Purification and Characterization Studies of Cadmium-Binding Proteins from the American Oyster, Crassostrea virginica

Abstract

The previously reported low molecular weight cadmium-binding protein (CdBP) from the American oyster, Crassostrea virginica, has been further purified and characterized by improved technical methods. The internal organ distribution ofthe protein within the oyster and effects of life cycle/season on CdBP production also have been evaluated. CdBP isolated by extended ion-exchange gradients or double ion-exchange chromatography followed by HPLC analysis possesses an electrophoretic R,of about 0.7 and contains relatively little Zn, as previously reported. Cysteine, lysine, and glycine are the dominant amino acids. When ionexchange columns are developed with NaCl gradients, the aromatic residues tryptophan, tyrosine, and phenylalanine are found to be present, but these may be largely removed depending upon whether the protein is denatured and carboxymethylated prior to analysis. The ultraviolent absorption spectrum of CdBP also was variable, with 250/280 nm ratios ranging from 17:1 immediately after ion-exchange chromatography to 2:1 following concentration procedures. Internal organ distribution studies showed that the visceral mass contained most of the Cd present with lesser amounts in the gills and mantle. In contrast with mammals, CdBP accounts for only about 30%o of the total cell Cd burden in these tissues. Cu displacement of Cd from the protein is a particular problem during the summer spawning season and appears to stem from altered Cu metabolism during this period. Relative oyster dormancy during the winter also reduces CdBP production in response to Cd, and the protein is obtained most readily during the fall and spring. In summary, CdBP shares both similarities and differences with MT, and the magnitude of these parameters depends greatly on both the biology of the oyster and technical procedures used to isolate/characterize the protein.

Diversity of metallothioneins in the American oyster, Crassostrea virginica, revealed by transcriptomic and proteomic approaches

“…as in the αββ MT seen in the Pacific oyster, C. gigas [27]. The data reported here reveal a structural and functional diversity within the MT family of the American oyster ( C. virginica ) that, while proposed by prior studies at the protein level [19–21], has not previously been documented. In particular, it is clear that in this species of oyster, the family of canonical αβ‐domain‐containing MTs (the CvMT‐I subfamily) has undergone substantial expansion to include MTs that solely express α‐domains (the CvMT‐II subfamily).…”

“…While the induction of MTs by various metals, particularly cadmium, has been established in a variety of metazoan taxa [14–17], to date only one MT, a cadmium‐inducible isoform, has been identified from Crassostrea virginica [18], although biochemical studies indicated the presence of two cadmium‐binding proteins of 10 and 24 kDa [19,20]. Several metal‐rich proteins, representing putative MTs, have been identified in control and metals‐treated C. virginica larvae [21], and the presence of multiple MT isoforms has been demonstrated in other molluscan species [22–26], including bivalves (the blue mussel, Mytilus edulis [23] and the Pacific oyster, Crassostrea gigas [27]) and gastropods (the terrestrial snail, Helix pomatia [25,26]).…”

Intracellular compartmentation of metals in aquatic organisms: roles in mechanisms of cell injury.

“…For example, both mammalian (10,11) and nonmammalian species (28) show increased binding of cadmium to high molecular weight cytosolic proteins once the metal-binding capacity of the inducible metallothionein or metallothioneinlike protein pools are exceeded. The implication of these data is that those toxic metal ions which are not under the homeostatic control of binding proteins actually produce toxicity when available as free cations which can react with other sensitive high or low molecular weight target molecules (5,11). At this point, it should be noted that nonmammalian organisms generally bind less (-30% versus -90% for mammals) of their total cellular toxic metal burden to specialized soluble proteins than mammals (5,6).…”

“…The implication of these data is that those toxic metal ions which are not under the homeostatic control of binding proteins actually produce toxicity when available as free cations which can react with other sensitive high or low molecular weight target molecules (5,11). At this point, it should be noted that nonmammalian organisms generally bind less (-30% versus -90% for mammals) of their total cellular toxic metal burden to specialized soluble proteins than mammals (5,6). This may be a reflection of the generally lower dissociation constants (Kd), e.g., -10 6 M for cadmium reported for nonmammalian binding proteins (6) relative to mammalian metallothionein (_ 10-16 M), a lower production rate of these proteins in nonmammals and/or greater competition between intracellular compartments in nonmammals.…”