Isolation and characterisation of cadmium binding components of the scallop, Pecten maximus
1. The digestive gland of the scallop Pecten maximus naturally contains a very high concentration of cadmium (100 ppm wet weight), which does not show individual or seasonal variations. 2. About 60% of the tissue cadmium is soluble (i.e. is found in the supernatant when homogenate is centrifuged for 1 hour at 100,000g) and is bound to three main types of macromolecule. Most (about 60%) of this metal is associated with a component of molecular weight 55,000, the rest being bound to a low molecular weight species and, probably non-specifically, to components of very high molecular weight (greater than 100,000 molecular weight). The latter components were not further characterised. 3 The major binding component complexes the Cd2+ via sulphydryl groups, and so has a high cysteine content, but the binding is weaker than that of cadmium to metallothionein. The component has a high content of glutamate and aspartate (or their amides) and contains aromatic amino acids. It may also have a small carbohydrate content.4. The major cadmium binding component was susceptible to degradation by endogenous proteolytic enzymes. A major digestive enzyme was identified as a chymotrypsin which could be inactivated with phenylmethanesulphonylfluoride. Addition of PMSF to the homogenate reduced the total proteolytic activity of the digestive gland cytosol by up to 75%. Attempts to further inhibit, or remove, the remaining protease activity were largely unsuccessful. Probably as a result of the action of the proteolytic enzymes on the major cadmium component efforts to isolate the latter were characterised by irreproducibility, and satisfactory purification was not achieved. 5. The low molecular weight cadmium binding component binds 10-15% of the total soluble cadmium and exhibits many of the characteristics of a metallothionein. It has an apparent molecular weight of 10,000 on gel exclusion chromatography, high cadmium and cysteine contents and a high A250/A280 ratio. It is also heat stable and contains copper and zinc as well as cadmium. It can be detected by the metallothionein assay of Eaton & Toal (1982). 6. Preparations of both the major Cd binding component and metallo-thionein-like component contained relatively large amounts of carbohydrate, but the latter was probably not associated with these proteins. Its origin is unknown.