Isolation of novel metal-binding proteins distinct from metallothionein from spotted seatrout (Cynoscion nebulosus) and Atlantic croaker (Micropogonias undulatus) ovaries

Abstract

Metal-binding proteins were isolated from ovaries of the spotted seatroutCynoscion nebulosus and the Atlantic croakerMicropogonias undulatus collected in 1988 near Port Aransas, Texas, USA. Gel-filtration analysis of spotted seatrout trout ovarian cytosolic fraction on Sephadex G-75 revealed the presence of three zincbinding protein fractions. A major zinc/calcium-binding protein fraction had a low molecular weight (Mr)(6 000 to 10 000), similar to mammalian hepatic metallothionein (MT). All the metals were displaced from this fraction following saturation with exogenous cadmium. After exposure of Atlantic croaker to 2 mg cadmium l−1 seawater for 2 mo, the majority of the cadmium in the ovarian cytosolic fraction was associated with a similar low molecular weight protein fraction. These proteins were further purified by heat treatment and sequential acetone precipitation. Three isoforms were isolated by reversephase high-performance liquid chromatography. All the isoforms were found to be distinct from mammalian MT, based on amino acid composition. The major isoform contained low amounts of cysteine (approximately 5 residues per molecule) and aromatic amino acids, compared to high amounts of cysteine (typically 17 to 20 residues/molecule) and a lack of aromatic amino acids for mammalian MT. All the ovarian protein isoforms contained more glutamate than mammalian MT. The spotted seatrout and Atlantic croaker ovarian isoforms showed a high degree of homology with metal-binding proteins isolated from mammalian gonadal tissues. The results suggest a physiological role for these metal-binding proteins in developing vertebrate ovaries as well as an involvement in the sequestration of cadmium following environmental exposure.