The effect of dithiothreitol on the subcellular distribution of zinc in the cytosol of mussel kidney ( Mytilus Edulis): Isolation of metallothionein and a unique low molecular weight zinc-binding ligand

Abstract

1. A reducing agent such as dithiothreitol (DTT) was found to be necessary for the isolation of metallothionein-like zinc-binding proteins (MLP) from the cytosol of the kidney of the mussel, Mylilus edulis using Sephadex G-75 gel filtration chromatography. 2. When DTT-free buffer was used, little or no zinc was found to elute in the region usually associated with metallothionein-like proteins (MLP) in mussels. However, when a small amount of DTT was added to the buffer (0.7 mM), a substantial amount of zinc was found to be eluted in the MLP zone. When the DTT concentration was increased (4 mM), serious artefacts were seen. 3. Both in the presence and absence of DTT, most cytosolic kidney zinc from uncontaminated mussels was bound to a unique non-metallothionein low mol. wt ligand with a mol. wt around 1000. This very low mol. wt zinc-binding ligand was responsible for nearly all of the increase in cytosolic zinc when mussels were exposed to 400 ppb zinc.