Quantification of metallothioneins in the common asteroid Asterias rubens (Echinodermata) exposed experimentally or naturally to cadmium

Abstract

Uptake and intracellular fate of Cd was assessed in the pyloric caeca of the common asteroid Asterias rubens exposed experimentally or naturally to Cd. Cd partitioning among intracellular metal-binding pools was studied by graphite furnace atomic absorption spectrometry of heat-treated cytosolic fractions which had been separated by gel filtration chromatography. Low molecular mass proteins bound > 80% of cytosolic Cd and appeared to become saturated with the metal after experimental exposure to 20 μg Cd 1 −1 for 10–20 days. These proteins presented several features of metallothioneins (MTs), namely molecular mass (13–22 kDa), heat-stability (90 °C, 10 min), high Cd content, and high thiolic content as determined by differential pulse polarography. Analysis of Chromatographie fractions indicated that the majority of thiolic groups (66–73%) present in heat-treated cytosol was associated with the MT pool. Subsequent determinations of MT concentrations were made using whole cytosol extracts after correcting for interference from thiolic groups present in residual, heat-stable, high molecular mass proteins. Asteroids collected from unpolluted sites in SW England, SW Netherlands and SW Norway contained basal levels of 2.5 – 4.5 mg MT g −1 dw. Asteroids experimentally exposed to Cd (20 μg Cd 1 −1) responded by a 2-fold increase in the production of MT after 30 days. Turnover of MTs was rapid in A. rubens and probably accounted for the low net increase in protein and characteristic rapid loss of Cd from the pyloric caeca. Alkaline phosphatase (AP) activity was measured as a marker of the functional state of the pyloric caeca. It was not significantly affected during Cd exposure in the laboratory. Asteroids collected in the heavy metal polluted Sørfjord (SW Norway) contained significantly higher MT concentrations (5 – 5.6 mg MT g −1 dw) and significantly lower AP activity in the most heavily contaminated region, though these two parameters were not correlated.