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KMID : 0382619960160020197
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Hanyang Journal of Medicine
1996 Volume.16 No. 2 p.197 ~ p.207
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Purification and amino acid composition analysis of cadmium binding protein from rat liver
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Abstract
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In order to investigate whether the newly synthesized cadmium(Cd)-bound metallothionein(MT) accumulates in livers of rats which have received subcutaneous injections of Cd, isolation and purification of these protein were performed using Sephacryl S-200 and DEAE-Sephadex column chromatographies, and SDS-PAGE. And PICO.TAG HPLC analysis of amino acids was carried out to find out amino acid composition of MT-Ⅱ. To confirm that the newly synthesized components are protein in nature, the Cd-bound fractions were examined for incorporation of ［^(35)S］cysteine. The effect of actinomycin-D on the synthesis of MT was also studied.
1. Cd induced newly synthesized peak which was identified by gel filtration on a Sephacryl S-200 column. This peak was detectable only by measurement of Cd and zinc by atomic absorption analysis, and was labelled with［^(35)S］cysteine. These results indicated that the peak components were protein which bound with Cd and zinc, e.g. MT.
2. MT was resolved into two isoforms i.e. MT-Ⅰ and MT-Ⅱ that had an apparent molecule mass of 10,000 by ion-exchange chromatography on a DEAE-Sephadex A-25 column
3. Formation of MT was completely abolished by actinomycin-D treatment
4. SDS-polyacrylamide gel electrophoresis of MT-Ⅱ showed the presence of a single band which appeared as a single peak on scanning at 590nm.
5. The most typical characteristic of MT-Ⅱ was a extremely high cysteine content(32.8%) followed by lysine, serine and alanine. Also MT-Ⅱ showed completely lacking in aromatic amino acids and histidine.
These results suggested that newly synthesized MT-Ⅱ containing high cysteine content was the major isometallothionein and might have protective role against the toxic effects of Cd through the formation of metal-thiolate complexes and that the induction of formation of MT was due to the de novo synthesis of the protein after the induction of mRNA synthesis by Cd.
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