Studies on the uptake of radioactive copper by sheep erythrocytes in vitro

From previous studies different mechanisms for uptake of Cu by mammalian tissues from buffer and media containing proteins have been proposed. The interpretation of some of these investigations may have been complicated by the binding of Cu to proteins in the media.The uptake of ⁶⁴Cu from both buffer and plasma has therefore been studied using sheep erythrocytes in order to determine the mechanism and the effects of protein. Cu uptake was proportional to concentration of added Cu and the kinetics were those of a first order reaction for both media. There was no evidence for the participation of a membrane carrier and studies with inhibitors indicated that active transport was not involveed. For a given concentration of added Cu the rate of uptake was much slower in plasma and the effective concentration was calculated to be about 13% of that added. Efflux from labelled cells was much faster into plasma than into buffer. The addition of histidine to the medium increased uptake from dialysed plasma but not from buffer.It is concluded that Cu is taken up by the erythrocyte by simple diffusion, and the reduced rate in plasma is due to the binding of Cu to plasma protein thus reducing the effective concentration. The effect of histidine is attributed to the formation of a Cu-histidine complex which exists in equilibrium with Cu bound to albumin and facilitates removal of Cu from the latter.     

Copper and molybdenum absorption by rats given ammonium tetrathiomolybdate

Previous studies have shown that the tetrathiomolybdate ion [MoS_4^2?] is a potent antagonist of Cu metabolism. Effects of orally administered MoS_4^2? on the absorption and tissue distribution of ⁶⁴Cu in rats have now been investigated. Four or 12 mg Mo/kg diet, when given as MoS_4^2?, strongly inhibited ⁶⁴Cu absorption and modified the fate of absorbed Cu, decreasing hepatic and renal uptake but increasing plasma retention of ⁶⁴Cu. These effects were not induced by equivalent dietary concentrations of Mo as MoS_4^2? or when S^2? was given as CaS. Clinical and biochemical effects induced by orally administered MoS_4^2? were abolished by increasing dietary concentrations of Cu. Such treatment also inhibited the absorption and tissue retention of ⁹⁹Mo derived from ⁹⁹MoS_4^2?. Intraperitoneal administration of Cu ameliorated clinical effects attributable to MoS_4^2? but neither inhibited ⁹⁹Mo absorption nor the appearance of systemic defects in Cu metabolism. Since the absorption of MoS_4^2? (or its derivatives) from the gastrointestinal tract is inhibited by Cu, it is evident that the site of its action as an antagonist influencing either the absorption or the subsequent metabolic fate of Cu depends upon the ratio Cu/MoS_4^2? in the diet.     

Resolution of the effects of sulfhydryl-blocking reagents on hormone-and DNA-binding activities of the chick oviduct progesterone receptor

The differential effects of sulfhydryl (SH)-blocking agents on hormone and DNA binding by the chick oviduct progesterone receptor were investigated. Previous studies have demonstrated inhibition of steroid-receptor interaction by SH-blocking agents and protection against inhibition by bound hormone. The present results indicate that the SH group required for steroid binding is within or near the hormone-binding site itself, and that a second SH group (or groups) is involved in the binding of receptor to DNA. Three findings relate to the site of action of SH-blocking agents on hormone binding. First, glycerol decreased the rate of hormone dissociation and the rate of hormone displacement by mercurial reagents by 75 to 90%. Second, mercurial reagents displaced [³H]progesterone bound to the mero-receptor, a M_r 23,000 proteolytic fragment containing the hormone-binding site, but not the site of interaction with DNA. Third, hormone displacement was still present after a 10,000-fold purification of the progesterone receptor. Mercurial reagents also inhibited binding of progesterone receptor to DNA, whereas the SH-alkylating agents N-ethylmaleimide and iodoacetamide had no effect. It is likely that distinct sulfhydryl groups are required for steroid receptor interaction with hormone and with DNA, since brief treatment with mercurial reagents blocked DNA binding, but caused only a slight displacement of bound hormone. The SH group required for hormone binding probably lies within or near the hormone-binding site, is sensitive to mercurials, alkylating agents, and 5,5′-dithiobis(2-nitrobenzoate) (DTNB), and is protected by bound hormone. The SH group required for DNA binding, in contrast, is sensitive to mercurials but not to alkylating agents, is only partially sensitive to DTNB, and is not protected by bound hormone.     

Inactivation of Copper,Zinc superoxide dismutase by H2O2 : Mechanism of protection

Cu,Zn SOD is known to be inactivated by HO₂⁻ and to be protected against that inactivation by a number of small molecules including formate, imidazole, and urate. This inactivation has been shown to be due to oxidation of a ligand field histidine residue by a bound oxidant formed by reaction of the active site Cu(II) with HO₂⁻. We now report that protective actions of both formate and NADH increase as the pH was raised in the range 8.0–9.5. This is taken to indicate increased accessibility of the Cu site with rising pH and/or increased reactivity of the bound oxidant toward exogeneous substrates at high pH. Formate appears to act as a sacrificial substrate that protects by competing with the endogenous histidine residue for reaction with the bound oxidant, or that repairs the damage by reducing the histidyl radical intermediate. The same is likely also true of NADH.     

The effect of cadmium om intestinal copper absorption and binding in the rat

A study has been made on the effects of dietary Cd on Cu absorption in rats. When Cd was included in diets containing 2.6 mg Cu/kg to give Cd:Cu molar ratios of 0:1, 1:1, 2:1 and 4:1, the absorption of a tracer dose of ⁶⁴Cu added to the diets was reduced at the highest level of Cd supplementation. At all levels tested dietary Cd increased the amount of ⁶⁴Cu retained by the intestinal tissue. In a subsequent experiment it was found that dietary Cd increased the amount of ⁶⁴Cu recovered in a low molecular form (mol wt 10,000) although there was an inverse relationship between dietary Cd content and amount of ⁶⁴Cu bound. The possible roles of this Cu binding fraction in intestinal Cu absorption are discussed.     

Pyridoxal-5'-phosphate as a cofactor for rat brain histidine decarboxylase.

The cofactor requirements of brain histidine decarboxylase activity have been studied. Preincubation with carbonyl reagents caused inhibition of the activity ranging from 90% for 10^?2m -semicarbazide, 10^?3m -phenylhydrazide and 10^?3m -hydroxylamine to 50% for isonicotinic acid hydrazide. Sodium borohydride, a reducing agent, also caused complete inhibition of activity. The histidine decarboxylase activity was maximal at 10^?4m -pyridoxal-P concentration and was inhibited at higher concentrations of the cofactor. The cofactor-apoenzyme mode of binding was studied by dialysing brain homogenates against several media. Neither the dialysis against buffers alone nor against buffers containing semicarbazide nor cysteine plus EDTA caused a total loss of activity. A 50% of the activity dialysed easily while the other 50% remained ‘tightly’ bound to the apoenzyme. The dialysable and non dialysable activity is evenly distributed between the soluble and particulate activity.     

The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by,and stable complex formation with,Src

The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3^src. Co-expression of activated Src (pp60^527F) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 a nd stable complex formation with pp60^527F. However, co-expression of the SH3 null-binding mutant (AFAP^71A) with pp60^527F revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP^71A was phosphorylated on tyrosine, AFAP^71A could not be detected in stable complex with pp60^527F, unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60^527F. (Mol Cell Biochem 175: 243–252, 1997)     

The amyloidogenic region of the human prion protein contains a high affinity (Met)2(His)2 Cu(I) binding site

The prion protein (PrP^c) is a cuproprotein implicated in a number of human neurodegenerative diseases. Although many physiological functions have been ascribed to PrP, its potential to act as a neuronal antioxidant, based in part on its copper binding ability, is controversial and unresolved. A number of studies have shown that copper bound to PrP^c is not redox silent, and recent data shows that the Cu(II) sites at histidines 96 and 111 display reversible electrochemistry. Reversible electrochemistry implies redox cycling whilst the metal remains bound and with the absence of permanent oxidation or reduction of the protein. Despite this indirect evidence of Cu(I) binding to PrP, the nature of the Cu(I) binding site/s is unclear, although previous extended X-ray absorption fine structure (EXAFS) data has implicated methionines in the Cu(I) binding site. Using spectroscopic techniques we find that the PrP region encompassing histidines 96 and 111 can bind a Cu(I) ion in a site comprising His 96, His 111, Met 109 and Met 112. The four-coordinate (His)₂(Met)₂ Cu(I) site has a K_d = 10⁻¹⁵–10⁻¹² M indicative of high affinity. Mutation of histidine residues reduces the Cu(I) affinity. Although alluding to the fact the PrP could act in a direct superoxide dismutase-like fashion, the Cu(I)–PrP(91–124) site and affinity is comparable to that observed for bacterial periplasmic Cu(I) transporters.     

Comparison of plasma corticosterone- and progesterone-binding activity in rat and human

Corticosterone-and progesterone-binding activity were measured by saturation analysis, with dextran-charcoal separation, in plasma obtained from male and female rats, and a normal male and female human. In plasma from normal male and female rats, progesterone was much less effective than corticosterone in displacing ³H-corticosterone from plasma protein binding sites although the parallelism of the displacement curves indicated competition for the same binding sites. In plasma from the normal male human, corticosterone and progesterone were equally effective in displacing ³H-corticosterone. However, ³H-progesterone showed no apparent binding to either rat or human plasma proteins, suggesting that dextran-charcoal effectively removed progesterone from transcortin binding sites at 4°C. This observation was confirmed by multiple equilibrium dialysis. In dialysis, ³H-corticosterone and ³H₃-progesterone were bound equally by human plasma, but rat plasma bound ³H-corticosterone to a much greater extent than it did ³H-progesterone. These data indicate that, in contrast to human plasma, rat plasma has much greater affinity for corticosterone than for progesterone.     

Inhibition of copper-induced LDL oxidation by vitamin C is associated with decreased copper-binding to LDL and 2-oxo-histidine formation

Oxidatively modified low-density lipoprotein (LDL) has numerous atherogenic properties, and antioxidants that can prevent LDL oxidation may act as antiatherogens. We have previously shown that vitamin C (L-ascorbic acid, AA) and its two-electron oxidation product dehydro-L-ascorbic acid (DHA) strongly inhibit copper (Cu)-induced LDL oxidation. These findings are unusual, as AA is known to act not only as an antioxidant, but also a pro-oxidant in the presence of transition metal ions in vitro, and DHA has no known reducing capacity. Here we report that human LDL (0.4 mg protein/ml) incubated with 40 μM Cu²⁺ binds 28.0 ± 3.3 Cu ions per LDL particle (mean ± SD, n = 10). Co-incubation of LDL with AA or DHA led to the time- and concentration-dependent release of up to 70% of bound Cu, which was associated with the inhibition of LDL oxidation. Incubation of LDL with Cu and AA or DHA also led to the time-dependent formation of 2-oxo-histidine, an oxidized derivative of histidine with a low affinity for Cu. Addition of free histidine prevented the formation of the LDL-Cu complexes and inhibited LDL oxidation, despite the fact that Cu remained redox-active. Interestingly, histidine was more effective than AA or DHA at limiting Cu binding to LDL, but at low concentrations AA and DHA were more effective than histidine at inhibiting LDL oxidation. These data suggest that there are at least two types of Cu binding sites on LDL: those that bind Cu in a redox-active form critical for initiation of LDL oxidation, and those that bind Cu in a redox-inactive form not contributing to LDL oxidation. The former sites may be primarily histidine residues of apolipoprotein B-100 that are oxidized to 2-oxo-histidine in the presence of Cu and AA or DHA, thus explaining, at least in part, the unusual inhibitory effect of vitamin C on Cu-induced LDL oxidation.     

Analytical variables affecting exchangeable copper determination in blood plasma

To resolve discrepancies observed in the determination of plasma exchangeable Cu (also called direct reacting Cu or loosely bound Cu) by several methods, plasma storage techniques and various aspects of a stable isotope dilution procedure for exchangeable Cu were evaluated. Results indicated that the exchangeable Cu fraction of plasma increased with storage at room temperature, at 5°C and when subjected to repeated freeze/thaw cycles. Samples could be safely stored at −65°C. Exchange between added ⁶⁵Cu²⁺ and endogenous plasma Cu rapidly went to completion in the isotope dilution procedure. Analytical results were unaffected by shaking method, sample size or the presence of heparin. A small difference was observed between serum and plasma. The determination of exchangeable Cu did not vary over a period of 4 h when plasma was exposed to 1.6 × 10⁻⁴-M sodium diethyldithiocarbamate (used in the isotope dilution method) but steadily increased when exposed to 1.1 × 10⁻²-M sodium diethyldithiocarbamate, which suggested that tightly bound Cu (probably in ceruloplasmin) was exchanging with isotopic tracer at the higher concentration. Determination of exchangeable Cu was constant from pH 7.2–8.5 but increased substantially at higher pH. Complete recovery of natural Cu added to plasma was obtained. Studies in solution indicated that ⁶⁵Cu²⁺ exchanged readily with albumin- and amino acid-bound Cu. Ultrafiltration of plasma yielded a Cu fraction about half that of the exchangeable Cu fraction. We conclude that the stable isotope dilution procedure for plasma exchangeable Cu yields reliable, physiologically meaningful results.     

Mn(II) binding sites of calf liver arginase

Commercial calf liver arginase was further purified through gel filtration column chromatography. The enzyme is nearly homogeneous in SDS-PAGE; it contains 4 manganese atoms per molecule of enzyme. By dialysis against 1,10-o-phenanthroline at 4°C it is possible to obtain an arginase apo-form containing 2 manganese atoms per molecule of enzyme (apo-2 form) while a treatment of the pur enzyme with o-phenanthroline at 37°C followed by dialysis against 0.1 M NaCl is capable of producing an apoenzyme with only 1 manganese atom per molecule (apo-1 form). The apo-2 and apo-1 arginases retain respectively about 50% and 25% of the full enzymatic activity. NMR titrations of both apo-arginases with increasing concentrations of manganese allowed us to determine the affinity constants for the binding of Mn²⁺ to the protein. It was shown that in this enzyme two manganese atoms are weakly bound, one is more strongly bound and the fourth one is bound so tightly that it is not removed under the experimental conditions used.     

Possible involvement of plasma histidine in differential brain permeability to zinc and cadmium

Zinc gets into the brain parenchyma across the blood-brain and the blood-cerebrospinal fluid barriers, while cadmium hardly gets into the brain parenchyma. Because histidine may be involved in zinc transport across the brain barrier systems, the binding to histidine was compared between zinc and cadmium to understand the difference in brain permeability to both metals. Sephadex G-10 gel filtration indicated that ¹⁰⁹Cd, unlike ⁶⁵Zn, does not bind to histidine. When the plasma incubated with ⁶⁵Zn or ¹⁰⁹Cd was dialyzed in physiological saline containing histidine (0-10 mM), ⁶⁵Zn concentration in the dialysate was increased with the increase of the histidine concentration, suggesting the transfer of zinc from plasma proteins to histidine. The low affinity of zinc to plasma proteins may be important for brain permeability to this metal. On the other hand, ¹⁰⁹Cd was not detected in the dialysate in the presence of 0.1 mM histidine, which is equal to the concentration in the plasma, suggesting no transfer of cadmium from plasma proteins to histidine. These results suggest that the avid binding of cadmium to plasma proteins is related to brain impermeability to this metal.     

The oxidation of cat,human, and the cat-human hybrid hemoglobins α2Humanβ2Cat and α2Catβ2Human by copper(II)

The heme iron of the β chains of mammalian hemoglobins are rapidly and selectively oxidized in the presence of excess Cu(II) ions in a reaction that requires the presence of a free -SH groups on the β globin chain. The presence of freely reactive -SH groups on the α chains of cat and sheep hemoglobins does not alter the course of this reaction: only the β hemes are oxidized rapidly by Cu(II) in these hemoglobins. Two equivalents of copper are required for the rapid oxidation of the two β chain hemes per mole of cat hemoglobin, in contrast with the four equivalents that are required for reaction with human hemoglobin. The human-cat hybrid hemoglobins, α₂^Humanβ₂^Cat and α₂^Catβ₂^Human, required two and four equivalents of copper/mol, respectively, for the reaction. Thus, the kinetics and stoichimetry of the reaction are determined by the nature of the β subunit. Analysis of the esr spectra of the products of the reaction of Cu(II) with these hemoglobins indicate that human hemoglobin and the hybrid α₂^Catβ₂^Human contain tight binding sites for two equivalents of Cu(II) that are not involved in the oxidation reaction and are not present in cat hemoglobin or α₂^Humanβ₂^Cat. Cat β globin like others (sheep, bovine) that lack the tight binding site, has no histidine residue at 2β. It has phenylalanine in this position. These results support the suggestion of Rifkind et al. (Biochemistry 15,5337[1976]) that the tight binding site is near the amino terminal region of the β chain and is associated with histidine 2β.     

Structural Basis of Heme Binding in the Cu,Zn Superoxide Dismutase from Haemophilus ducreyi

The Cu,Zn superoxide dismutase from Haemophilus ducreyi is characterized by the unique ability to bind heme at its dimer interface. Here we report the high-resolution crystal structures of this protein in the heme-loaded (holo) and heme-free (apo) forms. Heme is asymmetrically bound between the two enzyme subunits, where heme iron is coordinated by two histidine residues, His64 and His 124, provided by the two subunits. Moreover, the binding of heme to the protein is ensured by stabilizing contacts between the prosthetic group and a limited number of other residues, most of which are not present in other bacterial enzyme variants. We show that the introduction of only three mutations at the dimer interface of the enzyme from Haemophilus parainfluenzae, a closely related bacterial species, is sufficient to induce heme-binding ability by this enzyme variant. Heme binding does not alter protein activity. Moreover, the binding of the prosthetic group does not induce any significant structural perturbation at the subunit level and requires only limited local structural rearrangements that widen the cleft at the dimer interface and cause a limited shift in the relative orientation between the subunits. The presence of a preformed heme-binding pocket and the significant solvent exposure of the cofactor to the solvent are compatible with the suggested protective role of the enzyme against heme toxicity or with its involvement in heme trafficking in the periplasmic space.     

Modeling the interplay of glycine protonation and multiple histidine binding of copper in the prion protein octarepeat subdomains

The octarepeat region of the prion protein can bind Cu²⁺ ions up to full occupancy (one ion per octarepeat) at neutral pH. While crystallographic data show that the HGGG octarepeat subdomain is the basic binding unit, multiple histidine coordination at lower Cu occupancy has been reported by X-ray absorption spectroscopy, EPR, and potentiometric experiments. In this paper we investigate, with first principles Car–Parrinello simulations, the first step for the formation of the Cu low-level binding mode, where four histidine side chains are coordinated to the same Cu²⁺ ion. This step involves the further binding of a second histidine to an already HGGG domain bonded Cu²⁺ ion. The influence of the pH on the ability of Cu to bind two histidine side chains was taken into account by simulating different protonation states of the amide N atoms of the two glycines lying nearest to the first histidine. Multiple histidine coordination is also seen to occur when glycine deprotonation occurs and the presence of the extra histidine stabilizes the Cu–peptide complex. Though the stabilization effect slightly decreases with the number of deprotonated glycines (reaching a minimum when both N atoms of the two nearest glycines are available as Cu ligands), the system is still capable of binding the second histidine in a 4N tetrahedral (though slightly distorted) coordination, whose energy is very near to that of the crystallographic square-planar 3N1O coordination. This result suggests that at low metal concentration the reorganization energy associated with Cu(II)/Cu(I) reduction is small also at pH ~ 7, when glycines are deprotonated. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.

Development of a generic approach to native metalloproteomics: application to the quantitative identification of soluble copper proteins in Escherichia coli

A combination of techniques to separate and quantify the native proteins associated with a particular transition metal ion from a cellular system has been developed. The procedure involves four steps: (1) labeling of the target proteins with a suitable short-lived radioisotope (suitable isotopes are ⁶⁴Cu, ⁶⁷Cu, ¹⁸⁷W, ⁹⁹Mo, ⁶⁹Zn, ⁵⁶Mn, ⁶⁵Ni); (2) separation of intact soluble holoproteins using native isoelectric focusing combined with blue native polyacrylamide gel electrophoresis into native–native 2D gel electrophoresis; (3) spot visualization and quantification using autoradiography; and (4) protein identification with tandem mass spectrometry. The method was applied to the identification of copper proteins from a soluble protein extract of wild-type Escherichia coli K12 using the radioisotope ⁶⁴Cu. The E. coli protein CueO, which has previously been only identified as a multicopper oxidase following homologous overexpression, was now directly detected as a copper protein against a wild-type background at an expression level of 0.007% of total soluble protein. The retention of the radioisotope by the copper proteins throughout the separation process corroborates the method to be genuinely native. The procedure developed here can be applied to cells of any origin, and to any metal having suitable radioisotopes. The finding that the periplasmic protein CueO is the only major form of soluble protein bound copper in E. coli strengthens the view that the bacterial periplasm contains only a few periplasmic copper proteins, and that the cytosol is devoid of copper proteins. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Presence of nucleoside triphosphates and calcium associated with mycobacteriophage I3

The association of nucleoside triphosphate molecules and calcium ions with purified particles of mycobacteriophage I3 has been documented. The content of nucleoside triphosphate has been determined to be 118 molecules per phage particle by equilibrium dialysis against labelled ATP or 148 molecules per phage particle by the direct determination of labelled nucleoside triphosphate. The concentration of bound Ca²⁺ exhibited a high degree of variation between different batches, which may be due to the nonspecific binding of Ca²⁺ by the virus particles. However, the tightly bound Ca²⁺ not removable by dialysis against calciumspecific chelating agent, showed a constant value of 2985 atoms/phage particle.Abbreviations EGTA Ethylene glycol-bis (-aminoethylether)-N,N¹ tetraacetic acid - PFU plaque forming unit - NTP nucleoside triphosphate     

Copper(I)-incorporation into spinach apoplastocyanin

Spinach plastocyanin was converted into the apoprotein. CuSO₄ and oxidized Cu(II)- thionein reacted with the apoprotein to Cu(II) plastocyanin. Cu(I) transfer from Cu(I)0-thionein was only 15%. The structural analogue of the copper thiolate chromophore [Cu(I)(thiourea)₃]Cl as well as [Cu(CH₃CN)₄]ClO₄ successfully formed the Cu(I)- holoprotein. Characteristic circular dichroism bands at θ₂₈₄ (?5300 deg·cm²·dmol^?1 and θ₃₁₀ (+3300 deg·cm²·dmol^?1) were seen. Upon oxidation with ferricyanide and dialysis against phosphate buffer the correct Cu(II) binding into the active centre of Cu(II) plastocyanin was confirmed by EPR-measurements. The use of [Cu(I)(thiourea)₃] Cl as a convenient Cu(I) source for reconstitution studies on copper proteins is highly recommended.     

Binding of active and inactive hemolytic factor of sea anemone nematocyst venom to red blood cells

Sea anemone nematocyst venom, in the presence of Ca²⁺, induced the lysis of red blood cells after an induction period. In the absence of Ca²⁺, however, no lysis occurred, but the hemolytic factor was shown to bind to the cells. This binding was shown to be requisite for the Ca²⁺ dependent lysis to ensue. After freeze thawing, the venom proteins responsible for lysis lost their hemolytic activity, yet still bound to the cells. The freezethawed inactivated venom competitively blocked hemolysis by active venom.