Heavy metal complexation in polluted molluscs II. Oysters (Ostrea edulis and Crassostrea gigas)

Cadmium, zinc and copper, accumulated from polluted habitats by the oysters Ostrea edulis and Crassostrea gigas, were studied. Three distinct low molecular weight (less than 3000 daltons) zinc complexes were separated from highly contaminated C. gigas, one of which may be a complex with the amino acid taurine. Only one of these zinc complexes was present in less contaminated specimens of O. edulis. On gel permeation chromatography of oyster extracts, copper was eluted together with amino acids (principally taurine) and the betaine homarine. No evidence of metallothionein type proteins was found.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO : Relationship to Large Subunit-Binding Protein Complex Dissociation

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coli. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of in vitro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.     

Detection of low molecular weight copper(II) and zinc(II) binding ligands in ultrafiltered milks—the citrate connection

Low molecular weight zinc(II) and copper(II) binding ligands were detected in ultrafiltered human, bovine, and goat milk by the application of the method of modified gel chromatography. Human milk contains at least three detectable low molecular weight copper binders, whereas bovine and goat milk contain at least two. All three milks show two copper binding peaks with the same elution volumes. Zinc chromatograms were less specific than copper. Zinc showed only a single detectable low molecular weight binding ligand common to all three milks. Elution volumes for both zinc(II) and copper(II) citrate and picolinate systems were measured. Elution volumes of both copper(II) and zinc(II) citrate complexes are identical to elution volumes of an intense peak observed with all three milks; it is reasonable to assume that at least part of this peak corresponds to citrate. Human milk alone has a relatively intense binding peak for copper(II) at the same elution volume as the glutamate complex. Human and goat milk have another low intensity copper(II) binding ligand peak at the same elution volume; a number of amino acid complexes have binding peaks at this position. No peak characteristic of the zinc(II) or copper(II) picolinate systems could be found with any of the milks.     

Partitioning of zinc and copper within subnuclear nucleoprotein particles.

Nuclei from frozen calf thymus suspended in buffer were analyzed for metal content prior to and after repeated washing. After three such extractions about 0.1 micrograms Zn/mg DNA and 0.025 micrograms Cu/mg DNA remained tightly associated with chromatin. This level of metal was essentially unchanged with subsequent washings. Digestion of extracted nuclei with micrococcal nuclease yielded soluble nucleoprotein containing zinc and copper. Metal enriched regions of chromatin appeared to be preferentially solubilized by digestion, and the solubilized metal was only partially dializable either with or without EDTA. Metal profiles generated from gel (A-5m) chromatography analysis of chelated and non-chelated solubilized chromatin were distinctive in that copper was undetectable (by flame AA) while zinc was associated only with low molecular weight products when EDTA was used. In contrast, both metals were detected with higher molecular weight oligonucleosomes in the absence of chelating agents. Additionally, the two metals localized within nucleoprotein peaks and these metal-containing regions were only resolved by gel chromatography when EDTA was omitted throughout the procedure. A discrete Cu-rich species in a region of the profile suggests a subset of Cu-rich nucleoprotein complexes.     

Metabolism of AGEs – Bacterial AGEs Are Degraded by Metallo-Proteases

Advanced Glycation End Products (AGEs) are the final products of non-enzymatic protein glycation that results in loss of protein structure and function. We have previously shown that in E. coli AGEs are continually formed as high-molecular weight protein complexes. Moreover, we showed that AGEs are removed from the cells by an active, ATP-dependent secretion and that these secreted molecules have low molecular weight. Taken together, these results indicate that E. coli contains a fraction of low molecular weight AGEs, in addition to the high-molecular weight AGEs. Here we show that the low-molecular weight AGEs originate from high-molecular weight AGEs by proteolytic degradation. Results of in-vitro and in vivo experiments indicated that this degradation is carried out not by the major ATP-dependent proteases that are responsible for the main part of bacterial protein quality control but by an alternative metal-dependent proteolysis. This proteolytic reaction is essential for the further secretion of AGEs from the cells. As the biochemical reactions involving AGEs are not yet understood, the implication of a metalloprotease in breakdown of high molecular weight AGEs and their secretion constitutes an important step in the understanding of AGEs metabolism.     

A re-investigation of the reactions between superoxide anion and metal picolinate complexes

Rate constants for the reactions of superoxide with the α-picolinate ion and its complexes with copper(II), iron(III) and zinc(II), and for the reaction of α-picolinate with the hydrated electron, were measured using pulse radiolysis. The rate constant for the reaction of superoxide with copper(II)picolinate at pH 9 [(4.1 ± 0.4) × 10⁷l mol⁻¹ s⁻¹] was an order of magnitude higher than that determined previously (W. H. Bannister, J. V. Bannister, A. J. F. Searle and P. J. Thornally, Inorg. Chim. Acta, 78, 139 (1983)) using a less direct competitive inhibition method. The corresponding rate constant for iron(III)picolinate [(7.5 ± 1.5) X 10³ l mol⁻¹ s⁻] was an order of magnitude lower than a previous pulse radiolysis determination (same reference as above). We are not able to reconcile these two values for iron(III)picolinate, although a possible source of spuriously high results is contamination with the kinetically active copper(II) complex. The likely roles of iron(III)picolinate and other low molecular weight iron complexes as potential catalysts of an in vivo superoxide-driven Fenton reaction are discussed in the light of present measurements.     

Bis(thiosemicarbazone) copper complexes: mechanism of intracellular accumulation

The molecular basis of Alzheimer’s disease has not been clearly established, but disruption of brain metal ion homeostasis, particularly copper and zinc, might be closely involved in the pathogenesis of this disease and its characteristic β-amyloid neuropathological features. The use of complexes of copper with bis(thiosemicarbazones) ([Cu(btsc)]) has been proposed for the treatment of Alzheimer’s disease. Their mode of action could involve modulation of the concentration of copper or zinc, and it has been suggested that the compounds can modulate the production of β-amyloid peptide at the neuron level. Furthermore, it has been reported that [Cu(btsc)] complexes can be reduced inside the cells. However, to our knowledge the intracellular reduction of these compounds has never been demonstrated. Thus, the goal of our study was to increase understanding of the mechanism of intracellular accumulation of [Cu(btsc)] complexes. Our results reveal that the intracellular concentration of copper inside the cells is very high and that these compounds are not P-glycoprotein substrates. This protein is a key element of the low permeability properties of the blood–brain barrier. Furthermore, no intracellular reduction of cupric ions was detected. Finally, once inside the cells, the complexes undergo aggregation, strongly suggesting that aggregation of complexes is the driving force responsible for their intracellular accumulation.     

Structural Aspects of Photosystem I from Dunaliella salina

A native PSI complex and a PSI core complex have been isolated from the halophilic green alga, Dunaliella salina. The composition and properties of these complexes are similar to previously described PSI complexes from spinach membranes. By growth on ¹⁴C-NaHCO₃, it has been possible to isolate uniformly labeled ¹⁴C-PSI complexes in order to determine PSI subunit stoichiometry. This analysis has shown a ratio of one copy of three low molecular weight subunits (22,000; 15,000; 8,000) per two copies of high molecular weight subunits (84,000). Using a ¹⁴C-labeled cytochrome b₆-f complex as an internal protein standard, it has been possible to estimate the molecular weight of a PSI core complex as about 330,000. This complex contains one P700, two 84,000 subunits, and one subunit of 22,000, 15,000, and 8,000.     

Biosynthesis of transfer RNA: isolation and characterization of precursors to transfer RNA in the posterior silkgland of Bombyx mori.

Distinct low molecular weight RNA species that have properties expected for the precursor to tRNA have been isolated from the posterior silkglands of the silkworm Bombyx mori. These RNAs migrate between 4 S and 5 S markers on acrylamide gels and are labeled preferentially in vivo in relation to tRNA. The precursor RNAs can be converted specifically into molecules indistinguishable in size from tRNA upon incubation with “cleavage” enzymes isolated from the silkgland ribosomes. Two of the three low molecular weight RNAs contain the modified residues, pseudouridine, dihydrouridine and ribothymidine, and are methylated in vivo, suggesting that these base modifications occur while the tRNA is still in its precursor stage.     

Nuclear magnetic relaxation of 19F as a novel assay method of superoxide dismutase.

The 19F longitudinal nuclear magnetic relaxation rate (T-1(1p)) of F- solutions is greatly enhanced by copper, zinc or manganese superoxide dismutase. The measured T-1(1p) values are at least an order of magnitude, and in most cases 2 orders of magnitude, higher than those of other metal-containing proteins and low molecular weight complexes. This property is suitable for a direct, specific, and sensitive assay of superoxide dismutase, free of interference by other molecules. For mixtures of the copper, zinc and manganese enzymes, addition of CN- permits quantification of the relative amounts of each enzyme. In the case of copper, zinc enzyme, the method proved exquisitely sensitive to the native state of the active site. Results are reported to show that the 19F relaxation method can be used to assay biological fluids and crude homogenates for copper, zinc and manganese superoxide dismutases, under conditions that offer considerable advantages in comparison to other assay methods.     

Imidazole-containing ternary complexes of N-benzyloxycarbonyl-aminoacids. Crystal and molecular structure of bis(N-benzyloxycarbonyl-alaninato)bis-(N-methylimidazole)copper(II) ethanol solvate

Mixed ligand complexes of the type Cu(Z-aminoacidato)₂(B₂) (Z = benzyloxycarbonyl group, Z-aminoacidate = Z-glycinate (Zgly), Z-alaninate (Zala); Z-valinate (Zval), Z-leucinate (Zleu) ion, B = imidazole (Im), N-methylimidazole (MeIm)) were synthesized and characterized by means of electronic, infrared and EPR spectroscopies. For one of them, bis(Z-alaninato)bis(N-methylimidazole)copper(II) ethanol solvate, the crystal and molecular structure was also determined by the single crystal X-ray diffraction method. The complex crystallizes in the monoclinic space group P2₁/c, with cell dimensions a = 11.1119(6), b = 18.8398(7), c = 8.9652(5) Å, β = 105.380(2)° and Z = 2. The structure was solved by conventional Patterson and Fourier methods and refined by full-matrix least-squares to an R value of 0.045. The complex has square-planar coordination via two centrosymmetric carboxylic oxygens and two N-methylimidazole nitrogens. The second carboxylate oxygen is 2.731(5) Å from the copper atom in an ‘out of plane’ position. Packing is mainly determined by hydrogen bondings between amide nitrogen and amide carboxyl oxygen. Electronic, infrared and EPR spectra are consistent with this type of coordination geometry for anhydrous complexes, while for hydrate complexes are suggestive of tetragonal bipyramidal geometry.     

Assessment of copper,iron, zinc and manganese status and speciation in patients with Parkinson’s disease: A pilot study

BackgroundThe objective of this pilot study was to assess iron (Fe), copper (Cu), zinc (Zn), and manganese (Mn) status (hair, serum, and urine) and speciation (serum) in Parkinson’s disease (PD) patients.MethodsA pilot study involving a total of 27 subjects (13 PD patients, 14 controls) was performed. Serum, urine, and hair metal content was assessed using ICP-MS. Speciation analysis of Cu, Zn, Fe, and Mn was performed using a hybrid HPLC-ICP-MS system.ResultsGroup comparisons did not reveal any significant group difference in serum Cu, Zn, Fe, and Mn total metal level between PD patients and controls. Speciation analysis revealed a significant decrease in Cu/ceruloplasmin copper in association with elevation of low-molecular weight species (amino acids)-bound copper. It is proposed that in PD, binding of Cu(II) ions to ceruloplasmin is reduced and free copper ions coordinate with low molecular weight ligands. The level of Mn-albumin complexes in PD patients was more than 4-fold higher as compared to the respective value in the control group. The observed difference may be considered as a marker of redistribution between high and low molecular weight ligands.ConclusionsMetal speciation is significantly affected in serum of PD-patients. These findings are indicative of the potential role of metal metabolism and PD pathogenesis, although the exact mechanisms of such associations require further detailed studies.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Stereoelectronic properties of metalloenzymes. 10. a refined model that mimics the type II copper(II) site in galactose oxidase2

A number of copper(II) complexes of tridentate ligands with various donor atoms have been studied in an attempt to duplicate the unusual reactivity patterns and accompanying spectral changes of the copper(II) center in galactose oxidase. Results indicate that in order to match the optical and electron spin resonance spectral change observed upon CN^? binding by the enzyme, an equatorial, negative ligand must be displaced in a small molecule model. The crystal and molecular structure of the best model complex was solved by a single crystal X-ray diffraction study. The compound, monoacetato-1,3-bis(2-(4-methyl-pyridyl)imino)isoindolatocopper(II), crystallizes in the centro-symmetric triclinic space group Pī with a = 7.392(3) Å, b = 13.782(5) Å, c = 23.422(12) Å, α = 92.08(3)°, β = 104.11(5)°, γ = 109.98(4)°, V = 2156(1) ų, d(obsd.)(calc.)=(1.43)(1.44) g/cm^?3 for mol wt of 466.7 and Z = 4. Diffraction data were collected with a Syntex Pl diffractometer using graphite-monochromatized Cu radiation (λ = 1.5418 Å). The copper atoms were located from a Patterson synthesis; all other nonhydrogen atoms were located via difference. Fourier techniques, and hydrogen atoms were placed in calculated positions. Final refinement resulted in discrepancy indices of R = 0.089 and “Goodness to Fit” = 3.68 for all 3608 reflections having (I) ? 3σ(I) (5°<2θ<100°). There are two unique molecules in the asymmetric unit that are monomeric and well separated. The geometry around the copper atom is approximately square pyramidal, with the coordination sphere derived from three nitrogens of the tridentate ligand, one oxygen from the acetate unit, and an oxygen atom of a water molecule occupying an axial position. The structure is surprising both in that an axial water molecule is present and that the remaining four ligand atoms to the copper atom are rather distorted from a planar configuration. The plane defined by the copper, N5, and N3 atoms intersects the plane defined by the copper, Nl, and Ol, atoms forming a “twist angle” of 25.0° (0.0° would be ideal for a planar inner coordination sphere). The stereoelectronics of the inner coordination spheres of the type II Cu(II) enzymes galactose oxidase and superoxide dismutase are discussed and appropriate comparisons are made with emphasis on the origin of spectral changes observed upon anion binding.     

Synthesis,characterization, toxicity,cytogenetic and in vivo antitumor studies of 1,1-dithiolate Cu(II) complexes with di-, tri-, tetra- amines and 1,3-thiazoles. Structure–activity correlation

A series of new mixed-ligand neutral copper(II) complexes of the general type [Cu(amine)(i-MNT)] and [Cu(tz)(i-MNT)] was prepared and characterized by elemental, spectroscopic methods, μ_eff, Λ_μ measurements and molecular modeling studies. The acute toxicity, the cytogenetic and the in vivo antitumor activity of the new complexes, is related to their chemical and physicochemical properties. Among the Cu(II) compounds tested the complex with 2-amino-5-methyl thiazole increases significantly the life span of leukemia P388 bearing mice in vivo.     

Interaction between transition metals and nucleic acids. Part III. Synthesis and structural studies of copper(II) complexes with Schiff base ligands derived from barbituric acid

Schiff bases have been prepared from 5-formyl-barbituric acid and 5-formyl-1,3-dimethyl-barbituric acid and various di- or tri-amines. The structures of the corresponding copper(II) complexes have been established by elemental analysis and spectroscopic methods. The molecular structure of one of the complexes, Cu(DiMeBardpt), was determined by X-ray diffraction. Electrochemical study shows that these complexes are reduced at slightly more negative potentials than the corresponding complexes obtained from uracil, which suggests that these new ligands are better electron-donors.     

Molecular distribution of zinc in biliary and pancreatic secretions

Rat bile and pancreatic fluid were examined for the presence of low molecular weight zinc complexes. Fluids were collected separately by cannulation, and zinc distribution in collected samples was analyzed by gel filtration on Sephadex G-50. Most of the zinc in bile was associated with low molecular weight zinc complexes; only a small amount of zinc was present in the high molecular weight fraction. In contrast, pancreatic secretions did not contain low molecular weight zinc complexes, but there were considerable amounts of zinc bound to high molecular weight compounds. The addition of zinc to bile resulted in an increased amount of zinc in the low molecular weight fraction, while the addition of zinc to pancreatic fluid resulted primarily in an increase in zinc bound to the high molecular weight components. Like pancreatic fluid, homogenates of pancreatic tissue had no low molecular weight zinc complex. In rats whose bile and pancreatic fluid were removed and not returned into the intestine, the amount of zinc bound to low molecular weight complexes in intestinal homogenates was reduced. This alteration of the molecular distribution of zinc in intestinal homogenates by removal of bile and pancreatic fluid suggests the potential importance of low molecular weight zinc complexes for zinc homeostasis.     

The isolation and partial characterisation of the light-harvesting pigment-protein complement of Rhodopseudomonas acidophila

The photosynthetic membranes of two strains of Rhodopseudomonas acidophila (7750 and 7050) have been resolved into their constituent light-harvesting pigment-protein complexes. Four different types of antenna complexes (B880, B800–830 and two types of B800–850) have been isolated and partially purified. In each case the light-harvesting pigments (bacteriochlorophyll a and carotenoids) are bound to rather low molecular weight polypeptides (in the 5000–9000 region).