Phosphoenolpyruvate carboxykinase (guanosine 5'-triphosphate) from rat liver cytosol. Divalent cation involvement in the decarboxylation reactions

The presence of a divalent metal ion together with a catalytic amount of inosine 5'-diphosphate (IDP) is essential for the formation of pyruvate from oxalacetate catalyzed by purified rat liver cytosol phosphoenolpyruvate carboxykinase (PEPCK). With decreasing order of effectiveness, this pyruvate-forming activity was supported by micromolar levels of Cd2+, Zn2+, Mn2+, and Co2+. At the same concentrations, Mg2+ or Ca2+ was not effective. Combinations of Cd2+ with either Zn2+, Mn2+ or Co2+ were not additive with respect to the pyruvate-forming activity of PEPCK. Kinetic determination, with Cd2+ as the supporting cation, showed a 1:1 stoichiometry of interaction between each enzyme molecule and the nonconsumable substrate IDP. With 10 muM added Cd2+, the apparent Km for oxalacetate was 41 muM, and the apparent Ka for IDP was 0.25 muM. With Zn2+ or Mn2+, the apparent Ka for IDP was 0.2 or 0.13 muM, respectively. The effect of divalent transition-metal ions on PEPCK-catalyzed formation of phosphoenolpyruvate from oxalacetate was also investigated. Under steady-state conditions, the basal activity with MgITP was effectively enhanced with micromolar levels of Mn2+, Cd2+, or Co2+ included in the assay. The Vm increased 7- and 3.6-fold, and the apparent Km for MgITP changed by about a factor of 2 with the optimal concentrations of Mn2+ and Co2+, respectively. The most striking changes were in the apparent Km values for oxalacetate, which decreased to one-third and one-tenth when either Mn2+ or Co2+ was present in the assay together with Mg2+. The possible physiological importance of this kinetic effect is discussed.     

Accumulation and storage of Zn2+ by Candida utilis.

Starved cells of Candida utilis accumulated Zn2+ by two different processes. The first was a rapid, energy- and temperature-independent system that probably represented binding to the cell surface. The cells also possessed an energy-, pH-, and temperature-dependent system that was capable of accumulating much greater quantities of the cation than the binding process. The energy-dependent system was inhibited by KCN, Na2HAsO4, m-chlorophenyl carbonylcyanide hydrazone, N-ethylmaleimide, EDTA and diethylenetriaminepenta-acetic acid. The system was specific inasmuch as Ca2+, Cr3+, Mn2+, Co2+ or Cu2+ did not compete with, inhibit, or enhance the process, Zn2+ uptake was inhibited by Cd2+. The system exhibited saturation kinetics with a half-saturation value of 1.3 muM and a maximum rate of 0.21 (nmol Zn2+) min(-1) (mg dry wt(-1)) at 30 degrees C. Zn2+ uptake required intact membranes since only the binding process was observed in the presence of nystatin, toluene, or sodium dodecyl sulphate. Cells did not exchange recently accumulated toluene, or sodium dodecyl sulphate. Cells did not exchange recently accumulated 65Zn following the addition of a large excess of non-radioactive Zn2+. Similarly, cells pre-loaded with 65Zn did not lose the cation during starvation, and efflux did not occur when glucose and exogenous Zn2+ were supplied after the starvation period. Efflux was only observed after the addition of toluene or nystatin, or when cells were heated to 100 degrees C. Cells fed a large quantity of Zn2+ contained a protein fraction resembling animal cell metallothionein. In batch culture, cells of C. utilis accumulated Zn2+ only during the lag phase and the latter half of the exponential-growth phase.     

Induction of metallothionein-I mRNA in cultured cells by heavy metals and iodoacetate: evidence for gratuitous inducers.

A mouse hepatocyte cell line selected for growth in 80 microM CdSO4 (Cdr80 cells) was used to test the role of metallothioneins in heavy metal detoxification. The cadmium-resistant (Cdr80) cells have double minute chromosomes carrying amplified copies of the metallothionein-I gene and accumulate ca. 20-fold more metallothionein-I mRNA than unselected cadmium-sensitive (Cds) cells after optimal Cd stimulation. As a consequence, the amount of Cd which inhibits DNA synthesis by 50% is ca. 7.5-fold higher in Cdr80 cells than in Cds cells. Cds and Cdr80 cells were compared in terms of their resistance to other heavy metals. The results indicate that although Zn, Cu, Hg, Ag, Co, Ni, and Bi induce metallothionein-I mRNA accumulation in both Cdr80 and Cds cells, the Cdr80 cells show increased resistance to only a subset of these metals (Zn, Cu, Hg, and Bi). This suggests that not all metals which induce metallothionein mRNA are detoxified by metallothionein and argues against autoregulation of metallothionein genes. Metallothionein-I mRNA is also induced by iodoacetate, suggesting that the regulatory molecule has sensitive sulfhydryl groups.     

Maleate induced change in the kidney binding of mercury in rats pretreated with cadmium

The kidney uptake of Hg2+ was increased by Cd2+-pretreatment when Hg2+ was given intraperitoneally but not subcutaneously. Subsequent s.c. administration of maleate increased Hg2+ release from the kidneys only if Hg2+ was given subcutaneously. Neither the effect of Cd2+, nor that of maleate, on the distribution of Hg2+ among the renal soluble protein fractions was affected by the route of Hg2+ administration. The protective effect of Cd2+-pretreatment against the nephrotoxic effect of Hg2+ was also independent of the route of Hg2+ administration. Maleate given in nephrotoxic doses removed Hg2+ and copper, but not Cd2+ from the renal metallothionein fraction. Mercury in the urine, however, was not complexed by proteins with the molecular weight of thionein, but was bound to high molecular weight proteins and diffusible molecules. These findings are discussed in relation to the role of metallothionein in the interaction between Cd2+ and Hg2+.     

Uptake of cadmium is diminished in transfected mouse NIH/3T3 cells enriched for metallothionein.

To determine the relationship between cellular uptake of cadmium and content of metallothionein, we measured uptake of 109Cd in cells that differed in content of metallothionein (MT). MT cells were derived from NIH/3T3 cells by transfection with a plasmid containing the genome of bovine papilloma virus and the mouse metallothionein-I gene, driven by the promotor for the glucose-regulated protein of 78 kDa. Control cells were similarly transfected with bovine papilloma virus-based plasmids with the gene for metallothionein inverted and thus separated from the promoter (TM), or deleted, along with the promoter (BPA). The number of copies of bovine papilloma virus-based plasmids was similar in MT, TM, and BPA cells, approximately 100 per cell. MT cells were more than 10 times more resistant to the lethal effect of cadmium than were the control cells. Synthesis of metallothionein was 15-fold greater in the MT cells than in the TM or BPA cells. The uptake of 109Cd by the cells enriched in metallothionein was 4-fold less than by the control cells. These data suggest that an increased content of metallothionein may protect some cells from the toxic effects of cadmium, in part, by diminishing uptake of the metal.     

Metallothioneins and resistance to cadmium poisoning in Drosophila cells

Toxicity of cadmium on Drosophila cell lines has been studied. Maximal tolerance for cadmium chloride is 10 microM. Metallothioneins are induced in Drosophila cells following cadmium addition. A stable cadmium resistant cell line (Cd R200) has been selected starting from the haploid D clone. The Cd R200 cells are diploid and display metallothionein levels 22 times higher than cells of the original line fully induced with cadmium. The 200 microM CdCl2 tolerance upper limit in Cd R200 line is overcome if L-cysteine is supplemented to the medium. It is thus possible, in the presence of 5 mM L-cysteine, to select cells able to resist 800 microM CdCl2. These cells produce 4 times more metallothioneins than Cd R200 cells.     

Subcellular changes of essential metal shown by in-air micro-PIXE in oral cadmium-exposed mice

To clarify the relation of essential metals to cadmium (Cd) toxicity, we evaluated metallothionein expression and analyzed the subcellular distribution of essential metals using in-air micro-Particle-Induced X-ray Emission (PIXE). Four mice were dosed orally with 100 mg/L of Cd in drinking water for 1.5 or 2 years. Frozen samples of organs were used for micro-PIXE analysis and formalin-fixed samples were used for metallothionein staining. Immunohistochemically, metallothionein induction by 1.5y-Cd exposure was higher in the renal cortex than in the liver. Metallothionein expression was reduced after 2y-Cd administration compared to the 1.5y-Cd-exposed mice. Cd-induced tissue damage became marked in the 2y-Cd-exposed mice compared to the 1.5y-Cd-exposed mice, in which nephrotoxicity was more prominent than hepatotoxicity. Cd yield was higher in the renal cortex of the 2y-Cd-exposed mouse than in that of the 1.5y-Cd-exposed mouse, whereas no such increasing tendency was found in the liver. Compared to the control, the Cd-exposed mice markedly accumulated zinc in the liver and renal cortex. In the Cd-exposed mice, iron was mildly accumulated in the renal cortex and was slightly deprived in the liver. Elemental maps showed that a large amount of Cd was spatially combined with zinc in the 1.5y-Cd mouse. Free Cd became abundant in the 2y-Cd-exposed mouse. In addition, a small amount of Cd was colocalized with iron. The data suggest that zinc may contribute to protect against oral-administrated Cd toxicity, and impaired induction of MT may participate in hepato-nephrotoxicity of the 2y-Cd-exposed mouse.     

Domain-specificity of Cd2+ and Zn2+ binding to rabbit liver metallothionein 2. Metal ion mobility in the formation of Cd4-metallothionein alpha-fragment.

The yield of the alpha-fragment of rabbit liver metallothionein 2 was used to test the domain-specificity and mobility of Cd2+ and Zn2+ when bound to metallothionein. Increasing molar ratios of Cd2+ were added to either Zn7-metallothionein or the metal-ion-free apo-metallothionein. The enzyme subtilisin was used to digest those parts of the peptide chain that were not bound to Cd2+. Analysis of the digestion products was carried out by separation by polyacrylamide-gel electrophoresis. The chelation agent EDTA was used as a competitive chelator. It was found that the presence of excess EDTA greatly enhances the formation of the Cd4-metallothionein alpha-fragment, and catalyses the complete digestion of all other the metal-ion-containing peptides, so that even Cd7-metallothionein, formed when 7 molar equivalents of Cd2+ are added to Zn7-metallothionein, is digested to the alpha-fragment. These results suggest that the Cd2+ bound in the beta-sites is very labile, much more labile than the kinetics of the off-reaction would suggest. The observation of significant amounts of alpha-fragment on the gels, even when the stoichiometry of the metal ions initially present in the protein should not have resulted in much concentration of Cd4-alpha-fragment clusters, indicates that as the digestion proceeds the metal ions move to sites that form complete clusters and therefore selectively protect that part of the peptide chain from digestion. We also find that rabbit Cd4-metallothionein 2 alpha-fragment stains near to the top of the gel, in complete contrast with the location of rat Cd4-metallothionein 2 alpha-fragment. This difference in the mobilities suggests that the alpha-fragment prepared from rabbit metallothionein 2 is much less negatively charged than the analogous protein fragment prepared from rat liver metallothionein 2.     

Bacterium-based heavy metal biosorbents: enhanced uptake of cadmium by E. coli expressing a metallothionein fused to beta-galactosidase

We investigated the potential utility of a recombinant E. coli that expresses the human metallothionein II gene as a fusion protein with beta-galactosidase as a heavy metal biosorbent. E. coli cells expressing the metallothionein fusion demonstrated enhanced binding of Cd2+ compared to cells that lack the metallothionein. It was shown that the metallothionein fusion was capable of efficiently removing Cd2+ from solutions. Approximately 40% of the Cd2+ accumulated by the recombinant cells free in suspension was associated with the outer cell membrane, and 60% of that was present in the cytoplasm.     

The occurrence of cadmium in sub-cellular particles in the kidney of the marine mussel, Mytilus edulis, exposed to cadmium. The use of electron microprobe analysis

In mussels (Mytilus edulis) chronically exposed to cadmium, 85% of the Cd2+ was found to be associated with membrane-limited granular structures when elemental analyses were carried out on cryo-sectioned tissue by electron probe X-ray microanalysis. These granules also contained high concentrations of sulphur and phosphorus as well as other metalions, including Ca2+, iron and Zn2+. In contrast, after homogenisation and fractionation by differential centrifugation, the major proportion of the Cd2+ was found in the cytoplasmic fraction. However, many lysosomes were also ruptured by this treatment. Gel filtration chromatography of this fraction indicated the presence of a Cd2+-binding component of similar molecular weight to the metallothionein purified from the digestive gland of the same animals. It is therefore proposed that metallothionein may be associated with particulate structures which would thus reduce its cellular toxicity.     

Expression of human metallothionein III and its metalloabsorption capability in Escherichia coli

Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.     

Metal-responsive elements act as positive modulators of human metallothionein-IIA enhancer activity.

The human metallothionein IIA (hMT-IIA) gene contains two enhancer elements whose activity is induced by heavy-metal ions such as Cd2+. To determine the nature of the relationship between the metal-responsive elements and the element(s) responsible for the basal activity of the enhancers, the basal-level enhancer element(s), the hMT-IIA enhancers were subjected to mutational analysis. We show that deletion of the metal-responsive elements had no effect on the basal activity of the enhancer but prevented further induction by Cd2+. On the other hand, replacement of the basal-level enhancer element with linker DNA led to inactivation of the enhancer both before and after treatment with Cd2+. Therefore, the metal-responsive elements seems to act as a positive modulator of enhancer function in the presence of heavy-metal ions. In addition to the two enhancers, the hMT-IIA promoter contained one other element, the GC box, required for its basal expression. Unlike deletion of the basal-level enhancer element, replacement of the GC box with linker DNA had no effect on the ability of the promoter to be induced by Cd2+.     

不同应激因子对小鼠肝脏金属硫蛋白诱导合成的影响

目的筛选出小鼠肝脏金属硫蛋白（MT）合成量最大的诱导方式。方法从时间-效应和剂量-效应两方面研究了重金属元素（Cd）、微量元素（Zn）、重金属与微量元素的组合（Cd＋Zn）、生理因子（饥饿）及创伤因子等五大类组合应激因子、19种诱导方式对小鼠肝脏中MT诱导合成的影响及效果。结果生理因子诱导MT量最小,饥饿诱导小鼠肝脏MT的量随饥饿程度的加重而增加,但各组间差异不显著（P〉0.05）;创伤因子诱导产生MT的量最高,其诱导量随创伤恢复时间的增加而降低,各组之间差异显著（P〈0.01）,本实验诱导峰值（9.0241±0.6441μmol/g）出现在创伤后6 h;重金属元素和微量元素诱导量居中,且两者混合诱导量比单独诱导量之和要大。结论成功筛选出诱导小鼠肝脏MT合成最有效的因子和最佳时间,为进一步大量合成MT及研究其功能等奠定基础。     

Identification of a cadmium-binding protein from a cadmium-resistant variant of human lymphoblastoid cells (WI-L2)

Cadmium-binding protein synthesis and induction by cadmium chloride were studied in the human lymphoblastoid cell line WI-L2. Lymphoblasts were adapted to growth in 5 microM cadmium chloride (Cdr) and these cells were 2.5-fold more resistant to cadmium than the parental line. There was no difference in the cellular protein profile between the parental line and lymphoblasts grown for a short period, less than 10 days, in cadmium chloride as measured by [35S]cysteine labelling and SDS-polyacrylamide gel electrophoresis. A basal level of cadmium binding protein was apparent, however, by gel filtration. The Cdr lymphoblasts were found to synthesize a substantial amount of cadmium-binding protein, approximately 25-fold more than the parental line. The cadmium-binding protein has the following properties which are consistent with its being a metallothionein: (1) [35S]Cysteine-labelled protein eluted at a Ve/Vo = 2.1 on a Sephadex G-75 column; (2) the molecular weight was estimated as 11 kDa on 7-17% SDS polyacrylamide gels; (3) the protein was heat-stable; (4) the unlabelled protein bound 109Cd2+.     

Construction and expression of functional multi-domain polypeptides in Escherichia coli: expression of the Neurospora crassa metallothionein gene

A system for the construction of polymeric peptides in Escherichia coli was utilized to prepare a library of plasmids coding for tandem repeats of the Neurospora crassa metallothionein gene. Selected oligomeric metallothionein clones were expressed and targeted to the periplasm as a fusion with the maltose-binding protein. Bacterial cells harbouring the expressed oligopeptides were characterized for their ability to bind 109Cd2+. The metal-binding ability was enhanced for all the oligomeric constructs tested and, in the best case, a 6.5-fold increased capacity for metal uptake was achieved with cells expressing a tandem 9-mer in comparison with cells expressing a monomer. Plateauing of the metal uptake ability occurred at between six and nine tandem repeats, possibly due to a combination of lowered translation levels, inefficient export and prematurely terminated translation products. The overall enhancement of the heavy metal removal capacity was approximately 65-fold relative to non-recombinant cells. The use of this strategy for the design and expression of de novo polypeptides containing multiple functional domains for use in bioremediation is discussed.     

Enhanced production of hepatitis B virus surface antigen in mouse C127 cell on a bovine papillomavirus-metallothionein vector

We have constructed a recombinant plasmid pCPS12 containing the hepatitis B viral surface antigen (HBsAg) gene linked to the mouse metallothionein promoter on a BPV-pML2 vector. Two stable clones S12-8 and S12-2, obtained by transfection of the mouse C127 cells with pCPS12 propagated in dam+ dcm+ and dam- dcm- Escherichia coli respectively, exhibited different types of response to 5-azacytidine (5-aza-CR) and cadmium (Cd) induction. In S12-8, the productivity of HBsAg was enhanced by 5-aza-CR or 5-aza-CR plus Cd, but not by Cd alone. In S12-2, the expression of HBsAg was not affected by 5-aza-CR but was induced by Cd in the presence or absence of 5-aza-CR. This suggests that methylation may be important in controlling the HBsAg expression and the inducibility of Cd in the transfectants.     

Induction of metallothionein in a human trophoblast cell line by cadmium and zinc

The induction of metallothionein (MT) in a cell line derived from a malignant trophoblastic tumor (JAr cells) was demonstrated using the Cd/heme radioassay following exposure to Cd or Zn. Cd at an optimal concentration of 1 microM produced a 30-fold increase in MT following a 24 hr incubation. Induction by Cd was both time and dose dependent, with a significant increase in MT noted as early as 3 hr, with levels continuing to increase up to 24 hr. Zn was also quite effective in inducing MT synthesis in this cell line. Exposure to 80 microM Zn for 24 hr produced a 70-fold increase in MT. Although Cd was a more potent inducer of MT, exposure to Zn resulted in a greater magnitude of induction. The magnitude of MT induction in JAr cells was much greater than that seen in cultured trophoblasts from term chorion laeve. The degree of induction seen in this cell line makes it an interesting model for the study of MT's role in trophoblast function. MT induction in trophoblasts may reflect a protective mechanism against heavy metal toxicity and/or an integral aspect of normal zinc homeostasis.     

Development of a retroviral vector for inducible expression of transforming growth factor beta 1.

A retroviral vector system for the expression of exogenous genes under the control of an inducible promoter was developed. By utilizing this system, the cDNA for human transforming growth factor beta 1 (TGF-beta 1) was inserted into a retroviral vector under the control of an internal mouse metallothionein promoter and introduced via infection into normal rat kidney fibroblasts (NRK-49F) and epithelial cells (NRK-52E), Chinese hamster ovary cells (CHO), and the human monocytic cell line U937. Control of TGF-beta 1 expression, achieved by Cd2+ induction of vector-encoded TGF-beta 1 mRNA, was cell line specific and resulted in a concomitant increase in neutralizable TGF-beta 1 production by the cells. Autocrine stimulation of vector-containing cells by vector-encoded TGF-beta 1 was detected by an increase in soft-agar colony formation of NRK-49F infectants compared with that of the control cells. In addition, the use of a second internal promoter in a retroviral vector of similar design allowed isolation of stable infectants from a cell line (CHO) in which the viral long terminal repeat does not function efficiently.