Cadmium binding to metallothioneins. Domain specificity in reactions of alpha and beta fragments, apometallothionein, and zinc metallothionein with Cd2+

The cadmium-binding properties of rabbit liver Zn7-metallothionein (MT) 2 and apo-MT, rat liver apo-alpha MT and Zn4-alpha MT, and calf liver apo-beta MT, have been studied using circular dichroism (CD) and magnetic circular dichroism (MCD) spectroscopies. Both sets of spectra recorded during the titration of Zn7-MT 2 with Cd2+ exhibit a complicated pattern that is quite unexpected. Such behavior is not found at all in sets of spectra recorded during titrations of the apo-species (apo-MT, apo-alpha MT, and apo-beta MT), and is observed to a much lesser extent in the titration of Zn-alpha MT. Comparison between the band centers of the Cd-alpha MT and Cd-beta MT indicates that the CD spectrum of Cd7-MT is dominated by intensity from transitions that originate on Cd-S chromophores in the alpha domain, with little direct contribution from the beta domain. Analysis of the spectra recorded during titrations of Zn7-MT 2 with Cd2+ suggests: (i) that Cd2+ replaces Zn2+ in Zn7-MT isomorphously; (ii) that cadmium binds in a nonspecific, "distributed" manner across both domains; (iii) that cluster formation in the alpha domain only occurs after 4 mol eq of cadmium have been added and is indicated by the presence of a cluster-sensitive, CD spectral feature; (iv) that the characteristic derivative CD spectrum of native Cd4,Zn3-MT is only obtained from "synthetic" Cd4,Zn3-MT following a treatment cycle that allows the redistribution of cadmium into the alpha domain; warming the synthetic "native," Cd4,Zn3-MT, to 65 degrees C results in cadmium being preferentially bound in the alpha domain; and (v) Zn7-MT will bind Cd2+ quite normally at up to 65 degrees C but with greater specificity for the alpha domain compared with titrations carried out at 25 degrees C. These results suggest that the initial presence of zinc in both domains is an important factor in the lack of any domain specificity during cadmium binding to Zn-MT which contrasts the domain specific manner observed for cadmium binding to apo-MT.     

兔肝金属硫蛋白结合铅离子的圆二色性光谱研究

从锌诱导的家兔肝脏中分离纯化得到金属硫蛋白两种亚型：ＺｎＭＴ－Ⅰ和ＺｎＭＴ－Ⅱ．在酸性条件下脱金属,经Ｓｅｐｈａｄｅｘ Ｇ－２５柱层析得到脱金属硫蛋白（ａｐｏＭＴｓ）．用圆二色性（ＣＤ）光谱法研究,发现两种亚型ａｐｏＭＴｓ 与Ｐｂ２＋ 的结合依赖于Ｐｂ２＋ 的加入比例及ｐＨ 值．ａｐｏＭＴ－Ⅰ在ｐＨ３～５之间,ａｐｏＭＴ－Ⅱ在ｐＨ４～６之间与Ｐｂ２＋ 结合形成特征簇合物Ｐｂ７ＭＴｓ,其ＣＤ谱图特征峰位于３１６ｎｍ （－ ）,２７０ ｎｍ （＋ ）,２４５ ｎｍ （＋ ）及２２５ ｎｍ （－ ）,提示解铅中毒的最佳条件应控制在弱酸性环境．不同亚型ａｐｏＭＴｓ 与Ｐｂ２＋ 的结合方式各不相同：Ｐｂ２＋ 与ａｐｏＭＴ－Ⅰ的结合采取平均分配的方式,而与ａｐｏＭＴ－Ⅱ则为选择性结合方式,表明这两种亚型在解铅毒功能上存在差异．     

Cu+ distribution in metallothionein fragments

The differential distribution of Cu+ between separate alpha and beta domains of metallothionein (the isolated peptide fragments) and the rate of transfer of Cu+ between the two domains using copper-thiolate specific emission spectroscopy are reported. Kinetic data show the rate of transfer of Cu+ from the Cu6alpha to the Cd3beta domain is 2 x 10(-1) s(-1) while the transfer from Cu6beta to the Cd4alpha domain is much slower at 8 x 10(-3) s(-1), indicating the greater binding affinity of Cu+ for the MT beta domain. We report that the emission intensity of Cu6beta is 0.45 the emission intensity of Cu6alpha-MT. Lambda(max) is shown to be a probe of the environment of the Cu+. A series of copper-containing domain intermediates to the formation of the filled Cu6S9-beta and Cu6S11-alpha-clusters are identified. A mechanism is proposed for the formation of Cu12(betaalpha)-MT that involves metal exchange reactions of Cu+ ions from the alpha to the beta domain with initial formation of a Cu4beta-cluster.     

Circular dichroism, kinetic and mass spectrometric studies of copper(I) and mercury(II) binding to metallothionein

The metalloprotein metallothionein (MT) is remarkable in its metal binding properties: for the mammalian protein, well-characterized species exist for metal to sulfur ratios of M7S20, M12S20, and M18S20, where M = Cd(II), Zn(II), Hg(II), Ag(I), Au(I), and Cu(I). Optical spectra in general, and circular dichroism (CD) and luminescence spectra in particular, provide rich detail of a complicated metal binding chemistry when metals are added directly to the metal-free or zinc-containing protein. CD spectral data unambiguously identify key metal to protein stoichiometric ratios that result in well-defined structures. Electrospray ionization-mass spectrometry data are reported for reactions in which Hg(II) binds to apo-MT 2A as previously described from CD data. Emission spectra in the 450-750 nm region have been reported for metallothioneins containing Ag(I), Au(I), and Cu(I). The luminescence of Cu-MT can also be detected directly from mammalian and yeast cells. We report both steady-state and new dynamic data for titrations of Zn-MT with Cu(I). Analysis of kinetic data for the addition of the first two Cu(I) atoms to Zn-MT indicates a first-order mechanism over a concentration range of 5-50 microM. Three-dimensional modeling was carried out using the results of the CD and EXAFS studies, model calculations for Zn7-MT, Hg7-MT, and Cu12-MT are described.     

以FITC作为荧光探针研究金属硫蛋白的构象变化

本文以异硫氰基荧光素（ＦＩＴＣ）作为荧光探针标记于金属硫蛋白分子上,用荧光光谱研究了Ｃｄ＾２＋及Ａｇ＾＋离子与ＺｎＭＴ２－ＦＩＴＣ进行金属交换及与ＡｐｏＭＴ２－ＦＩＴＣ进行金属重组时的构象变化。结果表明,标记后ＭＴ与Ｃｄ＾２＋离子进行金属交换及金属重组时不具有明显的结构域特征,而Ａｇ＾＋离子进行金属交换及金属重组时,分别在Ａｇ６ＭＴ、Ａｇ１２ＭＴ及Ａｇ１８ＭＴ处具有明显的结构域形成特征。此外高温下     

Zinc(II) is required for the in vivo and in vitro folding of mouse copper metallothionein in two domains

We postulate that zinc(II) is a keystone in the structure of physiological mouse copper metallothionein 1 (Cu-MT 1). Only when Zn(II) is coordinated does the structure of the in vivo- and in vitro-conformed Cu-MT species consist of two additive domains. Therefore, the functionally active forms of the mammalian Cu-MT may rely upon a two-domain structure. The in vitro behaviour of the whole protein is deduced from the Cu titration of the apo and Zn-containing forms and compared with that of the independent fragments using CD, UV-vis, ESI-MS and ICP-AES. We propose the formation of the following Cu, Zn-MT species during Zn/Cu replacement in Zn7-MT: (Zn4)alpha(Cu4Zn1)beta-MT, (Cu3Zn2)alpha(Cu4Zn1)beta-MT and (Cu4Zn1)alpha(Cu6)beta-MT. The cooperative formation of (Cu3Zn2)alpha(Cu4Zn1)beta-MT from (Zn4)alpha(Cu4Zn1)beta-MT indicates that the preference of Cu(I) for binding to the beta domain is only partial and not absolute, as otherwise accepted. Homometallic Cu-MT species have been obtained either from the apoform of MT or from Zn7-MT after total replacement of zinc. In these species, copper distribution cannot be inferred from the sum of the independent alpha and beta fragments. The in vivo synthesis of the entire MT in Cu-supplemented media has afforded Cu7Zn3-MT [(Cu3Zn2)alpha(Cu4Zn1)beta-MT], while that of alpha MT has rendered a mixture of Cu4Zn1-alpha MT (40%), Cu5Zn1-alpha MT (20%) and Cu7-alpha MT (40%). In the case of beta MT, a mixture of Cu6-beta MT (25%) and Cu7-beta MT (75%) was recovered [1]. These species correspond to some of those conformed in vitro and confirm that Zn(II) is essential for the in vivo folding of Cu-MT in a Cu-rich environment. A final significant issue is that common procedures used to obtain mammalian Cu6-beta MT from native sources may not be adequate.     

Probing structural changes in the α and β domains of copper- and silver-substituted metallothionein by emission spectroscopy and electrospray ionization mass spectrometry

Steady-state emission spectra, excited-state lifetimes, kinetic data, and mass spectroscopic properties are reported for Ag(I)- and mixed Ag(I)/Cu(I)-substituted α and β domains of recombinant human metallothionein (MT1a). Kinetic analysis of the changes in the Cu(I) emission spectra during the stepwise displacement of Cu(I) ions by Ag(I) at room temperature shows that the rate of displacement of Cu(I) is unexpectedly slow. Although the first Ag(I) added results in major changes in the Cu(I)-MT binding site, Cu(I) displacement by Ag(I) does not take place until the addition of the third Ag(I), and is completed by the addition of the seventh Ag(I). The emission from Ag(I) and mixed Cu(I)/Ag(I)-MT species at 77 K shows that the band maxima shift as a function of Ag(I) loading, which can be correlated with shifts in coordination geometry from trigonal to digonal. Two phosphorescence lifetimes were detected for the Ag(I)-substituted α and β domains of MT, which are attributed to the presence of Ag(I) ions in two different environments. The lifetime of Ag(I)-substituted MT was found to be shorter when the Ag(I)-MT species were formed by Ag(I) additions to the Cu(I)-substituted α and β fragments than when the Ag(I)-MT species were formed from the apo-α and apo-β fragments, suggesting the formation of structurally different Ag(I)-MT clusters. Electrospray ionization mass spectrometric studies suggest the metallation reactions of Ag(I) with MT take place in a series of steps to form a series of Ag(I)-substituted MT species. Ag(I)-substituted MT species are not detected until past the addition of 3 mol equiv of Ag(I), suggesting that cluster formation begins only at this point, stabilizing the metallated species sufficiently to survive ionization.     

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.     

Cadmium-thiolate clusters in metallothionein: spectrophotometric and spectropolarimetric features

Cd-thiolate cluster formation in rabbit liver metallothionein 1 (MT) has been followed at pH 8.4 by monitoring spectroscopic features below 300 nm as a function of increasing Cd-to-apometallothionein (apoMT) ratio. The emerging absorption profiles form a family of closely similar spectra attributable to tetrahedral Cd-tetrathiolate coordination previously established for Cd7-MT [Vasák, M., K?gi, J.H.R., & Hill, H.A.O. (1981) Biochemistry, 20, 2852-2856]. However, there is a 6-nm red shift of the unresolved lowest energy absorption band when greater than 3 equiv of Cd(II) is incorporated. This shift is paralleled by a changeover in the circular dichroism (CD) features of MT from a broad monophasic positive CD profile with ellipticity bands near 240 and 220 nm to a biphasic CD spectrum characterized by positive ellipticity bands at 260 and 224 nm and an interposed negative band at 240 nm. Both features can be attributed to a changeover from separate Cd-tetrathiolate units formed at low metal-to-apoMT ratio to Cd-thiolate clusters when the supply of cysteine ligands becomes limiting. A comparable red shift signaling the transition from the mononuclear to a trinuclear tetrahedral Cd-tetrathiolate complex is also observed upon titration of the synthetic tetrathiol dodecapeptide N-Ac-Pro-Cys-Orn-Cys-Pro-Glu-Cys-Glu-Cys-Arg-Arg-Val with Cd(II). The latter studies also provide evidence for the predominantly ligand (sulfur) character of the lowest energy Cd-tetrathiolate ligand-metal charge-transfer transition. As a corollary it is inferred that the biphasic CD profile arises from excitonic coupling of these sulfur-centered transition dipole moments dissymmetrically oriented within the Cd(II)-thiolate clusters.     

Chemical foundation of the attenuation of methylmercury(II) cytotoxicity by metallothioneins.

To elucidate the chemical interactions underlying the role of metallothioneins (MTs) in reducing the cytotoxicity caused by MeHg(II), we monitored in parallel by electronic absorption and CD spectroscopies the stepwise addition of MeHgCl stock solution to mammalian Zn(7)-MT1 and the isolated Zn(4)-alphaMT1 and Zn(3)-betaMT1 fragments. The incorporation of MeHg(+) into Zn(7)-MT and Zn(3)-betaMT entails total displacement of Zn(II) and unfolding of the protein. However, both features are only partial for Zn(4)-alphaMT. The different behavior observed for this fragment, whether isolated or constituting one of the two domains of Zn(7)-MT, indicates interdomain interactions in the whole protein. Overall, the binding properties of Zn(7)-MT, Zn(4)-alphaMT and Zn(3)-betaMT toward MeHg(+) are unprecedented. In addition, the sequestration of MeHg(+) by Zn(7)-MT and the concomitant release of Zn(II) are probably two of the main contributions in the detoxifying role of mammalian MT.     

Alpha beta TCR+ cells are a minimal fraction of peripheral CD8+ pool in MHC class I-deficient mice

MHC class I molecules play a role in the maintenance of the naive peripheral CD8+ T cell pool. The mechanisms of the peripheral maintenance and the life span of residual CD8+ cells present in the periphery of beta 2-microglobulin-deficient (beta 2m-/-) mice are unknown. We here show that very few CD8+ cells in beta 2m-/- mice coexpress CD8 beta, a marker of the thymus-derived CD8+ T cells. Most of the CD8 alpha+ cells express CD11c and can be found in beta 2m/RAG-2 double-deficient mice, demonstrating that these cells do not require rearranged Ag receptors for differentiation and survival and may be of dendritic cell lineage. Rare CD8 alpha+CD8 beta+ cells can be detected following in vivo alloantigenic stimulation 2 wk after the adult thymectomy. Selective MHC class I expression by bone marrow-derived cells does not lead to an accumulation of CD8 beta+ cells in beta 2m-/- mice. These findings demonstrate that 1) thymic export of CD8+ T cells in beta 2m-/- mice is reduced more severely than previously thought; 2) non-T cells expressing CD8 alpha become prominent when CD8+ T cells are virtually absent; 3) at least some beta 2m-/- CD8+ T cells have a life span in the periphery comparable to wild-type CD8+ cells; and 4) similar ligands induce positive selection in the thymus and survival of CD8+ T cells in the periphery.     

Gamma delta T cells assist alpha beta T cells in adoptive transfer of contact sensitivity.

Cutaneous immune responses to contact sensitizers such as picryl chloride or oxazolone, are classical manifestations of T cell-mediated immunity in vivo. In fact, the first documentation of T cell-mediated immunity was the ability to adoptively transfer contact sensitivity (CS) responses. Although it is now clear that Ag/MHC-restricted alpha beta TCR positive effector T cells are responsible for 24 to 48 h CS responses, other subsets of Thy-1+ cells in mice also participate in the elicitation of CS. Thus, Thy-1+, CD5+, CD3-, B220+, hapten-specific, non-MHC-restricted early-acting cells are required to initiate CS responses by leading to local serotonin release, which allows for extravascular recruitment of the late-acting, alpha beta TCR+, CS effector T cells. This study describes another T cell population that is needed for the adoptive transfer of CS by alpha beta T cells. In vitro treatment of a mixture of CS effector cells with hamster mAb to gamma delta TCR, together with rabbit complement, or by panning on anti-hamster Ig-coated dishes, diminished substantially the subsequent transfer of CS reactivity without affecting either CS-initiating cells, or the later-acting, alpha beta TCR+ CS effector T cells. Immune cells treated with anti-alpha beta TCR mAb, or recovered as adherent cells from petri dishes after anti-gamma delta TCR panning (i.e., gamma delta TCR-enriched cells), reconstituted the ability of anti-gamma delta TCR-treated immune cells (i.e., alpha beta TCR-enriched cells) to transfer 24-h CS responsiveness. The phenotype of the gamma delta T cells that assisted CS effector alpha beta T cells was: CD3+, CD4-, and CD8+. The gamma delta T cells that assisted alpha beta T cells were not Ag-specific since anti-alpha beta-TCR-treated cells (gamma delta T-enriched) from picryl chloride immunized donors aided alpha beta T cells (anti-gamma delta TCR-treated) from oxazolone-immunized donors, and conversely gamma delta T cells from oxazolone-immunized donors aided alpha beta T cells from picryl chloride immunized donors. Furthermore, the CS-regulating gamma delta T cells were not MHC-restricted because gamma delta T cells from H2d or H2b donors could assist alpha beta T cells from H2k donors. It was concluded that a regulatory population of non-Ag specific, non-MHC-restricted gamma delta T cells was needed to assist immune effector, Ag/MHC-specific alpha beta T cells in the adoptive transfer of CS.     

The Alzheimer beta-peptide shows temperature-dependent transitions between left-handed 3-helix, beta-strand and random coil secondary structures

The temperature-induced structural transitions of the full length Alzheimer amyloid beta-peptide [A(beta)(1-40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left-handed 3(1) (polyproline II; PII)-helix at 0 degrees C to a random coil state at 60 degrees C, with an average DeltaH of 6.8 +/- 1.4 kJ.mol(-1) per residue, obtained by fitting a Zimm-Bragg model to the CD data. The transition is noncooperative for the shortest N-terminal fragment A(beta)(1-9) and weakly cooperative for A(beta)(1-40) and the longer fragments. By analysing the temperature-dependent 3J(HNH(alpha)) couplings and hydrodynamic radii obtained by NMR for A(beta)(1-9) and A(beta)(12-28), we found that the structure transition includes more than two states. The N-terminal hydrophilic A(beta)(1-9) populates PII-like conformations at 0 degrees C, then when the temperature increases, conformations with dihedral angles moving towards beta-strand at 20 degrees C, and approaches random coil at 60 degrees C. The residues in the central hydrophobic (18-28) segment show varying behaviour, but there is a significant contribution of beta-strand-like conformations at all temperatures below 20 degrees C. The C-terminal (29-40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of A(beta) may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.     

Platinum(II) binding to metallothioneins

The reaction of equine renal metallothionein (MT) with excess K2PtCl4 at pH 2 results in a polymeric adduct containing 17 +/- 2 mol Pt/mol MT. A monomeric adduct containing 7 mol Pt/mol MT is obtained at neutral pH. Rates of reaction of Pt7MT with DTNB and iodoacetic acid are consistent with Pt2+ to cysteine thiolate coordination, and the extent of reaction in both cases is 11 +/- 2 mol cys/mol MT. Adducts from the reaction of K2PtCl4 with apoMT chemically modified at the N-terminal methionine residue, Cd7MT, and native MT are also reported. A structural model of Pt7MT is proposed in which the square planar tetrathiolate Pt(II) unit is incorporated into a three-metal beta cluster. Implications for the metabolism of platinum anticancer drugs are discussed.     

Cadmium binding and metal cluster formation in metallothionein: a differential modification study

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively [Otvos, J.D., & Armitage, I. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. The formation of these oligonuclear complexes designated as Cd4 and Cd3 clusters has now been monitored in MT reconstituted with varying amounts of Cd by using differential chemical modification of Cys with [14C]iodoacetamide. At ratios below 2-3 mol of Cd/mol of MT bound, no differential protection of Cys by the metal, and hence no preferred binding, is detectable. At Cd-to-protein ratios between 3 and 5 mol of Cd/mol of MT, the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain, indicating formation of the Cd4 cluster. At still higher ratios, formation of the Cd3 cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labeling of the different Cys in Cd7-MT indicates that the ligands of the Cd3 cluster are 3 times as accessible to iodoacetamide than those of the Cd4 cluster, suggesting a greater thermodynamic or kinetic stability of the latter.     

Spectral changes upon subunit association in valency hybrid hemoglobins

The absorption, circular dichroism (CD) and magnetic circular dichroism (MCD) spectra of valency hybrid hemoglobins and their constituents (alpha + and beta chains for alpha 2+beta 2, alpha and beta + chains for alpha 2 beta 2+: + denotes ferric heme) were measured in the Soret region for F-, H2O, N3- and CN- derivatives. Absorption and MCD spectra of valency hybrid hemoglobins were very similar to the arithmetic mean of respective spectra of their corresponding component chains in all derivatives. The Soret MCD intensity around 408 nm for various complexes of valency hybrid hemoglobins seems to reflect the spin state of ferric chains. Upon ferric and deoxy ferrous subunit association to make the deoxy valency hybrid hemoglobins, only the high-spin forms bound with F- and H2O of alpha 2+beta 2 displayed a blue shift in the peak position around 430 nm and those of alpha 2 beta 2+ an increase in intensity around 430 nm. The blue shift and the increase in intensity were considered to be caused by the structural changes in deoxy beta chains of alpha 2+beta 2 and deoxy alpha chains of alpha beta 2+, respectively. These spectral changes were interpreted on the basis of their oxygen-equilibrium properties. In contrast to absorption and MCD spectra, the CD spectra of valency hybrid hemoglobins were markedly different from the simple addition of those of their component chains in all derivatives examined. The large part of CD spectral changes upon subunit association were interpreted as changes in the heme vicinity accompanied by formation of the alpha 1 beta 1 subunit contact.     

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.     

M?ssbauer studies on the metal-thiolate cluster formation in Fe(II)-metallothionein

The stepwise 57Fe(II)-thiolate cluster formation in rabbit liver metallothionein-2 (MT) has been followed at pH 8.5 using M?ssbauer spectroscopy. The zero-field spectra recorded at 4.2 K exhibit at all stages of filling one virtually identical single quadrupole splitting delta EQ and isomer shift delta as found for reduced rubredoxin (Rdred) or the model compound [Fe(II)(SPh)4]2-, thus indicating an Fe(II)-tetrathiolate coordination. A similar conclusion was reached also in previous electronic absorption studies [M. Good and M. Vasák (1986) Biochemistry 25,8353--8356]. The M?ssbauer spectra obtained in the presence of a magnetic field were analyzed on the basis of a spin-Hamiltonian formalism resulting in M?ssbauer parameters similar to those for Rdred and the inorganic model compound [Fe(II)(SPh)4]2-. The identity of the M?ssbauer parameters of partially and fully metal-occupied MT suggests that a comparable distortion of the metal binding sites must exist. Simulation of the spectra revealed that the Fe(II) ions in the partially metal-occupied 57Fe(II)4-MT form appear to be magnetically isolated, whereas in the fully metal-saturated 57Fe(II)7-MT form a ratio of 3:4 of paramagnetic to diamagnetic subspectra was obtained. The latter result suggests the existence of three isolated metal binding sites and a metal-thiolate cluster containing four metal ions. In the light of structure determinations of MT containing Zn(II) and/or Cd(II) [W. Braun et al. (1986) J. Mol. Biol. 187, 125-129, and W. F. Furrey et al. (1986) Science (Wash. DC) 231, 704-710], which revealed two metal-thiolate clusters containing three and four metal ions, respectively, and involving all 20 cysteine residues in metal binding, the appearance of M?ssbauer parameters characteristic of three isolated Fe(II) sites in 57Fe(II)7-MT is peculiar and deserves further studies. It is concluded, moreover, that the four-metal cluster is diamagnetic with the four Fe(II) ions being antiferromagnetically coupled. The appearance of magnetic coupling above four Fe(II) equivalents bound to apoMT indicates that the cluster formation occurs in a two-step process.     

Expression and function of a 90-kilodalton homodimeric molecule (10D1 antigen) on human thymocytes

The 10D1 Ag is a 90-kDa homodimeric molecule specifically expressed on a subpopulation of human T cells, and is involved in an alternative pathway of T cell activation. In the present study, we have examined the expression and function of the 10D1 Ag on human thymocytes. Three-color FMF analysis showed that the 10D1 Ag was highly expressed on minor but distinct subpopulations of double-negative and CD4 single-positive thymocytes, and weakly on a part of double-positive thymocytes, but not on CD8 single-positive thymocytes. In double-negative thymocytes, the vast majority of 10D1+ cells were immature thymocytes of CD7+2+3- phenotype. Interestingly, 10D1 mAb could induce the proliferation of CD4 single-positive thymocytes in the presence of goat anti-mouse Ig to cross-link the 10D1 Ag. The treatment of thymocytes with OKT4 mAb plus C but not with OKT8 mAb plus C totally abrogated the proliferative response induced by 10D1 mAb, indicating that the 10D1-responsible thymocytes were of CD4+8- phenotype. This 10D1 mAb-induced thymocyte proliferation was perfectly dependent on the endogenous IL-2/IL-2R system since a complete inhibition was observed with anti-IL-2 and anti-IL-2R mAb. The proliferating CD4 single positive thymocytes predominantly expressed the IL-2R alpha (p55) but not a detectable level of the IL-2R beta (p75). These results indicate that, although the 10D1 Ag can be detected on the CD7+2+3-4-8- thymocytes, its functional expression is restricted to a minor more mature CD4+ thymocyte population as well as in peripheral blood T cells, and the implications of these findings are discussed.