88MHz 113Cd-n.m.r. studies of native rat liver metallothioneins.
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ABSTRACT:
SUBMITTER: Nicholson JK
PROVIDER: S-EPMC1154349 | biostudies-other | 1983 Apr
REPOSITORIES: biostudies-other
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Project description:Two electrophoretically distinguishable metallothioneins were isolated from the livers of Cd2+-treated rats and had thiol group/metal ratios of 3:1, a total metal content, in each of these proteins, of 3.6 atoms of Cd2+ + 2.4 atoms of Zn2+/molecule and 4.2 atoms of Cd2+ + 2.8 atoms of Zn2+/molecule and respective apoprotein mol.wts. of 5844 and 6251. Studies with 1 h pulse labels of [3H]cysteine, given after a single injection of ZnCl2 or CdCl2, showed that these metals stimulated radioactive isotope incorporation into the metallothioneins over the control value by 10- and 15-fold respectively. This stimulation was maximal at 4 h after a single CdCl2 injection and decreased to control values by 16 h, suggesting that either a translational event is responding to free intracellular Cd2+ or a short-lived mRNA is being produced or stabilized in response to the metal treatment. In rats chronically exposed to CdCl2, the metallothioneins increased to 0.2% of the liver wet weight from a control value of 2--4 mumol/kg of liver, with a maximum rate of accumulation of 2--3 mumol/h per kg of liver. The turnover of these proteins in control animals was 0.3--0.6 mumoles/h per kg of liver, measured by the rate of disappearance of 203Hg2+, which binds irreversibly to the metallothioneins. Pretreatment with CdCl2 completely stopped the rapid 203Hg turnover observed in untreated animals. Unlike CdCl2, treatment with ZnCl2 increased the concentration of metallothioneins to a new steady-state pool, 11 mumole/kg of liver, after 10 h. The increase in the zinc-thionein pool by exposure to ZnCl2 in vivo was determined to be primarily due to a stimulation of metallothionein biosynthesis.
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Project description:Reaction of rat liver cadmium-metallothionein-II(Cd-MT-II) with p-hydroxymercuribenzoate(pHOHgBzO-) causes displacement of bound Cd. When pHOHgBzO- -induced displacement of 109Cd is observed after dialysis of the reaction mixture, the stoichiometry is consistent with stepwise displacement of tetraco-ordinate Cd atoms by non-random entry of reagent into the polynuclear clusters. 113Cd n.m.r. allows direct observation of the effects on bound Cd of stepwise titration of 113Cd-MT-II with pHOHgBzO-. The first equivalent reduces all resonances approximately equally. Subsequently differential reactivity of the protein thiolates towards the reagent gives rise to differential decreases in the 113Cd signal intensities. Resonances previously attributed to a three-metal cluster are lost before those arising from the four-metal cluster. These results are interpreted in terms of current models of the MT structure. They are distinct from the results of reaction of MT with 5,5'-dithiobis-(2-nitrobenzoic acid), which distinguishes between only two classes of thiolates, terminal and bridging. Such different patterns of reactivity of the protein thiolates may underlie a biological activity of this protein.
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