ABCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 170, 242-252 (19%) Metal-Induced Formation of Metallothionein in Rat Liver1 DENNIS R. WINGE,2 R. PREMAKUMAR, AND K. V. RAJAGOPALAN Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 Received February 14, 1975 Metallothionein, which accumulates in livers of rats which have received subcuta- neous injections of cadmium, has been purified to homogeneity by the use of Sephadex chromatography, acetone fractionation and chromatography on diethyl aminoethyl (DEAE)-cellulose. Anion exchange chromatography of Cd-thionein yielded two compo- nents differing in amino acid composition. Both forms of purified Cd-thionein displayed such characteristic features as high cysteine content and lack of aromatic amino acids. A molecular weight of 10,200 and a typical metal content of five atoms of cadmium and two atoms of zinc per protein molecule were common features of the two forms. The low molecular weight proteins which are induced in rats exposed to zinc, mercury or silver have been purified by the same procedure as was used for Cd-thionein. In each case the thionein was resolved into what appeared to be the same two fractions on DEAE-cellulose. The two forms of each metalloprotein exhibited mobilities identical to those of the corresponding Cd-thionein on polyacrylamide-gel electrophoresis. The amino acid compositions of the more anionic forms of Hg-thionein and Zn- thionein were quite similar to that of the corresponding Cd-thionein. Thus, the identity of the proteins induced in rats by zinc, mercury and silver with the previously known metallothionein induced by cadmium has been established. Metallothionein was first isolated as a cadmium-containing protein from equine renal cortex by Margoshes and Vallee in 1957 (1). Subsequent studies with this cyto- plasmic protein from a variety of sources including human kidney (21, equine kid- ney and liver (3, 4), rabbit liver (51, chicken liver (61, and rat liver (6, 7) re- vealed an amino acid composition marked by an unusually high cysteine content, ranging from 26-33 mol%, and absence of aromatic amino acids. Metallothioneins from various sources have been shown to contain cadmium, zinc and traces of mer- cury and copper (l-7). In rabbits (51, chickens (61, and rats (6- 9), accumulation of large amounts of metal- lothionein in the liver is accomplished by 1 This work was supported by Grant No. GM 00091 from the United States Public Health Service. * Supported by Predoctoral Traineeship, Grant No. GM 00233 from the National Institutes of Health. Present address: Department of Biochemis- try, Universite de Geneve, Case 76, 1211 Geneve 8, Switzerland. injection of cadmium. Other reports have indicated that copper (10, 111, zinc (8, 121, and mercury (13 -15) also induce the forma- tion of low molecular weight soluble pro- teins. These proteins have usually been assumed to be metallothioneins, although no definitive proof has been advanced to that effect. The protein induced in rat liver by copper has been established to be dis- tinct from metallothionein and has been termed copper-chelatin (16, 17). In view of the possible multiplicity of related pro- teins, it was of interest to characterize the proteins induced by zinc and mercury in rat liver and to determine their relation- ship to metallothionein. In this paper we present evidence for the identity of the proteins induced in rat liver by zinc, mercury and silver with cadmium- induced metallothionein. MATERIALS AND METHODS Sephadex G-75 was purchased from Pharmacia. Spectrophotometric grade acetone was obtained from Matheson, Coleman and Bell. DE-52 cellulose 242 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.