PubMed Health⌕ Search

Biomedical subjects

U Weser

Publications and source records attributed to U Weser.

At least 19 recordsLinked to original sources
PubMed1992

Analogous copper(I) coordination in metallothionein from yeast and the separate domains of the mammalian protein.

The three-dimensional structures of both vertebrate Cu12-metallothionein (class 1) and yeast Cu8-thionein (class 2) are still unknown. The different copper:protein stoichiometry compared with that of the (ZnCd)7-metallothioneins was expected to alter the metal-thiolate cluster structure considerably. In order to avoid possible domain interactions in the hepatic rat metallothionein, separate chemically synthesized alpha- and beta-domains were used rather than the apoprotein. Apo yeast thionein, and the alpha- and beta-domains of rat liver metallothionein-2 were reconstituted by Cu(I) titration. Reconstitution steps were monitored using spectroscopic methods including luminescence emission and circular dichroism. Upon UV irradiation a linear increase in intensity of the orange-red luminescence was observed near 600 nm up to 6 Cu eq using either compound regardless of the different cysteine sulfer content (yeast thionein 12S, alpha-domain 11S, beta-domain 9S). The characteristic dichroic properties of the yeast copper-protein between 240 and 400 nm were in good agreement with those of the respective class 1 metallothionein domains. All observed Cotton bands were of similar shape and appeared in the same wavelength regions. However, the molar ellipticities were less pronounced in the alpha- and beta-fragments employed. There appears to be a striking similarity between the oligonuclear Cu(I) binding centers in all metallothionein species.

Animals↗
PubMed1991

Increase of Cu,Zn-superoxide dismutase activity during differentiation of human K562 cells involves activation by copper of a constantly expressed copper-deficient protein.

Cu,Zn-superoxide dismutase activity, expressed on the basis of cell number, increased by 50% during sodium butyrate-induced differentiation of human K562 erythroleukemia cells. The increased enzyme activity was found to be concomitant with constant Cu,Zn-superoxide dismutase mRNA and immunoreactive protein levels and was accompanied by a rise in intracellular copper and glutathione. Incubation of K562 cell homogenates with copper caused an increase of Cu,Zn-superoxide dismutase activity which reached the levels observed after differentiation in the presence of sodium butyrate. The same treatment led to no significant activity increase in homogenates derived from differentiated cells. Externally added ceruloplasmin increased both intracellular copper levels and Cu,Zn-superoxide dismutase activity in undifferentiated cells to a level comparable with that observed after induction of differentiation. Both increments were abolished by depletion of cell glutathione. Cu,Zn-superoxide dismutase purified from control cells had both a lower kcat and a lower copper content than the enzyme purified from differentiated cells. From these data we conclude that: 1) Cu,Zn-superoxide dismutase is present in K562 cells also under the form of a less active copper-deficient enzyme, 2) the extent of enzyme activation is regulated post-translationally by differential delivery of copper as a function of differentiation stage, and 3) glutathione is likely to play a role in delivering copper to the copper-deficient protein in intact K562 cells.

Catalase↗
PubMed1991

Oxidative stress induced by a di-Schiff base copper complex is both mediated and modulated by glutathione.

The reductive activation of N,N'-bis(2-pyridylmethylene)-1-4-butanediamine (N,N',N",N"')-Cu(II)-diperchlorate (CuPUPY), a di-Schiff base copper complex with antineoplastic properties, was investigated in vitro in the presence of glutathione, ascorbate, NADH or NADPH. Glutathione and ascorbate but not the pyridine dinucleotides were able to reduce the compound. The apparent second order rate constants of the reduction reaction (9.6 +/- 2.0 M-1 sec-1 for ascorbate and 94.7 +/- 1.9 M-1 sec-1 for glutathione) indicate that glutathione is more effective by about one order of magnitude in reducing CuPUPY than ascorbate. Reduction by glutathione triggered a CuPUPY-supported redox-cycle with oxygen yielding H2O2. Whereas reduction by ascorbate was reversible, CuPUPY reduced by glutathione reacted with excess reduced glutathione (GSH) in a ligand exchange reaction yielding a GSH-Cu(I) complex which was reoxidized by O2, forming a complex between copper(II) and oxidized glutathione. These results suggest a dual role for the reduced thiol tripeptide; promoting oxidative stress induced by CuPUPY at low concentrations and inhibiting it at high concentrations. This hypothesis was verified by showing that optimum glutathione/CuPUPY ratios are needed in order to obtain maximum oxidative damage to both DNA and albumin in vitro. Evidence was obtained for the occurrence of the same reaction pathway in human K562 erythroleukemia cells: CuPUPY was more toxic to cells in which glutathione synthesis was inhibited by buthionine sulfoximine. Moreover, ESR spectroscopy revealed alterations in the hyperfine structure of the Cu(II) spectrum, consistent with the occurrence of ligand-exchange reactions in K562 cells.

Albumins↗
PubMed1991

Chemiluminescence assays of Cu2Zn2 superoxide dismutase mimicking Cu-complexes.

The aqueous decay of K3CrO8 was used to compare the reactivity of Cu2Zn2 superoxide dismutase and two active centre analogues where the first shell atoms around the copper are four unsaturated nitrogens. Unlike the acetate or biuret type Cu(II) chelates these di-Schiff-base complexes had an identical reactivity compared to that of the intact enzyme. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence by 50%. Furthermore, a lucigenin amplified chemiluminescence assay based on isolated polymorph nuclear leucocytes in the absence and presence of whole, unseparated blood was developed and successfully employed. CuPu(Im)2 and CuPu(Py)2 equivalent to 0.5 and 0.8 SOD units, only, were required to inhibit the photon emission by 50% in the absence of bovine serum albumin. Even in the presence of 600 microM albumin mimicking the competitive copper chelation in biological fluids Cu-Pu(Py)2 and CuPu(Im)2 remained active, whereas the carboxylate- and biuret type chelates Cu(Sal)2 and Cu(Ser)2 reacted like CuSO4. The same reactivity of these low M, SOD mimics was seen in human blood.

Acridines↗
PubMed1990

Antiinflammatory reactivity of copper(I)-thionein.

In unseparated human blood the reactivity of yeast copper (I)-thionein on TPA-activated polymorphonuclear leukocytes was evaluated and compared with low Mr copper chelates exerting Cu2Zn2 superoxide dismutase mimetic activity. Cu, 18 microM, in the form of Cu-thionein was sufficient to inhibit the superoxide production of activated human blood phagocytes by 50%. Furthermore, the scavenging of hydroxyl radicals and singlet oxygen by Cu(I)-thionein was determined, using the 2-deoxyribose fragmentation assay induced by decaying K3CrO8 and the NADPH oxidation caused by UVA illuminated psoralen, respectively. The inhibitory reactivity of Cu-thionein in both assays was compared with that of serum proteins including albumin, ceruloplasmin, transferrin, and ferritin. The galactosamine/endotoxin-induced hepatitis in male NMRI mice was used to evaluate the antiinflammatory reactivity of Cu-thionein in vivo. The serum copper, superoxide dismutase, and sorbitol dehydrogenase concentrations, as well as the activity of polymorphonuclear leukocytes in unseparated blood seemed most appropriate to quantify the protective capacity of Cu-thionein in the course of an oxidative stress-dependent liver injury. The intraperitoneal application of 32.5 mumols/kg thionein-Cu limited this damage to 45%.

Animals↗
PubMed1990

An EXAFS study of the copper accumulated by yeast cells.

X-ray absorption spectroscopy has been applied to the in vivo examination of copper-resistant yeast cells. The in vivo structure of the metal-binding site of the accumulated copper has been compared to that of the purified yeast thionein. Analysis of the EXAFS spectra performed on intact yeast cells indicates that the accumulated copper is univalent and is exclusively coordinated to sulfur atoms at a distance of 219 pm with an average coordination number of 2. In contrast, the purified protein indicates a univalent copper trigonally coordinated to sulfur at a distance of 221 pm. These discrepancies are discussed in terms of copper location in the resistant yeast cells.

Carrier Proteins↗
PubMed1990

Copper transfer through the intestinal wall. Serosal release of metallothionein.

The elucidation of the molecular side of copper transport in biological systems is a promising task. In this context the transfer of ingested copper into the portal blood plasma was examined. Intralumenal addition of 200 microM copper caused the release of Cu-thionein into the venous effluent. This Cu-thionein became detectable after prior perfusion of the porcine small bowel using a modified isotonic phosphate-buffered saline (Pi/NaCl) medium. The protein was characterized by gel chromatography, luminescence, electronic absorption and immunological identification. ELISA and immunoblotting employing a murine monoclonal antibody to rat liver metallothionein-I proved to be most convenient. Using buffer-loaded sacs of porcine jejunum into which Cu2+, Zn2+ and Cd2+ were added, the release of metallothionein into the serosal fluid was successfully seen by ELISA. The observed excretion of metallothionein into the portal compartment may be a genuine metal transport system for many biochemically active metals.

Animals↗
PubMed1990

Monoclonal antibodies to monomeric rat liver metallothionein-I: the immunoreactivity of lysine residues in metallothionein.

Regardless of the weak immunological response against the low-Mr metallothioneins (MTs) the production of murine monoclonal antibodies (mAbs) to monomeric rat liver MT-I was successful. ELISA revealed two groups of mAbs which exhibited different specificities as examined on the native and the lysine-residue modified antigen (Ag). One "lysine-directed mAb" group, consisting of three mAbs, exhibited a specific immunoreactivity with the lysine-containing epitopes of MTs. Their role in the antigenicity of MTs was examined by modifying these residues using glutaraldehyde (GA). Titration with GA resulted in a progressive decline in Ag recognition in the immunoblot; this was completely leveled off when equimolar concentrations were reached. A similar response employing the GA-modified protein in the ELISA was noticed. The second group of mAb cross-reacted with various MTs of different origin, indicating that the common, lysine-free NH2-terminus is exclusively recognized. In direct ELISA of cross-linked MTs, the observed reactivities were much more pronounced. Iodoacetamide (IA) modification of the lysines confirmed the above observations of the GA-derived Ag. Notably, the immunoreactivity was not affected when the cysteine residues were IA-carboxymethylated, nor did the subsequent loss of metals diminish the immunological response in the immunoblot.

Animals↗
PubMed1990

Phagocytic response modifying reactivity of enzymatic cell wall digests of Nocardia opaca.

Aqueous extracts (ENOCW) and enzymatic digests of purified Nocardia opaca cell wall fragments, virtually free of muramyl peptides, were monitored for their phagocytic response modifying reactivity on polymorphonuclear leucocytes, separated or unseparated in whole human blood. In the presence of ENOCW a 74% increased production of superoxide during the respiratory burst of TPA-activated polymorphonuclear leukocytes was observed, as compared to the unprimed control. Delipidation of this preparation resulted in a further increase in reactivity (144%). Even in the presence of whole human blood, as a model for competitive binding in biological fluids, an enhanced generation of superoxide by TPA activated blood phagocytes remained detectable. A 37-75% decreased phagocytic reactivity in samples of HIV-seropositive blood was considerably restored in the presence of ENOCW.

Acridines↗
PubMed1990

Anticarcinogenic reactivity of copper-dischiffbases with superoxide dismutase-like activity.

CuPu(Py)2 and CuPu(Im)2, two novel dischiffbase coordinated low Mr active centre analogues of Cu2Zn2 superoxide dismutase, were shown to effectively catalyze the production of hydroxyl radicals in the presence and absence of TPA-activated polymorphonuclear leukocytes. These stable copper chelates exhibited a pronounced anticarcinogenic reactivity in male Sprague Dawley rats implanted with Walker 256 carcinosarcoma cells. When four doses of 5 mumol/kg CuPu(Py)2 and CuPu(Im)2, respectively, were administered intratumorally, reduction in tumor size, delay of metastasis and a significant increase in survival of the hosts were observed, resulting in 75% of total remissions. 60% of the animals recovered totally from the carcinosarcoma, when CuPu(Py)2 was applicated intravenously.

Animals↗
PubMed1990

Reactivity of active centre analogues of Cu2Zn2 superoxide dismutase.

Active centre analogues of Cu2Zn2 superoxide dismutase were devised and successfully employed. Emphasis was placed on the flexible nature of the superoxide mimicking compounds. Di-Schiff-bases proved most appropriate to fulfil these requirements. Both structural and functional aspects of the copper binding centre of the intact enzyme were met by these complexes. Nanomolar concentrations of copper coordinated in these complexes were sufficient to inhibit the K3CrO8 induced chemiluminescence identical to the reaction of Cu2Zn2 superoxide dismutase.

Animals↗
PubMed1979

Oxidation-reduction reactions of copper-thiolate centres in Cu-thionein.

Cu-thionein from yeast was investigated by EPR spectroscopy to probe the oxidation state of copper, and the effects on it of oxidizing and reducing agents. At pH 0.2 the copper was released, but no EPR signal from Cu(II) was observed, unless air was present. Optical experiments did not detect any disulphide groups which might have been formed during anaerobic release of copper. The mercurial, p-hydroxymercuribenzoate caused the release of EPR-detectable copper only under aerobic conditions, and EDTA caused release of Cu(II) on heating. No reduction of the copper-thiolate units in Cu-thionein by ascorbate was detected. Potentiometric titrations with hexachloroiridate(IV) or hexacyanoferrate(III) produced several different Cu(II) EPR signals at various stages of oxidation. The former oxidizing agent required a lower oxidation-reduction potential (+350 mV) to oxidize the copper, than the latter (+410 mV) and neither titration was fully reversible. The EPR signal from Cu(II) oxidized by hexachloroiridate(IV) resembled that produced by p-hydroxy-mercuribenzoate in air, suggesting that the copper was released from its thiolate ligands. It is concluded that the EPR non-detectable copper in the native protein is Cu(I). Oxidation-reduction of the copper-thiolate clusters of Cu-thionein is proposed to be decisive for controlling storage and transport of cellular copper.

Biological Transport↗
PubMed1979

Functional aspects of the superoxide dismutative action of Cu-penicillamine.

The superoxide dismutative action of Cu-penicillamine was examined by pulse radiolysis. The second order rate constand of the reaction wpith superoxide was 0.4 +/- o.2.10(9) M-1.s-1, comparable to the action of Fe and Mn-superoxide dismutases. No marked pH-dependence was seen. Neither ethylene diamine tetraacetic acid nor cyanide affected the catalytic action of Cu-penicillamine. The cyanide resistant reactivity as well as further X-ray photoelectron spectrometric measurements supported the suggestion of a Cu(I) stabilized sulphur radical being the active species involved in the catalysis of superoxide dismutation.

Binding Sites↗