1H NMR study of the interaction of ATP with Escherichia coli RNA polymerase containing in vivo-incorporated Co(II)

The DNA-dependent RNA polymerase containing two intrinsic cobalt ions (Co2-RPase) instead of the naturally occurring zinc was purified from Escherichia coli cells grown in zinc-depleted, cobalt-enriched media. Longitudinal NMR relaxation rates of the H2 and H8 protons of ATP were measured in the absence and presence of up to 92 microM Co2-RPase. No enhancement of the proton relaxation rates was observed in the presence of cobalt-containing enzyme, suggesting that the ATP substrate does not undergo rapid exchange at a site close to either of the intrinsic cobalt ions. This result is in contrast to that previously observed when Co2+ was incorporated into RPase by an in vitro procedure involving partial urea denaturation of the protein.     

Cytotoxicity of zinc in vitro

The effect of zinc ions on B16 mouse melanoma lines, HeLa cells and I-221 epithelial cells was investigated in vitro in order to ascertain whether sensitivity to Zn2+ is a general feature of cells in vitro and in an attempt to elucidate the mechanism(s) of zinc cytotoxicity. The proliferation of B16, HeLa and I-221 cell lines was inhibited by 1.25 x 10(-4), 1.50 x 10(-4) and 1.50 x 10(-4) mol/l Zn2+, respectively. The free radical scavengers, methimazole and ethanol, did not suppress the toxicity of Zn2+, neither did superoxide dismutase or catalase. The addition of the chelating agent EDTA reduced the zinc cytotoxicity. It was possible to suppress the cytotoxicity of zinc by increasing the concentration of either Fe2+ or Ca2+ but not Mg2+, which suggests that a prerequisite for the toxic action of zinc is entry into cells using channels that are shared with iron or calcium. This view was supported by experiments in which transferrin intensified the cytotoxic action of zinc in serum-free medium. Another agent facilitating zinc transport, prostaglandin E2, inhibited the proliferation of the B16 melanoma cell line. There were no conspicuous differences in zinc toxicity to pigmented and unpigmented cells. The toxic effect of zinc in the cell systems studied exceeded that of iron, copper, manganese and cobalt in the same concentration range. In vitro, Zn2+ should be regarded as a dangerous cation.     

Effect of calcium ion uptake on Candida albicans morphology

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.     

Zinc Ions Stabilise the Association of Basic Protein with Brain Myelin Membranes

Myelin basic protein (MBP) dissociated from brain myelin membranes when they were incubated (37 degrees C; pH 7.4) at physiological ionic strength. Zinc ions inhibited, and calcium promoted, this process. Protease activity in the membrane preparations cleaved the dissociated MBP into both small (less than 4 kilodaltons) and large (greater than 8 kilodaltons) fragments. The latter were detected, together with intact MBP, by gel electrophoresis of incubation media. Zinc ions appeared to act in two distinct processes. In the presence or absence of added CaCl2, zinc ions in the range 0.1-1 mM inhibited MBP-membrane dissociation. This process was relatively insensitive to heat and Zn2+ could be substituted by either copper (II) or cobalt (II) ions. A second effect was evident only in the presence of added calcium ions, when lower concentrations of Zn2+ (less than 0.1 mM) inhibited MBP-membrane dissociation and the accumulation of intact MBP in incubation media. This process was heat sensitive and only copper (II), but not cobalt (II), ions could replace Zn2+. To determine whether endogenous zinc in myelin membranes is bound to MBP, preparations were solubilised in buffers containing Triton X-100/2 mM CaCl2 and subjected to gel filtration. Endogenous zinc, as indicated by a dithizone-binding method, eluted with fractions containing both MBP and proteolipid protein (PLP). Thus, one means whereby zinc stabilises association of MBP with brain myelin membranes may be by promoting its binding to PLP.     

The influence of a specific microelemental environment in alginate gel beads on the course of propionic acid fermentation

In this work the exchange of calcium, cobalt, iron, magnesium, zinc and manganese ions between alginate gel beads and casein medium was investigated. The high release of calcium ions from alginate to the medium and the biosorption of some metal ions were observed. The pure alginate gel adsorbed all the metal ions examined, from a fermentative medium. Gel with immobilized cells of two strains of Propionibacterium freudenreichii subsp. shermanii showed an active ability to adsorb only cobalt, iron and zinc ions. In this way, a special microelemental environment was created in the alginate gel. This resulted in an increase of propionic acid production and a decrease of vitamin B₁₂ biosynthesis. Received: 30 April 1997 / Received revision: 2 July 1997 / Accepted: 4 July 1997     

Effect of pH on the modulation of rat osseous plate alkaline phosphatase by metal ions.

1. Metal ions other than zinc and magnesium were effective in modulating the activity of rat osseous plate alkaline phosphatase. 2. Increasing pH had remarkable effects on the modulation of rat osseous plate alkaline phosphatase. 3. The modulation of enzyme activity by zinc, manganese and cobalt ions was slightly affected by pH variations. 4. Zinc ions were stimulatory for the enzyme at very low concentrations (50 nM). Above 50 nM zinc ions inhibited the enzyme by displacing magnesium ions. 5. Calcium ions were inhibitors of alkaline phosphatase (Kd = 10 microM) whereas manganese (Kd = 1.3 microM) and cobalt (Kd = 0.2 microM) ions were stimulatory in the pH range 8.0-10.0.     

A new type of Alcaligenes eutrophus CH34 zinc resistance generated by mutations affecting regulation of the cnr cobalt-nickel resistance system.

Spontaneous mutants that were resistant to zinc were isolated from Alcaligenes eutrophus CH34 containing either the native plasmid pMOL28 or a derivative derepressed for its self-transfer, pMOL50. With the cured plasmid-free derivative of CH34, strain AE104, such mutants were not detected. The mutations, which were shown to be located in the plasmid, increased the level of the nickel and cobalt resistance determined by the cnr locus. The chromate resistance closely linked to the cnr locus was not affected by these mutations. In the Znr mutants, the resistance to zinc and nickel was constitutively expressed. Uptake studies showed that the zinc resistance in a Znr mutant resulted from reduced accumulation of zinc ions in comparison with that in the plasmid-free strain. Reduced accumulation of zinc was also observed to a lesser degree in the parental strain induced with nickel, suggesting that zinc interferes with the Ni2+ and Co2+ efflux system. A 12.2-kb EcoRI-XbaI restriction endonuclease fragment containing the cnr locus was cloned from plasmid pMOL28 harboring the mutation and shortened to an 8.5-kb EcoRI-PstI-PstI fragment conferring resistance to zinc, nickel, and cobalt. The 12.2-kb EcoRI-XbaI fragment was also reduced to a 9.7-kb BamHI fragment still encoding weak resistance to nickel and cobalt but not to zinc. Complementation studies demonstrated the recessivity of the cnr mutations with a Znr phenotype. Such mutations thus allow positive selection of mutants affected in the expression of the cnr operon.     

The possible occurrence of zinc in ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279

The activity of polyhydric alcohol dehydrogenases in Mycobacterium sp. 279 was studied under limitation of zinc in the growth medium. It was found that the activity of ribitol and sorbitol dehydrogenases were markedly lowered and that of D-arabinitol dehydrogenase remained unchanged in the Zn2+-deficient cells. Other ions tested i.e., Co2+, Cu2+, Ni2+ and Mn2+ failed to substitute Zn2+ ions in their effect on the enzyme activities. The Zn2+-responsive enzymes were sensitive to the chelating agents (1,10-phenanthroline, 2,2'-dipyridyl), whereas D-arabinitol dehydrogenase was insensitive. The results indicate possible existence of a zinc component in the ribitol and sorbitol dehydrogenases from Mycobacterium sp. 279.     

The effect of zinc and cobalt ions on the chromogenesis of Pseudomonas aeruginosa in the presence of methylphosphonic acid]

Adding zinc or cobalt sulphate into synthetic culture media of Pseudomonas aeruginosa was followed by partial or total inhibition of pyocyanine production, varying with the nature of the chemical source of phosphorus. An antagonism between Zn2+ and Co2+ ions was shown.     

Selective Electrodiffusion of Zinc Ions in a Zrt-, Irt-like Protein,ZIPB

All living cells need zinc ions to support cell growth. Zrt-, Irt-like proteins (ZIPs) represent a major route for entry of zinc ions into cells, but how ZIPs promote zinc uptake has been unclear. Here we report the molecular characterization of ZIPB from Bordetella bronchiseptica, the first ZIP homolog to be purified and functionally reconstituted into proteoliposomes. Zinc flux through ZIPB was found to be nonsaturable and electrogenic, yielding membrane potentials as predicted by the Nernst equation. Conversely, membrane potentials drove zinc fluxes with a linear voltage-flux relationship. Direct measurements of metal uptake by inductively coupled plasma mass spectroscopy demonstrated that ZIPB is selective for two group 12 transition metal ions, Zn²⁺ and Cd²⁺, whereas rejecting transition metal ions in groups 7 through 11. Our results provide the molecular basis for cellular zinc acquisition by a zinc-selective channel that exploits in vivo zinc concentration gradients to move zinc ions into the cytoplasm.     

A direct spectrophotometric method for the simultaneous determination of zinc and cobalt in metalloproteins using 4-(2-pyridylazo)resorcinol

An assay involving the direct and simultaneous determination of low micromolar concentrations (1-10 μM) of both zinc and cobalt ions suitable for metal content analyses of metalloproteins is described. The procedure exploits differences in the visible absorption spectra of the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) resulting from its complexation to Zn²⁺ and/or Co²⁺ ions and is based on the fit of experimental spectra to a linear addition of Beer-Lambert law. The method eliminates the need for separating or masking one of the metal ions prior to their quantification and could prove to be particularly useful in studies on Co²⁺-substituted zinc proteins.     

Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

Transient fluctuations of intracellular zinc ions in cell proliferation

Zinc is essential for cell proliferation, differentiation, and viability. When zinc becomes limited for cultured cells, DNA synthesis ceases and the cell cycle is arrested. The molecular mechanisms of actions of zinc are believed to involve changes in the availability of zinc(II) ions (Zn²⁺). By employing a fluorescent Zn²⁺ probe, FluoZin-3 acetoxymethyl ester, intracellular Zn²⁺ concentrations were measured in undifferentiated and in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Intracellular Zn²⁺ concentrations are pico- to nanomolar in PC12 cells and are higher in the differentiated than in the undifferentiated cells. When following cellular Zn²⁺ concentrations for 48 h after the removal of serum, a condition that is known to cause cell cycle arrest, Zn²⁺ concentrations decrease after 30 min but, remarkably, increase after 1 h, and then decrease again to about one half of the initial concentration. Cell proliferation, measured by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, decreases after both serum starvation and zinc chelation. Two peaks of Zn²⁺ concentrations occur within one cell cycle: one early in the G1 phase and the other in the late G1/S phase. Thus, fluctuations of intracellular Zn²⁺ concentrations and established modulation of phosphorylation signaling, via an inhibition of protein tyrosine phosphatases at commensurately low Zn²⁺ concentrations, suggest a role for Zn²⁺ in the control of the cell cycle. Interventions targeted at these picomolar Zn²⁺ fluctuations may be a way of controlling cell growth in hyperplasia, neoplasia, and diseases associated with aberrant differentiation.     

Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH₂-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH₂-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.     

Evidence that two zinc fingers in the methionine aminopeptidase from Saccharomyces cerevisiae are important for normal growth

Limited proteolysis of intact yeast methionine aminopeptidase (MAP1) with trypsin releases a 34 kDa fragment whose NH₂-terminal sequence begins at Asp70, immediately following Lys69. These results suggest that yeast MAP may have a two-domain structure consisting of an NH₂-terminal zinc finger domain and a C-terminal catalytic domain. To test this, a mutant MAP lacking residues 2–69 was generated, overexpressed, purified and analyzed. Metal ion analyses indicate that 1 mol of wild-type yeast MAP contains 2 mol of zinc ions and at least 1 mol of cobalt ion, whereas 1 mol of the truncated MAP lacking the putative zinc fingers contains only a trace amount of zinc ions but still contains one mole of cobalt ion. These results suggest that the two zinc ions observed in the native yeast MAP are located at the Cys/His rich region and the cobalt ion is located in the catalytic domain. The k.at and Km values of the purified truncated MAP are similar to those of the wild-type MAP when measured with peptide substrates in vitro and it appears to be as active as the wild-type MAP in vivo. However, the truncated MAP is significantly less effective in rescuing the slow growth phenotype of map mutant than the wild-type MAP. These findings suggest that the zinc fingers are essential for normal MAP function in vivo, even though the in vitro enzyme assays indicate that they are not involved in catalysis. In addition, a series of single mutations were generated by changing the cysteines and the histidines in the zinc finger region to serines and arginines, respectively. Analyses of these point mutations provide further evidence that the cysteines and histidines are important for the growth promotion function of yeast MAP.     

Inhibition of 2'-5' oligoadenylate synthetase by divalent metal ions.

OAS1 is the small form and OAS2 is the medium form of the human interferon-induced 2'-5' oligoadenylate synthetases. The p42 isoform of OAS1 and the p69 isoform of OAS2 have been expressed in insect cells and purified to give pure, highly active 2'-5' oligoadenylate synthetase. The catalysis of 2'-5' oligoadenylate synthesis is strictly dependent on double-stranded RNA and magnesium ions. We have examined the effect of a series of divalent metal ions: copper, iron and zinc ions strongly inhibited the enzymatic activity, cobalt and nickel ions were partly inhibitory whereas calcium and manganese ions were without effect. However, manganese ions can replace magnesium ions as activator. The inhibitory effect of zinc ions was characterised in detail. The inhibitory constants of Zn(2+) were estimated to be 0.10 mM for OAS1p42 and to 0.02 mM for OAS2p69. Cross-linking experiments showed that zinc ions can control the oligomerisation by enhancing the formation of tetrameric forms of OAS1p42     

Characterization of a cobalt-resistant mutant of Neurospora crassa with transport block

A cobalt-resistant strain of Neurospora crassa (cor) was obtained by repeated subculturing of the wild type on cobalt-containing agar medium. N. crassa cor is twentyfold more resistant to cobalt ions compared with the wild type. Resistance was stable on repeated subculturing of cor on cobalt-free media. N. crassa cor is also cross-resistant to nickel (fourfold), but not to zinc or copper. Higher concentrations of iron and magnesium ions are required to reverse growth inhibition due to cobalt toxicity in N. crassa cor, compared with the wild type. Germinating conidia and mycelia of the cor strain accumulated lower levels of cobalt ions compared with the parent N. crassa. The partial transport block for cobalt uptake is shown to be primarily due to decreased surface binding of cobalt to mycelia and cell walls. Efflux of mycelial cobalt was also observed in wild type and cobalt-resistant N. crassa. The characteristics of cor in comparison with wild type N. crassa are discussed in relation to the mechanisms of cobalt resistance.     

Cobalt-dependent transcription of the nitrile hydratase gene in Rhodococcus rhodochrous M8

Abstract The effects of cobalt ions on the activities of Rhodococcus rhodochrous M8 enzymes for nitrile utilization, nitrile hydratase and amidase, were investigated. In contrast to amidase, synthesis of nitrile hydratase and its activity required cobalt ions in the growth medium. Northern blot analysis showed that in the presence of cobalt ions, the level of mRNA for nitrile hydratase genes was several times higher than that under cobalt-limited conditions. It was assumed that the low nitrile hydratase activity in cells grown in the absence of cobalt ions is connected either with the weak expression of nitrile hydratase genes or with the rapid degradation of nitrile hydratase mRNA.     

Effects of Metals on Streptomyces coelicolor Growth and Actinorhodin Production

Actinorhodin production by Streptomyces coelicolor was used as a model system to study the effects of metals on growth and polyketide synthesis in a streptomycete. Numerous metals were tested in cultures grown in liquid media. Mercury and cadmium were highly toxic, and copper, nickel, and lead were less so, but all tended to inhibit both growth and antibiotic synthesis to a similar extent. Unexpectedly, manganese, cobalt, zinc, and, to a lesser extent, chromium caused complex effects that in general resulted in some enhancement of growth yield but a reduction in antibiotic titers. These complex effects meant that cobalt, manganese, and zinc had lower 50% inhibitory concentrations for antibiotic yields compared with those for biomass. The physiologically active divalent cations calcium and magnesium were also tested. Calcium at high concentrations was particularly effective in reducing antibiotic titers and enhancing growth yields. By adding calcium at different phases of growth, it could be demonstrated that it was most effective in reducing the antibiotic yield when added during the early growth phase. Addition during the antibiotic-producing phase resulted in little reduction of final actinorhodin titers.