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Isolation and partial characterization of the low-molecular-mass zinc/cadmium-binding protein from the testes of the patas monkey (Erythrocebus patas). Distinction from metallothionein.

ABSTRACT: The mammalian testes are generally quite susceptible to cadmium. A deficiency of metallothionein (MT), a metal-binding protein linked to Cd tolerance, has been observed in rat testes and may explain the sensitivity in rats. Little is known about the metal-binding proteins in primate testes. Thus this study examined the nature of these proteins in a non-human primate species, the patas monkey (Erythrocebus patas). In all cases proteins isolated from testes were compared with authentic MT isolated from the liver of a zinc-treated monkey. A low-molecular-mass Zn/Cd-binding protein was seen in testicular and hepatic cytosol after gel filtration. Neither protein had substantial amounts of associated copper. These proteins could be partially purified from both sources by heat treatment and acetone precipitation. When such extracts were further separated by reverse-phase h.p.l.c., four hepatic forms were isolated, all of which proved to be authentic MT by amino acid analysis. However, only two testicular forms were separated by h.p.l.c., both of which had amino acid compositions quite unlike that of MT, having a much lower cysteine content and amino acids which are absent from MT (leucine and phenylalanine). The testicular protein appeared to be uninducible by Zn treatment. These results suggest that the low-molecular-mass Cd/Zn-binding proteins in the patas testes are not MTs and further support the hypothesis that a MT deficiency may be an important determinate of the marked testicular sensitivity to Cd toxicity.

SUBMITTER: Waalkes MP

PROVIDER: S-EPMC1135378 | biostudies-other | 1988 Nov

REPOSITORIES: biostudies-other

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Cadmium-binding proteins of rat testes. Characterization of a low-molecular-mass protein that lacks identity with metallothionein.

Project description:Cadmium-binding proteins in the cytosol of testes from untreated rats were separated by Sephadex G-75 gel filtration. Three major testicular metal-binding proteins (TMBP), or groups of proteins, with relative elution volumes of approx. 1.0 (TMBP-1), 1.7 (TMBP-2) and 2.4 (TMBP-3) were separated. Elution of Zn-binding proteins exhibited a similar pattern. TMBP-3 has previously been thought to be metallothionein (MT), and hence this protein was further characterized and compared with hepatic MT isolated from Cd-treated rats. Estimation of Mr by gel filtration indicated a slight difference between MT (Mr 10000) and TMBP-3 (Mr 8000). Two major forms of MT (MT-I and MT-II) and TMBP-3 (TMBP-3 form I and TMBP-3 form II) were obtained after DEAE-Sephadex A-25 anion-exchange chromatography, with the corresponding subfractions being eluted at similar conductances. Non-denaturing polyacrylamide-gel electrophoresis on 7% acrylamide gels indicated that the subfractions of TMBP-3 had similar mobilities to those of the corresponding subfractions of MT. However, SDS (sodium dodecyl sulphate)/12% (w/v)-polyacrylamide-gel electrophoresis resulted in marked differences in migration of the two corresponding forms of MT and TMBP-3. Co-electrophoresis of MT-II and TMBP-3 form II by SDS/polyacrylamide-gel electrophoresis revealed two distinct proteins. Amino acid analysis indicated much lower content of cysteine in the testicular than in the hepatic proteins. TMBP-3 also contained significant amounts of tyrosine, phenylalanine and histidine, whereas MT did not. U.v.-spectral analysis of TMBP-3 showed a much lower A250/A280 ratio than for MT. Thus this major metal-binding protein in testes, which has been assumed to be MT is, in fact, a quite different protein.

| S-EPMC1153700 | biostudies-other

Zinc and low-dose of cadmium protect sertoli cells against toxic-dose of cadmium: The role of metallothionein.

Project description:BackgroundThe impact of cadmium (Cd) on male infertility may be related to the interaction with metal-binding proteins known as metallothioneins (Mts). Trace elements like zinc (Zn) have protective effects on testicular damage induced by Cd.ObjectiveWe determined the effect of Zn and low-dose Cd pre-treatment on the expression of Mt1 and Mt2 genes on testicular Sertoli cells.Materials and methodsThe cultured TM4 mouse sertoli cells were treated with 50 μM ZnSO4 (Zn pre-treated group; ZnPG), 2 μM CdCl2 (Cd pre-treated group; CdPG), or distilled water (DW pre-treated group; DWPG). After 18 hour, all of these groups were exposed to 100 μM CdCl2 for different periods of time (1, 2, 3, and 6 hours). There was also a control group for all three groups, which was treated only with distilled water (without Cd or Zn pre-treatment). Cellular viability, Zn and Cd concentrations and gene expression were assessed by MTT, atomic absorption spectrometry and real time PCR methods, respectively.ResultsThe expression of Mt1 and Mt2 genes in ZnPG, CdPG, and DWPG was greater than the control group (p=0.02 and p=0.01, respectively). Cd concentrations in CdPG and DWPG were greater than the control group (p=0.00). Expression of both genes in ZnPG and CdPG increased after 3 hours of treatment and Cd concentration decreased simultaneously, which was more obvious in ZnPG.ConclusionZn and short term low-dose Cd pre-treatment might reduce the adverse effects of Cd by increasing expression of Mts genes in Sertoli cells. The protective effect of Zn was stronger than Cd.

| S-EPMC3941315 | biostudies-literature

Properties of cadmium-binding proteins in rat testes. Characteristics unlike metallothionein.

Project description:Since the exposure of rats to cadmium causes zinc to accumulate in metallothionein in liver and kidney but not in a similar protein in the testes, the properties of the low-Mr cadmium-binding proteins were investigated in rat testes. Weanling rats that had been given dietary cadmium for 6 weeks were injected with 109CdCl2 and subsequently killed, and the 109Cd-labelled low-Mr proteins from testes were purified. The pooled low-Mr cadmium-containing fractions from the gel-filtration (Sephadex G-75) columns were eluted through DEAE-Sephacel columns, yielding two peaks. Each of the individual peaks from this Sephacel column was further purified by rechromatography on DEAE-Sephacel and on Bio-Gel P-10 columns. Amino acid analysis of the two purified proteins revealed a low cysteine (about 3%) content, with aspartate, glutamate and glycine as the predominant amino acids. Thus these low-Mr cadmium-binding proteins induced by cadmium in rat testes do not appear to be metallothionein.

| S-EPMC1152742 | biostudies-other

Mechanism of cadmium ion substitution in mammalian zinc metallothionein and metallothionein alpha domain: kinetic and structural studies.

Project description:Cellular metallothionein (MT) protects against Cd(2+) exposure through direct binding of the metal ion. The model reaction between rabbit liver Zn(7)-MT-2 with Cd(2+) was studied with stopped flow kinetics. Four kinetic steps were observable. Comparison of this reaction with an analog utilizing the MT Zn(4)-alpha domain revealed that only the fastest step involved the Zn(3)-beta domain. Each step of the Zn(4)-alpha domain reaction with Cd(2+) displayed hyperbolic dependence of the observed rate constant on Cd(2+) concentration, with the first step comprising 50% of the total reaction and each of the other two, 25%. The two constants extracted from each of these relationships were interpreted as the equilibrium constant for the initial binding of Cd(2+) to the Zn((4-n)),Cd(n)-thiolate cluster (n = 0-3) of the alpha domain and the first order rate constant for the exchange of Cd(2+) for Zn(2+) in the cluster. Activation enthalpies and entropies were determined for each constant. A suite of Zn((4-n)),Cd(n)-thiolate clusters (n = 0-3) was prepared by titration of the Zn(4)-alpha domain with (113)Cd(2+). The products were analyzed by one-dimensional (113)Cd(2+) NMR spectroscopy to define the distribution of (113)Cd(2+) among the four cluster binding sites. Each of these species was also reacted with Cd(2+). The properties of these reactions were similar to those extracted from the reaction of Cd(2+) with the overall domain. Thus, the kinetic results were linked to (113)Cd(2+) occupancy among the cluster metal binding sites. In turn, this linkage permitted the interpretation of the various constants determined for the reaction of Cd(2+) with the Zn(4)-alpha domain in relation to the alpha domain cluster structure.

| S-EPMC2935266 | biostudies-literature

Cadmium-binding proteins of rat testes. Apparent source of the protein of low molecular mass.

Project description:Fractionation of rat testicular cytosolic proteins by gel filtration indicates three major metal-binding proteins, or groups of proteins, termed testicular metal-binding protein (TMBP) 1, 2 and 3 by order of elution. The major heat-stable, metal-binding proteins in testes is TMBP-2, which has an Mr of approx. 25000. In most tissues, metallothionein (MT) is the major heat-stable, metal-binding protein, but it has an Mr of 6000. This testicular protein (TMBP-2) is much larger than MT, and since polymeric forms of MT have been previously reported, further characterization of TMBP-2 was performed. TMBP-2 was separated into two forms by DEAE-Sephadex A-25 anion-exchange chromatography. Amino acid analysis of both forms of TMBP-2 revealed that they differed markedly from MT, having particularly low cysteine contents. However, amino acid analysis showed that TBMP-2 was strikingly similar to TMBP-3, with an approximate stoichiometric relationship of 4:1. Therefore, experiments were conducted to determine if TMBP-3 could be a breakdown product of TMBP-2. Heat treatment of testicular cytosol in room air before gel filtration resulted in a marked increase in TMBP-3 and loss of TMBP-2. Storing intact testes at -20 degrees C for 2 weeks before processing for gel filtration also resulted in an increase in TMBP-3 and a loss of TMBP-2. Addition of a reducing agent (dithiothreitol) or proteinase inhibitor (N-ethylmaleimide) in processing of samples before gel filtration inhibited the appearance of TMBP-3. Results suggest that the low-Mr Cd-binding protein (TMBP-3) of rat testes results from either proteolytic or oxidative breakdown of a higher-Mr species, or from a combination of such factors.

| S-EPMC1153701 | biostudies-other

Genetic variation in metallothionein and metal-regulatory transcription factor 1 in relation to urinary cadmium, copper, and zinc.

Project description:Metallothionein (MT) proteins play critical roles in the physiological handling of both essential (Cu and Zn) and toxic (Cd) metals. MT expression is regulated by metal-regulatory transcription factor 1 (MTF1). Hence, genetic variation in the MT gene family and MTF1 might influence excretion of these metals.321 women were recruited in Seattle, WA and Las Cruces, NM and provided demographic information, urine samples for measurement of metal concentrations by mass spectrometry and creatinine, and blood or saliva for extraction of DNA. Forty-one single nucleotide polymorphisms (SNPs) within the MTF1 gene region and the region of chromosome 16 encoding the MT gene family were selected for genotyping in addition to an ancestry informative marker panel. Linear regression was used to estimate the association of SNPs with urinary Cd, Cu, and Zn, adjusted for age, urinary creatinine, smoking history, study site, and ancestry.Minor alleles of rs28366003 and rs10636 near the MT2A gene were associated with lower urinary Cd, Cu, and Zn. Minor alleles of rs8044719 and rs1599823, near MT1A and MT1B, were associated with lower urinary Cd and Zn, respectively. Minor alleles of rs4653329 in MTF1 were associated with lower urinary Cd.These results suggest that genetic variation in the MT gene region and MTF1 influences urinary Cd, Cu, and Zn excretion.

| S-EPMC4689293 | biostudies-literature

Zinc protects against cadmium-induced toxicity in neonatal murine engineered cardiac tissues via metallothionein-dependent and independent mechanisms.

Project description:Cadmium (Cd) is a nonessential heavy metal and a prevalent environmental toxin that has been shown to induce significant cardiomyocyte apoptosis in neonatal murine engineered cardiac tissues (ECTs). In contrast, zinc (Zn) is a potent metallothionein (MT) inducer, which plays an important role in protection against Cd toxicity. In this study, we investigated the protective effects of Zn against Cd toxicity in ECTs and explore the underlying mechanisms. ECTs were constructed from neonatal ventricular cells of wild-type (WT) mice and mice with global MT gene deletion (MT-KO). In WT-ECTs, Cd (5-20 μM) caused a dose-dependent toxicity that was detected within 8 h evidenced by suppressed beating, apoptosis, and LDH release; Zn (50-200 μM) dose-dependently induced MT expression in ECTs without causing ECT toxicity; co-treatment of ECT with Zn (50 µM) prevented Cd-induced toxicity. In MT-KO ECTs, Cd toxicity was enhanced; but unexpectedly, cotreatment with Zn provided partial protection against Cd toxicity. Furthermore, Cd, but not Zn, significantly activated Nrf2 and its downstream targets, including HO-1; inhibition of HO-1 by a specific HO-1 inhibitor, ZnPP (10 µM), significantly increased Cd-induced toxicity, but did not inhibit Zn protection against Cd injury, suggesting that Nrf2-mediated HO-1 activation was not required for Zn protective effect. Finally, the ability of Zn to reduce Cd uptake provided an additional MT-independent mechanism for reducing Cd toxicity. Thus, Zn exerts protective effects against Cd toxicity for murine ECTs that are partially MT-mediated. Further studies are required to translate these findings towards clinical trials.

| S-EPMC7471469 | biostudies-literature

Induction of metallothionein synthesis by cadmium and zinc in cultured rabbit kidney cells (RK-13).

Project description:The effects of increasing concentrations of Zn(II) and Cd(II) on the expression of the four isometallothioneins (isoMTs), namely MT-1a, MT-2a, MT-2d and MT-2e, in rabbit kidney cells (RK-13) and the development of cellular tolerance to these metal ions were studied. The results showed that, whereas in parental cells MT concentration was low and composed nearly exclusively of MT-2a and MT-1a, all four isoMTs increased massively in abundance when the cells were exposed to toxic concentrations of Zn(II) or Cd(II), the relative increase being largest in the two minor isoforms MT-2d and MT-2e. While the response of the four isoMTs to the challenge by Zn(II) or Cd(II) was qualitatively comparable, there were differences in sensitivity and delay time, Cd(II) being the more efficient inducer and much faster in eliciting the onset of isoMT synthesis. An even larger production of isoMTs resulted when RK-13 cells were cultured in the presence of a series of metal concentrations yielding sub-lines of increased metal tolerance. In this instance too, there were marked differences in the response to Cd(II) and Zn(II). Thus, in cells of sub-lines selected for tolerance to moderate concentrations of Cd(II) the kinetic analysis of isoMT accretion gave indications of a saturable induction process while no such evidence was forthcoming for Zn(II). In sub-line cells selected for tolerance to the highest concentrations of Cd(II) or Zn(II) isoMT formation was increased by another order of magnitude, reaching for some isoforms a 100- to 1000-fold augmentation over the amounts measured in cells of the unexposed parental cells. A potentiation of this magnitude goes beyond the range of ordinary regulation of gene expression. It is to be viewed instead as an enlargement of the capacity of isoMT synthesis acquired by a variety of mechanisms in the surviving cells.

| S-EPMC1134254 | biostudies-other

Ion mobility mass spectrometry and molecular dynamics simulations unravel the conformational stability of zinc metallothionein-2 species.

Project description:Ion mobility-mass spectrometry (IM-MS) unraveled different conformational stability in Zn4-7-metallothionein-2. We introduced a new molecular dynamics simulation approach that permitted the exploration of all of the conformational space confirming the experimental data, and revealed that not only the Zn-S bonds but also the α-β domain interactions modulate protein unfolding.

| S-EPMC10089061 | biostudies-literature

Drivers of metallothionein-mediated detoxification of cadmium in C.elegans

Project description:Cadmium is toxic and intricate pathways linked to metallothioneins (MT) drive the detoxification process. Whist the mechanisms are well understood in mammalian systems, detailed knowledge is still elusive in invertebrates (which notably differ to mammalian systems). The model nematode Caenorhabditis elegans is ideally suited for assessing metallothionein mediates detoxification in invertebrates as is relatively short-lived, can be easily exposed and its genome is fully sequenced and widely annotated. The aim of the experiment was to identify new MT mediated target genes involved in Cd toxicosis.

2014-06-01 | GSE29951 | GEO

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