metallothionein and cuprous-chloride

Room temperature luminescence attributable to Cu(I)-thiolate clusters has been used to probe the transfer of Cu(I) from Cu(I)-glutathione complex to rabbit liver thionein-II and plant metal-binding peptides phytochelatins (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly and (gamma-Glu-Cys)4Gly. Reconstitutions were also performed using CuC1. The Cu(I)-binding stoichiometry of metallothionein or phytochelatins was generally independent of the Cu(I) donor. However, the luminescence of the reconstituted metallothionein or phytochelatins was higher when Cu(I)-GSH was the donor. This higher luminescence is presumably due to the stabilizing effect of GSH on Cu(I)-thiolate clusters. As expected, 12 Cu(I) ions were bound per molecule of metallothionein. The Cu(I) binding to phytochelatins depended on their chain length; the binding stoichiometries being 1.25, 2.0 and 2.5 for (gamma-Glu-Cys)2Gly, (gamma-Glu-Cys)3Gly and (gamma-Glu-Cys)4Gly respectively at neutral pH. A reduced stoichiometry for the longer phytochelatins was observed at alkaline pH. No GSH was found to associate with phytochelatins by a gel-filtration assay. The Cu(I) binding to (gamma-Glu-Cys)2Gly and (gamma-Glu-Cys)3Gly occurred in a biphasic manner in the sense that the relative luminescence increased approximately linearly with the amount of Cu(I) up to a certain molar ratio whereafter luminescence increased dramatically upon the binding of additional Cu(I). The luminescence intensity declined once the metal-binding sites were saturated. In analogy with the studies on metallothioneins, biphasic luminescence suggests the formation of two types of Cu(I) clusters in phytochelatins.

Topics: Amino Acid Sequence; Animals; Biological Transport; Chromatography, Gel; Copper; Drug Stability; Glutathione; Liver; Luminescent Measurements; Metalloproteins; Metallothionein; Molecular Sequence Data; Phytochelatins; Plant Proteins; Rabbits; Spectrophotometry, Ultraviolet

A progressive reduction in the size of rat metallothionein-1 mRNA following induction by copper chloride or dexamethasone was demonstrated on RNA blots, and was shown to be due to shortening of the poly(A)-tail. The rate of poly(A) removal was the same in rat liver and kidney following copper chloride induction, in rat liver following dexamethasone induction, and in mouse liver following copper chloride induction. In mouse liver metallothionein-1 and 2 mRNAs were shortened at the same rate. The reduction of the poly(A) tail was more rapid in the first 5 hours (approximately 20 nucleotides/h) but much slower (approximately 3 nucleotides/h) after the poly(A)-tail had been reduced to about 60 residues. Metallothionein mRNA molecules with poly(A) tail sizes less than 30-40 nucleotides were not observed. Exonuclease digestion of the poly(A)-tail is suggested, at least in the initial rapid phase. It is hypothesized that poly(A)-tails longer than 30 are required for mRNA stability and that much longer poly(A) tails may give newly synthesized mRNA molecules a competitive advantage in protein synthesis.

Topics: Animals; Copper; Dexamethasone; Gene Expression Regulation; Kidney; Liver; Male; Metallothionein; Mice; Molecular Weight; Nucleic Acid Hybridization; Poly A; Rats; Rats, Inbred Strains; RNA Processing, Post-Transcriptional; RNA, Messenger

The kinetics of the increase of metallothionein mRNA in rat liver and kidney after CuCl2 injection was determined by cell-free translation and dot-blot hybridization of total RNA isolated at various times after the injection. Both assay procedures gave essentially the same result: a 16-fold increase in hepatic metallothionein mRNA was observed 7h after CuCl2 injection, with a decline to basal values by 15 h. The response in the kidney was less dramatic, with a 6-fold increase in metallothionein mRNA 5 h after injection, and basal values were attained by 12h. The rise in Cu2+ concentration in both organs was closely correlated with the increase in metallothionein mRNA; hepatic Cu2+ was increased 5.9-fold by 5h after injection and renal Cu2+ was increased 4.3-fold 5h after injection. The Zn2+ concentration in the liver had not risen significantly within 5h of Cu2+ injection. Renal Zn2+ concentrations did not alter appreciably in the Cu2+-treated animals. These results support the conclusion that Cu2+ is acting as a primary inducer of metallothionein mRNA in the rat.

Topics: Adrenalectomy; Animals; Copper; Kidney; Liver; Male; Metallothionein; Nucleic Acid Hybridization; Protein Biosynthesis; Rats; Rats, Inbred Strains; RNA, Messenger; Sulfates; Zinc; Zinc Sulfate