Role of lysine side chains in metallothionein

Summary pK_a-Values of lysine residues of mammalian metallothionein were determined by chemical titration measurements of -CH₂ lysine resonances in the¹H-NMR spectra. They are about 0.5 pH-unit higher than the average pK_a-value of a metal-free derivative, suggesting interaction of the positively charged residues with the two three-fold negatively charged metal-thiolate clusters of the metal-containing form. Deprotonation of the lysines leads to circular dichroism changes attributable to an electrostatically induced structural transition of the protein.To whom correspondence concerning this paper should be addressed.     

Radioresistance in cells with high content of metallothionein

Summary An endogenous cytoplasmic protein, metallothionein (MT) apparently gave rise to radioresistance in 2 different cell lines. A dose reduction factor of 1.9 was achieved in MT-containing cells. MT accounted for a 3–4-fold increase of total sulfhydryl groups in the resistant cell strains, compared to the non-resistant lines from which they were derived. The protein is rich in cysteine (30%), an amino acid known to give radioprotection when administered exogeneously. Glutathione levels and cell-cycle phase distribution showed no marked difference between resistant and corresponding non-resistant cells.We thank Dr F. Devik, Dr L. Eldjarn and Dr E. Jellum for stimulating discussions, and Miss A. K. Syversen and Mrs T. Nysted for skillful technical assistance. This work was partly supported by the Norgwegian Council for Science and the Humanitites. LE is a fellow of the Norwegian Society for Fighting Cancer.     

Restraint stress induced changes in rat liver and serum metallothionein and in Zn metabolism

24 h of a psychogenic stress (restraint) caused a strong increase of liver metallothionein (MT) levels. 3 h of stress were sufficient to induce an increase in liver MT, measured 21 h later, but the increase was much lower than in continuously restrained rats. Stress induction of liver MT was not due to food deprivation, since rats deprived for 24 h showed lower MT levels than stressed ones. Zn on MT presented the same qualitative but not quantitative pattern of response as MT protein. Liver cytosolic Zn was increased by restraint in spite of their being no decrease in serum Zn. Any treatment altered serum MT. Liver and serum MT were not correlated. The present results demonstrate that basically psychogenic stresses increased liver but not serum MT levels. No positive evidence for a relationship between corticosterone secretion and MT induction was found.     

Restraint stress induced changes in rat liver and serum metallothionein and in Zn metabolism

Summary 24 h of a psychogenic stress (restraint) caused a strong increase of liver metallothionein (MT) levels. 3 h of stress were sufficient to induce an increase in liver MT, measured 21 h later, but the increase was much lower than in continuously restrained rats. Stress induction of liver MT was not due to food deprivation, since rats deprived for 24 h showed lower MT levels than stressed ones. Zn on MT presented the same qualitative but not quantitative pattern of response as MT protein. Liver cytosolic Zn was increased by restraint in spite of their being no decrease in serum Zn. Any treatment altered serum MT. Liver and serum MT were not correlated. The present results demonstrate that basically psychogenic stresses increased liver but not serum MT levels. No positive evidence for a relationship between corticosterone secretion and MT induction was found.     

The enhanced induction of metallothionein by zinc,its half-life in the marine fishPleuronectes platessa,and the influence of stress factors on metallothionein levels

Summary Intraperitoneal injection of zinc raised levels of a hepatic metallothionein-like species. Assuming that this species was metallothionein (MT) then levels were raised from approximately 20 g/g to 300 g/g in 7 days, and levels thereafter remained high for the next 4 weeks. The half-lives of the protein in liver and kidney from starved fish, measured using in vivo incorporation of³⁵S cysteine at 11°C, were approximately 27 days and 32 days respectively. The following agents failed to stimulate synthesis of MT in plaice: stress (due to catching), endotoxin, dexamethasone, cortisol and turpentine.     

The enhanced induction of metallothionein by zinc, its half-life in the marine fish Pleuronectes platessa, and the influence of stress factors on metallothionein levels

Intraperitoneal injection of zinc raised levels of a hepatic metallothionein-like species. Assuming that this species was metallothionein (MT) then levels were raised from approximately 20 micrograms/g to 300 micrograms/g in 7 days, and levels thereafter remained high for the next 4 weeks. The half-lives of the protein in liver and kidney from starved fish, measured using in vivo incorporation of 35S cysteine at 11 degrees C, were approximately 27 days and 32 days respectively. The following agents failed to stimulate synthesis of MT in plaice: stress (due to catching), endotoxin, dexamethasone, cortisol and turpentine.     

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein

The incorporation of 35S-cysteine and 3H-glutamic acid was studied in mouse hepatic and renal metallothionein and in testicular cadmium-binding protein of similar molecular weight. Preferential incorporation of 35S-cysteine over 3H-glutamic acid was observed not only in hepatic and renal metallothionein, but also in testicular cadmium-binding protein. When the antigenic reactivity of these proteins was compared, all three proteins reacted with the metallothionein antibody. These similarities suggest that the low molecular weight testicular cadmium-binding protein is apparently metallothionein.     

Similarities between metallothionein and low molecular weight testicular cadmium-binding protein

Summary The incorporation of³⁵S-cysteine and³H-glutamic acid was studied in mouse hepatic and renal metallothionein and in testicular cadmium-binding protein of similar molecular weight. Preferential incorporation of³⁵S-cysteine over³H-glutamic acid was observed not only in hepatic and renal metallothionein, but also in testicular cadmium-binding protein. When the antigenic reactivity of these proteins was compared, all three proteins reacted with the metallothionein antibody. These similarities suggest that the low molecular weight testicular cadmium-binding protein is apparently metallothionein.     

Chronic cadmium intake results in dose-related excretion of metallothionein in urine

Urinary excretion of metallothionein was measured by radioimmunoassay in rats given drinking water containing 5 or 50 mg cadmium/l for up to 2 years. The metallothionein levels corresponded to the concentration of cadmium in the drinking water and increased linearly over the course of the study. These results demonstrate that urinary metallothionein is a sensitive biological indicator of oral cadmium exposure.     

Increased demyelination and axonal damage in metallothionein I+II-deficient mice during experimental autoimmune encephalomyelitis

Metallothioneins I+II (MT-I+II) are antioxidant, neuroprotective factors. We previously showed that MT-I+II deficiency during experimental autoimmune encephalomyelitis (EAE) leads to increased disease incidence and clinical symptoms. Moreover, the inflammatory response of macrophages and T cells, oxidative stress, and apoptotic cell death during EAE were increased by MT-I+II deficiency. We now show for the first time that demyelination and axonal damage are significantly increased in MT-I+II deficient mice during EAE. Furthermore, oligodendroglial regeneration, growth cone formation, and tissue repair including expression of trophic factors were significantly reduced in MT-I+II-deficient mice during EAE. Accordingly, MT-I+II have protective and regenerative roles in the brain. Received 31 October 2002; received after revision 23 November 2002; accepted 26 November 2002 RID="*" ID="*"Corresponding author. M. Penkowa and C. Espejo contributed equally to this paper.     

Copper metabolism in the LEC rat: Involvement of induction of metallothionein and disposition of zinc and iron

The Cu concentration was about 40 times higher in the liver of LEC (Long-Evans with a cinnamon-like coat color) rats aged 77 days (227.5±21.6 g/g liver) than in Fischer rats (5.2±0.1 g/g liver). However, in the kidney and brain of the LEC rats, Cu concentrations were lower than in these organs of the Fischer rats. Cu concentration in the hepatic metallothionein fraction was about 130 times higher in the LEC rats than in the Fischer rats. The LEC rats showed markedly low concentrations of Cu in the serum and bile. It seems likely that excretion of Cu from the liver into the bile and blood (as ceruloplasmin) is inherently lacking in the LEC rat.     

Isolation,characterization, and determination of human liver (copper/zinc) metallothionein

Summary A copper-containing protein was purified from the liver of a patient with primary biliary cirrhosis by a combination of gel filtration and anion exchange chromatography. This copper-protein had UV absorption and emission spectra, an amino acid composition, and a molecular mass which were characteristic for metallothionein (MT).From 8 livers (3 control, 1 fetal and 4 primary biliary cirrhosis) MT was extracted with non-reducing buffer and centrifuged, and the pellets were re-extracted with a 1% 2-mercaptoethanol-containing buffer. The non-reducing buffer extracted a predominantly copper-containing MT from the livers of patients with primary biliary cirrhosis and a predominantly zinc-containing MT from control lives and the fetal liver. Only from the fetal liver was a copper/zinc containing MT solubilized during the re-extraction with 2-mercaptoethanol-containing buffer. These results indicate that human MT is a unique metalloprotein with age and disease-dependent characteristics.     

Isolation, characterization, and determination of human liver (copper/zinc) metallothionein

A copper-containing protein was purified from the liver of a patient with primary biliary cirrhosis by a combination of gel filtration and anion exchange chromatography. This copper-protein had UV absorption and emission spectra, an amino acid composition, and a molecular mass which were characteristic for metallothionein (MT). From 8 livers (3 control, 1 fetal and 4 primary biliary cirrhosis) MT was extracted with non-reducing buffer and centrifuged, and pellets were re-extracted with a 1% 2-mercaptoethanol-containing buffer. The non-reducing buffer extracted a predominantly copper-containing MT from the livers of patients with primary biliary cirrhosis and a predominantly zinc-containing MT from control livers and the fetal liver. Only from the fetal liver was a copper/zinc containing MT solubilized during the re-extraction with 2-mercaptoethanol-containing buffer. These results indicate that human MT is a unique metalloprotein with age and disease-dependent characteristics.     

Recombinant synthesis of mouse Zn3-β and Zn4-α metallothionein 1 domains and characterization of their cadmium(II) binding capacity

Genetic engineering, coupled with spectro scopic analyses, has enabled the metal binding proper ties of the α and β subunits of mouse metallothionein 1 (MT) to be characterized. A heterologous expression system in E.coli has led to high yields of their pure zinc-complexed forms. The cadmium(II) binding properties of recombinant Zn₄-αMT and Zn₃-βMT have been studied by electronic absorption and circular dichroism. The former binds Cd(II) identically to α fragments obtained from mammalian organs, showing that the recombinant polypeptide behaves like the na tive protein. Titration of Zn₃-βMT with CdCl₂ results in the formation of Cd₃-βMT. The addition of excess Cd(II) leads to Cd₄-βMT which, with the extra loading of Cd(II), unravels to give rise isodichroically to Cd₉-βMT. The effect of cadmium-displaced Zn(II) ions and excess Cd(II) above the full metal occupancy of three has been studied using Chelex-100. The Cd₃-βMT species is stable in the presence of this strong metal-chelating agent. Received 20 May 1997; received after revision 7 July 1997; accepted 9 July 1997