 cells was purified by affinity chromatography with monoclonal anti-receptor antibodies. The purified radiolabeled receptor was incubated with EGF and then analyzed by gel electrophoresis under nondenaturing conditions. In these gels, the EGF receptor migrates in two forms: a fast-migrating (low) form and an EGF-induced slow-migrating (high) form. On the basis of the various control and calibration experiments described, it is concluded that the low form represents the monomeric 170-kilodalton EGF receptor and the high form represents an EGF receptor dimer. The binding of EGF causes a rapid, temperature-sensitive dimerization of the EGF receptor. Receptor dimerization is fully reversible and involves saturable, noncovalent interactions that are stable at neutral pH and in nonionic detergents. Both the monomeric and dimeric forms of the receptor bind EGF and undergo self-phosphorylation. The dimeric form of the receptor may possess higher ligand binding affinity, and it seems to be phosphorylated earlier than the monomeric form following the addition of EGF and [gamma-32P]ATP. On the basis of these results, it is concluded that receptor oligomerization is an intrinsic property of the occupied EGF receptor and that it may play a role in the activation of the kinase function and the subsequent transmembrane signaling process.     

Isolation and catalytic properties of the soluble monomeric form of inorganic pyrophosphatase from baker's yeast

Data from sedimentation analysis suggest that modification of about 40% of free amino groups of inorganic pyrophosphatase by maleic anhydride, pH 10.5, results in a loss of the enzyme ability to form dimers at neutral values of pH. The specific activity of monomeric pyrophosphatase is 50-80% of that of the dimeric form. The monomer has a pH optimum of about 7, requires metal ions for activation of both enzyme and substrate and is capable of exergonic synthesis of PPi in the active center. The enzyme binding to PPi is strongly stabilized by fluoride. The experimental data indicate that the individual subunit of inorganic pyrophosphatase possesses all the main catalytic properties of native dimeric molecule.     

Reactivity of Cd7-metallothionein with Cu(II) ions: evidence for a cooperative formation of Cd3,Cu(I)5-metallothionein

Reaction of Cd7-metallothionein-2 (MT) with Cu(II) ions has been studied by a variety of spectroscopic techniques including UV-absorption, circular dichroism (CD) and luminescence spectroscopy. The addition of up to 5 Cu(II) equivalents to Cd7-MT resulted in a cooperative formation of the monomeric Cd3,Cu5-MT form, as revealed by the analytical data and the presence of isosbestic or isodichroic points in the respective UV and CD spectra. The presence of Cu(I) luminescence and the absence of Cu(II) EPR signal indicated that copper is bound in the Cu(I) oxidation state, i.e., Cd3,Cu(I)5-MT. Consequently, the reduction of Cu(II) ions is accompanied by the oxidation of thiolate ligands of the protein. The absorption features and the luminescence data at 77 K are consistent with the presence of an air-stable Cu(I)-cluster in Cd3,Cu(I)5-MT. The participation of other ligands, besides cysteine thiolates, in metal coordination cannot be ruled out. With more than 5 Cu(II) equivalents added a mixture of unstable MT metalloforms were formed. The concomitant reduction and binding of copper ions by metallated MT represent a new aspect of the MT structure.     

Two EGF molecules contribute additively to stabilization of the EGFR dimer.

Receptor dimerization is generally considered to be the primary signaling event upon binding of a growth factor to its receptor at the cell surface. Little, however, is known about the precise molecular details of ligand-induced receptor dimerization, except for studies of the human growth hormone (hGH) receptor. We have analyzed the binding of epidermal growth factor (EGF) to the extracellular domain of its receptor (sEGFR) using titration calorimetry, and the resulting dimerization of sEGFR using small-angle X-ray scattering. EGF induces the quantitative formation of sEGFR dimers that contain two EGF molecules. The data obtained from the two approaches suggest a model in which one EGF monomer binds to one sEGFR monomer, and that receptor dimerization involves subsequent association of two monomeric (1:1) EGF-sEGFR complexes. Dimerization may result from bivalent binding of both EGF molecules in the dimer and/or receptor-receptor interactions. The requirement for two (possibly bivalent) EGF monomers distinguishes EGF-induced sEGFR dimerization from the hGH and interferon-gamma receptors, where multivalent binding of a single ligand species (either monomeric or dimeric) drives receptor oligomerization. The proposed model of EGF-induced sEGFR dimerization suggests possible mechanisms for both ligand-induced homo- and heterodimerization of the EGFR (or erbB) family of receptors.     

Differential scanning calorimetry of Cd(II) alkaline phosphatases

Differential scanning calorimetry has been employed to monitor structural alterations induced in the dimeric enzyme alkaline phosphatase on binding of Cd(II) (to the metal-free apoenzyme) and phosphate (Pi) (to the Cd(II) enzyme). Cd(II) addition to the apoenzyme at pH 6.5 results in an increased transition temperature, suggesting a stabilizing effect of the bound metal ion. Two distinct structural forms of the protein are detected as discrete calorimetric transitions (Tm = 69-84 degrees C; 87-94 degrees C, respectively). Distribution of the enzyme between these forms is found to depend on the exogenous Cd(II) concentration and the protocol of Cd(II) addition. These results indicate that conversion between the conformational forms is a slow process which appears to require specific levels of metal ion site occupancy. These studies, in which the exogenous Cd(II) concentration was varied from 10(-5) M to 10(-3) M suggest a structural basis for previously observed hysteretic phenomena observed on Cd(II) binding to the enzyme. Even at a minimum stoichiometry of Cd(II) (2 eq/mol of dimer) a single equivalent of Pi is sufficient to accelerate assumption of a stabilized form of the protein (Tm = 90 degrees C). This is followed by a slow structural change paralleling the time course of formation of the functional 2 Cd(II) phosphoryl enzyme which displays two calorimetric transitions (Tm = 65 degrees C, 88 degrees C). The low temperature transition does not appear if Pi is initially present at millimolar concentrations and is abolished on addition of Pi at concentrations in excess of 0.1 mM. These observations suggest the presence of a second, distinct Pi binding site on the 2 Cd(II) phosphoryl enzyme. This is supported by the changes observed in the 31P NMR chemical shift of Pi added to comparable enzyme samples. These data, including assessment of the effect of the presence of Mg(II), are discussed in terms of the mechanism of metal ion association to the enzyme and rearrangement of bound metal ions induced by Pi binding.     

113Cd NMR. Arsenate binding to Cd(II) alkaline phosphatase

Alkaline phosphatase from Escherichia coli contains three metal binding sites (A, B, and C) located at sites forming a triangle with sides of 4, 5, and 7 A (Wyckoff, H.W., Handschumacher, M., Murthy, K., and Sowadski, J.M. (1983) Adv. Enzymol. 55, 453). When all three sites are occupied by Cd(II) the enzyme has a very low turnover; at least 10(3) slower than the native Zn(II) enzyme. The slow turnover number has made the Cd(II) enzyme useful in NMR studies of the mechanism of alkaline phosphatase. The binding of arsenate to two forms of Cd(II) alkaline phosphatase (Cd(II)2alkaline phosphatase and Cd(II)6alkaline phosphatase) has been studied by 113Cd NMR. Cd(II)2alkaline phosphatase, pH 6.3, binds arsenate at only one monomer of the dimeric enzyme and causes migration of Cd(II) from the A site of one monomer to the B site of the arsenylated monomer. This same migration has previously been observed to accompany metal ion-dependent phosphate binding, but is much more rapid in the case of arsenate. The acceleration of migration induced by arsenate supports the conclusion based on the phosphate data that the substrate anion binds to the A site metal ion of one monomer prior to migration and that only the metal ion at A site is required for phosphorylation (arsenylation) of serine 102. The 113Cd chemical shifts of A and B site metal ions are very sensitive to the form of the bound arsenate, i.e. covalent (E-As) or noncovalent (E X As) complex. Like the analogous phosphate derivatives, the change of chemical shift of A site (to which phosphate is coordinated in the E X P complex) is much greater than that of the B site metal ion, when the arsenate shifts between the two intermediates, suggesting that arsenate is also coordinated to A site in the E X As intermediate. The chemical shifts of A and B site 113Cd(II) ions are considerably different in the arsenate and phosphate derivatives, while the C site 113Cd(II) ions have nearly identical chemical shifts. Thus the substrate appears to interact closely with both A and B sites, while C site appears relatively unimportant in phosphomonoester hydrolysis. The analogous behavior of arsenate and phosphate at the active center as evaluated by 113Cd NMR supports the validity of using the heavier arsenate derivative in x-ray diffraction studies.     

Dimerization of the insulin-like growth factor II/mannose 6-phosphate receptor

The insulin-like growth factor II/mannose 6-phosphate receptor (IGF2R) interacts with lysosomal enzymes through two binding domains in its extracytoplasmic domain. We report in the accompanying article (Byrd, J. C., and MacDonald, R. G. (2000) J. Biol. Chem. 275, 18638-18646) that only one of the two extracytoplasmic mannose 6-phosphate (Man-6-P) binding domains is necessary for high affinity Man-6-P ligand binding, suggesting that, like the cation-dependent Man-6-P receptor, oligomerization of the IGF2R contributes to high affinity interaction with lysosomal enzymes. In the present study, we have directly characterized both naturally occurring and engineered forms of the IGF2R for their ability to form oligomeric structures. Whereas gel filtration chromatography suggested that purified bovine IGF2R species exist in a monomeric form, native gel electrophoresis allowed for the separation of dimeric and monomeric forms of the receptors with distinct phosphomannosyl ligand binding characteristics. The ability of the IGF2R to form oligomeric complexes was confirmed and localized to the extracytoplasmic domain through the use of epitope-tagged soluble IGF2R constructs bearing deletions of the transmembrane and cytoplasmic domains. Finally, chimeric receptors were engineered containing the extracytoplasmic and transmembrane domains of the IGF2R fused to the cytoplasmic domain of the epidermal growth factor receptor with which dimerization of the chimeras could be monitored by measuring autophosphorylation. Collectively, these results show that the IGF2R is capable of forming oligomeric complexes, most likely dimers, in the absence of Man-6-P ligands.     

Chloride binding to alkaline phosphatase. 113Cd and 35Cl NMR

Chloride binding to alkaline phosphatase from Escherichia coli has been monitored by 35Cl NMR for the native zinc enzyme and by 113Cd NMR for two Cd(II)-substituted species, phosphorylated Cd(II)6 alkaline phosphatase and unphosphorylated Cd(II)2 alkaline phosphatase. Of the three metal binding sites per enzyme monomer, A, B, and C, only the NMR signal of 113Cd(II) at the A sites shows sensitivity to the presence of Cl-, suggesting that Cl- coordination occurs at the A site metal ion. From the differences in the chemical shift changes produced in the A site 113Cd resonance for the covalent (E-P) form of the enzyme versus the noncovalent (E . P) form of the enzyme, it is concluded that the A site metal ion can assume a five-coordinate form. The E-P form of the enzyme has three histidyl nitrogens as ligands from the protein to the A site metal ion plus either two water molecules or two Cl- ions as additional monodentate ligands. In the E . P form, there is a phosphate oxygen as a monodentate ligand and either a water molecule or a Cl- ion as the additional monodentate ligand. The shifts of the 113Cd NMR signals of the unphosphorylated Cd(II)2 enzyme induced by Cl- are very similar to those induced in the E-P derivative of the same enzyme, supporting the conclusion that the phosphoseryl residue is not directly coordinated to any of the metal ions. Specific broadening of the 35Cl resonance from bulk Cl- is induced by Zn(II)4 alkaline phosphatase, while Zn(II)2 alkaline phosphatase is even more effective, suggesting an influence by occupancy of the B site on the interaction of monodentate ligands at the A site. A reduction in this quadrupolar broadening is observed upon phosphate binding at pH values where E . P is formed, but not at pH values where E-P is the major species, confirming a specific interaction of Cl- at the A site, the site to which phosphate is bound in E . P, but not in E-P. For the zinc enzyme, a significant decrease in phosphate binding affinity can be shown to occur at pH 8 where one monomer has a higher affinity than the other.     

Impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau fragment corresponding to the third repeat of microtubule-binding domain

Environmental exposure to some heavy metals such as cadmium appears to be a risk factor for Alzheimer's disease (AD), however, definite mechanism of their toxicity in AD remains to be elucidated. Previous studies largely focused on the metal ions binding to beta-amyloid, however, very few papers concerned the interaction between tau and metal ions. For the first time, we investigated the impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau peptide R3, corresponding to the third repeat of microtubule-binding domain. The initial state of R3 was proven to be dimeric linked by intermolecular disulfide bond, in the non-reducing buffer (Tris-HCl buffer pH7.5, containing no reducing reagent). In this paper, we show that Cd(II) can accelerate heparin-induced aggregation of R3 or independently induce the aggregation of R3, as monitored by ThS fluorescence. In the presence of Cd(II), the resulting R3 filaments became much smaller, as revealed by electron microscopy. Binding to the Cd(II) ion, the dimeric R3 partially lost its random coil, and converted to alpha-helix structure, as revealed by CD and Raman spectrum. Stoichiometric analysis of CD signal against the ratio of [Cd(II)]/[R3] suggested that the coordination intermediate consisted of two R3 dimers binding to a central cadmium ion. As the seed, the coordination intermediate could extensively accelerate the self-aggregation of R3 via promoting the nucleation step. On the other hand, gain in alpha-helix structure on the peptide chain, by coordinating with Cd(II), could be a critical role to promote self-aggregation, as revealed by Raman spectrum. These results provide a further insight into the mechanism of tau filament formation and emphasize the possible involvement of Cd(II) in the pathogenesis of AD.     

Monomeric 14-3-3ζ Has a Chaperone-Like Activity and Is Stabilized by Phosphorylated HspB6

Members of the 14-3-3 eukaryotic protein family predominantly function as dimers. The dimeric form can be converted into monomers upon phosphorylation of Ser(58) located at the subunit interface. Monomers are less stable than dimers and have been considered to be either less active or even inactive during binding and regulation of phosphorylated client proteins. However, like dimers, monomers contain the phosphoserine-binding site and therefore can retain some functions of the dimeric 14-3-3. Furthermore, 14-3-3 monomers may possess additional functional roles owing to their exposed intersubunit surfaces. Previously we have found that the monomeric mutant of 14-3-3ζ (14-3-3ζ(m)), like the wild type protein, is able to bind phosphorylated small heat shock protein HspB6 (pHspB6), which is involved in the regulation of smooth muscle contraction and cardioprotection. Here we report characterization of the 14-3-3ζ(m)/pHspB6 complex by biophysical and biochemical techniques. We find that formation of the complex retards proteolytic degradation and increases thermal stability of the monomeric 14-3-3, indicating that interaction with phosphorylated targets could be a general mechanism of 14-3-3 monomers stabilization. Furthermore, by using myosin subfragment 1 (S1) as a model substrate we find that the monomer has significantly higher chaperone-like activity than either the dimeric 14-3-3ζ protein or even HspB6 itself. These observations indicate that 14-3-3ζ and possibly other 14-3-3 isoforms may have additional functional roles conducted by the monomeric state.     

Significance of 14-3-3 self-dimerization for phosphorylation-dependent target binding

14-3-3 proteins via binding serine/threonine-phosphorylated proteins regulate diverse intracellular processes in all eukaryotic organisms. Here, we examine the role of 14-3-3 self-dimerization in target binding, and in the susceptibility of 14-3-3 to undergo phosphorylation. Using a phospho-specific antibody developed against a degenerated mode-1 14-3-3 binding motif (RSxpSxP), we demonstrate that most of the 14-3-3-associated proteins in COS-7 cells are phosphorylated on sites that react with this antibody. The binding of these phosphoproteins depends on 14-3-3 dimerization, inasmuch as proteins associated in vivo with a monomeric 14-3-3 form are not recognized by the phospho-specific antibody. The role of 14-3-3 dimerization in the phosphorylation-dependent target binding is further exemplified with two well-defined 14-3-3 targets, Raf and DAF-16. Raf and DAF-16 can bind both monomeric and dimeric 14-3-3; however, whereas phosphorylation of specific Raf and DAF-16 sites is required for binding to dimeric 14-3-3, binding to monomeric 14-3-3 forms is entirely independent of Raf and DAF-16 phosphorylation. We also find that dimerization diminishes 14-3-3 susceptibility to phosphorylation. These findings establish a significant role of 14-3-3 dimerization in its ability to bind targets in a phosphorylation-dependent manner and point to a mechanism in which 14-3-3 phosphorylation and dimerization counterregulate each other.     

Studies on the aggregation reactions and basic dye binding of tobacco mosaic virus. I. Variation of pH,particle asymmetry,acid and base titration results,irreversible binding of methylene blue,ultraviolet absorption,and extent of heat denaturation in tobacco mosaic virus solutions with time of standing

1. Aqueous solutions of tobacco mosaic virus were found to undergo a number of spontaneous changes on standing in the cold. The results of pH measurements, acid and base titrations, intrinsic viscosity determinations, studies on the irreversible binding of methylene blue with the virus, ultraviolet absorption, and the extent of nucleic acid splitting by heat denaturation indicated the occurrence of two successive reactions, the first one causing the release of hydrogen ions and a greater lability of the nucleic acid, and the second one, which involved end-to-end dimerization and which took place after 8 days of standing, requiring hydrogen ions. 2. The first over-all reaction was found to be a mixture of various types of reversible disaggregation and aggregation reactions, the nature of which depended on the pretreatment, the TMV concentration, the time of standing, and the phosphate concentration. For longer times of standing at high protein concentration a sudden drop in ultraviolet absorption is noted after dilution; also the drops in viscosity and pH are largest with a steep rise following, indicating the greatest breakup of end-to-end aggregates with formation of the side-to-side type. For concentrated solutions of TMV in water which have not stood long no drop in ultraviolet absorption is noted on dilution; the decrease in the other quantities is less, indicating that only a less extensive breakdown of end-to-end aggregates occurs. Addition of phosphate to concentrated solutions of TMV causes formation of side-to-side aggregates which break up on dilution. 3. Using the results for the pH increase and the viscosity increase in a given time interval for a given TMV preparation and also the slope of the corresponding titration curve at the pH mean, a value for the number of hydrogen ions taken up per TMV monomer in the formation of the end-to-end dimer was finally calculated. The average result obtained for two preparations was 3300. 4. Methylene blue, in the polymeric form, was demonstrated to cause complete irreversible conversion of TMV monomers to end-to-end dimers. At dye concentrations above 10(-4)M, higher TMV polymers are formed, but these are broken down to dimers on removal of free dye by dialysis. The irreversible binding ratios were shown to be decreased in accordance with the extent of the end-to-end aggregation of the preparation at the time of the experiment, which is in agreement with the concept that the irreversibly bound dye polymers go into the junction formed between two interacting TMV monomers. On the basis that only the monomers initially present in solution can react, maximum binding ratios corresponding to complete conversion of monomers to dimers were calculated from the observed irreversible binding ratios and from the fraction of dimers initially present which was obtained from viscosity data. The average result for three preparations in different states of aggregation was calculated to be 6565 for tetrameric binding or 3230 for dimeric binding, which agrees closely with the result obtained for the uptake of hydrogen ions per TMV monomer in the spontaneous dimerization.     

Polydispersity of Bacillus thuringiensis Cry1 toxins in solution and its effect on receptor binding kinetics

Dynamic light scattering and surface plasmon resonance techniques were used to investigate the influence of ionic strength, buffer composition and pH on the multimerization of trypsin-activated Cry1Ac and Cry1C toxins over time and the subsequent effects of the different multimers on receptor binding models. In carbonate buffer at pH 10.5, Cry1Ac and Cry1C assumed a monomeric state. After 24 h, a complete conversion of monomeric toxin to a dimeric or trimeric form was observed only for Cry1Ac under low ionic strength condition. Cry1C and Cry1Ac in high ionic strength buffer remained monomeric. Substitution of CAPS pH 11 for carbonate buffer suppressed this Cry1Ac oligomerization effect. Once Cry1Ac toxin was in an aggregated form, increases in ionic strength failed to revert the aggregated toxin back to a monomeric form. Monomeric Cry1Ac bound to a purified 115 kDa aminopeptidase N receptor from Manduca sexta in a 2:1 molar ratio thus confirming the existence of two binding sites on this receptor. Binding rates of dimeric or higher aggregated Cry1Ac toxin forms were different from those generated using the monomeric form and could not be fitted to existing binding models. In summary, our results confirm that the M. sexta 115 kDa aminopeptidase N receptor possesses two Cry1Ac binding sites. They further suggest that although high pH and low salt conditions promote Cry1Ac aggregation, this observation cannot be applied universally to other members of the Cry family.     

Metal binding and antioxidant properties of chimeric tri- and tetra-domained metallothioneins

An unusual tri-domained (alpha-beta-beta) natural oyster metallothionein (MT) is known, and non-oxidative MT dimers occur in vivo in mollusk species and in mammals. To assess the respective role of the MT domains, two chimeric MTs were constructed: a tetra-domained oyster MT corresponding to the alpha-beta-alpha-beta structure, in order to mimic the natural non-oxidative dimeric form, and a tri-domained alpha-beta-alpha oyster MT. Metal binding and putative antioxidant properties of these two chimeric MTs were investigated using expression of the related genes in the bacteria Escherichia coli. In a wild-type strain these MTs could efficiently bind Cd. In a superoxide dismutase (sodA sodB) null mutant, the tri-domained MT was found to exacerbate Cd toxicity whereas the tetra-domained MT efficiently protected bacteria from Cd. The paradoxical toxicity displayed by the tri-domained MT upon Cd contamination was linked to the generation of superoxide radicals generated by a mechanism which most probably involves a copper-redox cycling reaction, since a Cd-contaminated sodA sodB strain expressing this MT produced 4 times more O2(-) than the control bacteria, and MT toxicity disappeared in the presence of bathocuproine disulfonic acid, a copper chelator. In contrast, the tetra-domained form did not. Interestingly, in bacteria producing superoxide dismutase but hypersensitive to oxidative stress due to either mutations in thioredoxin and glutathione reductase pathways (WM104 mutant) or to a lack of gamma-glutamylcysteine synthetase (gshA mutant), both chimeric MTs were protecting against Cd toxicity. However, an unexpected lack of antioxidant function was observed for both chimeric MTs, which were found to enhance the toxicity of hydrogen peroxide in WM104, or that of menadione in QC1726. Altogether, our results suggest that superoxide dismutase activity counteracts the potential prooxidative effect of the tri-domained MT mediated by Cu ions and that the tetra-domained form is a very efficient protector against metal toxicity in vivo.