J. Biol. Chem., Vol. 260, Issue 18, 10032-10038, 08, 1985
(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties
K Munger, UA Germann, M Beltramini, D Niedermann, G Baitella-Eberle, JH Kagi and K Lerch
Two metallothioneins (MTs) from bovine fetal liver were purified by a
combination of gel filtration and ion-exchange chromatography. The primary
structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and
amino acid sequence analysis. The amino-terminal part was deduced from
automated Edman degradations of the pyridylethylated CNBr- cleaved
derivatives. The remaining part of the sequence was established by a
comparison of the carboxamidomethylated tryptic peptides to those from
equine liver MT-1A and MT-2B. Peptides differing in either amino acid
composition or retention time from high pressure liquid chromatography were
further subjected to manual Edman degradations or carboxypeptidase Y
digestion. The two isoproteins consist of 61 amino acids and show a
sequence identity of 90%. When compared with the primary structures of
other mammalian MTs, the 20 cysteinyl residues are totally conserved, in
agreement with their function as metal ligands. The two isoproteins contain
Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption
properties typical for both Cu- and Zn-thiolate transitions. The marked
differences of MT-1 and MT-2 in the Cu-thiolate CD features can be
attributed to the six amino acid substitutions occurring exclusively in the
amino-terminal parts of the molecules. It is proposed that in bovine fetal
MTs also the three copper ions are preferentially bound to the first 9
cysteinyl residues (cluster B) and the four zinc ions to the remaining 11
cysteinyl residues (cluster A) suggested previously by 113Cd NMR
spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982)
J. Biol. Chem. 257, 1259-1262).
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