A Naturally Occurring Cu-Thionein inSaccharomyces cerevisiae

Abstract

A naturally occurring monodisperse Cu-thionein was prepared using ammonium sulfate precipitation followed by ion exchange (DEAE 23) and gel chromatography (Sephadex G-75). The Chromatographie steps were repeated at least twice, or until the Cu-thionein remained homogeneous when subjected to analytical polyacrylamide disc electrophoresis. The molecular weight of this copper protein was 9500 ± 500. Up to 24.3 % cysteine residues were determined, indicating the relationship to the metallothioneins. Aromatic amino acids were virtually absent, while there were about three times as many acidic amino acid residues, including aspartate and glutamate, as in metallothioneins. 10 g atoms of Cu were measured per mole of protein. The copper binding strength of thionein was extremely high. Displacement by protons (pH 1.5) and gel chromatography or dialysis employing EDTA were not effective. Dialysis against diethyldithiocarbamate produced a protein essentially free of copper. Both the ultraviolet properties and the circular dichroism measurements proved identical with those properties reported for artificially prepared Cu-thionein (see refJ 1 ). The major absorption was in the far ultraviolet region with a weak shoulder at 270 nm attributable to copper charge-transfer transitions. 6 Cotton extrema were seen at 213, 283 and 302 nm (negative) and 245, 328 and 359 nm (positive). The possible role of Cu-thionein as an electron transport system was discussed. Isolierung von Cu-Thionein aus Saccharomyces cerevisiaeZusammenfassung: Ein natürlich vorkommendes monodisperses Cu-Thionein konnte aus Saccharomyces cerevisiae isoliert werden. Die aufgeschlossenen Zellen wurden mit (NH 4 ) 2 SO 4 versetzt und der suspendierte Niederschlag chromatographisch (DEAE-23-Cellulose und Sephadex G-75) aufgetrennt. Die chromatographischen Schritte wurden wiederholt, bis das Cu-Thionein sich als homogen erwies. Es zeigte bei der analytischen Polyacrylamid-Disk-Elektrophorese nur eine einzige Bande. Das Molekulargewicht dieses Cu-Proteins war 9 500 ± 500. Bis zu 24.3% Cysteinreste wurden bestimmt. Dieser hohe Anteil ließ eine Beziehung zu den Metallothioneinen

Efficient expression of the yeast metallothionein gene in Escherichia coli

“…The levels of copper needed to induce metallothionein synthesis in yeasts suggest that copper must saturate the metal-binding sites of the protein. Thus, the yeast molecule must be isolated in the copper-bound state and the copper must be removed to examine the apothionein form (28,36). By this method, it was found that the metallothionein from S. cerevisiae is capable of binding cadmium and zinc (36).…”

“…The Escherichia coli-synthesized yeast metallothionein bound copper, cadmium, and zinc, indicating that the protein was functional. Furthermore, E. coli cells expressing CUP) acquired a new, inducible ability to selectively sequester heavy metal ions from the growth medium.Metallothioneins consist of a class of small, cysteine-rich proteins that bind heavy metals and are found in a wide variety of organisms ranging from bacteria to vertebrates (10,12,15,18,25,(27)(28)(29)35). It has been shown in a number of studies that cells that synthesize increased levels of metallothionein display an increased resistance to the toxic effects of heavy metals, such as cadmium, zinc, copper, and mercury (1,5,6,(11)(12)(13)26).…”

Structure of Mammalian Metallothionein

“…Proteins satisfying this liberal definition have now been found to occur not only in many animal species but also in cexrtain eukaryotic microorganisms (6)(7)(8), in some plants (9,10), and even in prokaryotes (11,12). Table 1 lists the species and tissues in which the natural occurrence of metallothionein has been demonstrated and for which data on metal composition are available.…”

Crystallization of soluble proteins in vapor diffusion for x-ray crystallography

“…Crystallization attempts of Cu-MT date back to the protein's discovery in 1975 (ref. 108) and have always failed. NMR measurements on Cu-MT solutions provided evidence that 4 residues at the N-terminus and 13 residues at the C-terminus were disordered and that two Cys located in the disordered C-terminal part were not involved in copper coordination.…”