Antigenicity of metallothionein.

Abstract

The antigenic determinants of vertebrate metallothionein have been determined by a competitive-binding double-antibody radioimmunoassay to consist of two immunologically dominant regions in the NH2-terminal domain (residues 1-29). The COOH-terminal domain (residues 30-61) exhibited trivial immunoreactivity in competitive binding assays. The tryptic peptide encompassing residues Metallothionein is a 6,800-dalton metal-binding protein of unknown function isolated originally by Margoshes and Vallee (1). The protein is found in many cell types and is inducible by various metal ions (2-7) and glucocorticoids (8,9). Two sequence variants exist in the vertebrates thus far studied, and both are inducible. The protein is unusual in that it contains 32 mol % cysteine (20 of 61 residues in vertebrates), and all cysteine residues are involved in metal chelation (3,4,(10)(11)(12)(13)(14)(15)(16)(17)(18). Metallothionein has a stable tertiary conformation with the apoprotein being a random coil with possibly some short-range order (19,20). The structure appears to consist of two domains, each containing a metal-cysteine cage (21-23). Recently, Winge and Mildossy (24) showed that the two domains of Cd,Zn-thionein with seven metal atoms are separable by limited proteolysis. The COOH-terminal half of the molecule (residues 30-61) contained the four-metal atom-cysteine center (cluster A) and therefore the three-metal atom-cysteine center (cluster B) must be enfolded by the NH2-terminal half of the molecule (residues 1-29).With the isolated domains and peptide fragments of rat metallothionein available, we were interested in determining the immunoreactive properties of the domains and fragments in comparison with those of the native protein. (24). The a-fragments were prepared from both isoforms of metallothionein, designated Cd,Zn-MT-1 and Cd,Zn-MT-2. Purity of the a-peptides was assessed by amino acid analysis and NH2-terminal sequence analysis (24).The tryptic peptide OT1 (residues 1-25) was prepared by proteolysis of performic acid-oxidized thionein 2 with trypsin at a protease/thionein ratio (wt/wt) of 1:50 in 0.1 M pyridine acetate (pH 5) at 37°C for 48 hr. The tryptic peptides were resolved by gel filtration on a Sephadex G-50 column (1.5 X 160 cm) equilibrated with 1 M acetic acid as described by Huang et al. (15). The effluent fractions (1.5 m per tube) were monitored by the fluorescamine reaction after alkaline hydrolysis. The fluorescence emission at 475 nm was monitored in a Perkin-Elmer 650-10S spectrophotometer with excitation at 390 nm. The peptide eluting first was shown to be tryptic peptide OT1, in that its amino acid composition was the same as that for OTl reported by Huang et al. (16) (Table 1).Modification Reactions. Metallothionein was modified by incubation with 30% H202/88% formic acid, 1:9 (vol/vol), for 4 hr at 4°C (27). The cysteine -* cysteic acid conversion yield was 70-85%. Thionein was carboxymethylated by treatment with iodoacetate in 6 M guanidine HCI/l mM EDTA/0.1 M Tris HCl, pH 8....

Cloning and specific polymerised‐chain‐reaction amplification of a third charge‐separable human metallothionein isoform

“…The weaker interaction of MT-0, as compared to MT-1 and MT-2, with murine monoclonal antibodies to human MT-1 and MT-2 [24], as well as with rabbit polyclonal antibodies to a total MT fraction of human fetal liver (Van Houdt, K. and Soumillion, A., unpublished results), moreover suggests an important modification in the structure of MT-O and confirms once again the importance of the N-terminus and the Lys-containing sequences in antibody recognition of MT [25,261. Concomitant alterations in the strength of metal-ion binding in this cluster, however, remain to be investigated.…”

Copper‐binding proteins in ectomycorrhizal fungi

“…I'hey have been widely implicated in detoxification and storage of cadmium, zinc and copper ions (Lerch, 1980;Winge & Miklossy, 1982;Kagi & Kojima, 1987). Three classes of metallothionein proteins are now recognized.…”

Isolation and characterisation of metallothionein from the clam Ruditapes decussatus

“…Suggestions included different specificity of the promoter region of the gene (Schmidt et al, 1985), different metal-binding affinities (Winge and Miklossy, 1982) or simply the need for a large number of copies to facilitate a rapid response when necessary (Olafson et al, 1979). Considering the NH 2 -terminal sequence results (Table 2), the absence of methionine is consistent with other aquatic invertebrate MTs (Roesijadi et al, 1989;Mackay et al, 1993) indicating that MTs in this types of organisms are subject to NH 2 -terminal modifications following initial synthesis that does not occur in the mammalian MTs.…”