Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10kDa and 20kDa. The 1 0-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and Smethylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.Despite the discovery of the small cysteine-rich metalbinding-protein metallothionein (MT) more than 30 years ago in equine kidney cortex, the function of this protein is still not fully understood. MT is known to bind metals such as Zn and Cu (which are essential for cell growth and development) and Cd and Hg (which are toxic), which suggests that MT might have more than one function.The search for putative functions for these proteins is exacerbated by the existence of numerous inducers and isoforms. The range of factors which regulate MT synthesis and degradation together with the ubiquity and sequence conservation of MT perhaps suggests that these proteins have a central role in cell metabolism. The proposed functions for MT include the detoxification of heavy metals and the metabolism of essential metals. Zeng et al. (1991) have suggested that thionein may be the active form of the protein and it may be involved in the control of gene expression by virtue of its ability to remove Zn from Zn-finger proteins. Moreover, a tissue-specific isoform of MT in astrocytes (also called growth-inhibitory factor) which inhibits the survival Correspondence to

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Metallothioneins (MT) are metalloproteins expressed tissue specifically during the development of the sea urchin, Strongylocentrotus pururutus. To explore their structural and functional features and to compare them with those of the evolutionary distant mammalian MTs, one isoform (MTA) was obtained as the cadmium-containing form, from synthetic cDNA heterologously expressed in Escherichia coli. The purified protein was identified as the desired product by a combination of peptide-map analysis, amino acid sequence analysis and ion-spray mass spectroscopy. The existence of seven " T d NMR resonances revealed that the recombinant protein binds seven Cd ions/molecule. The position of the NMR resonances (605 -695 ppm) and the electronic absorption features suggest that the sea urchin MTA, like the mammalian MTs, possesses tetrahedrally coordinated cadmiumthiolate clusters. With its large Stokes' radius, sea urchin MTA resembles the mammalian forms, suggesting a comparable elongated molecular shape. Measurements by spectrophotometric pH titration of cadmium binding by the recombinant protein suggest that it possesses two metal-thiolate clusters of distinctly different stability. At pH 7 the average apparent association constant for Cd2' in the clusters is about 20-times weaker in sea urchin MTA than in rabbit MT-2.Metallothioneins (MT) are widely found low-molecularmass, cysteine-rich proteins distinguished by an exceptionally high content of d' O metal ions (Zn", Cd2+ and Cu'+) (see Roesijadi, 1993, andKagi, 1993, for reviews). MTs have traditionally been thought to be involved in cellular detoxification of metals but more central functions such as an involvement in the regulation of Zn-dependent processes in gene expression (Zeng et al., 1991a and and in development (Nemer et al. 1984;De et al., 1991) have also been suggested.The sea urchin Strongylocentotus pupurutus like many other species, contains a family of MT genes. Two of these genes, designated MTA and MTB,, have been well characterised. These genes show a high degree of identity, but probes specific for their mRNAs have been constructed and used to show that the expression patterns of the genes are quite distinct. Both mRNAs begin to accumulate at the early blastula stage (12 h after fertilization) but the MTB, mRNA becomes localized in the embryonic gut and in the oral ectoderm while the MTA mRNA is spatially restricted to the aboral ectoderm (Nemer et al., 1991). As found for mammalian MTs, both forms are readily inducible by zinc although with unequal thresholds (Wilkinson and Nemer, 1987). This responsiveness to zinc supply is accounted for by the occurrence of at least six metal-regulatory elements (MRE) in the 5' upstream region of both the sea urchin genes (Bai et al., 1993). The two MREs of the MTA gene (designated a and b) nearest to the transcription start site are identical in both sequence and position (relative to the TATA box) to those which give maximal activity in mammalian systems (Harlow et al., 1989). In addition to this conservation of...

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