| Summary: | 碩士 === 國立高雄師範大學 === 生物科技系 === 96 === Earthworms can be used to evaluate soil pollution, due to their they response to heavy metals by inducing the expression of metal-induced metallothionein-2 (MT-2). I report the sequences of MT-2 cDNA from two species of Megascoleidae earthworms, Metaphire posthuma and Polypheretima elongata, by mRNA differential display after exposure of the organisms to cadmium. Metal induced complementary DNA (cDNA) was verified as the MT-2 gene by the characteristics of its predicted translation product, with a high cysteine content, conserved CXC motifs, and a molecular weight of around 8 kDa. Amino acid sequence alignment revealed a conserved TKCCG in the cloned MT-2s between both megascolecid earthworms instead of the corresponding conserved TQCCG found in lumbricid earthworms. The two megascolecid MT-2 proteins were expressed from their cDNAs and purified. The binding of heavy metals was assayed using 5-5' dithiobis (2-nitrobenzoic) acid. The binding of Cu2+ exhibited monophasic kinetics and those of Zn2+ and Cd2+ showed biphasic kinetics. The proteins bound more tightly to Cd2+ than to Zn2+ and more tightly still to Cu2+. Zn-MT showed the most effective to scavenge free radicals, followed by Cd-MT. Cu-MT exhibited different kinetics in reacting with free radicals in comparison with apo-, Zn-, or Cd-MT, suggesting that the sulfhydryl groups of Cu-MT become inaccessible after one round of reaction. Despite the similarity of their primary sequences, the two Megaloscoleidae MT-2 proteins showed different in biochemical properties characteristics of heavy metal binding and free radical scavenging.
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