Mitsuru Yamamura

Cadmium-binding proteins induced in the earthworm.

Earthworms,Eisenia foetida, grown in composts of different cadmium concentrations accumulated cadmium in a dose-dependent manner and the cadmium was bound to three different molecular weight cadmium-binding proteins induced in the earthworm. The three proteins were stable to heat treatment and accompanied by a concomitant increase of absorbance at 254 nm and not at 280 nm. Each of the three proteins is a mixture of isoproteins; the molecular weight of one of the three proteins was estimated to be about 7,000 daltons, on the assumption that the cadmium-binding protein is similar in Chromatographic properties to metallothionein.

Isolation and characterization of metallothionein dimers

The dimeric forms of metallothionein were isolated from livers of rabbits which accumulated cadmium in large quantities. The dimers were separated into three isoproteins, both on a DEAE Sephadex A-25 column and on a gel permeation column. Reduction of each dimer with mercaptoethanol yielded monomeric metallothionein-I, an equivalent mixture of metallothionein-I and -II, and metallothionein-II. The three isoproteins were identified as a dimer of metallothionein-I, a dimer of metallothionein-I and -II, and a dimer of metallothionein-II, respectively.

Tolerance to cadmium and cadmium-binding proteins induced in the midge larva, Chironomus yoshimatsui (diptera, chironomidae)

Abstract 1. The midge larva ( Chironomus yoshimatsui ) was exposed to cadmium (10 μg Cd/ml) for 2 days. 2. A large portion of cadmium taken up rapidly into the insects was bound to the high molecular weight proteins and was rapidly discharged in control water. 3. Low molecular weight cadmium-binding protein was slowly induced in the larva by cadmium exposure. This protein was a mixture of four isoproteins and showed the characteristic properties of metallothionein. 4. The high tolerance of the midge larva to acute cadmium exposure was not explainable by induction of the cadmium-binding proteins.

Gel permeation, ion-exchange and reversed-phase columns for separation of metallothioneins by high-performance liquid chromatograph—atomic absorption spectrophotometry

Metallothioneins were separated on three kinds of HPLC column that differ in separation principle: gel permeation, ion exchange and reversed phase. Elution of the columns at neutral to weakly basic buffer conditions prevented dissociation of the metals, and the metals bound to metallothioneins were quantitatively eluted out. The metals in the eluate were continuously determined on an atomic absorption spectrophotometer by introducing the eluate directly to the nebulizer tube. Prerequisites for the on-line separation-detection system are discussed.

Separation of metallothioneins in rat liver, kidney, and spleen using SW and Sephadex columns.

Abstract Metallothioneins and other metalloproteins in cadmium-exposed rat liver, kidney, and spleen were separated both on SW 3000 and Sephadex G-75 columns. The former column was used as a gel permeation column equipped with a high-speed liquid chromatograph and the concentration of metals in eluate was simultaneously determined by directly connecting to a flame atomic absorption spectrophotometer. Metalloproteins were generally eluted at the same elution orders on the two columns except for the separation of metallothioneins into isometallothioneins and several unidentified zinc proteins. Slower elution and poorer resolution of metallothioneins were observed on a SW column for neutral and acidic buffer solutions, and it was explained by the cation exchange chromatographic property caused by the dissociation of hydroxyl groups of polyhydroxylated gel materials at alkaline pH. Although metallothioneins were also separated into the isometallothioneins on a Sephadex column, the proteins were apparently eluated as a single peak due to shortage in the numbers of theoretical plates. The pH value of eluting buffer solution was concluded to be a main factor which affects retention times and resolution of metallothioneins.

Metallothionein induced in the earthworm

One of the 3 different molecular weight cadmium-binding proteins induced in the earthworm was characterized as a metallothionein; this characterization was based on a high cysteine and cadmium content, low molecular weight, heatstability, and mercaptide bonding.

Induction of zinc-thionein in rat liver and kidneys by zinc loading as studied at isometallothionein levels

Zinc acetate was injected i.p. into female rats to try to clarify the detailed induction and degradation pathways of zinc-thionein (Zn-th) at isometallothionein levels in liver and kidneys. Injected zinc in serum decreased with the induction of Zn-th in the liver and kidneys, and the amounts of Zn-th in both tissues attained maxima 24 h after injection. The relative ratios of isometallothioneins changed with time in both tissues. The changes are compared with those of cadmium (Cd) loading.

Distribution of cadmium in liver and kidneys by loadings of various Cd-complexes and relative metal ratios in the induced metallothioneins.

Abstract The effects of chelating agents and pre-treatment on the distribution of cadmium in the liver and kidneys and on the relative amounts of cadmium, zinc and copper in the induced metallothioneins were compared by intraperitoneally injecting cadmium with various chelating agents. The co-injections of nitrilotriacetic acid or d -(−)-penicillamine resulted in a distribution of cadmium similar to the injection of the free cadmium ion. The co-injections of 2,3-dimercapto-1-propanol or ethylenediamine tetra-acetate resulted in a distribution intermediate between that of the free cadmium ion and the thionein-bound cadmium. Cadmium was recovered mainly from the kidneys for the injection of cadmium-thionein. The changes in the distributions between liver and kidneys were explained tentatively by the stability constants of the complexes from the distribution patterns of cadmium among protein fractions in serum after adding cadmium in vitro with those chelating agents. The copper content in the kidney metallothionein changed depending on the injected form of cadmium and pre-treatment method.

Gel and anion exchange chromatographic properties of copper-containing metallothioneins

Chromatographic properties of copper-binding proteins (copper-containing metallothioneins) induced in rat liver and kidney by the injection of either copper in the liver and kidney or cadmium in the kidney were compared with those of metallothioneins, with low or no copper content, induced by the injection of cadmium or zinc in the liver both on a Sephadex G-75 column and on a DEAE Sephadex A-25 column. The copper-containing metallothioneins were eluted at a slightly slower rate on a Sephadex G-75 column and at a higher buffer concentration on an anion exchange column than the metallothioneins with low or no copper content regardless of the inducing metals, copper or cadmium. The different Chromatographie properties of the metallothioneins with high and low copper contents may be explained by the more compact conformation of the proteins with high copper contents than those with low or no copper contents and by the changes of electrostatic charge with coordination by copper.

Induction and characterization of metallothionein in the liver and kidney of Japanese quail

Abstract 1. 1. The low mol. wt cadmium-binding protein induced in the liver and kidney of Japanese quail by cadmium injection was a mixture of two isoproteins. 2. 2. The injection of cadmium induced the two isoproteins at a relative ratio of 6:1 regardless of single or multiple injections. 3. 3. The isoproteins had an apparent mol. wt of about 12,000 daltons and were abundant in half-cystine (36.8 and 38.3 mol%) without aromatic amino acids, indicating that the two isoproteins are metallothionein (MT). 4. 4. The major isoform, MT-II, contained one residue of histidine and the amino acid composition was similar to that of chicken metallothionein, while that of the minor isoform, MT-I, was slightly different from that of chicken.