Abstract
A family of synthetic genes was constructed encoding a rainbow trout metallothionein (MT), a human MT, and two chimeric molecules which contained respectively (i) the N-terminal (or head) domain of human MT followed by the C-terminal (or tail) domain of a fish MT (termed mermaid MT) and (ii) the head domain of fish MT fused with the tail domain of human MT (denoted fishman MT). These were expressed in Escherichia coli and the four recombinant proteins were purified to homogeneity therefrom. All four were found to bind 7 g atoms of Cd(II) per mol; at pH 7.0, but not at pH 8.6, four Cd(II) ions were sequestered preferentially in the tail (or alpha) domain. Reciprocally, copper was found to bind preferentially in the head (or beta) domain. The human and fishman MTs displayed a stoichiometry of 12 g atoms of Cu(I) per mol, while rainbow trout and mermaid MTs bound only 10. The significance of these findings is discussed in relation to the different positional organization of cysteine residues close to the N and C termini of mammalian and piscine metallothioneins.
Highlights
Determination of the nucleotide sequences of the gene(s) encoding MT in the rainbow trout (7,8)and the single genes for MT in other piscine species (8, 9) has indicated a significance of these findings is discussed in relation to substantial degree of homology between the fish the different positional organization of cysteine resi- proteins and with mammalian MTs
The metals are liganded by cysteinyl thiolates to form an Construction of synthetic genes encoding a rainbow trout MT, a human MT, and twochimericmoleculeswhich contained, respecinorganic metal-sulfur core in both domains with the poly- tively, (i) the N-terminal domain of human M T followed by the C-terminal domaionf fish M T
The homogeneity of each of the four preparations was established from their migration as single bands on SDS-PAGE and by Edman degradation
Summary
’11 To whom correspondence should be addressed. grown in E. coli JMlOl as described previously (11). The abbreviationsused are: MT, metallothionein; HEPES, 4-(2- Expression studieswere performed adsescribed previously for hydroxyethy1)-1-piperazineethanesulfonicacidH;PLCh,igh-perrainbow trout MT(11)in Escherichia coli 1B392 Lon A 1 grown in L formance liquid chromatography. Stock solutions of Cu(1) were prepared anaerobically as (CuC12)-by dissolving cuprous chloride in 0.1 M HC1 containing 4% NaCl by weight. Each metal/protein mixture was neutralized in an anaerobic environment with an equal volume of 0.5 M HEPES-acetic acid buffers at either pH 7.0 or 8.6. Cadmium sequestration was analyzed by removal of each protein/metal mixture from the anaerobic environment and measurement of its absorbance spectrum between 220 and 320 nm. Proteolytic digestion of MT samples containing different amounts of metals (copper or cadmium) was carried out by overnight incubation at 4 ‘C with proteinase K (molar ratio of MT:proteinase of approximately 1001)
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