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D H Petering

Publications and source records attributed to D H Petering.

At least 37 records · Page 2Linked to original sources
PubMed1996

Different conformations and site selectivity of HO-2-Co(III)-bleomycin A2 and Co (III)-bleomycin A2 bound to DNA oligomers.

Conformational properties of HO2(-)-Co(III)-bleomycin A2 (Form I) and Co(III)-bleomycin (Form II) bound to DNA oligomers offering either principal cleavage site for the drug, d(GGAAGCTTCC)2 or d(AAACGTIT)2, have been studied by NMR methods. Form I binds in slow exchange to these oligomers. It retains most of its solution nuclear Overhauser effects (NOEs) upon binding to either oligomer. Pyrimidinyl methyl protons from the metal domain of the drug make an NOE connection with a G5 2-amino proton on DNA. The bithiazole intercalates between base pairs involving either C6 and T7 or T6 and T7 of the two DNA molecules, according to NOE connections between the bithiazole protons and protons from these bases and changes in the positions of their chemical shifts. Form II also retains most of its solution NOEs upon association with the first oligomer. However, in contrast to Form I it binds to DNA in fast exchange on the NMR time scale over the temperature range of 5-35 degrees C and does not break the degeneracy of the DNA proton chemical shifts. No intermolecular NOEs between Form II and the 10-mer have been detected. Likewise, the major perturbation in chemical shift of the histidine H2 and guanine G5 protons seen in Form I-DNA adducts is absent in Form II-DNA. The association constant of Form II with d(GGAAGCTTCC)2 in 20 mM HEPES buffer at pH 7.4 and 25 degrees C is 1.7 x 10(5) M(-1), and 1.0 mol of Form II bind per mol of 10-mer.

Antibiotics, Antineoplastic↗
PubMed1996

Metal coordination environment and dynamics in 113cadmium bleomycin: relationship to zinc bleomycin.

The 13C chemical shifts of Cd- and ZnBlm A2 are almost identical throughout the entire molecule, suggesting that these structures adopt similar conformations. Nuclear magnetic resonance experiments with 113Cd-bleomycin have defined part of the metal-ligand environment of the molecule. Nitrogen atoms from the primary amine, pyrimidine, and imidazole are bound to 113Cd according to 13C spectra showing 113Cd-13C spin-spin couplings. Bound and free forms of the secondary amine nitrogen may be in equilibrium, as suggested by temperature-dependent 13C studies with Cd-bleomycin. In addition, a number of other carbon resonances are in chemical exchange over the temperature range 5-54 degrees C. The temperature dependence of the line widths of carbon atoms of Zn-bleomycin strongly resembles that of Cd-bleomycin. Examination of the 113Cd resonance as a function of temperature also supports the presence of at least two differently coordinated forms of cadmium in the molecule. According to the position of the 113Cd chemical shift, at most four nitrogen atoms are bound to Cd at low temperature. Titrations of 113Cd-bleomycin with chloride or acetate demonstrate that these anions can bind to major and minor forms of the structure and that a minor species exists which does not associate with chloride.

Antibiotics, Antineoplastic↗
PubMed1996

Comparative effects of Cd2+ and Cd-metallothionein on cultured kidney tubule cells.

The effects of Ca2(+) and Cd-metallothionein on two cultured cells with proximal tubule characteristics, mouse kidney cortical cells and pig kidney LLC-PK1 cells, have been compared. Cd2+ inhibits Na(+)-glucose cotransport in LLC-PK1 cells and in the process decreases the number of binding sites for [3H]phlorizin, a competitive inhibitor of glucose for the Na(+)-glucose cotransporter. During 24 hr incubation and over a range of concentrations in the two cell types, only Cd2+ inhibited Na(+)-glucose cotransport even when approximately equal concentrations of intracellular Cd resulted from these treatments. Indeed, at low concentrations of Cd-metallothionein in mouse cells, transporter activity was elevated. Extension of incubations to 72 hr in mouse cells led to increased Cd uptake and reduction in cell density with both sources of Cd but only a progressive decline in Na(+)-glucose cotransport activity with Cd2+. Zn-metallothionein was without effect under comparable conditions. Both forms of Cd were accumulated by these cells, with the large majority of the metal ion localizing in metallothionein as a Cd, Zn-protein in LLC-PK1 cells. Under equal exposure conditions, the net uptake of Cd from Cd-metallothionein in the two cell types. It is evident that the mechanisms of toxicity of Cd2+ and Cd-metallothionein as well as their modes of uptake differ in these two cell types.

Animals↗
PubMed1996

Depletion of cellular iron by bps and ascorbate: effect on toxicity of adriamycin.

A new method was developed that reduces the intracellular iron content of cells grown in serum-containing culture without involving the significant uptake of iron-chelating agents into cells. Negatively charged bathophenanthrolinedisulfonate (BPS), together with ascorbate, caused cells to lose much of their cellular iron without causing much depression in HL-60 or H9c2 (2-1) cell proliferation over a 48-h period. When added to serum supplemented RPMI-1640 culture media, BPS and ascorbate efficiently reduced and competed for iron in Fe(III) transferrin to form Fe(II)(BPS)3. The reaction also occurred with purified human iron-transferrin. When cells were incubated with growth medium containing serum that had been treated with BPS and ascorbate for 24 h, little or no BPS2- or Fe(II)(BPS)(4-)3 entered the cells, according to direct measurements and in agreement with the highly unfavorable 1-octanol/water partition coefficients for these molecules. However, iron was mobilized out of both cell types. After 24 h incubation of cells in this medium, there was no change in the activities of catalase and superoxide dismutase, or in the concentration of glutathione. Glutathione peroxidase was elevated 9%. Using HL-60 and H9c2 (2-1) cells made iron deficient with BPS and ascorbate, HL-60 cells grown in defined-growth media in the absence of iron-pyridoxal isonicotinoyl hydrazone, or Euglena gracilis cells maintained in a defined medium that was rigorously depleted of iron, it was shown that the cytotoxicity of adriamycin is markedly dependent on the presence of iron in each type of cell. Similar results were obtained when HL-60 cells were grown in RPMI-1640 culture medium and serum that had been incubated for 24 h in BPS and ascorbate and then chromatographed over a Bio-Rad desalting column to remove small molecules including BPS, ascorbate, and Fe(II)(BPS)3.

Animals↗
PubMed1995

Reaction of 111Cd7-metallothionein with EDTA. A reappraisal.

The ligand substitution reaction of EDTA with Cd7-metallothionein (Cd7-MT) has been reinvestigated. NMR titration of the 111Cd-protein with EDTA showed that the ligand interacts preferentially and cooperatively with Cd2+ ions in the beta-domain cluster. NMR and ultrafiltration kinetic analysis of this reaction using 5.6 mM Cd2+ as 111Cd7-MT and 56 mM EDTA indicated that cadmium-EDTA formed less rapidly than 111Cd peak intensity declined. Spectrophotometric and gel filtration studies of the reaction with 20 microM Cd2+ as Cd7-MT with various concentrations of EDTA revealed biphasic kinetics with much larger rate constants than observed in the NMR experiments. The fraction of total ligand substitution occurring in each kinetic step varied with EDTA concentration. The EDTA concentration dependence of both kinetic steps was consistent with the initial formation of protein.EDTA adducts, followed by their breakdown into products. Kinetic measurements were also made for the reactions of the isolated Cd4-alpha- and Cd3-beta-domains with EDTA. The Cd4 domain reacted with EDTA with biphasic kinetics, in which one Cd2+ was removed rapidly with first-order kinetics, which were zero-order in EDTA. The other three reacted with kinetics like those for the slower step of the holoprotein. Cd3-beta reacted with EDTA like the faster rate process associated with the Cd7-protein. The observed rate constants for the reaction of Cd7-metallothionein with EDTA and the fraction of reaction in the faster rate process were sensitive to protein concentration. These results are consistent with the hypothesis that the monomer-dimer equilibrium of the protein controls its kinetic reactivity with EDTA.

Animals↗
PubMed1994

Reaction of Cd7-metallothionein with cis-dichlorodiammine platinum (II).

The reaction of Cd7-metallothionein with cis-dichlorodiammine platinum (II) has been examined in titration and kinetic experiments. Titrations followed by 111Cd NMR spectroscopy and analysis of cadmium and platinum in the products indicated that platinum displaced cadmium in approximate 1:1 fashion, producing Pt7-metallothionein. According to NMR analysis, the overall reaction involved cooperative exchange of platinum for 111Cd in each domain of the protein. There was a modest preference for the reaction of the drug with the Cd3-containing beta-domain of the protein. The product had an apparent molecular weight that was nearly the same as that of Cd7-metallothionein as determined by Sephadex G-75 chromatography. Kinetic analysis also supported a cooperative displacement mechanism. The reaction was biphasic; neither step could be identified solely with the reaction of one cluster as indicated by the biphasic reaction of the Cd4-containing alpha-domain with the platinum complex.

Cadmium↗
PubMed1994

Iron requirement for cellular DNA damage and growth inhibition by hydrogen peroxide and bleomycin.

Studies with Euglena gracilis and HL-60 cells have assessed the need for intracellular iron in the mechanisms of inhibition of cell growth and DNA damage by H2O2 and bleomycin. Cell culture media were directly depleted of iron in order to deprive cells of nutrient iron. Major pools of cellular iron were reduced in both cell types. Nevertheless, iron bound in e.s.r.-observable haem protein and ribonucleotide diphosphate reductase in HL-60 cells was not decreased. In both control cell populations, there was a concentration-dependent reduction in proliferation and cell survival caused by H2O2. In comparison, the proliferation rates of both iron-deficient cell types were significantly less sensitive to H2O2. H2O2 caused concentration-dependent single-strand breakage in DNA in control HL-60 and Euglena gracilis cells. Iron deficiency reduced the amount of strand breaks in HL-60 cells at each concentration of H2O2 used. Single-strand breakage caused by H2O2 in Euglena gracilis was a direct function of the concentration of iron in which the cells had been grown. Growth inhibition and both single- and double-strand DNA damage caused by bleomycin were substantially reduced or eliminated in iron-deficient cells. Copper bleomycin behaved like metal-free bleomycin when assayed for the capacity to cause DNA damage in iron-normal and iron-deficient HL-60 cells. In contrast, iron bleomycin was equally active under the two conditions in these cells.

Animals↗
PubMed1994

DNA strand breakage in isolated nuclei subjected to bleomycin or hydrogen peroxide.

The sources of iron (Fe) and reductant required for DNA strand breakage by the antitumor drug bleomycin (Blm), H2O2 and ascorbate were investigated using nuclei instead of whole cells in order to study a simpler, related system that was subject to better control and easier chemical manipulation. Ehrlich ascites tumor cells were isolated and treated directly on filters, and analysed for DNA damage by alkaline and nondenaturing elution. Extraction and treatment buffers were depleted of trace Fe by passage through Mg(OH)2 gel. Nuclei were treated for 1 hr at 37 degrees. High levels of single- and double-strand breakage were obtained using Fe(III)Blm in the range 0.01 to 0.08 microM. In contrast, Blm was effective only at two orders of magnitude greater concentration. Cu(II)Blm was totally ineffective in causing damage. Depletion of nuclear protein thiols with N-ethylmaleimide reduced double-strand breakage at the upper end of the FeBlm concentration-response curve. A 1 mM concentration of NADPH or NADH greatly increased the extent of double-strand breakage by 0.01 microM FeBlm, suggesting roles for cytochrome P450 or cytochrome b5 reductase in strand breakage. Fe(III)ATP (1:20 metal to ligand and 50 microM in Fe) and Fe(III)EDTA (1:2 metal to ligand and 50 microM in Fe) did not cause single-strand breaks. In the absence of added Fe, H2O2 or ascorbic acid (50 microM) caused less than one Gy-equivalent single-strand breakage. Addition of ascorbate plus Fe(III)ATP or Fe(III)EDTA produced breakage beyond the capacity of alkaline elution to analyse (5-6 Gy). Overall, the results indicate that Fe, which may contribute to DNA damage by Blm and forms of activated oxygen within cells, is not strongly bound in the nucleus and that nuclear thiols other than glutathione contribute reducing equivalents to Fe(III)Blm for the DNA damaging chemistry.

Animals↗
PubMed1994

Sequential proton resonance assignments and metal cluster topology of lobster metallothionein-1.

NMR studies of 111Cd6-MT 1 from lobster have been conducted to determine coordination structure of Cd-thiolate binding in the protein. Sequential proton resonance assignments were made using standard two-dimensional 1H NMR methods. Two-dimensional 1H-111Cd HMQC experiments were then carried out to determine the cadmium-cysteine connectivities in the protein. With this information, it was established that the six Cd ions exist in two different Cd3S9 clusters, each involving three bridging and six terminal thiolate ligands. Sequential cysteines in the sequence provide the sulfhydryl ligands for each cluster and do not overlap, as has been found in mammalian metallothionein. Comparison of the N-terminal, Cd3S9 B-type cluster of lobster MT 1 with the Cd3S9 cluster from rabbit MT 2 shows that while eight of the nine cysteine residues occupy homologous positions in their sequences, three of the 12 Cd-thiolate connectivities are different. Similarly, the C-terminal B-cluster of lobster MT 1 was compared with the Cd4S11 cluster of mammalian MT 2, excluding the two terminal cysteine sulfhydryl groups that convert this cluster from A- to B-type. As above, eight of nine cysteine positions are identical, yet five of 12 Cd-sulfhydryl connections are different. These differences are expanded when the role of each cysteine as bridging or terminal ligands in the clusters is considered.

Amino Acid Sequence↗
PubMed1994

Reaction of DNA-bound ferrous bleomycin with dioxygen: activation versus stabilization of dioxygen.

The properties of binding of dioxygen to ferrous bleomycin [Fe(II)Blm] in the presence of DNA have been examined. Dioxygen reacts rapidly with Fe(II)Blm.DNA, forming an adduct that is increasingly stable to oxidation-reduction as the ratio of base pairs to drug becomes larger. This species has a spectrum similar to that reported for a proposed dioxygenated intermediate in the solution reaction of Fe(II)Blm with O2. It contains Fe(II) as measured with bathophenanthroline disulfonate (BPS) and O2 according to direct observation of stoichiometric release of O2 upon chelation of Fe(II) by BPS. The dioxygenated form O2-Fe(II)Blm.DNA, is highly stable; bound O2 dissociates upon purging with N2 with a rate constant of 0.16 min-1. Although Fe(II)Blm or Fe(II)Blm.DNA reacts almost completely with BPS within the time of mixing, O2-Fe(II) Blm.DNA reacts much slower in a process that is first order in Fe(II) and in BPS at constant DNA concentration. The rate is inversely related to DNA concentration reaching a limiting value at 50-100 base pairs per drug molecule. The kinetics of oxidation of Fe(II)Blm bound to DNA by O2 are biphasic and depend on the base pair to drug ratio. After formation of O2-Fe(II)Blm.DNA, another reaction occurs in which O2 is released, which is second order in dioxygen donor, and in which the second-order rate constant declines with increasing base pairs to FeBlm ratio. The rate of this second-order process accelerates at higher ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

Aerobiosis↗
PubMed1994

Basal metallothionein in tumors: widespread presence of apoprotein.

A survey has been conducted of solid and ascites tumors from mice and solid tumors in rats for the presence of metallothionein or metallothionein-like protein. In most tumors, a positive identification was made on the basis of Sephadex G-75 and HPLC-DEAE chromatography followed by competitive radioimmunoassay for metallothionein. Apometallothionein was revealed in a number of tumors for the first time by comparing the Sephadex G-75 chromatographic profiles of Zn in native cytosol and Cd in cytosol incubated briefly with CdCl2 to saturate free binding sites on the protein before Sephadex G-75 chromatography. In two cases unsaturation of metallothionein was correlated with a lack of zinc in the ascites fluid which supplies the tumor with zinc.

Animals↗
PubMed1994

NMR determination of the structures of peroxycobalt(III) bleomycin and cobalt(III) bleomycin, products of the aerobic oxidation of cobalt(II) bleomycin by dioxygen.

The oxidation of Co(II) bleomycin A2 by dioxygen leads to two products, HO2-Co(III) bleomycin A2 (form I) and Co(III) bleomycin A2 (form II). 1H NMR chemical shift assignments for protons of both forms have been made by two-dimensional NMR spectral techniques. The chemical shifts of protons throughout forms I and II differ from each other and from apobleomycin A2. NOESY spectra reveal a number of intermediate and long-range 1H-1H couplings within the metal-binding domain, between the metal-binding domain and the peptide linker, which connects it and the DNA-binding region of the molecule, and, in form I, between the DNA- and metal-binding domains. Molecular dynamics calculations were carried out based on the NOESY results and an adjustable square pyramidyl ligand geometry around Co(III) composed of nitrogen atoms of the primary and secondary amine groups, pyridine (N5), and amide and imidazole (N1) of the hydroxyhistidine residue. In form I, the bithiazole group folds back across the square pyramid forming a compact structure. Although this conformational feature was not observed in form II, the peptide linker between the metal- and DNA-binding domains in both species shows extensive folding based on a large number of intramolecular interactions.

Amides↗
PubMed1994

Inhibition of Na(+)-glucose cotransport in kidney cortical cells by cadmium and copper: protection by zinc.

Properties of the inhibition of Na(+)-glucose cotransport by Cd2+ in mouse kidney cortical cells have been determined. In no case was any inhibition observed before 3 hr. The extent of inhibition was dependent upon both the concentration of Cd2+ and the length of exposure. Kinetic studies showed that metallothionein mRNA induction by Cd2+ was initiated within 1 hr after incubation with Cd2+ began and peaked by 3-6 hr. Metallothionein protein increased more slowly, beginning at 3 hr and continuing for at least 9 hr. The protein had both Cd2+ and Zn2+ bound to it throughout this period. Nevertheless, a pool of nonmetallothionein Cd2+ appeared after 3 hr, coinciding with the onset of inhibition of Na(+)-glucose cotransport, and increased over the next 9 hr. Pretreatment of cells with Zn2+ protected them from the effects of Cd2+ on Na(+)-glucose cotransport. It delayed the onset of inhibition of transport as well as the extent of inhibition. Detailed analysis of the distribution of Cd2+ and Zn2+ in the soluble fraction of these cells showed that the concentration of non-metallothionein bound Cd2+ was not suppressed by the presence of Zn-metallothionein after the onset of exposure to Cd2+. Incubation of cells with larger concentration of Zn2+ and Cu2+ also inhibited Na(+)-glucose cotransport.

Animals↗
PubMed1994

DNA double-strand breakage by bleomycin in Ehrlich ascites tumor cells as measured by nondenaturing filter elution.

DNA double-strand breakage by bleomycin (Bleo) in Ehrlich ascites tumor cells was examined by nondenaturing filter elution using 0.2% SDS at pH 9.6 as the eluant. The majority of damaged DNA from cells treated with 5 to 100 microM Bleo and for durations up to 6 h eluted in the first two or three 1.5-h fractions. This was significantly different from the multiphasic elution pattern of DNA from cells irradiated with 30 or 50 Gy 137Cs gamma rays, which showed a more gradual elution. Relative elution with respect to 50 Gy 137Cs gamma rays produced by 1-h treatments (about 0.35) showed no differences over the range of concentrations from 5 to 50 microM. Elution at pH 7.2 did not detectably reduce the fraction of DNA eluting from Bleo-treated cells, while reducing the elution of DNA from 50 Gy-irradiated cells by 35%. An experiment examining specificity for double-strand breakage by Bleo in different stages of the cell cycle demonstrated no differences in relative elution between G1, S and G2/M phases. Overall, the results of 1-h treatments are consistent with net production of high levels of double-strand breaks restricted to a portion of cellular DNA. However, extended treatments (up to 4 h) produced a nearly proportional increase in relative elution.

Animals↗
PubMed1993

Metallothionein reacts with Fe2+ and NO to form products with A g = 2.039 ESR signal.

The interaction of metallothionein, Fe2+, and nitric oxide has been examined by electron spin resonance spectroscopy. When either apoMT 2 or Zn-MT 2 from rabbit liver was mixed with Fe2+ and NO, paramagnetic products with g values of 2.013 and 2.039 were found. These are characteristic of iron nitrosyl thiolates with stoichiometry, Fe(NO)2(SR)2. This product was not generated when beef heart mitochondrial aconitase was used as the source of iron.

Animals↗
PubMed1993

The oxidation of rabbit liver metallothionein-II by 5,5'-dithiobis(2-nitrobenzoic acid) and glutathione disulfide.

Because metallothionein (MT) may undergo thiol-disulfide or other redox reactions under certain cellular conditions, the partially and completely oxidized products of the reactions of Cd7-MT-II with the electrophile 5,5'-dithiobis(2-nitrobenzoic acid), ESSE, and oxidized glutathione, GSSG, were characterized. Reaction with the stoichiometric quantity of ESSE (1 ESSE per MT thiolate) generates monomeric and polymeric MTs with three types of disulfide bonds: intra- and intermolecular CyS-SCy linkages and a small number (2-3/MT) of mixed disulfides, CyS-SE, involving thionitrobenzoate (ES-). Reaction with substoichiometric quantities of ESSE (0.02 or 0.1 per MT thiolate) causes the formation of intra- and intermolecular CyS-SCy disulfides, but no mixed disulfides. In the latter reactions, two equivalents of ES- are released per mole of ESSE, but the release is described by a single first-order rate constant (k = 3.0 +/- 0.5 sec-1). Substantial amounts of cadmium remained bound to the MT monomers and polymers after reaction with the substoichiometric quantities. Despite the Cd bound to the MT after reaction with 0.1 ESSE per MT thiolate, no 111Cd NMR signals were detected, indicating rapid equilibration of the remaining metal ions among the disrupted binding sites. Large excesses of the endogenous aliphatic disulfide, GSSG, displace Zn+2 from Zn7-MT slowly. The reaction is complete after 24 hours with 5000 microM GSSG, but only 25% complete after 72 hours with 250 microM GSSG. Approximately one Cd+2 is displaced rapidly from Cd7MT by 5000 microM GSSG and half as much by 250 microM GSSG, but no further reaction occurs. It is unlikely that GSSG oxidation of MTs would be physiologically significant.

Animals↗
PubMed1993

Repair of bleomycin-induced DNA double-strand breakage in Ehrlich ascites tumor cells.

The effect of 1,10-phenanthroline (OP) on repair of bleomycin (Bleo)-induced double-strand breaks in Ehrlich ascites tumor cells was studied using nondenaturing filter elution. 1,10-Phenanthroline is a metal chelator which is believed to inhibit strand breakage by Bleo through competition for intracellular iron. Cells were treated with 25 microM Bleo for 1 h, washed free of unincorporated drug, and then reincubated in the absence or presence of OP. In the absence of OP, relative elution (with respect to cells irradiated with 50 Gy and used as an internal standard) decreased in a first-order process with a half-time for repair of 2.4 h. 1,10-Phenanthroline at 10 nmol/10(5) cells (50 microM) accelerated the net decrease in relative elution, producing a biphasic response with half-times of 5.3 h and less than 30 min for the two components. Thus functionally active Bleo remained in cells after they were washed free of unincorporated drug. 1,10-Phenanthroline at a concentration of 3.1 nmol/10(5) cells did not result in a similar net acceleration of repair of double-strand breakage, though it increased the rate of repair of single-strand breakage as measured by alkaline elution. The differences in repair observed in response to different OP concentrations are discussed in terms of models for double-strand breakage by Bleo. After 4-5 h repair, relative elution from Bleo-treated cells remained at about 40% of that achieved at the end of 1-h Bleo treatment in either the presence or absence of the 10 nmol OP/10(5) cells, demonstrating that some double-strand breaks were resistant to repair.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗