Toxic heavy metal ions activate the heme-regulated eukaryotic initiation factor-2 alpha kinase by inhibiting the capacity of hemin-supplemented reticulocyte lysates to reduce disulfide bonds.

Addition of toxic heavy metal ions (Cd2+, Hg2+, and Pb2+) to hemin-supplemented rabbit reticulocyte lysate brings about the activation of the heme-regulated eukaryotic initiation factor 2 alpha kinase (HRI) and the inhibition of protein chain initiation. In this report we examined the effects of monothiol and dithiol compounds, metal ion-chelating agents, and metallothioneins (MT) on metal ion-induced inhibition of protein synthesis. The dithiol compounds dithiothreitol and 2,3-dimercaptopropane sulfonic acid prevented and relieved the inhibition of protein synthesis caused by Cd2+ and Hg2+ in hemin-supplemented lysates, but the monothiol compounds 2-mercaptoethanol, cysteamine, D-(-)penicillamine, and glutathione had no effect. The inhibition of protein synthesis caused by Cd2+ was reversed by the addition of excess EDTA but not by the addition of excess nitrilotriacetic acid. Toxic heavy metal ions inhibited the capacity of hemin-supplemented lysate to reduce disulfide bonds. Addition of excess EDTA to Cd(2+)-inhibited lysates restored the capacity of the lysate to reduce disulfide bonds and inhibited the phosphorylation of eukaryotic initiation factor eIF-2. MTs and their apoproteins (apoMTs) inhibited the activation of HRI and protected protein synthesis from inhibition by Cd2+, Hg2+, and Pb2+. Addition of apoMTs to heavy metal ion-inhibited lysates restored the capacity of lysates to reduce disulfide bonds. The restoration of the lysate's thioredoxin/thioredoxin reductase activity was accompanied by the inactivation of HRI and the resumption of protein synthesis, indicating that apoMTs can "detoxify" metal ions already bound to proteins. Several observations presented in this report suggest that the binding of metal ions to the alpha-domain of MT is responsible for the ability of MT to sequester bound metal in a non-toxic form. Addition of glucose 6-phosphate or NADPH had no effect on protein synthesis in metal ion-inhibited lysates, and NADPH concentrations in Cd(2+)-inhibited and hemin-supplemented control lysates were equivalent. The data suggest that the metal ions cause the inhibition of protein synthesis by binding to vicinal sulfhydryl groups present in some critical protein(s), possibly the dithiols present in the active site of thioredoxin and (or) thioredoxin reductase, which leads to the activation of HRI.     

Evidence for more than two metallothionein isoforms in primates

Two isoforms of metallothionein (MT) have in general been identified in mammalian cells. We have analyzed Cd2+-induced MTs of primate origin and demonstrated the presence of more than two isoforms. Four low molecular weight Cd2+-binding proteins were separated from Cd2+-exposed HeLa cells by gel filtration and ion-exchange chromatography and identified as MTs by amino acid analysis. These were carboxymethylated and analyzed by electrophoresis under denaturing conditions. Three of these proteins were found to be distinct molecules. We also analyzed hepatic MTs from Cd2+-exposed rhesus monkeys, which have previously been partially separated. In this case, five distinct isomers were detected.     

Cadmium-resistant Chinese hamster V79 cells with decreased accumulation of cadmium

Cells resistant to 3 x 10(-5) M CdCl2 (Cdr cells) were isolated from cultures of Chinese hamster V79 cells by a procedure that involved stepwise increase in the concentration of Cd2+ and subsequent mass selection. Cdr cells grew as fast as wild-type cells (Cds) in medium without cadmium. Cdr cells were not cross-resistant to other divalent metal ions, such as Hg2+, Ni2+, Pb2+, and Zn2+. Both Cds and Cdr cells induced similar levels of metallothioneins (MT) in response to zinc. Depletion of glutathione (GSH) did not significantly influence the sensitivity of Cdr cells to Cd2+ but markedly enhanced the sensitivity to Cd2+ of Cds cells. Furthermore, the rate of synthesis of GSH after depletion did not differ greatly between sensitive and resistant cells. The rate of uptake of 109Cd2+ by Cdr cells was only 10-15% that by Cds cells. The difference in rates of uptake between Cds and Cdr cells was observed irrespective of the presence or absence of serum in the culture medium. These results indicate that, in this system, resistance to Cd2+ is attributable neither to increased inducibility of MT nor to increases in intracellular levels of GSH, and that only a decrease in the rate of uptake of Cd2+ contributes to the acquisition of resistance to Cd2+. Uptake of Cd2+ by cells was dependent on temperature and the rate of uptake of Cd2+ by Cdr cells was lower at all temperatures examined than the rate of uptake by Cds cells. Cycloheximide did not suppress the uptake of Cd2+, suggesting that uptake does not require synthesis of cell proteins de novo. Preincubation of cells with N-ethylmaleimide suppressed the uptake of Cd2+ to some extent, a result that suggests the involvement of surface SH groups in the uptake of Cd2+ by these cells.     

Induction of cadmium-thionein in isolated rat liver cells.

The uptake of cadmium by isolated liver cells was linearly related to the cadmium concentration to which the cells were exposed in the medium. Cadmium-treated cells synthesized proteins de novo with the characteristics of cadmium-thionein induced in the liver of cadmium-treated animals. Thionein from liver cells incorporated cadmium and [35S]cysteine, had a Ve/Vo (Sephadex G-50) of 1.8-1.9, and was separated into two subfractions by DEAE-cellulose ion-exchange chromatography. Cycloheximide and actinomycin D when added after a cadmium exposure prevented the synthesis of thionein. However, addition of actinomycin D after synthesis had started only decreased the total amount of thionein synthesized. The concentration of cadmium to which the cells were exposed affected the amount of cadmium-thionein synthesized in 6h. The maximum response occurred when cells were exposed to 0.5 microgram of cadmium/ml; at higher metal concentrations the total amount of cadmium-thionein synthesized declined. The system described in the present paper can be used to study the mode of metal toxicity and the mechanism of cadmium-thionein synthesis.     

镉中毒大鼠睾丸与肝脏金属硫蛋白表达的时相研究

啮齿目动物睾丸对镉毒性较肝脏更敏感.为阐明睾丸的镉毒性分子机制,比较了肝脏与睾丸金属硫蛋白（MT）表达的时相变化.mRNA采用RT-PCR技术分析并用光密度扫描定量;蛋白质定量用ELISA方法.结果显示,睾丸中存在MT,镉中毒后MT1与MT2 mRNA明显升高,但MT没有相应增加;肝脏镉中毒后MTmRNA与MT均明显升高.结果提示：镉虽然能诱导睾丸MTmRNA的转录,但没有促进其MT的合成,这可能是睾丸对镉毒性与致癌作用较肝脏更敏感的重要原因.     

Binding properties and stoichiometries of a palladium(II) complex to metallothioneins in vivo and in vitro

This paper will be the first to discuss the in vivo and in vitro properties of a Pd(II) complex, K2PdCl4, interacting with metallothioneins (MTs). In vivo experiments revealed that intraperitoneal injections of K2PdCl4 into rabbits led to the simultaneous synthesis of Pd-MT in the kidney and Zn7MT in the liver. The renal Pd-MT complex contains 3.6 +/- 0.3 Pd, 2.1 +/- 0.2 Zn, and 1.0 +/- 0.1 Cu per mole protein. It was found that pre-treatment with Zn(NO3)2 before K2PdCl4 injections significantly enhanced renal Pd-MT level. The same pre-treatment also increases hepatic Zn-MT levels. These results strongly suggest that Pd(II) ions can be bound in vivo by MT existing in the rabbit kidneys to form Pd-MT. Gel-filtration chromatographic studies after the incubation of either native Cd5Zn2MT2 or Zn7MT2 with K2PdCl4 in vitro demonstrate that Pd(II) ions promote the non-oxidative oligomerization of native MTs. Increasing the level of Pd(II) relative to MT led to a concomitant increase in the apparent yield of MT oligomers. At relatively low Pd-MT ratio, Pd(II) is found predominantly in the oligomers while the monomeric products are chiefly composed of the reactants, Cd5Zn2MT2 or Zn7MT2. Based on our experimental data, the mechanisms of the reactions between Pd(II) and MTs in vivo and in vitro are discussed.     

Immunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.     

Effects of heavy metals and other trace elements on the fermentative activity of the rumen microflora and growth of functionally important rumen bacteria

The inhibitory effects of high concentrations of essential and non-essential trace elements were tested on the rumen microflora using the rate of fermentation in vitro as the assay. The elements (and the concentration causing 50% inhibition) in decreasing order of toxicity were Hg2+ (20 microgram/ml), Cu2+ (21 microgram/ml), Cr6+ (70 microgram/ml), Se4+ (73 microgram/ml), Ni2+ (160 microgram/ml), Cd2+ (175 microgram/ml), As3+ (304 microgram/ml) and As5+ (1610 microgram/ml). The elements tested that were either weak or noninhibitory at concentrations greater than 400 microgram/ml included Zn2+, Cr2+, Fe2+, Mn2+, Pb2+, and Co2+. Methylmercury was as inhibitory as mercuric chloride to the fermentation. When the inhibitory effect of Cd2+ was tested on separated bacterial and protozoal fractions, it was more inhibitory to the bacteria. The inhibitory effects of trace elements were also determined for a number of axenic cultures of rumen bacteria. The bacteria which most frequently exhibited the greatest sensitivity were Bacteroides succinogenses, Ruminococcus albus, Bacteroides amylophilus, and Eubacterium ruminantium. Those often exhibiting intermediate sensitivities included Butyrivibrio fibrisolvens, Selenomonas ruminantium, and Megasphera elsdenii, while Streptococcus bovis was very refractory to all elements tested. Rumen fluid provided a modest protective effect for the bacteria.     

Cadmium-induced synthesis of metallothioneins in human lymphocytes and monocytes

Cd2+-binding proteins of peripheral blood lymphocytes and monocytes have not well been characterized so far, although they are expected to be a clue for understanding Cd2+ toxicity in those immune competent cells. We separated a family of Cd2+-binding proteins from Cd2+-exposed human peripheral blood lymphocytes by gel filtration chromatography, and characterized them by SDS-gel electrophoresis. The proteins showed electrophoretic behaviours closely similar to metallothioneins (MTs) of HeLa cells derived from human cervical carcinoma. The proteins were also found in Cd2+-exposed monocytes, and were inducible by Cd2+ in both lymphocytes and monocytes. Anti-MT serum specifically precipitated these proteins, which were thus identified as MTs. These results suggest that the two classes of the cells involved in the immune system possess a protective mechanism against Cd2+ through MTs. A variety of human lymphoid cell lines derived from both T and B cells were also found to have capacity to synthesize MTs in response to Cd2+.     

Thionein gene expression in Cd++-variants of the CHO cell: correlation of thionein synthesis rates with translatable mRNA levels during induction, deinduction, and superinduction.

The relationship of thionein synthesis rates to translatable cytoplasmic thionein mRNA levels was investigated for the first time in a cultured cell system. Thionein synthesis was induced in Cdr, a cadmium-resistant variant of CHO, by exposure to 2 microM CdCl2. Following a short (1.5 hr) lag, thionein synthesis increases to a rate that is at least 30 times the uninduced rate 7-8 hr after addition of Cd++. This increase is blocked by the coincident addition of a actinomycin D. Cytoplasmic thionein mRNA levels, measured by translation in a modified wheat germ system, increase rapidly following induction to values approximately 25 times uninduced levels within 6-8 hr. The increase in thionein mRNA precede proportionate increases in thionein synthesis by 0.5-1.0 hr. Continued exposure to Cd++ results in a decreased thionein synthesis rate after 8 hr. By 30 hr, the rate is one-half that seen 6-8 hr after induction. Removal of Cd++ after 8 hr results in a rapid decrease in thionein synthesis (t 1/2 approximately 4 hr). Both decreases are inhibited by the addition of actinomycin. In all instances--induction, deinduction, and actinomycin-mediated "super-induction"--translatable thionein mRNA levels and thionein synthesis rates increase, decrease, or are maintained coordinately. The results suggest that thionein synthesis in Cdr is controlled primarily by the level of translatable cytoplasmic thionein mRNA.     

Metallothionein induction in human peripheral blood lymphocytes by heavy metals

Human peripheral blood lymphocytes have the capacity to produce metallothioneins (MTs) as a protective response to cadmium exposure. To define the range of metal species inducing lymphocyte MTs, cellular proteins synthesized after exposure to each of 11 heavy metals were analyzed by gel electrophoresis. Toxic metals such as cadmium, mercury and silver were found to induce thioneins (apoproteins of MTs) at relatively low concentrations (maximum at approximately 10 microM), whereas less toxic metals such as zinc, copper and nickel were inductive at relatively high concentrations (maximum at approximately 200 microM). Tin, lead, iron, cobalt, and manganese did not induce thioneins. The heavy metal specificity of MT induction in the lymphocyte resembles that in the liver, and the regulatory mechanism of MT production seems to be similar in both of these tissues. In the cells exposed to highly toxic metals such as cadmium and mercury, expression of cytotoxicity (represented by decline of cysteine uptake) was remarkable at the metal concentrations higher than those saturating thionein induction, supporting the protective role of MTs against heavy metals.     

脱金属硫蛋白与镉离子的络合作用及构象研究

用圆二色（ＣＤ）谱地研究兔肝脱金属硫属蛋白的两个亚型与Ｃｄ＾２＋的络合作用及对重组ＭＴ构象的影响。观测了ａｐｏ－ＭＴ垢巯基在空气和室温下的稳定性。在ＰＨ４．７１,镉重组ＭＴ１的ＣＤ谱特征峰在２５７ｎｍ（＋）,２３８ｎｍ（－）,２２６ｎｍ（＋）与镉诱导的天然ＭＴ１相同。在空气存在和ＰＨ７．９０的ＣＤ谱只有２４３ｎｍ（＋）一个峰。向两亚型分别加入７ｅｑＣｄ＾２＋测定ＣＤ谱随ＰＨ值的变化,发现在ＰＨ２．     

Induction of two major isoforms of metallothionein in crucian carp (Carassius cuvieri) by air-pumping stress,dexamethasone, and metals

The induction of metallothionein (MT) by physical and chemical stress was assessed using the fresh-water fish, crucian carp (Carassius cuvieri Temminck et Schlegel). The fish exposed to violent air-pumping stress for 6 days revealed time-dependent induction of MT-like metal-binding proteins in both their livers and kidneys. Their hepatic contents after exposure to stress were elevated to twice the basal level with 24 h, resulting in more than a 3-fold increase at 144 h, whereas their renal contents gradually increased after 24 h and reached the same level as that in the liver around 96 h. Two major inducible proteins were purified from livers of fish exposed to stress and were shown to be MT based upon their chromatographic behavior, UV absorption spectra and their molecular weights. Consequently, they were termed ccMT-1 and ccMT-2, according to their elution sequence upon anion-exchange chromatography. Both proteins mainly bound zinc in their endogenous forms and showed different immunogenicity to rat and rabbit MTs. Dexamethasone, a potent inducer for MT synthesis in mammals, induced the production of both isoforms in crucian carp, whereas cadmium and zinc ions prominently induced the synthesis of ccMT-2. These results indicate that crucian carp have the ability to produce MTs in response to various kinds of environmental stress and that violent air-pumping stress in crucian carp may induce MT synthesis, in part, via the release of endogenous factor(s), such as glucocorticoids.     

Localization effect on the metal biosorption capability of recombinant mammalian and fish metallothioneins in Escherichia coli

In this study, we examined the expression of mammalian and fish metallothioneins (MTs) in Escherichia coli as a strategy to enhance metal biosorption efficiency of bacterial biosorbents for lead (Pb), copper (Cu), cadmium (Cd), and zinc (Zn). In addition, MT proteins were expressed in either the cytoplasmic or periplasmic compartment of host cells to explore the localization effect on metal biosorption. The results showed that MT expression led to a significant increase (5-210%) in overall biosorption efficiency (eta(ads)), especially for biosorption of Cd. The MT-driven improvement in metal biosorption relied more on the increase in the biosorption rates (r(2), a kinetic property) than on the equilibrium biosorption capacities (q(max), a thermodynamic property), despite a 10-45% and 30-80% increase in q(max) of Cd and Zn, respectively. Periplasmic expression of MTs appeared to be more effective in facilitating the metal-binding ability than the cytoplasmlic MT expression. Notably, disparity of the impacts on biosorption ability was observed for the origin of MT proteins, as human MT (MT1A) was the most effective biosorption stimulator compared to MTs originating from mouse (MT1) and fish (OmMT). Moreover, the overall biosorption efficiency (eta(ads)) of the MT-expressing recombinant biosorbents was found to be adsorbate-dependent: the eta(ads) values decreased in the order of Cd > Cu > Zn > Pb.     

Characterization and measurement of metallothionein messenger RNA of C57BL mouse liver

Liver poly(A)+RNA of Cd2+-treated C57BL mouse was characterized by cell-free translation, particularly intending to establish a procedure to measure the levels of messenger RNA coding for metallothioneins (MT-mRNA). Intact polysomes were obtained by Mg2+ precipitation from the liver cytoplasm of mice injected with 1 mg Cd2+/kg body wt. Poly(A)+RNA isolated from the polysomes was translated by a wheat germ cell-free system and the [35S]cysteine-labeled translation products were analyzed by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis and fluorography. MTs were identified in the translation products directed by the RNA from the Cd2+-treated mice, but not in the translation products directed by the RNA from untreated mice. Relative incorporation of [35S]cysteine into MTs was determined by densitometrical quantification of the MT bands, and was found to be linear up to a RNA concentration of 150 micrograms/ml in the translation reaction mixture, showing that this system is suitable for the measurement of translatable MT-mRNA levels. Cd2+ stimulated the total levels of cell-free translation (1.4-fold at 20-60 micrograms/ml), not specifically to MT-mRNA. MT-mRNA sedimented at 9S in a sucrose gradient, and its size was comparable with rat and human MT-mRNAs.     

In vivo metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells

Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.     

Microtubule reassembly from nucleating fragments during the regrowth of amputated neurites

We have proposed that stable microtubule (MT) fragments that resist depolymerization may serve as nucleating elements for the local control of MT dynamics in the axon (Heidemann, S. R., M. A. Hamborg, S. J. Thomas, B. Song, S. Lindley, and D. Chu, 1984, J. Cell Biol., 99:1289-1295). Here we report evidence that supports this proposal in studies on the role of MTs in the regrowth of neurites from the distal segments of amputated chick sensory neurites. Amputated neurites collapse to "beads" of axoplasm that rapidly regrow (Shaw, G., and D. Bray, 1977, Exp. Cell Res., 104:55-62). We examined both unarrested regrowth and regrowth after MT disassembly by either cold (-5 degrees C for 2 h) or nocodazole (0.1 microgram/ml for 15-20 min). In all these cases regrowth occurred at 3.5-4.5 micron/min with no delay times other than the times to reach 37 degrees C or rinse out the nocodazole. Electron micrographs of untreated beads show many MTs of varying lengths, while those of cold- and nocodazole-treated beads show markedly shorter MTs. The robust regrowth of neurites from beads containing only very short MTs argues against unfurling of intact MTs from the bead into the growing neurite. Electron micrographs of cold-treated beads lysed under conditions that cause substantial MT depolymerization in untreated intact neurites show persistent MT fragments similar to those in unlysed cold-treated beads. We interpret this as evidence that the MT fragments in cold-treated beads are somehow distinct from the majority of the MT mass that had depolymerized. Collapsed neurites treated with a higher dose of nocodazole (1.0 microgram/ml for 15-20 min) were completely devoid of MTs and regrew only after a 15-20 min delay in two cases but never regrew in 11 other cases. We found that MTs did not return in beads treated with 1.0 microgram/ml nocodazole even 30 min after removal of the drug. It was unlikely that the inability of these beads to reassemble MTs was due to incomplete removal of nocodazole in that a much higher dose (20 micrograms/ml nocodazole) could be quickly rinsed from intact neurites. Beads treated with 1.0 microgram/ml nocodazole could, however, be stimulated to reassemble MTs and regrow neurites by treatment with taxol. We conclude that the immediate, robust regrowth of neurites from collapsed beads of axoplasm requires MT nucleation sites to support MT reassembly.(ABSTRACT TRUNCATED AT 400 WORDS)     

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+.     

Regulation of metallothionein synthesis in HeLa cells by heavy metals and glucocorticoids

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins that bind heavy metals. MT induction occurs in liver in response to either heavy metal (Zn++ or Cd++) administration or stress. The synthesis of MT can also be induced by either heavy metals or glucocorticoid hormones in HeLa cells cultured in serum-free medium. Induction of MT by zinc is subject to "desensitization." In contrast, dexamethasone (dex) induction results in a continued elevation in the rate of MT synthesis. The stability of MT is dependent on the availability of metal; consequently, MT induced by dex is degraded much more rapidly (half-life of 11 to 12 hours) than MT induced by elevated zinc levels (half-life of 36 to 38 hours). Removal of either inducer results in biphasic degradation curves, as apothionein and zinc come into balance. In contrast, deinduction kinetics for MT synthesis following removal of the two inducers (zinc and dex) are the same, with a half-life of two and one-half hours. Inhibition of RNA synthesis blocks deinduction after removal of inducer. Induction of MT occurs in a wide variety of species, from blue-green algae to man. This system should provide an excellent model for the comparative biochemistry of regulation of gene expression.