Winge D R, Gray W R, Zelazowski A, Garvey J S
Arch Biochem Biophys. 1986 Feb 15;245(1):254-62. doi: 10.1016/0003-9861(86)90212-2.
Metallothionein isoform II was purified from calf liver. The protein had a metal content of 1.2-1.9 Cu ions and 5.6-6.2 Zn ions per molecule in different preparations. The complete amino acid sequence of the molecule was determined by automatic Edman degradation of CNBr and tryptic peptides of the carboxymethylated protein. The positions of the 20 cysteines were identical to those in other mammalian metallothioneins. The calf molecule exhibited one position of microheterogeneity. The homology in amino acid sequence of the calf protein to horse and human metallothioneins exceeded 87%. Attempts to isolate the Cu-binding domain by selective destabilization of the Zn-binding region followed by proteolysis revealed that the beta domain is the predominant site of Cu ligation, but significant quantities of the alpha domain peptide were also recovered. Therefore, the native CuZn-metallothionein must contain separate populations of molecules with Cu distributed differently. The immunoreactivity of the calf protein and the two corresponding domain peptides was analyzed. Analogous to the situation with rat metallothionein, the antigenic epitopes reside in the amino-terminal beta domain with the alpha domain region containing only minimal antigenicity.
