Factors affecting copper metallothionein turnover
Investigations concerning metallothionein (MT) have covered 4 main areas. Initial studies were carried out to develop an immunocytochemical technique for the detection of MT. An indirect peroxidase technique was used to localise MT within the livers and kidneys of rats injected with CuSO4. An increase in immunocytochemical staining was observed following Cu injection which was consistent with the increase in MT-I concentrations as detected by RIA. To establish whether degradation of MT in vitro is influenced by prior exposure of protein to oxygen free radicals. (Cu,Zn)-MT with Cu:Zn ratio 1:1, purified from pig liver following injection with diethylamine copper oxyquinoline sulphonate (Cujec), was found to be extensively degraded after incubation with a free radical generating system (xanthine/xanthine oxidase) and subsequently with trypsin. However proteins with Cu:Zn ratios of 2:1 or 5:1 were not greatly affected. This indicates that an oxidative step may be involved in the degradation pathway and/or aggregation of MT but the magnitude of the effect is ultimately determined by the ratio of metals present within MT. To establish whether the turnover rate of hepatic CuMT is increased in vivo in animals subjected to oxidant stress. Iron overload was used to initiate oxidant stress in rats prior to injection of Cu using a mixture of Cujec and CuSO4. It could not be concluded, however, if in vivo degradation of CuMT was influenced by the application of iron-induced oxidant stress. Subcellular localisation of MT by fractionation of liver and kidney homogenates using preformed Percoll gradients did, however, demonstrate that MT was not associated with the lysosomal fraction but within the nuclear fraction in correlation with previous studies. To identify specific chelators for selective removal of Cu from CuMT in vitro and to establish the effect of administration of such chelators on the turnover of CuMT in vivo. Ammonium terathiomolybdate [(NH4)2MoS4] was incubated with (Cu,Zn)-MT and Cd, resulting in the complete removal of Cu from protein and replacement with Cd. The effect of this chelating action for Cu was then studied in vivo by administration of (NH4)2MoS4 to rats following injection of Cu using a mixture of Cujec and CuSO4. The turnover and degradation of induced CuMT, however, could not be said to be increased by the addition of the Cu chelator, (NH4)2MoS4, conclusively.