Isolation and characterization of naturally and artificially induced hepatic metallothioneins from two species of flatfish: Limanda limanda and Microstomus kitt

Abstract

The separation of hepatic metallothioneins of Limanda limanda and Microstomus kitt by gel-filtration chromatography, anion-exchange high-performance liquid chromatography (HPLC) and reversed-phase HPLC is described. Two isoforms of metal-binding proteins were isolated by DEAE HPLC chromatography from fish caught in the field and fish injected intraperitoneally with Cd. The amino acid composition of these proteins further purified by reversed-phase HPLC chromatography revealed the existence of metallothioneins. The route of contamination (natural or artificial) of the fish has no effect on the nature of these proteins and one of the two protein isoforms from each species has similar ionic properties.

Acute effects of cadmium on liver phase I and phase II enzymes and metallothionein accumulation on sea bream Sparus aurata

“…intraperineal injection, may not necessarily simulate the same biological response as exposure to heavy metals in the field but was considered as a beginning to study the effects of acute exposure to single and mixed pollution on fish. Duquesne and Richard (1994) observed that the route of metal uptake does not affect the nature of induced MT in Limanda Iimanda and Mictwstomus kitt caught in the wild or injected with Cd.…”

Quantification of Metallothionein Isoforms in Fish Liver and Its Implications for Biomonitoring

“…Figure 1 depicts representative GPC chromatograms of an untreated and an acetone-treated cytosol obtained from Cdexposed dabs without further Cd saturation during the sample preparation. These two proteins in dab liver were investigated by Duquesne and Richard [21] using similar isolation procedures. Furthermore, it is obvious that cytosol treatment with acetone reduces the load of proteins with molecular weight higher than 15 kDa (eluted between 150 and 220 min) for the most part.…”

“…The chromatographic profile obtained by moni- toring UV absorbance and Cd content indicated in both cases the occurrence of a Cd binding protein with an apparent molecular weight of 13 kDa eluting at about 250 min. Duquesne and Richard [21] separated three cadmium-containing proteins from cadmium-exposed dab livers by IEC. Application of concentrated and desalted Cd-containing fractions (about 13 kDa molecular weight) from GPC of an acetone-precipitated cytosol to IEC resulted in the separation of two cadmium-containing proteins ( Fig.…”

“…Furthermore, it is obvious that cytosol treatment with acetone reduces the load of proteins with molecular weight higher than 15 kDa (eluted between 150 and 220 min) for the most part. Addition of 2-mercaptoethanol at each isolation step avoided this problem [21]. 2; designated as MT-I and MT-II).…”

“…Application of GPC fractions from nonacetone-treated cytosol to IEC resulted in comparable chromatograms (not shown). These two proteins in dab liver were investigated by Duquesne and Richard [21] using similar isolation procedures. They confirmed that these two proteins are the major MT isoforms MT-I and MT-II.…”