Structure of mammalian metallothionein.

All mammalian metallothioneins characterized contain a single polypeptide chain of 61 amino acid residues, among them 20 cysteines providing the ligands for seven metal-binding sites. Native metallothioneins are usually heterogeneous in metal composition, with Zn, Cd, and Cu occurring in varying proportions. However, forms containing only a single metal species, i.e., Zn, Cd, Ni, Co, Hg, Pb, Bi, have now been prepared by in vitro reconstitution from the metal-free apoprotein. By spectroscopic analysis of such derivatives it was established that all cysteine residues participate in metal binding, that each metal ion is bound to four thiolate ligands, and that the symmetry of each complex is close to that of a tetrahedron. To satisfy the requirements of the overall Me7(Cys-)20 stoichiometry, the complexes must be combined to form metal-thiolate cluster structures. Experimental proof for the occurrence of such clusters comes from the demonstration of metal-metal interactions by spectroscopic and magnetic means. Thus, in Co(II)7-metallothionein, the Co(II)-specific ESR signals are effectively suppressed by antiferromagnetic coupling of juxtaposed paramagnetic metal ions. By monitoring changes in ESR signal size occurring on stepwise incorporation of Co(II) into the protein, it is possible to follow the building up of the clusters. This process is biphasic. Up to binding of four equivalents of Co(II), the ESR amplitude increases in proportion to the metal content, indicating generation of magnetically noninteracting high-spin complexes. However, upon addition of the remaining three equivalents of Co(II), these features are progressively suppressed, signaling the formation of clusters. The same mode of cluster formation has also been documented for Cd and Hg. The actual spatial organization of the clusters and the polypeptide chain remains to be established. An attractive possibility is the arrangement of the tetrahedral metal-thiolates in adamantane-like structures surrounded by properly folded segments of the chain providing the ligands. 1H-NMR data and infrared absorption measurements are consistent with a tightly folded structure rich in beta-type conformation.

Paint and tape: collection and storage of microtraces of paint in adhesive tape.

The collection and preservation of microtrace evidence with the aid of an adhesive tape is a method of choice in forensic science. This technique is rapid and easy and allows the concentration of microtraces on a carrier, which facilitates further investigations in the laboratory. Adhesive tapes are currently used to secure microtraces of fibers and glass, but hardly for traces of automotive paint and other lacquers for fear of interference with the analysis of binders. A collection of automotive paint consisting of original and repair lacquers collected by tape has been evaluated. After various times of storage within the tape, these samples were compared with untreated references by microscope FT-IR and microspectrophotometry (MSP). Another set of paints was collected in 1984, stored within the tape until 1995, and examined the same way. About 170 layers of lacquer with various types of binder were examined. With the exception of one clear lacquer no difference between treated samples and references was detected. This small difference observed could be correlated to the exposure to xylene (extractant) and was not caused by the storage within the adhesive tape.

Coidentity of putative amylase inhibitors from barley and finger millet with phospholipid transfer proteins inferred from amino acid sequence homology.

A class of small polypeptides, isolated from seeds of barley and millet, which had been previously identified as putative amylase inhibitors has been found to have striking amino acid sequence identity with phospholipid transfer proteins. In addition, both classes of proteins have the same molecular weight and appear to be produced by proteolytic cleavage of an amino-terminal peptide of similar size. These properties, and the lack of any known activity for the barley protein, suggest that the putative amylase inhibitors are lipid transfer proteins.

Purification and characterization of atypical cadmium-binding polypeptides from Zea mays.

Exposure of corn seedlings to high concentrations of Cd ions induces formation of large quantities of polypeptidic compounds which are composed almost exclusively of Glu, Cys and Gly and which bind Cd through thiolate coordination. These polymers occur in classes with molecular weights of 4000 and 8000, respectively, and are made up of chains of about 2000 crosslinked by disulfide bridges. By derivatization and HPLC separation four different molecular species of similar amino acid composition were resolved. In the "native" state all Cys are either coordinated to Cd or linked in intra- and intermolecular disulfide bridges. The overall thiol-to-Cd ratio is close to 2. Judged from the refractoriness to endopeptidase digestion it is inferred that as in glutathione Glu is linked to the adjoining amino acid through its gamma-carboxyl group.

Cadmium binding and metal cluster formation in metallothionein: a differential modification study.

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively [Otvos, J.D., & Armitage, I. M. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7094-7098]. The formation of these oligonuclear complexes designated as Cd4 and Cd3 clusters has now been monitored in MT reconstituted with varying amounts of Cd by using differential chemical modification of Cys with [14C]iodoacetamide. At ratios below 2-3 mol of Cd/mol of MT bound, no differential protection of Cys by the metal, and hence no preferred binding, is detectable. At Cd-to-protein ratios between 3 and 5 mol of Cd/mol of MT, the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain, indicating formation of the Cd4 cluster. At still higher ratios, formation of the Cd3 cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labeling of the different Cys in Cd7-MT indicates that the ligands of the Cd3 cluster are 3 times as accessible to iodoacetamide than those of the Cd4 cluster, suggesting a greater thermodynamic or kinetic stability of the latter.

Cadmium binding and metal cluster formation in metallothionein: a differential modification study.

Mammalian metallothioneins (MT) contain 20 Cys in a total of 61 amino acid residues and bind 7 Cd and/or Zn ions. The metal is localized in two clusters made up of three and four metal-thiolate complexes in the NH2- and COOH-terminal half of the chain, respectively. The formation of these oligonuclear complexes designated as Cd4- and Cd3-cluster has now been monitored in MT reconstituted with varying amounts of Cd using differential modification of Cys with 14C-iodoacetamide. At ratios below 3 moles Cd/mole MT bound, no differential protection of Cys by the metal and, hence, no preferred binding is detectable. At Cd-to-protein ratios between 3 and 5 moles Cd/mole MT the modification profiles reveal preferred and cooperative binding in the COOH-terminal half of the chain indicating formation of the Cd4-cluster. At still higher ratios formation of the Cd3-cluster is initiated in the NH2-terminal section of the polypeptide chain. Comparison of the differential modification data of Cd6-MT and Cd7-MT suggests that the last Cd to be bound is coordinated to Cys ligands located mainly between positions 20 and 30 of the sequence. The extent of labelling of the different Cys in Cd7-MT indicates that the ligands of the Cd3-cluster are three times as accessible to iodoacetamide as those of the Cd4-cluster, suggesting a greater thermodynamic stability of the latter.

Isolation of a cDNA Clone for Spinach Lipid Transfer Protein and Evidence that the Protein Is Synthesized by the Secretory Pathway.

A cDNA clone encoding a nonspecific lipid transfer protein from spinach (Spinacia oleracea) was isolated by probing a library with synthetic oligonucleotides based on the amino acid sequence of the protein. Determination of the DNA sequence indicated a 354-nucleotide open reading frame which encodes a 118-amino acid residue polypeptide. The first 26 amino acids of the open reading frame, which are not present in the mature protein, have all the characteristics of a signal sequence which is normally associated with the synthesis of membrane proteins or secreted proteins. In vitro transcription of the cDNA and translation in the presence of canine pancreatic microsomes or microsomes from cultured maize endosperm cells indicated that proteolytic processing of the preprotein to the mature form was associated with cotranslational insertion into the microsomal membranes. Because there is no known mechanism by which the polypeptide could be transferred from the microsomal membranes to the cytoplasm, the proposed role of this protein in catalyzing lipid transfer between intracellular membranes is in doubt. Although the lipid transfer protein is one of the most abundant proteins in leaf cells, the results of genomic Southern analysis were consistent with the presence of only one gene. Analysis of the level of mRNA by Northern blotting indicated that the transcript was several-fold more abundant than an actin transcript in leaf and petiole tissue, but was present in roots at less than 1% of the level in petioles.