Multiple DNA-binding estrogen receptor forms resolved by interaction with immobilized metal ions. Identification of a metal-binding domain

Immobilized metal ions have been used to characterize and locate metal ion-specific binding domains on the surface of the DNA-binding form of the estrogen receptor protein. Soluble estrogen receptors in calf uterine cytosol were labeled with [3H]estradiol and transformed to the DNA-binding configuration by brief exposure (30 min) to 3 M urea at 0-4 degrees C. The transformed receptors were purified in the presence of 3 M urea using single-stranded calf thymus DNA-agarose and characterized by high-performance size-exclusion chromatography (Stokes radius of 7.0-7.5 nm) and sucrose density gradient centrifugation (4.25 S) as dimers of 130,000 Da. Such receptor preparations subsequently labeled with [3H]desmethylnafoxidine aziridine by ligand exchange revealed one major peak of radioactivity (67 kDa) by sodium dodecyl sulfate polyacrylamide gradient gel electrophoresis. When analyzed by immobilized metal ion affinity chromatography on iminodiacetate (IDA)-agarose loaded with Cu(II), Ni(II), or Zn(II) ions, the receptor was bound with various degrees of affinity and metal interaction heterogeneity even in the presence of 0.5 M NaCl to neutralize electrostatic interactions. The intact DNA-binding receptor dimers were most tightly bound to IDA-Cu(II) and IDA-Ni(II), but were eluted with 100-200 nM imidazole. The receptors were bound less tightly to IDA-Zn(II), and four separate peaks of receptor activity were resolved by elution with 10, 15, 30, and 100 mM imidazole (n = 27). Limited trypsin digestion of the DNA-binding receptor forms resulted in the generation of a 2.8-nm fragment with both the DNA-binding and metal-binding domains removed or destroyed. These results demonstrate that DNA-binding estrogen receptor dimers have high affinity metal ion-binding sites which are located at the DNA-binding domain. We have found (Zn(II) interaction chromatography to be unique thus far in its ability to resolve separate DNA-binding receptor forms.     

Immobilized metal ion affinity chromatography of serum albumins.

The interaction of several serum albumins with chelated (iminodiacetate, IDA) and immobilized (agarose-IDA) metal ions, Co2+, Ni2+, Cu2+ and Zn2+, was studied. There was no retention of human, bovine, porcine, murine and avian albumins on IDA-Zn(II) and IDA-Co(II) columns. However, all albumins studied, i.e., those of: man, cow, pig, dog, rabbit, rat, mouse, chicken and pigeon were retained on IDA-Cu(II) columns, and all except dog albumin were retained also on IDA-Ni(II). The recognition of albumins by chelated and immobilized transition metals seems to be related to an affinity for the imidazole side chains. It is postulated that one to three imidazoles is involved in this interaction, under the employed experimental conditions (pH 7.0; 1 M sodium chloride). There is no evidence for any significant contribution of tryptophan or cysteine (Cys 34) residues to the chromatographic event. The retention of defatted albumin and albumin oligomers (human), on IDA-Cu(II) columns was not significantly different from that of non-defatted albumin or albumin monomer, respectively.     

Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography

High-performance immobilized metal ion affinity chromatography was utilized to evaluate the adsorption properties of 67 synthetic, biologically active, peptides ranging in size from 5 to 42 residues. The metal ions, Cu(II), Ni(II) and Zn(II), were immobilized by iminodiacetic acid (IDA) coupled to TSK gel 5PW (10 microns). Two types of gradient elution (imidazole and pH) were used to evaluate peptide retention by the metal ions. A decreasing pH gradient and an increasing imidazole gradient eluted the peptides in similar order. IDA-Cu(II) and IDA-Zn(II) showed very similar selectivities for the peptides analyzed; however, IDA-Zn(II) displayed a weaker affinity for the peptides. IDA-Ni(II) showed a slightly different pattern of selectivity. Peptide adsorption effects contributed by the metal-free gel matrix were found to be relatively minor. The concentration and type of salt included in the mobile phase could affect the relative affinities of the peptides for the immobilized metal ions. Retention coefficients were assigned to individual amino acid residues by multiple linear regression analysis. Histidine showed the largest positive correlation with retention, followed by aromatic amino acid residues. Modified N-terminal residues resulted in negative contributions to retention. Analyses of peptide amino acid composition alone allowed prediction of peptide retention behavior on immobilized metal ion affinity columns.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

Surface topography of histidine residues in lysozymes.

Several avian and mammalian c-type lysozymes were chromatographed on chelated (to iminodiacetate) and immobilized transition metal ions (Co2+, Ni2+, Cu2+ and Zn2+) under a variety of experimental conditions. The varied affinity of evolutionary variants of the lysozyme family for chelated metal ions, IDA-M(II), can be rationalized primarily in terms of the presence, multiplicity and microenvironments of histidine residues. The chromatographic resolution of some of these closely related proteins attests to the analytical power of immobilized metal-ion affinity chromatography.     

Purification and properties of the synthetase catalyzing the biotination of the aposubunit of transcarboxylase from Propionibacterium shermanii

The synthetase that attaches biotin to the aposubunit of transcarboxylase (biotin-[methylmalonyl-CoA-carboxyltransferase]ligase) (EC 6.3.4.9) was purified to homogeneity by ion-exchange chromatography on cellulose DE-52 and CM-cellulose. The synthetase is a monomer of molecular weight 30,000. The pH and temperature optima for the synthetase are 6.0 and 37 degrees C, respectively. The apparent Km for the substrates ATP, biotin, and apo 1.3 S subunit of apotranscarboxylase are 38, 2.0, and 0.9 microM, respectively. Ni2+, Co2+, Zn2+, or Mn2+ could replace Mg2+ in the reaction. The affinity of synthetase toward metals is as follows: Zn2+ greater than Ni2+ greater than Mn2+ greater than Co2+ greater than Mg2+, and the activity with Zn2+ was much greater than that with the other divalent metals. EDTA completely inactivates the enzyme. The metals are necessary not only for the catalytic activity but also for the storage stability of the enzyme. The synthetase shows absolute specificity toward ATP.     

Detection of two Zn-finger proteins ofXenopus laevis,TFIIIA, and p43, by probing western blots of ovary cytosol with65Zn2+,63Ni2+, or109Cd2+

Two Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenile Xenopus laevis females by in vitro binding of radiolabeled divalent metals. Proteins fractionated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) were transferred by Western blotting onto nitrocellulose membranes, probed with 65Zn2+, 63Ni2+, or 109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 micrograms/well by 109Cd(2+)-probing, 0.13 micrograms/well by 65Zn(2+)-probing, and 0.26 mu/well by 63Ni(2+)-probing. Protein p43 was more clearly visualized by probing with 63Ni2+ than with 65Zn2+ or 109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide, 65Zn2+, 109Cd2+, and 63Ni2+ distinctly labeled the 22 kDa middle fragment; 65Zn2+ and 109Cd2+ also labeled the 11 kDa N-terminal fragment, but did not label the 13 kDa C-terminal fragment. These results are consistent with the notion that the radioligands were bound to finger-loop domains of TFIIIA, which occur in the middle and N-terminal fragments. Based on the abilities of nonradioactive metal ions to compete with 65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+ = Cu2+ greater than or equal to Hg2+ greater than Cd2+ greater than Co2+ greater than or equal to Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with 65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43.     

Removal of heavy metals from water effluents using supermacroporous metal chelating cryogels

Applications of IDA in, for example, immobilized metal ion affinity chromatography for purification of His-tagged proteins are well recognized. The use of IDA as an efficient chelating adsorbent for environmental separations, that is, for the capture of heavy metals, is not studied. Adsorbents based on supermacroporous gels (cryogels) bearing metal chelating functionalities (IDA residues and ligand derived from derivatization of epoxy-cryogel with tris(2-aminoethyl)amine followed by the treatment with bromoacetic acid (defined as TBA ligand)) have been prepared and evaluated on capture of heavy metal ions. The cryogels were prepared in plastic carriers, resulting in desired mechanical stability and named as macroporous gel particles (MGPs). Sorption and desorption experiments for different metals (Cu2+, Zn2+, Cd2+, and Ni2+ with IDA adsorbent and Cu2+ and Zn2+ with TBA adsorbent) were carried out in batch and monolithic modes, respectively. Obtained capacities with Cu2+ were 74 μmol/mL (TBA) and 19 μmol/mL gel (IDA). The metal removal was higher for pH values between pH 3 and 5. Both adsorbents showed improved sorption at lower temperatures (10°C) than at higher (40°C) and the adsorption significantly dropped for the TBA adsorbent and Zn2+ at 40°C. Desorption of Cu2+ by using 1 M HCl and 0.1 M EDTA was successful for the IDA adsorbent whereas the desorption with the TBA adsorbent needs further attention. The result of this work has demonstrated that MGPs are potential treatment alternatives within the field of environmental separations and the removal of heavy metals from water effluents.     

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.     

Unique molecular properties of a urea- and salt-stable DNA-binding estrogen receptor dimer covalently labeled with the antiestrogen [3H]desmethylnafoxidine aziridine. A comparison with the estrogen-receptor complex

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.     

Metal binding by citrus dehydrin with histidine-rich domains

Dehydrins are hydrophilic proteins that are responsive to osmotic stress, such as drought, cold, and salinity in plants. Although they have been hypothesized to stabilize macromolecules in stressed cells, their functions are not fully understood. Citrus dehydrin, which accumulates mainly in response to cold stress, enhances cold tolerance in transgenic tobacco by reducing lipid peroxidation. It has been demonstrated that citrus dehydrin scavenges hydroxyl radicals. In this study, the metal binding of citrus dehydrin is reported and the specific domain responsible is identified. The metal binding property of citrus dehydrin was tested using immobilized metal ion affinity chromatography (IMAC). Fe3+, Co2+, Ni2+, Cu2+, and Zn2+ bound to citrus dehydrin, but Mg2+, Ca2+, and Mn2+ did not. Among the bound metals, the highest affinity was detected for Cu(2+)-dehydrin binding, which showed a dissociation constant of 1.6 microM. Citrus dehydrin was able to bind up to 16 Cu2+ ions. IMAC indicated that His residues contributed to Cu(2+)-dehydrin binding. The amino acid sequence of CuCOR15 was divided into five domains, of which domain 1 bound Cu2+ most strongly. One portion of domain 1, HKGEHHSGDHH, was the core sequence for the binding. These results suggest that citrus dehydrin binds metals using a specific sequence containing His. Since citrus dehydrin is a radical-scavenging protein, it may reduce metal toxicity in plant cells under water-stressed conditions.     

Chromatographically distinguishable heme insertion isoforms of human hemopexin

Two spectroscopically distinct, non-interconverting forms of human hemopexin have been isolated by immobilized metal ion affinity chromatography and characterized spectroscopically. Form alpha (characterized by a bisignate Soret CD spectrum) and form beta (Soret CD characterized by a positive Cotton effect) exhibit different spectroscopic responses to addition of Zn2+ or Cu2+, yet both forms exhibit the same metal ion-induced decrease in Tm for the thermally induced release of the heme prosthetic group. Far UV-CD spectra indicate that the two isoforms possess essentially identical secondary structures, but their differential retention during metal ion affinity chromatography indicates slight differences in exposure of His residues on the protein surface. We propose that these observations result from the binding of heme in form beta with an orientation that differs from the crystallographically observed binding orientation for rabbit hemopexin by rotation of the heme prosthetic group by 180 degrees about the alpha-gamma meso-carbon axis and from interaction of metal ions at two separate binding sites.     

Ordered association of tobacco mosaic virus in the presence of divalent metal ions

The formation of ordered aggregates of tobacco mosaic virus (TMV) in the presence of divalent metal ions has been studied in concentrated (1-25 mg/ml) solutions of the virus. The divalent metal cations Cd2+, Zn2+, Pb2+, Cu2+, and Ni2+ have been found to promote TMV precipitation from solution at a critical concentration Ccrit, which for a given metal depends on the pH and the ionic strength of the solution, but is largely independent of the virus concentration. The TMV precipitate behaves as a nematic liquid crystal and on drying at a glass surface produces highly ordered, optically birefringent films. However, precipitation is not observed with alkali-earth metals such as Ca2+ and Mg2+. The experimental data suggest that, apart from two 'internal' metal-binding sites in each TMV subunit, the virus contains metal-binding sites of a lower affinity which promote cross-linking of TMV rods via metal bridges. The latter seem to be responsible for the precipitation of TMV in the presence of divalent cations at neutral pH. We propose that the metal-induced cross-linking may be the predominant mechanism to account for the limited solubility of a variety of proteins in solution containing metal cations with valence 2 and higher.     

Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris)

Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.     

Metal chelate affinity chromatography of Zn2+-inhibited protein Tyr(P) phosphatases

Metal chelate affinity chromatography using Zn2+-iminodiacetate agarose is shown to provide quantitative recoveries of Zn2+-inhibited protein Tyr(P) phosphatases. To elute adsorbed enzymes from immobilized Zn2+ three methods were compared: (1) removal of Zn2+ with chelators such as EDTA, (2) introduction of ligands to compete with enzyme for Zn2+ and (3) lowering pH to protonate sidechains in the enzyme that serve as ligands to Zn2+. Results show highest yields but poor purification for method 1, high purification but poor yields of active enzyme for method 3. It is concluded that gradients of competing Zn2+ ligands, such as imidazole, provide the best strategy for the purification of enzymes with retention of activity using metal chelate affinity chromatography.     

Purification and properties of urease from bovine rumen.

Urease (urea amidohydrolase, EC 3.5.1.5) was extracted from the mixed rumen bacterial fraction of bovine rumen contents and purified 60-fold by (NH4)2SO4 precipitation, calcium phosphate-gel adsorption and chromatography on hydroxyapatite. The purified enzyme had maximum activity at pH 8.0. The molecular weight was estimated to be 120000-130000. The Km for urea was 8.3 X 10(-4) M+/-1.7 X 10(-4) M. The maximum velocity was 3.2+/-0.25 mmol of urea hydrolysed/h per mg of protein. The enzyme was stabilized by 50 mM-dithiothreitol. The enzyme was not inhibited by high concentrations of EDTA or phosphate but was inhibited by Mn2+, Mg2+, Ba2+, Hg2+, Cu2+, Zn2+, Cd2+, Ni2+ and Co2+. p-Chloromercuribenzenesulfphonate and N-ethylmaleimide inhibited the enzyme almost completely at 0.1 mM. Hydroxyurea and acetohydroxamate reversibly inhibited the enzyme. Polyacrylamide-gel electrophoresis showed that the mixed rumen bacteria produce ureases which have identical molecular weights and electrophoretic mobility. No multiple forms of urease were detected.     

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions

Essentially all bacteria have genes for toxic metal ion resistances and these include those for Ag+, AsO2-, AsO4(3-), Cd2+ Co2+, CrO4(2-), Cu2+, Hg2+, Ni2+, Pb2+, TeO3(2-), Tl+ and Zn2+. The largest group of resistance systems functions by energy-dependent efflux of toxic ions. Fewer involve enzymatic transformations (oxidation, reduction, methylation, and demethylation) or metal-binding proteins (for example, metallothionein SmtA, chaperone CopZ and periplasmic silver binding protein SilE). Some of the efflux resistance systems are ATPases and others are chemiosmotic ion/proton exchangers. For example, Cd2+-efflux pumps of bacteria are either inner membrane P-type ATPases or three polypeptide RND chemiosmotic complexes consisting of an inner membrane pump, a periplasmic-bridging protein and an outer membrane channel. In addition to the best studied three-polypeptide chemiosmotic system, Czc (Cd2+, Zn2+, and Co2), others are known that efflux Ag+, Cu+, Ni2+, and Zn2+. Resistance to inorganic mercury, Hg2+ (and to organomercurials, such as CH3Hg+ and phenylmercury) involve a series of metal-binding and membrane transport proteins as well as the enzymes mercuric reductase and organomercurial lyase, which overall convert more toxic to less toxic forms. Arsenic resistance and metabolizing systems occur in three patterns, the widely-found ars operon that is present in most bacterial genomes and many plasmids, the more recently recognized arr genes for the periplasmic arsenate reductase that functions in anaerobic respiration as a terminal electron acceptor, and the aso genes for the periplasmic arsenite oxidase that functions as an initial electron donor in aerobic resistance to arsenite.