Metal-binding Metallothionein Research Articles

Rhizofungus Aspergillus terreus Mitigates Heavy Metal Stress-Associated Damage in Triticum aestivum L.

Industrial waste and sewage deposit heavy metals into the soil, where they can remain for long periods. Although there are several methods to manage heavy metals in agricultural soil, microorganisms present a promising and effective solution for their detoxification. We isolated a rhizofungus, Aspergillus terreus (GenBank Acc. No. KT310979.1), from Parthenium hysterophorus L., and investigated its growth-promoting and metal detoxification capabilities. The isolated fungus was evaluated for its ability to mitigate lead (25 and 75 ppm) and copper (100 and 200 ppm) toxicity in Triticum aestivum L. seedlings. The experiment utilized a completely randomized design with three replicates for each treatment. A. terreus successfully colonized the roots of wheat seedlings, even in the presence of heavy metals, and significantly enhanced plant growth. The isolate effectively alleviates lead and copper stress in wheat seedlings, as evidenced by increases in shoot length (142%), root length (98%), fresh weight (24%), dry weight (73%), protein content (31%), and sugar content (40%). It was observed that wheat seedlings possess a basic defense system against stress, but it was insufficient to support normal growth. Fungal inoculation strengthened the host's defense system and reduced its exposure to toxic heavy metals. In treated seedlings, exposure to heavy metals significantly upregulated MT1 gene expression, which aided in metal detoxification, enhanced antioxidant defenses, and maintained metal homeostasis. A reduction in metal exposure was observed in several areas, including normalizing the activities of antioxidant enzymes that had been elevated by up to 67% following exposure to Pb (75 mg/kg) and Cu (200 mg/kg). Heavy metal exposure elevated antioxidant levels but also increased ROS levels by 86%. However, with Aspergillus terreus colonization, ROS levels stayed within normal ranges. This decrease in ROS was associated with reduced malondialdehyde (MDA) levels, enhanced membrane stability, and restored root architecture. In conclusion, rhizofungal colonization improved metal tolerance in seedlings by decreasing metal uptake and increasing the levels of metal-binding metallothionein proteins.

Consequences of Arsenic Contamination on Plants and Mycoremediation-Mediated Arsenic Stress Tolerance for Sustainable Agriculture

Arsenic contamination in water and soil is becoming a severe problem. It is toxic to the environment and human health. It is usually found in small quantities in rock, soil, air, and water which increase due to natural and anthropogenic activities. Arsenic exposure leads to several diseases such as vascular disease, including stroke, ischemic heart disease, and peripheral vascular disease, and also increases the risk of liver, lungs, kidneys, and bladder tumors. Arsenic leads to oxidative stress that causes an imbalance in the redox system. Mycoremediation approaches can potentially reduce the As level near the contaminated sites and are procuring popularity as being eco-friendly and cost-effective. Many fungi have specific metal-binding metallothionein proteins, which are used for immobilizing the As concentration from the soil, thereby removing the accumulated As in crops. Some fungi also have other mechanisms to reduce the As contamination, such as biosynthesis of glutathione, cell surface precipitation, bioaugmentation, biostimulation, biosorption, bioaccumulation, biovolatilization, methylation, and chelation of As. Arsenic-resistant fungi and recombinant yeast have a significant potential for better elimination of As from contaminated areas. This review discusses the relationship between As exposure, oxidative stress, and signaling pathways. We also explain how to overcome the detrimental effects of As contamination through mycoremediation, unraveling the mechanism of As-induced toxicity.

Metal removal by metallothionein and an acid phosphatase PhoN, surface-displayed on the cells of the extremophile, Deinococcus radiodurans

The utility of surface layer proteins (Hpi and SlpA) of the radiation resistant bacterium, Deinococcus radiodurans, was investigated for surface display and bioremediation of cadmium and uranium. The smtA gene, from Synechococcus elongatus (encoding the metal binding metallothionein protein), was cloned and over-expressed in D. radiodurans, either as such or as a chimeric gene fused with hpi ORF (Hpi-SmtA), or fused to the nucleotide sequence encoding the SLH domain of the SlpA protein (SLH-SmtA). The expressed fusion proteins localized to the deinococcal cell surface, while the SmtA protein localized to the cytoplasm. Recombinant cells surface-displaying the SLH-SmtA or Hpi-SmtA fusion proteins respectively removed 1.5–3 times more cadmium than those expressing only cytosolic SmtA. The deinococcal Hpi protein layer per se also contributed to U binding, by conferring substantial negative charge to deinococcal cell surface. The ORF of an acid phosphatase, PhoN was fused with the hpi or SLH domain DNA sequence and purified. Isolated Hpi-PhoN and SLH-PhoN, immobilized on deinococcal peptidoglycan showed efficient uranium precipitation (446 and 160 mg U/g biomass used respectively). The study demonstrates effective exploitation of the deinococcal S layer protein components for (a) cell surface-based sequestration of cadmium, and (b) cell-free preparations for uranium remediation.

Copper toxicity of inflection point in human intestinal cell line Caco-2 dissected: influence of temporal expression patterns.

We previously described a non-monotonic dose response curve at low copper concentrations where 3.125 μM CuSO4 (the early inflection point) was more toxic than 25 μM CuSO4 in Caco-2 cells. We employed global proteomics to investigate this observation. The altered expression levels of a small number of proteins displaying a temporal response may provide the best indication of the underlying mechanism; more well-known copper response proteins including the metal binding metallothioneins (MT1X, MT1F, MT2A) and antioxidant response proteins including Heme oxygenase were upregulated to a similar level in both copper concentrations and so are less likely to underpin this phenomenon.The temporal response proteins include Granulins, AN1-type zinc finger protein 2A (ZFAND2A), and the heat shock proteins (HSPA6 and HSPA1B). Granulins were decreased after 4 h only in 25 μM CuSO4 but from 24 h, were decreased in both copper concentrations to a similar level. Induction of ZFAND2A and increases in HSPA6 and HSPA1B were observed at 24 h only in 25 μM CuSO4 but were present at 48 h in both copper conditions. The early expression of ZFAND2A, HSPs, and higher levels of α-crystallin B (CRYAB) correlated with lower levels of misfolded proteins in 25 μM CuSO4 compared to 3.125 μM CuSO4 at 48 h. These results suggest that 3.125 μM CuSO4 at early time points was unable to activate the plethora of stress responses invoked by the higher copper concentration, paradoxically exposing the Caco-2 cells to higher levels of misfolded proteins and greater proteotoxic stress.

Metallothioneins regulate ATP7A trafficking and control cell viability during copper deficiency and excess

Copper (Cu) is an essential, yet potentially toxic nutrient, as illustrated by inherited diseases of copper deficiency and excess. Elevated expression of the ATP7A Cu exporter is known to confer copper tolerance, however, the contribution of metal-binding metallothioneins is less clear. In this study, we investigated the relative contributions of ATP7A and the metallothioneins MT-I and MT-II to cell viability under conditions of Cu excess or deficiency. Although the loss of ATP7A increased sensitivity to low Cu concentrations, the absence of MTs did not significantly affect Cu tolerance. However, the absence of all three proteins caused a synthetic lethal phenotype due to extreme Cu sensitivity, indicating that MTs are critical for Cu tolerance only in the absence of ATP7A. A lack of MTs resulted in the trafficking of ATP7A from the trans-Golgi complex in a Cu-dependent manner, suggesting that MTs regulate the delivery of Cu to ATP7A. Under Cu deficiency conditions, the absence of MTs and / or ATP7A enhanced cell proliferation compared to wild type cells, suggesting that these proteins compete with essential Cu-dependent pathways when Cu is scarce. These studies reveal new roles for ATP7A and metallothioneins under both Cu deficiency and excess.

Annelid Coelomic Fluid Proteins.

The coelomic cavity is part of the main body plan of annelids. This fluid filled space takes up a considerable volume of the body and serves as an important site of exchange of both metabolites and proteins. In addition to low molecular substances such as amino acids and glucose and lactate, the coelomic fluid contains different proteins that can arise through release from adjacent tissues (intestine) or from secretion by coelomic cells. In this chapter, we will review the current knowledge about the proteins in the annelid coelomic fluid. Given the number of more than 20,000 extant annelid species, existing studies are confined to a relatively few species. Most studies on the oligochaetes are confined to the earthworms-clearly because of their important role in soil biology. In the polychaetes (which might represent a paraphyletic group) on the other hand, studies have focused on a few species of the Nereidid family. The proteins present in the coelomic fluid serve different functions and these have been studied in different taxonomic groups. In oligochaetes, proteins involved antibacterial defense such as lysenin and fetidin have received much attention in past and ongoing studies. In polychaetes, in contrast, proteins involved in vitellogenesis and reproduction, and the vitellogenic function of coelomic cells have been investigated in more detail. The metal binding metallothioneins as well as antimicrobial peptides, have been investigated in both oligochaetes and polychaetes. In the light of the literature available, this review will focus on lipoproteins, especially vitellogenin, and proteins involved in defense reactions. Other annelid groups such as the Pogonophora, Echiura, and Sipuncula (now considered polychaetes), have not received much attention and therefore, this overview is far from being complete.

Відмінності вмісту металотіонеїну у мозку гризунів за умов постнатального розвитку та кадмієвої інтоксикації

Introduction. Metal-binding metallothionein genes are found in a vast population of organisms. These proteins are non enzymatic and very rich in cysteine residues. The various metallothionein isoforms in different brain regions arguably change over time.The aim of the study – evaluating the distribution of metallothionein in different brain regions of gerbils and Wistar rats at different stages of postnatal development (PND), under standard and low dose Cd-induced conditions.Materials and Methods. 18 Mongolian gerbils and 36 Wistar rats were divided into 6 groups (n=6), by age and condition of experiment: groups 1, 2, 3, 4 – 1, 30, 90 and 180-days old were exposed to standard conditions; group 5 and 6 – 180-days old+0.1 or 1.0 µgµg Cd2+ per animal everyday for 36 days. The metallothionein content in the hippocampus, cerebellum, and thalamus were detected by the ELISA.Results and Discussion. Obtained data was shown the dynamic of metallothionein distribution in different brain regions of gerbils and Wistar rats depending on the stage of postnatal development and functional capacities. The content of metallothionein in the hippocampus continually decreased in both animal types but the cerebella metallothionein distribution pattern was different from that of the hippocampus, but identical in both rodents, rising from day one before decreasing on day 30. The low levels of metallothionein under the influence of Cd were proportional to the doses administered.Conclusions. The level of metallothionein in the brain varies depending on the stage of development of the functional capacities of the brain parts. The significant down-regulation of metallothionein in the investigated brain regions under Cd influence suggests that a decrease in metallothionein levels depends on the dose of Cd and on the time necessary for its accumulation.

Characterization of three distinct metallothionein genes of the Ag-hyperaccumulating ectomycorrhizal fungus Amanita strobiliformis

Mechanisms evolved in eukaryotes to handle heavy metals involve cytosolic, metal-binding metallothioneins (MTs). We have previously documented that the sequestration of silver (Ag) in the Ag-hyperaccumulating Amanita strobiliformis is dominated by 34-amino-acid (AA) AsMT1a, 1b, and 1c isoforms. Here we show that in addition to AsMT1a, 1b, and 1c isogenes, the fungus has two other MT genes: AsMT2 encoding a 34-AA AsMT2 similar to MTs known from other species, but unrelated to AsMT1s; AsMT3 coding for a 62-AA AsMT3 that shares substantial identity with as-yet-uncharacterized conserved peptides predicted in agaricomycetes. Transcription of AsMT1s and AsMT3 in the A. strobiliformis mycelium was specifically inducible by treatments with Ag or copper (Cu) and zinc (Zn) or cadmium (Cd), respectively; AsMT2 showed a moderate upregulation in the presence of Cd. Expression of AsMTs in the metal-sensitive Saccharomyces cerevisiae revealed that all AsMTs confer increased Cd tolerance (AsMT3 proved the most effective) and that, unlike AsMT1 and AsMT2, AsMT3 can protect the yeasts against Zn toxicity. The highest level of Cu tolerance was observed with yeasts expressing AsMT1a. Our data indicate that A. strobiliformis can specifically employ different MT genes for functions in the cellular handling of Ag and Cu (AsMT1s) and Zn (AsMT3).

Cadmium bioaccumulation in European flat oysters (Ostrea edulis) from Middle Adriatic Sea (San Benedetto del Tronto district, Italy)

Bivalve molluscs represent an important source of cadmium exposure in humans, in particular oysters, because of their high filter feeding capability and high concentration of metal-binding metallothionein in tissues. In this study the authors investigated the difference in cadmium bioaccumulation in European flat oysters harvested from production areas in the district of San Benedetto del Tronto (Ascoli Piceno province, Italy), as a function of their origin (farming or natural beds) and the time of gathering. The beds lie 3 nm off-shore at a depth of 20-40 m and are collected by dredging. In the farms, baskets are suspended in the water column 2.5-3 nm offshore at a depth of 4 m. The authors analysed the results of cadmium monitoring plan carried out in oyster natural beds for a total of 15 samples collected from 2004 to 2012 and in two oyster farms for a total of 11 samples from 2009 to 2012. Although the few data did not allow to find a significant statistical association, they suggested two findings: i) cadmium concentration in oysters from natural beds seemed to be lower than in farmed oysters; and ii) in farmed oysters cadmium concentration even exceeded allowed maximum level for human consumption, in particular in autumn. The vertical stratification in the water column of phytoplankton and a cadmium dilution at oyster gonadal maturation might cause changes in oyster cadmium accumulation.

C. elegans metallothioneins: response to and defence against ROS toxicity

The genome of the nematode Caenorhabditis elegans encodes for two multifunctional metal binding metallothioneins (MTs), CeMT-1 and CeMT-2. Here we applied qPCR to identify a transcriptional up-regulation following the exposure to free radical generators (ROS) paraquat or hydrogen peroxide, a trend that was confirmed with Pmtl::GFP expressing alleles. The deletion of the MT loci resulted in an elevation of in vivo levels of hydrogen peroxide and exposure to ROS caused a reduction in total egg production, growth and life span in wild type nematodes, effects that were particularly pronounced in the CeMT-2 and double knockout. Moreover, in vitro incubation of recombinant MTs with hydrogen peroxide demonstrated the presence of direct oxidation of the CeMTs, with zinc released from both isoforms and concomitant formation of intra-molecular disulfides. Finally, metabolic profiling (metabolomic) analysis of wild type and MT knockouts in the presence/absence of oxidative stressors, confirmed the overall trend described by the other experiments, and identified 2-aminoadipate as a potential novel small-molecule marker of oxidative stress. In summary, this study highlights that C. elegans metallothioneins scavenge and protect against reactive oxygen species and potentially against oxidative stress, with CeMT-2 being more effective than CeMT-1.

Astrocyte-derived metallothionein protects dopaminergic neurons from dopamine quinone toxicity

Our previous studies demonstrated the involvement of quinone formation in dopaminergic neuron dysfunction in the L-DOPA-treated parkinsonian model and in methamphetamine (METH) neurotoxicity. We further reported that the cysteine-rich metal-binding metallothionein (MT) family of proteins protects dopaminergic neurons against dopamine (DA) quinone neurotoxicity by its quinone-quenching property. The aim of this study was to examine MT induction in astrocytes in response to excess DA and the potential neuroprotective effects of astrocyte-derived MTs against DA quinone toxicity. DA exposure significantly upregulated MT-1/-2 in cultured striatal astrocytes, but not in mesencephalic neurons. This DA-induced MT upregulation in astrocytes was blocked by treatment with a DA-transporter (DAT) inhibitor, but not by DA-receptor antagonists. Expression of nuclear factor erythroid 2-related factor (Nrf2) and its binding activity to antioxidant response element of MT-1 gene were significantly increased in the astrocytes after DA exposure. Nuclear translocation of Nrf2 was suppressed by the DAT inhibitor. Quinone formation and reduction of mesencephalic DA neurons after DA exposure were ameliorated by preincubation with conditioned media from DA-treated astrocytes. These protective effects were abrogated by MT-1/-2-specific antibody. Adding exogenous MT-1 to glial conditioned media also showed similar neuroprotective effects. Furthermore, MT-1/-2 expression was markedly elevated specifically in reactive astrocytes in the striatum of L-DOPA-treated hemi-parkinsonian mice or METH-injected mice. These results suggested that excess DA taken up by astrocytes via DAT upregulates MT-1/-2 expression specifically in astrocytes, and that MTs or related molecules secreted specifically by astrocytes protect dopaminergic neurons from damage through quinone quenching and/or scavenging of free radicals.

The characterisation of the behaviour and gill toxicity of CdS/CdTe quantum dots in rainbow trout (Oncorhynchus mykiss)

The increasing commercial application of nanomaterials is raising concerns about their potential release to aquatic environments. The purpose of this study was to examine the behaviour in freshwater of capped CdS/CdTe quantum dots and the sublethal effects on gills of rainbow trout. Rainbow trout (Oncorhynchus mykiss) were exposed to increasing concentrations of CdS/CdTe or dissolved cadmium (CdSO4) for 48 h at 15°C. The initial Cd and Te concentration in the aquarium water and size fractionation were determined. After the exposure period, the gills were analysed for labile Cd content, changes in lactate and pyruvate, total and redox status of metallothioneins (MT), lipid peroxidation, protein chaperones of the heat shock protein (Hsp) 72 family and ubiquitin conjugates. QDs were mostly between 6.8 and 25 nm suggesting aggregates. Significant increases in total and metal-binding MT, gill lactate and pyruvate levels and Hsp 72 proteins were observed while ubiquitin protein conjugates were significantly decreased by the QDs only with dissolved Cd, LPO and lactate/pyruvate ratio were not affected by the QDs. A discriminant function analysis of the biomarker responses revealed that colloidal and dissolved Cd differed significantly from each other, suggesting different modes of action.

The resistance of metallothionein to proteolytic digestion: An LC‐MS/MS analysis

Metallothioneins (MTs) are a family of cysteine-rich metalloproteins which strongly bind to heavy metals, such as Cd(II), Zn(II), and Cu(I). Previous works by other group using gel electrophoresis and fluorescence showed MTs were resistant to proteolytic digestion by a variety of enzymes, raising the difficulties in proteomic identification of MTs. The present work was attempted to analyze the resistance of MTs to trypsin using LC with MS/MS (LC-MS/MS), which was able to determine the sequences of the produced peptides and thus precisely characterize the cleavages. The results showed that metal-saturated MTs were completely resistant to trypsin. This resistance problem could be overcome by the addition of EDTA to MT samples, which rendered MTs readily digested into peptides and identified by MS/MS. Interestingly, the partially metal binding MTs were digested into peptides predominantly with miss cleavages which were well dependent on the amount of heavy metals bound to MTs. An explanation for these observations was proposed. The potential applications of the MT's resistance to trypsin in isolation and identification of MTs in complex mixtures such as cultured cells was demonstrated. The preliminary data also showed the same proteomic approach of proteolytic digestion followed by MS/MS analysis may provide information on metal binding status of MTs, along with the identification of MTs in a mixture.

The fine‐tuning of heavy metals in mycorrhizal fungi

The fine‐tuning of heavy metals in mycorrhizal fungi

A sensitive negative-ion electrospray ionization mass spectrometry detection for metallothionein in tris(hydroxymethyl)aminomethane acetate buffer

Although positive-ion (PI) electrospray ionisation mass spectrometry (ESI-MS) has been usually applied for the analysis of native metallothioneins (MT) isoforms binding with metal ions, it suffers from the lack of insensitivity under neural conditions because of the low efficiency of protonation during ESI process. In this study, multiply deprotonated metallothionein (Zn 7-MT-2a), produced from tris(hydroxymethyl)aminomethane (TRIS) acetate solutions under near neutral condition, was analyzed by negative-ion (NI) ESI-MS. Compared with the ammonium acetate buffer system which has been normally used for ESI-MS experiments, the use of TRIS acetate buffer results in the formation of more abundant ions and higher charge states of MT-2a, and consequently higher intensity is attained. The sensitivity enhancement of the system could be explained by the high gas-phase proton affinity, small molecular volume of the anion (acetate), and the high hydrogen consumption by TRIS in the buffer system, which would all favor the deprotonation of the analyte during NI-ESI process. Factors that may affect the NI-ESI responses, such as acid added in buffer, pH and concentration of TRIS acetate had been evaluated, and the result showed that using acetic acid, lower pH under the pH range evaluated, higher concentration of TRIS acetate all favored the MS detection of MT-2a. Our finding sheds light on a buffer system that may offer substantial sensitivity advantages in the studying of weakly bound, non-covalent complexes such as metal binding MTs, which are usually analyzed under near neutral conditions.

Investigation of metal-binding metallothioneins in the tissues of rats after oral intake of cinnabar.

The multi-metal-binding MT fractions in rat tissues after oral intake of cinnabar were characterized by hyphenated size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). With the increase of both the feeding time and dosage of cinnabar the amounts of Hg-binding MT fractions in rat kidney of groups fed cinnabar increased significantly compared with the control group. Meanwhile, more Cu-binding MT were synthesized in the rat kidney, which confirmed the manipulating effect of MT in the homeostasis of Cu for detoxification of nephritic mercury. Although the Hg-binding MT fractions in rat liver of all cinnabar groups were almost independent of cinnabar dosage (2.5-5.0 g kg(-1) bw) and feeding time (2-4 weeks), the amount was much higher than that of the control group. The amounts of Hg-MT in other rat tissues of the cinnabar groups and the control group were compared and their significance is discussed.

Metallohistins: A New Class of Plant Metal-Binding Proteins

Two small multimeric histidine-rich proteins, AgNt84 and Ag164, encoded by two nodule-specific cDNAs isolated from nodule cDNA libraries of the actinorhizal host plant Alnus glutinosa, represent a new class of plant metal binding proteins. This paper reports the characterization of the purified in vitro-expressed proteins by size exclusion chromatography, circular dichroism, equilibrium dialysis, metal affinity chromatography coupled with mass spectrometry, and nuclear magnetic resonance spectroscopy. These analyses reveal that each polypeptide is capable of binding multiple atoms of Zn2+, Ni2-, Co2+, Cu2+, Cd2+ and Hg2+. A reversible shift in histidine Cepsilon1 and Cdelta2 protons in NMR analysis occurred during titration of this protein with ZnCl2 strongly suggesting that histidine residues are responsible for metal binding. AgNt84 and Ag164 are not related to metal binding metallothioneins and phytochelatins and represent a new class of plant metal binding proteins that we propose to call metallohistins. Possible biological roles in symbioses for AgNt84 and Ag164, and their potential for use in bioremediation are discussed.

Metallothionein and Heavy Metals in Brown Trout (Salmo trutta) and European Eel (Anguilla anguilla): A Comparative Study

The levels and the cellular distribution of heavy metals, and the extent by which the metals binds to metallothionein (MT) in brown trout (Salmo trutta) and European eel (Anguilla anguilla), were analyzed in order to assess the natural conditions of MT and heavy metals in these two fish species. There were no differences in heavy metals and MT concentrations between males and females of brown trout in a nonreproductive status and between adult brown trout individuals. Brown trout presented higher Cu content than European eel. The cellular distribution of Cu was also different between the two fish species; while in brown trout most of the Cu was in the noncytosolic fraction, Cu was mainly located in the cytosol in European eel. However, the cellular distribution of Zn, Cd, and Pb was similar in the two fish species. There was also an important difference in the metal content of MT between both species. Whereas, in brown trout, Cu-binding MT represented 75% of total metal-binding MT, this value was 25% in European eel. The between-species differences found in this study are intrinsic characteristics not associated with environmental factors. These results establish the basis to use MT as a bioindicator.

Mechanisms for protection against copper toxicity

Essential transition metals such as copper, molybdenum, and zinc and nonessential metals like cadmium, mercury, and lead can be toxic at the cellular, tissue, and organ levels when present in excess. To avoid metal-induced toxicity most organisms use a redundant combination of metal-regulated import inhibition, sequestration, and enhanced export mechanisms. Combinations of these mechanisms are used to form detoxification pathways controlled through metal-binding proteins at transcriptional, translational, or enzymatic levels. In mammalian pathways copper is partially detoxified by sequestration in the metal-binding metallothioneins or export via the copper-translocating ATPases. Copper regulation of these two mechanisms is afforded by specific conformational changes induced in regulatory proteins on metal binding.

Occurrence of various forms of metallothionein in the rat after a short-term cadmium injection regimen

1. Results are presented showing that the metal-binding metallothionein (Mt) species induced in rat liver in response to Cd administration consist of dimeric and trimeric forms of Mt. A monomeric form might be the first step in the polymerization process. 2. Two proteins (about 8 and 20 kDa mol.wt) are found in hippocampus, but not in brain cortex. 3. These proteins could not be demonstrated to cross-react with our Mt antibody, but the largest of them has strong Cd-binding capacity. 4. Our Mt antibody cross-reacts with a high metal affinity protein present in both brain cortex and hippocampus of twice the mol.wt (20 kDa) of our purified rat liver Mt standard. 5. The results indicate, however, that these Mt like proteins probably emerge from high molecular or membrane bound forms in the cells. 6. A theory is proposed that the predominant polyacrylamide gel band, matching the monomeric, rat liver Mt standard band, seen for all tissues studied in the present work originate from two sources, namely membrane bound and heavy metal induced monomeric form. 7. It is furthermore suggested that those tissues playing an active role in heavy metal metabolism and in protection against toxicity of such metals contain soluble Mts whose active metal-binding forms are oligomers.