Immunological and biochemical properties of metallothioneins of golden hamster,mouse and rat

Summary Golden hamster, mouse and rat hepatic cadmium metallothioneins (MT) were purified by Sephadex G-75 gel filtration, DEAE-Sephadex A-25 chromatography and activated Thiol-Sepharose 4B affinity chromatography. Metallothioneins were separated by DEAE-Sephadex A-25 chromatography into two forms: MT-1 and MT-2. In mouse and golden hamster liver, MT-1 was the major form. The purified proteins were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. In non-denaturing polyacrylamide gel electrophoresis, migration of mouse, rat and golden hamster hepatic metallothioneins were found to be different. Antibodies to mouse hepatic MT-1 was raised in rabbits. The antiserum cross reacted with mouse and hamster MT-1 and MT-2 giving a single precipitin band. Mouse, rat and hamster hepatic MTs are immunologically identical but electrophoretically different. The kidney and pancreatic MTs of rat and golden hamster were purified by Sephadex G-75 gel filtration. They were immunologically distinct. Pancreas MT formed a line of partial identity with hepatic MTs. Kidney MTs form two precipitin band one identical with the pancreatic form and another of complete identity with the hepatic MTs. This indicates the presence of tissue specific MTs.     

棕尾别麻蝇金属硫蛋白的分离纯化及性质分析

将棕尾别麻蝇Boettcherisca peregrina幼虫置于含800 μg/g CdCl₂的食物中取食48 h后,可诱导金属硫蛋白(MT)的合成。诱导处理后的幼虫匀浆上清液经Sephadex G-50分子筛柱、UNO^TM Q1阴离子交换柱和Bio-Gel P-6脱盐柱层析,纯化得到2个亚型,即MT-Ⅰ和MT-Ⅱ。MT-Ⅰ和MT-Ⅱ的分子量均为9 kD,每蛋白分子均含7个Cd和20个巯基,且具254 nm的Cd-SH特征吸收肩。两者的氨基酸组成中,以半胱氨酸含量最高,分别为36.6%和31.8%;而芳香族氨基酸和组氨酸含量甚少,约1%～2%。     

Metallothionein response to cadmium and zinc exposures compared in two freshwater bivalves, Dreissena polymorpha and Corbicula fluminea

Metallothionein (MT) response to cadmium (Cd) and zinc (Zn) bioaccumulation after single or combined direct exposure was compared in two freshwater bivalves, Dreissena polymorpha (zebra mussel) and Corbicula fluminea (Asiatic clam). Bivalves were exposed to 0.133 μM Cd and/or 15.3 μM Zn, with metal and MT concentrations analysed in the whole soft body after 1, 3, 10 and 24 days of exposure and compared with controls. Results showed significant increase in MT concentrations in both species exposed to Cd and Cd+Zn with a higher accumulation of the protein compared to the control in D. polymorpha for nevertheless similar Cd levels accumulated with time. Exposure to Zn alone led to a significant increase in MT concentrations only in C. fluminea, whereas there was a lack of MT gene induction in the zebra mussels which was confirmed by MT mRNA quantification in gills (RT-PCR). Mussel mortality after 10 days of exposure to Zn and Cd + Zn is discussed with regard to detoxification mechanisms, which include metallothioneins.     

Studies on the evolutionary relationships between hemoglobins inChironomus pallidivittatus andC. Tentans

Summary The monomeric hemoglobins ofChironomus tentans andC. pallidivittatus have been isolated and separated into their respective components by gel chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-Sephacel. The amino acid compositions of the purified components are given. The sequence of the 30 N-terminal amino acid residues of one of the monomeric components (Hb I fromC. pallidivittatus) was determined and found to be identical in almost all of its parts with the monomeric hemoglobins ofC. thummi (CTT III and CTT IV).Antibodies against the monomeric hemoglobins Hb I and Hb IIc and the dimeric fraction were highly specific and no cross reaction between dimeric and monomeric hemoglobins could be demonstrated. The antibodies against the monomers crossreact with the monomeric hemoglobins CTT III and CTT IV ofC. thummi. Taken together with genetic data, the immunological results indicate that divergence of monomeric from dimeric forms was an early event in the evolution of the various hemoglobins inChironomus.     

Metal-binding capacity of metallothioneins of the liver of rats poisoned with heavy metals

The functioning of metallothioneins in the liver of rats, poisoned with copper sulfate and cadmium sulfate has been investigated. By sequential chromatography on sephadex G-50 and DEAE-cellulose the authors obtained metallothioneins (MT-1, MT-1A, MT-2, MT-2a), which differ in molecular weight and composition of associated metals. Heavy metal poisoning leads to activation of synthesis and metal-binding function of metallothioneins, as well as to changes in the composition of their isoforms.     

Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.     

Staphylococcal Enterotoxin D

Purification of enterotoxin D from Staphylococcus aureus 494 was attempted by utilizing the following techniques: Concentration of bacterial culture supernatant with polyethylene glycol 20000, CM-cellulose column chromatography, Sephadex G-100 gel-filtration, chromatography on a DEAE cellulose column with concave gradient elution and gel-filtration with Sephadex G-50. The final preparation obtained gave a single precipitin line with anti-crude enterotoxin D serum and no precipitation with anti-enterotoxin A, B or C when it was tested by Ouchterlony's technique. It was capable of producing 100% emesis in monkeys at about 1.25 μg of protein per kg body weight. Antiserum prepared by injecting rabbits with the purified preparation gave a single precipitin line with concentrated crude toxin, and it also gave a single arc when tested in immunoelectrophoresis with concentrated crude toxin. A type-specific neutralization effect in monkeys was obtained. Thus, enterotoxin D has been identified with a purified preparation.     

Adenosine deaminase from pig thyroid gland purification and some properties

Adenosine deaminase was isolated from the pig thyroid gland and purified over 900-fold using DEAE Sephadex A-50 column chromatography, Sephadex G-100 gel filtration and DEAE Sephadex A-50 rechromatography. The enzyme was specific towards adenosine. The Michaelis constant based on the Lineweaver-Burk plot was 5 × 10^?5M. The optimum pH was about 7.0, and molecular weight 44 700.     

Purification and characterization of glycosyltransferases involved in anthocyanin biosynthesis in cell-suspension cultures ofDaucus carota L.

The major anthocyanins accumulated by an Afghan cultivar ofDaucus carota L. are cyanidin 3-(xylosylglucosylgalactosides) acylated with sinapic or ferulic acid. The formation of the branched triglycoside present as a common structural element requires an ordered sequence of glycosylation events. Two of these enzymic glycosylation reactions have been detected in protein preparations from carrot cell-suspension cultures. The first step is a galactosyl transfer catalyzed by UDP-galactose: cyanidin galactosyltransferase (CGT) resulting in cyanidin 3-galactoside. The putative second step is the formation of cyanidin 3-(xylosylgalactoside) catalyzed by UDP-xylose: cyanidin 3-galactoside xylosyltransferase (CGXT). Both enzyme activities were characterized from crude protein preparations. The CGT was purified 526-fold from the cytosolic fraction of UV-irradiated cell cultures by ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, affinity chromatography on Blue Sepharose CL-6B, gel permeation chromatography on Sephadex G-75 and elution from the gel matrix after non-dissociating PAGE. Its molecular mass was estimated by SDS-PAGE and by calibrated gel permeation chromatography on Sephadex G-75. In both cases a molecular mass of 52 kDa was determined, indicating that the native protein is a monomer of 52 kDa. The galactosyl transfer and the xylosyl transfer are presumed to be catalyzed by separate enzymes.Abbreviations CGT UDP-galactose: cyanidin galactosyltransferase - CGXT UDP-xylose: cyanidin 3-galactoside xylosyltrans-ferase - DEAE diethylaminoethyl This study was supported by a grant from the Deutsche Forschun-gsgemeinschaft and a fellowship (W.E.G.) from the Land Baden-Württemberg. The skilful technical assistance of Johannes Madlung is gratefully acknowledged.     

Evidence for Circulating Immune Complexes in Thyroid Disease

Anti-complementary activity was detected in the sera of 23 (59%) out of 39 patients with Hashimoto thyroiditis; in 10 (29%) out of 34 patients with primary hypothyroidism; in six (17%) out of 36 patients with thyrotoxicosis; and in only six (8%) out of 78 control subjects. Fractionation of Hashimoto sera by Sephadex G-200 chromatography showed that the anti-complementary activity was founding fractions of larger molecular size than monomeric IgG, being localized predominantly in the ascending limb of the second elution peak, suggesting that it was in the form of small immune complexes.     

Cadmium-2-acetylaminofluorene interaction in isolated rat hepatocytes

Cadmium (Cd) is a non-essential, highly toxic heavy metal and a ubiquitous environmental contaminant. Evidence exists that Cd can affect parameters which are of great importance in the response towards xenobiotics. However, there is a lack of information about the mechanisms that take place at the cellular and molecular levels upon dual exposure to Cd and other toxins. The purpose of the present work was therefore to examine the biochemical interactions between Cd and a well-known genotoxic hepatocarcinogen, 2-acetylaminofluorene (AAF) in isolated rat hepatocytes. The cells were incubated for 10 hr with a sub-cytotoxic concentration (0.22 M) of ¹⁰⁹Cd. This was followed by a 10 hr exposure to 1 M [³H]AAF. Cellular distribution of Cd and ³H was determined. Sephadex G-75 elution profiles of the cytosol showed that Cd was almost entirely associated with the intermediate molecular weight (IMW) fractions containing metallothionein (MT) (>80%), and with high molecular weight proteins. In parallel, the highest proportion of ³H was found in the low molecular weight components. Further analysis of IMW fractions by DEAE A-25 anion-exchange chromatography revealed that, in addition to Cd, there was some ³H which coeluted along with MT-I and MT-II isoforms, but preferentially with MT-I. Moreover, Cd pretreatment caused a 1.6-fold increase in MT level, as measured by the silver-saturation assay. Under these conditions, there was a 17% lower binding of ³H to the DNA. This reduced binding was neither accompanied by diminished AAF uptake nor by inhibition of cytochrome P-450 activity. Taken together, these results suggest that Cd exposure has a protective effect against the genotoxicity of AAF. MT, whose synthesis is induced, could play a role in the Cd-AAF interaction through scavenging of reactive metabolites.Abbreviations AAF 2-acetylaminofluorene - Cd cadmium - DMSO dimethyl sulfoxide - HBSS Hank's balanced salt solution - LDH lactate dehydrogenase - MT metallothionein - UDS unscheduled DNA synthesis     

Isolation and partial characterization of a cadmium-binding protein from the American oyster (Crassostrea virginica).

American oysters (Crassostrea virginica) were exposed to 0.1 ppm cadmium for 0--15 days in a flowing seawater system and then placed into clean flowing seawater for 24 h prior to sacrifice. Whole oysters were homogenized and a cadmium-binding protein isolated and purified by a process of centrifugation, heat-treatment, Sephadex G-75 chromatography, DEAE cellulose chromatography and disc gel electrophoresis. A highly anionic protein which is not present in control oysters was found to be present in cadmium-exposed animals after 3 days of treatment and to increase in concentration at succeeding time points. The protein does not extensively bind zinc or copper. Amino acid analysis of the purified protein disclosed an amino acid composition characterized by a high percentage of dicarboxylic amino acids and relatively little cysteine.     

镉诱导家兔肝脏含锌金属硫蛋白的纯化及鉴定

预先给家兔皮下注射CdCl_2四次,取肝脏经匀浆、离心、乙醇沉淀后,通过Sephadex G-50凝胶过滤层析,DEAE Sepharose Fast Flow离子交换层析和Sephadex G-25层析柱脱盐,得到MT的两个“亚型”—MT-1和MT-2,对其进行鉴定,其紫外扫描图谱示在280和250nm处无吸收峰,而最大吸收在220nm处。MT-1和MT-2均不含镉和铜,而每分子结合6个锌。用简化巯基试剂测定,MT-1含18.8个巯基,MT-2含19.1个巯基;HPLC分析MT主要以单体存在,也有少量二聚体。氨基酸组成与Cd-MT一致。整个纯化过程采用示波极谱法和火焰原子吸收光谱法测锌含量。结果表明前者可代替后者。     

The antigenic structure ofFrancisella tularensis

The ether antigen ofFrancisella tularensis was fractionated using DEAE-cellulose and CM-cellulose chromatography, and Sephadex G-200 gel filtration. The CM-cellulose chromatography did not appear to be a suitable method for separation of individual components of the complex ether antigen. Most of the polysaccharide material and substances with a high phosphorus content were eluted already in the first peak of the elution curve. This method could be used only to separate a component, yielding one immunoelectrophoretic precipitin line, localized towards the cathode. On the other hand, the DEAE-cellulose chromatography, and particularly the Sephadex G-200 gel filtration (especially when recycling was introduced), yielded a clear separation of relatively clean components of the ether antigen. The present work provides a comparison between the immunochemical (immunoelectrophoretic) properties of fractions obtained by these methods and components isolated by salting out with ammonium sulphate, ethanol or trichloracetic acid precipitation.     

Interactions of selenium and cadmium with metallothionein-like and other cytosolic proteins of rat kidney and liver

Following injection of rats with CdCl2 and [75Se]selenite using five different protocols, the metallothionein-like proteins (MTLPs) of kidney and liver cytosols were fractionated by Sephadex G-75 gel filtration and DEAE Sephacel ion-exchange chromatography. Cd and 75Se distribution in gel-filtration elution profiles was influenced mainly by the time that elapsed between administration of these elements and by the sequence of their administration. There was no Cd redistribution to high molecular weight proteins after long-term Cd injection when rats were killed 48 hr after 75Se injection. Cd was redistributed from MTLP to high molecular-weight proteins in the liver when Cd and 75Se were injected within 1-3 hr of each other. Incorporation of 75Se into MTLP of kidney and liver was independent of Cd injection. The strength of 75Se binding of MTLP was comparable to the covalent binding of 75Se to glutathione peroxidase. Cd and 75Se did not share binding sites on MTLP. In ligand-exchange studies, 1000 ppm Cd did not displace 75Se from MTLP, but 2% 2-mercaptoethanol displaced 10% of the presumably nonspecifically bound 75Se from kidney and liver MTLP. This study provides new information regarding the apparent covalent binding of Se to low molecular-weight, Cd-containing proteins in kidney and liver.     

Purification of low molecular weight copper proteins from copper loaded liver.

Purification of low molecular weight copper binding proteins from the livers of copper loaded male rats was achieved by sequential ultracentrifugation (186,000g, 2h), ultrafiltration (Amicon PM 30), gel filtration (Sephadex G-75) and anion exchange chromatography (DEAE - Biogel A) of soluble tissue extracts. The three major copper-associated polypeptides obtained which had molecular weights of about 7000, 9,000, and 12,000 daltons contained approximately 2.5g atoms of copper per mole. Amino acid analyses indicated a similarity between these proteins and the copper protein ‘L-6D’ isolated earlier from livers of Wilson's disease patients and distinguished them from metallothioneins which have been isolated from animals administered other trace metal ions.     

Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liver.

Recently we have demonstrated that the exposure of monomeric Cd7-metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers containing approximately two additional Cd(II) ions/monomer subunit [Palumaa, P., Mackey, E. and Vasák, M. (1992) Biochemistry 31, 2181-2186]. In this study, we demonstrate that inorganic phosphate participates in the Cd-induced dimerization of MT. In the absence of phosphate, Cd-induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an additional peak of MT tetramers were detected in gel-filtration experiments. Arsenate exhibits a similar effect to that of phosphate, whereas a number of other anions, i.e. F-, NO3-, SO4(2-), ClO4-, BO3-, SCN-, HCOO- and CH3COO- had no effect on Cd-induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equilibrium-dialysis experiments using potassium [32P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, Kd, of 23 +/- 3 microM (20 mM Tris/HCl and 0.1 M KCl, pH 8.0 at 25 degrees C), whereas binding of phosphate was not observed with the monomeric Cd7-MT. The noncovalent nature of phosphate binding to the Cd-induced MT dimers has been demonstrated. The presented data provide the first evidence for the binding of a nonmetallic cellular component to MT.     

Characterization of macrophage activation factor, a lymphokine that causes macrophages to become cytotoxic for tumor cells.

Macrophage activation factor (MAF) was isolated from PPD-stimulated, BCG-immune mouse spleen cell culture fluids. In nine gel filtration runs on Sephadex G-100 or G-200, MAF was eluted in a single peak corresponding to a MW of 55,000 ± 1600. Recovery of activity was about 65%. Since the relative concentration curve of eluted MAF was wider than that of a single protein, MAF activity may be due to more than one protein with similar molecular weights. This possibility is strengthened by a broad elution range on DEAE cellulose chromatography, from a specific conductance of 3.5 to 8.5 mmho/cm, at pH 7.9. MAF was labile at both pH 4 and 10, and was destroyed by proteolytic enzymes. Eighty percent was destroyed by heating at 56 °C for 30 min. In affinity chromatography experiments, MAF did not bind to Con-A Sepharose; but it was bound to insolubilized Cibacron-blue and was eluted by an increase in ionic strength.     

Heavy Metal Accumulation and Distribution in Organs of the Mussel Crenomytilus grayanus from Upwelling Areas of the Sea of Okhotsk and the Sea of Japan

The subcellular and cytosolic distribution of Zn, Fe, Ni, Cu, Mn, Cd, and Pb in the digestive gland and kidney of the mussel Crenomytilus grayanus from upwelling areas of the Sea of Okhotsk and the Sea of Japan was studied. Cd, Zn, Pb, and Ni have accumulated in the kidneys of mussels from these areas. When the concentrations of both essential and toxic metals in the mussel organs had increased significantly, their redistribution into the cytosol took place. Gel chromatography of cytosolic proteins in the kidneys of mussels sampled in the area with a strong stationary upwelling revealed metallothioneins. This is uncommon for bivalve mollusks from unpolluted areas. High molecular weight proteins act as preliminary ligands for Cd.