A single form of metallothionein is Present in both heavy metal induced and neonatal chicken liver

MultiPlicity of metallothionein and their genes in higher animals are documented extensively in recent literature. In contrast, chicken liver Produced aPParently a single form of metallothionein uPon heavy metal exPosure. This Protein was Purified by gel filtration and ion exchange chromatograPhy and another technique based on heat treatment and acetone fractionation, followed by ion exchange chromatograPhy. In adult uninduced chicken liver the Presence of metallothionein was below the detection limit. But, like mammalian system, chicken liver was found to contain high amount of metallothionein at neonatal stage. This naturally occurring neonatal chicken hePatic metallothionein was Purified and comPared with the heavy metal induced adult hePatic metallothionein. The biochemical and immunobiological comParative analysis of adult and neonatal hePatic metallothionein showed identical characteristics. The neonatal metaltothionein exPressed naturally was a zinc metallothionein and unlike few other mammalian neonatal metallothionein did not contain any coPPer. Metallothionein was undectable in unfertilized eggs, in early embryos, and in Postnatal chicken, from 4 weeks after birth. The highest level of this naturally occurring neonatal metallothionein was found in 1–4 day old neonatal liver, which was about 1.5% of the total cytosolic Protein. This is the first rePorted evidence for the Presence of ontogenically modulated exPression of metallothionein in avian system. Possible biological role of neonatal metallothionein and their cellular interactions has been discussed.     

Detoxification of cadmium Ultrastructural study and electron-probe microanalysis of the midgut in a cadmium-resistant strain ofDrosophila melanogaster

Summary The midgut of a cadmium-resistant strain ofDrosophila melanogaster has been studied at the ultrastructural level and by electronprobe microanalysis (EPMA). Chronic exposure to cadmium leads to a concentration of the metal in a lysosomal system developed in both anterior and posterior segments of the midgut, where it coexists with copper and sulfur. This mechanism apparently ensures a permanent cadmium detoxification and prevents cellular injury. Wild-type flies fed on a cadmium-contaminated medium manifest the same detoxification process. As a result of contamination, copper is stored along the entire length of the midgut, including a part of the middle-midgut previously named copper-accuumulating region. Our data demonstrate that the midgut, particularly the posterior segment, is an accumulative organ for both cadmium and copper. The involvement of the metallothionein system in the detoxification process is discussed.     

以FITC作为荧光探针研究金属硫蛋白的构象变化

本文以异硫氰基荧光素（ＦＩＴＣ）作为荧光探针标记于金属硫蛋白分子上,用荧光光谱研究了Ｃｄ＾２＋及Ａｇ＾＋离子与ＺｎＭＴ２－ＦＩＴＣ进行金属交换及与ＡｐｏＭＴ２－ＦＩＴＣ进行金属重组时的构象变化。结果表明,标记后ＭＴ与Ｃｄ＾２＋离子进行金属交换及金属重组时不具有明显的结构域特征,而Ａｇ＾＋离子进行金属交换及金属重组时,分别在Ａｇ６ＭＴ、Ａｇ１２ＭＴ及Ａｇ１８ＭＴ处具有明显的结构域形成特征。此外高温下     

Cyanobacterial metallothioneins: Biochemistry and molecular genetics

Summary Metallothioneins have been extensively studied in many different eukaryotes where they sequester, and hence detoxify, excess amounts of certain metal ions. However, the precise functions of many of these molecules are not fully understood. This article reviews literature concerning their namesakes in prokaryotes.Abbreviations MT metallothionein - MRE metal regulatory element     

大鼠再生肝抗四氯化碳损伤作用—粗面内质网的形态计量学研究

电镜形态计量学证明,大鼠再生肝对ＣＣｌ４损伤有明显抵抗作用。根据肝小叶外带肝细胞体密度、粗面内质网面密度、比表面及单位膜面附着核糖体数等参量的测算,再生肝接受ＣＣｌ４后肝细胞损伤减轻,粗面内质网不肿胀,膜面积及附着的核糖体数比正常肝ＣＣｌ４损伤后的相应数值增加,假手术动物对ＣＣｌ４肝损伤也有抵抗作用。这种细胞保护机制可能与应激与肝细胞内金属硫蛋白合成增加有关。     

Evidence for a high molecular weight cytosolic factor that binds brain and liver metallothionein

When brain extracts were fractionated in a Sephadex G-75 chromatography and MT levels were assayed by RIA or ELISA using polyclonal antibodies specific for the MT-I and MT-II isoforms, it was found that MT mostly eluted in the high molecular weight (HMW) peak even in reducing or anaerobic conditions. This was also the case for the liver extracts of control rats; in stressed animals MT immunoreactivity in the HMW peak (>80 Kd) was increased compared with undisturbed animals, but the major amount of the newly induced MT eluted, as expected from the current literature, in the low molecular weight (LMW) peak, around 10 Kd. The addition of purified MT to brain extracts precluded its binding to a DEAE-Sephadex column. Furthermore, immunoblot results of native PAGE showed that MT changed its electrophoretic mobility in the presence of HMW proteins from brain cytosol. Altogether, these results suggest that a cytosolic factor binds MT in a saturable manner, which may have strong physiological implications.     

Effect of zinc supplementation on metallothionein,copper, and zinc concentration in various tissues of copper-loaded rats

The present study was designed to investigate the effects of Zn administration on metallothionein concentrations in the liver, kidney, and intestine of copper-loaded rats. Male CD rats were fed a diet containing 12 mg Cu and 67 mg Zn/kg body wt. They were divided into either acute or chronic experimental protocols. Rats undergoing acute experiments received daily ip injections of either Cu (3 mg/kg body wt) or Zn (10 mg/kg body wt) for 3 d. Chronic experiments were carried out on rats receiving Cu ip injections on d 1, 2, 3, 10, 17, and 24, Cu injections plus a Zn-supplemented diet containing 5 g Zn/kg solid diet, or a Zn-supplemented diet alone. Rats injected Zn or Cu had increased MT concentrations in liver and kidney. Zn produced the most important effects and the liver was the most responsive organ. Rats fed a Zn-supplemented diet had significantly higher MT concentrations in liver and intestine with respect to controls. Increased MT synthesis in the liver may contribute to copper detoxification; the hypothesis of copper entrapment in enterocytes cannot be confirmed.     

Heterogeneity of antibodies to metallothionein isomers and development of a simple enzyme-linked immunosorbent assay

A competitive enzyme-linked immunosorbent assay (ELISA) for the measurement of metallothionein (MT) in tissues and body fluids has been developed. The ELISA employs the IgG fraction of a rabbit antiserum to rat liver Cd-MT-2 polymer, a biotinylated secondary antibody, and peroxidase conjugated avidin. With a 1:4000 dilution of the immunoglobulins, typical standard curves (logit-log regression) provide a linear range of 0.1–100 ng for MT-2 and 10–1000 ng for MT-1. Fifty percent inhibition is accomplished with 15 ng and 250 ng for MT-2 and MT-1, respectively. Rat liver MT-1 and MT-2 containing different metals (Ag, Cu, and Zn) inhibited the antibodies as effectively as CdMT. However, the antibodies exhibited greater affinity for both Apo-MT isoforms. Previously reported discrepancies between results obtained by metal binding assays (e.g., Ag-hem binding) and radioimmunoassay for MT levels in tissues have been largely resolved. By addition of 1% Tween 20 to samples, the ELISA routinely estimated the total MT in samples of rat, mouse, and human liver and kidney at 88% of the value obtained by the silver-hem binding assay. Specific antibodies to MT-2 were purified from our anti-serum by affinity purification using CH-Sepharose 4B coupled with rat liver MT-1. Estimation of MT in samples using purified MT-2 antibodies provided slightly lower values (72%) for MT in tissues as compared to the Ag-hem method. The predominant form of MT in tissues of control animals was found to be MT-2. Therefore, the MT-2 specific antibodies may be useful for the study of the functions of MT isoforms. Levels of total MT in tissues and biological fluids of rats injected with CdCl₂ (0.3 mg Cd/kg) and Cd-MT (0.3 mg Cd/kg) were estimated by ELISA. The results suggest urinary MT levels may be related to kidney damage.     

Liver, Brain, and Heart Metallothionein Induction by Stress

To date, stress has been reported to induce metallothionein (MT) synthesis in the liver only. In the present experiment, the effects of food and water deprivation alone or of immobilization stress plus food and water deprivation on liver, brain, and heart MT have been studied in adult male rats. Liver and brain MT levels were increased by immobilization stress as soon as 6 h after the onset of stress. Eighteen hours of immobilization, which is accompanied by food and water deprivation, further increased liver and brain MT levels and significantly increased heart MT content. A specific effect of immobilization was evident in all three tissues, because the effect of food and water deprivation alone was significantly lower than that of immobilization plus starvation. Changes in MT apparently were not related to changes in cytosolic Zn.     

Copper transfer through the intestinal wall Serosal release of metallothionein

Summary The elucidation of the molecular side of copper transport in biological systems is a promising task. In this context the transfer of ingested copper into the portal blood plasma was examined. Intralumenal addition of 200 M copper caused the release of Cu-thionein into the venous effluent. This Cu-thionein became detectable after prior perfusion of the porcine small bowel using a modified isotonic phosphate-buffered saline (P_i/NaCl) medium. The protein was characterized by gel chromatography, luminescence, electronic absorption and immunological identification. ELISA and immunoblotting employing a murine monoclonal antibody to rat liver metallothionein-I proved to be most convenient. Using buffer-loaded sacs of porcine jejunum into which Cu²⁺, Zn²⁺ and Cd²⁺ were added, the release of metallothionein into the serosal fluid was successfully seen by ELISA. The observed excretion of metallothionein into the portal compartment may be a genuine metal transport system for many biochemically active metals.