Purification and primary structure of metallothioneins induced by cadmium in the protists Tetrahymena pigmentosa and Tetrahymena pyriformis.
Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non-toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd(2+)-metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd(2+)-isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N-terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences. The Tetrahymena metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal-binding sites. Amino acid sequences of Tetrahymena metallothioneins have some similarity with other eukaryotic metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse metallothionein-1B.
Piccinni
E
E
Department of Biology, University of Padova, Italy.
Staudenmann
W
W
Albergoni
V
V
De Gabrieli
R
R
James
P
P
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology*; D002845:Chromatography; D003545:Cysteine / Q000032:analysis; D010450:Endopeptidases / Q000378:metabolism; D013058:Mass Spectrometry; D008668:Metallothionein / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis; D013056:Spectrophotometry, Ultraviolet; D013768:Tetrahymena / Q000378:metabolism*; D013769:Tetrahymena pyriformis / Q000378:metabolism*
D010446:Peptide Fragments; D002104:Cadmium; D008668:Metallothionein; D010450:Endopeptidases; D003545:Cysteine