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Biomedical subjects

D Beyersmann

Publications and source records attributed to D Beyersmann.

At least 55 records · Page 3Linked to original sources
PubMed1990

The DNA cleavage induced by a chromium(V) complex and by chromate and glutathione is mediated by activated oxygen species.

The number of strand breaks induced by the combination of chromate and glutathione (GSH) in PM2 DNA was effectively reduced upon addition of the hydroxyl radical scavengers dimethyl sulphoxide (DMSO), formate and benzoate. Administration of catalase also led to a depression of DNA degradation whereas superoxide dismutase (SOD) had very little influence. Essentially the same results were obtained in experiments employing a chromium(V) complex Na4(GSH)4Cr.8H20, which is an intermediate chromium species isolated from the reduction of chromate by glutathione. DNA cleavage was dependent on the presence of iron (FeCl3). When compared with the number of breaks produced by FeCl3 and GSH alone, chromate stimulated the generation of single-strand breaks. These findings suggest that hydroxyl radicals are one ultimate DNA cleaving agent in both reactions. A reaction scheme for the production of hydroxyl radicals is proposed.

Catalase↗
PubMed1990

Indirect mechanism of lead-induced genotoxicity in cultured mammalian cells.

The data concerning the mutagenic, clastogenic and carcinogenic properties of inorganic lead compounds have been conflicting. To investigate whether the genotoxicity of lead is due to indirect effects such as interference with DNA-repair processes, the induction of mutations, sister-chromatid exchanges and strand breaks by lead ions alone as well as in combination with UV light as a standard mutagen were determined. Lead acetate alone does not induce DNA-strand breaks in HeLa cells or mutations at the HPRT locus and sister-chromatid exchanges in V79 Chinese hamster cells. However, at all endpoints tested, lead ions interfere with the processing of UV-induced DNA damage. They inhibit the closing of DNA-strand breaks after UV irradiation and enhance the number of UV-induced mutations and sister-chromatid exchanges, indicating an inhibition of DNA repair. These data point out the necessity to consider such indirect effects when assessing the genotoxicity of metal compounds. As possible mechanisms of repair inhibition we suggest either the interaction with repair enzymes such as polymerase or ligase or else the interaction with calcium-regulated processes, for example with calmodulin.

Animals↗
PubMed1990

Probing of active site residues of the zinc enzyme 5-aminolevulinate dehydratase by spin and fluorescence labels.

5-Aminolevulinate dehydratase from bovine liver requires Zn(II) for its activity and is inhibited by micromolecular concentrations of Pb(II). To elucidate the structure of the active site and its interactions between the active site and the metal binding site we labeled the active site for fluorescence studies and ESR spectroscopy. o-Phthalaldehyde reacted with active site lysyl and cysteinyl residues to form a fluorescent isoindole derivative. The fluorescence energy was independent of the deprivation of Zn(II) and of its substitution by the inhibitory Pb(II). For ESR-studies five iodoacetamide and four isothiocyanate pyrrolidine-N-oxyl derivatives with various spacer lengths were used to label the active site cysteinyl and lysyl residues, respectively. The ESR spectra of the modified enzyme preparations exhibited a significant immobilization of all labels, even with the longest spacers employed. Obviously the reactive cysteine is buried more than 12 A, and the active site lysine more than 11 A in a cleft of the enzyme structure. Zn(II) deprivation from the iodoacetamide spin-labeled enzyme caused a marked reversible increase in label mobility, whereas the Pb(II) substituted enzyme exhibited a smaller mobilization of the label. These results are interpreted by a model of the active site where the reactive cysteinyl and the lysyl side groups are close enough to be crosslinked by o-phthalaldehyde within a distance of 3 A. A structural role is assigned to Zn(II) in the enzyme, since Zn(II) deprivation does not alter the fluorescence of the isoindole derivative and increases the mobility of the cysteine-bound spin labels in the active site cleft.

Apoenzymes↗
PubMed1989

Generation of PM2 DNA breaks in the course of reduction of chromium(VI) by glutathione.

The carcinogen chromate is efficiently taken up and reduced to chromium(III) compounds by various biological systems. To test the possible DNA damage induced in the course of chromium(VI) reduction, we used a combination of chromate with the reductant glutathione (GSH) as well as a green complex of chromium(V), which is formed in the reaction of chromate with GSH. The combination of chromate and glutathione was found to cause single-strand breaks in supercoiled circular DNA of the bacteriophage PM2. The green chromium(V) complex Na4(GSH)4Cr(V).8H2O, prepared from chromate and glutathione, also cleaved supercoiled PM2 DNA. No DNA-degrading effects were observed with either chromate or the final product of the reaction with GSH, a purple anionic chromium(III) GSH complex. The nature of the buffering agents revealed a strong influence on the extent of DNA strand breaks produced by chromate and GSH. A variation of the GSH concentration in the reaction with chromate and PM2 DNA, performed in sodium phosphate-buffered solutions showed an initial increase in the number of strand breaks at GSH concentrations up to 1 mM followed by a decline at higher GSH concentrations. Since neither chromate, when administered individually, nor the final product of chromium(VI) reduction, the purple chromium(III) GSH complex, produced any detectable DNA cleavage, the critical steps leading to DNA strand breaks occur in the course of the conversion of chromium(VI) to chromium(III) by GSH, the most abundant intracellular low molecular thiol. Moreover, the demonstration that DNA cleavage is induced in the presence of the chromium(V) complex identifies chromium(V) as the oxidation state of the metal, which is involved in the steps leading to DNA-damaging effects of chromate.

Bacteriophages↗
PubMed1989

Enhancement of UV-induced mutagenesis and sister-chromatid exchanges by nickel ions in V79 cells: evidence for inhibition of DNA repair.

With regard to contradictory results concerning the mutagenicity of nickel compounds in short-term assays, especially in bacterial test systems, Chinese hamster V79 cells were used to measure mutagenicity, comutagenicity and the induction of sister-chromatid exchanges (SCEs) by NiCl2. We confirmed the induction of mutations at the HGPRT locus as well as SCEs. In addition, NiCl2 shows a pronounced comutagenic effect towards UV. When using confluent cultures or resting cells due to serum deprivation, where more time is given for repair processes, the comutagenic effect is higher compared to logarithmically growing cells (10 and 4 times, respectively, compared to twice). Hence, we attribute this enhancement in mutagenicity to inhibition of DNA repair. Also the increase in induced SCEs after combined treatment with UV and NiCl2 supports this thesis. Furthermore, NiCl2 enhances the cyto-toxicity of cis-DDP about 12-fold. Since no comutagenic effect is observed in combination with MMS, we suggest that the inhibition of DNA repair by Ni(II) applies to all DNA changes that are repaired by the 'long-patch' excision repair system. This inhibition may occur via replacement of other divalent metal ions essential in repair and regulation processes.

Cell Line↗
PubMed1988

Chromate effects on human erythrocytes--investigations on sulphydryl groups, cross-linking of membrane proteins and electromechanical properties in the coulter-counter.

The carcinogen chromate inactivates its own carrier in the human erythrocyte membrane. This effect is paralleled by the inhibition of chromate uptake by the sulphydryl reagents N-ethylmaleimide and iodoacetate. However, no decrease in the sulphydryl content of erythrocyte membranes treated with up to 100 mM chromate was detected. By SDS gel electrophoresis, a limited cross-linking of red cell membrane proteins was found at 100 mM chromate, but not at cytotoxic concentrations up to 10 mM chromate. Erythrocytes treated with up to 100 mM chromate exhibited no change in the "dielectric breakdown", i.e. the sharp decrease of the apparent cellular volume at a critical detector current.

Biological Transport↗
PubMed1988

Small angle X-ray scattering study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase).

The quaternary structure of the native (zinc) porphobilinogen synthase (5-amino-laevulinate dehydratase) from bovine liver and its lead-substituted derivative is studied in solution by small angle X-ray scattering. In spite of the profound inhibitory effect of lead ions in the enzyme they do not produce a change in the quaternary structure detectable by small angle X-ray scattering. The most important molecular parameters of the native enzyme were found to be: radius of gyration Rg = 4.04 +/- 0.04 nm and maximum dimension Dmax = 12.0 +/- 0.5 nm. The corresponding values for the lead derivative are: Rg = 4.26 +/- 0.1 nm and Dmax = 12.5 +/- 0.5 nm. The quaternary structure of the enzyme in solution is described by a model, which fits the experimental scattering and distance distribution function.

Animals↗
PubMed1987

Zinc and cadmium 5-aminolevulinate dehydratase. Metal-dependent pH profiles.

Native 5-aminolevulinic acid dehydratase contains zinc ions, which are essential for the enzymatic activity. Replacement of zinc by cadmium yielded an active enzyme whose kinetic parameters (kkat and Km) are similar to those of the zinc enzyme in the neutral pH range. However, the pH profiles of kcat and Km were different due to different pKa values. Two groups both with pKa values of 6.5 in the free zinc enzyme, but with pKa values of 7.0 in the cadmium enzyme were calculated from plots of log (kcat/Km) versus pH. On the other hand, the enzyme-substrate complex is controlled by one acidic group (zinc pKa = 6.0, cadmium pKa = 6.4) and one basis group (zinc pKa = 8.2, cadmium pKa = 7.7) as calculated from plots of log kcat versus pH. The Arrhenius plots for kcat of the two enzymes show no significant difference, the free energies of activation are 77.1 kJ/mol for the zinc and 76.8 kJ/mol for the cadmium enzyme. From this and from previous work it is concluded that the metal ions are located near the active site and influence the ionisations of essential amino acid residues. From the pH profiles of the modifying reaction and inhibition by diethylpyrocarbonate a histidinyl residue is inferred as one of the ionisable groups of the active site.

Cadmium↗
PubMed1985

Extended-X-ray-absorption-fine-structure investigations of zinc in 5-aminolaevulinate dehydratase.

The zinc co-ordination in 5-aminolaevulinate dehydratase (5-aminolaevulinate hydro-lyase, EC 4.2.1.24) was investigated by recording and interpreting the extended X-ray-absorption fine structure (e.x.a.f.s.) associated with the zinc K-edge. The enzyme has a molecular mass of 280 000 Da and consists of eight subunits of 35 000 Da each; the samples studied contained approx. 1 g-atom of zinc/mol of subunit. Four forms of the enzyme were investigated and details of the zinc environment were elucidated, as follows. In the native enzyme, zinc is considered to be co-ordinated to three sulphur atoms at 0.228(2)nm [2.28(2)A] and a lower-Z atom at 0.192(5)nm [1.92(5)A] (if nitrogen) or 0.189(5)nm [1.89(5)A] (if oxygen). Reaction of the enzyme with the inhibitor 2-bromo-3-(imidazol-5-yl)propionic acid produced significant changes in the e.x.a.f.s., the nature of which are consistent with co-ordination by about three sulphur atoms at 0.222(2)nm [2.22(2)A], a nitrogen atom at 0.193(5)nm [1.93(5)A] and a nitrogen atom from the inhibitor at 0.214(5)nm [2.14(5)A]. Inactivation of the enzyme by air-oxidation of essential thiol groups and binding of the substrate produce slight changes in the e.x.a.f.s. consistent with slight re-arrangement of ligands with additional lighter ligands (nitrogen or oxygen). These results, when combined with previous findings, are taken to indicate that zinc has a structural rather than a direct catalytic role in 5-aminolaevulinate dehydratase.

Affinity Labels↗
PubMed1984

Affinity labelling of 5-aminolevulinic acid dehydratase with 2-bromo-3-(5-imidazolyl)propionic acid.

2-Bromo-3-(5-imidazolyl)propionic acid, a zinc-directed thiol reagent, inactivates the enzyme 5-aminolevulinic acid dehydratase from bovine liver (5-aminolevulinate hydro-lyase (adding 5-aminolevulinate and cyclizing, EC 4.2.1.24). The substrate, 5-aminolevulinic acid, completely protects against inactivation. The reagent inhibits the zinc-containing enzyme to a greater extent than the zinc-deprived enzyme; and it competes with the zinc chelator 1,10-phenanthroline. The reagent alkylates essential sulfhydryl groups of the enzyme, since the extent of the inactivation depends on the reduction of the enzyme protein by thiol compounds. It is concluded that the zinc site, the substrate site and the essential sulfhydryl groups are in close proximity in the active site.

Affinity Labels↗
PubMed1984

Zinc and cadmium in 5-aminolevulinic acid dehydratase. Equilibrium, kinetic, and 113Cd-nmr-studies.

5-Aminolevulinic acid dehydratase (ALAD) from bovine liver contains zinc that is partially lost during the isolation of the enzyme. ALAD has its maximal activity at 10(-5) M ZnCl2. It binds 7.4 Zn per octameric protein with an association constant of 5.3 X 10(6)M-1. ALAD is inactivated by 1,10-phenanthroline or ethylenediaminetetraacetic acid (EDTA) but not by monodentate anions like cyanide or sulfide. After removal of zinc by chelating agents, the enzyme activity may be restored by Zn2+ or Cd2+. Removal of zinc by EDTA increases KM 60-fold and decreases Vmax to about 1/2 of its original value. The 113Cd nuclear magnetic resonance spectrum of the enzyme reconstituted with 113Cd-acetate exhibits a single sharp resonance signal at 79 ppm. It does not change by the addition of substrate but disappears when the inhibitor lead acetate is added. Therefore, an immediate interaction between the metal ion of the enzyme and the substrate is excluded, whereas lead changes the environment of cadmium and probably of zinc too.

Animals↗
PubMed1983

Temperature dependence of activity and inhibition by lead with 5-aminolevulinic acid dehydratase.

5-Aminolevulinic acid dehydratase (EC 4.2.1.24) was purified 230-fold from human erythrocytes. The effects of temperature on various properties of the isolated enzyme were studied. The maximal activity is observed at 60 degrees C. The pH optimum is shifted from pH 7.3 at 5 degrees C to pH 6.6 at 55 degrees C. The Michaelis constant increases 5-fold from 5 to 55 degrees C. Inhibition by lead is more pronounced at elevated temperatures. This effect is in contradiction to the restoration of enzyme activity by heating hemolysate after inhibition by lead in vivo, which has been described in previous reports.

Humans↗
PubMed1974

Mutants of Escherichia coli B-r defective in deoxyribonucleic acid initiation: dnaI, a new gene for replication.

Mutagenized E. coli B/r cells were subjected to a procedure designed to select mutants temperature-sensitive for initiation of deoxyribonucleic acid (DNA) replication. Seventeen mutants exhibiting limited residual DNA synthesis at 42 C were obtained and the dna(-) sites were mapped genetically. Sixteen of the sites map near dnaA, dnaB, and dnaC. One mutant (dna-208) maps in a new location between the trp and his genes. We propose to call this mutant dnaI208. In complementation experiments dnaC(+) and dnaI(+) were dominant to dnaC(-) and dnaI(-) alleles, respectively. However, dnaA(-) was dominant to the wild-type allele dnaA(+). All dnaA mutants and four out of six dnaC mutants could be suppressed by F factor integration. The pattern of suppression was specific for each mutant.

Bacterial Proteins↗
PubMed1973

Prophage induction by high temperature in thermosensitive dna mutants lysogenic for bacteriophage lambda.

High-temperature treatment of thermosensitive dna mutants lysogenic for phage lambda leads to prophage induction and release of phage (at the permissive temperature) in elongation-defective mutants of the genotypes dnaB, dnaE, and dnaG. In initiation-defective mutants no prophage induction occurs at 42 C in mutants of the genotype dnaA, whereas with a dnaC mutant as well as with strain HfrH 252 (map position not yet known) phages are released at 42 C. DNA degradation at the replication fork at 42 C is observed in all dnaB(lambda) mutants tested, but not in mutants of the genotypes dnaE(lambda) and dnaG(lambda). Therefore, degradation of replication fork DNA is not a prerequisite for prophage induction.

Coliphages↗