Partial purification, characterization and translation in vitro of rat liver metallothionein messenger ribonucleic acid

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Abstract
Poly(A)+ (polyadenylated) mRNA coding for metallothioneins was purified 13-fold from rat liver polyribosomes and was identified by its ability to direct the biosynthesis of these proteins in a wheat-germ cell-free system. The carboxymethylated products of the protein-synthesizing system in vitro were analyzed with sodium dodecyl sulfate/20% polyacrylamide-gel electrophoresis. The labeled compounds [3H]serine and [35S]cysteine were incorporated at high specific radioactivity into proteins that co-migrated with authentic metallothioneins. No [3H]leucine incorporation was found, in agreement with the amino acid composition of the metallothioneins. Metallothionein mRNA had a sedimentation coefficient of 9 S and carried a maximum of 4 ribosomes. At 5h after a s.c. injection of ZnCl2 or CdCl2 (10 .mu.mol/kg body wt), the amount of this mRNA increased approximately 2- and 4-fold, respectively, on the basis of translation in vitro. The increase in metallothionein mRNA (defined by translation in the wheat-germ system) was transient and, after CdCl2 treatment, fell back to control values by 17 h. Metallothioneins constituted a maximum of 0.8% of the total protein products synthesized in the wheat-germ system by total mRNA isolated from rat liver after CdCl2 treatment.