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D R Winge

Publications and source records attributed to D R Winge.

At least 91 records · Page 5Linked to original sources
PubMed1986

Sequence and antigenicity of calf metallothionein II.

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.

Amino Acid Sequence↗
PubMed1986

Crystal structure of Cd,Zn metallothionein.

The anomalous scattering data from five Cd in the native protein were used to determine the crystal structure of cadmium, zinc (Cd,Zn) metallothionein isoform II from rat liver. The structure of a 4-Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by iterative single-wavelength anomalous scattering. The structure is folded into two domains. The amino terminal domain (beta) of residues 1 to 29 enfolds a three-metal cluster of one Cd and two Zn atoms coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The carboxyl terminal domain (alpha) of residues 30 to 61 enfolds a 4-Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, and the diameter is 15 to 20 A; there is limited contact between domains. The folding of alpha and beta is topologically similar but with opposite chirality. Redundant, short cysteine-containing sequences have similar roles in cluster formation in both alpha and beta.

Animals↗
PubMed1986

Cooperative cluster formation in metallothionein.

An ion-exchange chromatography procedure was used to resolve apometallothionein from the metallo- form in a study of metal-thiolate cluster formation. Chromatography of metallothionein reconstituted with Cd(II), Zn(II), or Cu(I) at neutral pH on carboxymethyl-cellulose led to removal of apoprotein from a solution without effect on recovery of the metalloprotein. Analysis of the effluent revealed apparent cooperative binding of these metal ions to the protein. Addition of 1-4 mol eq Cd(II) ions led to the recovery of metallothionein with around 4 mol eq Cd bound. The yield of this form increased with increasing starting metal ion equivalency. These results were obtained with two different ion-exchange resins. The cooperativity of binding was not total, but was initially confined to the carboxyl-terminal alpha domain. The results of metal and protein yields are inconsistent with random, noninteractive binding. Similar data were obtained with Zn(II) and Cu(I) ions although Cu(I) exhibited initial cooperative binding within the amino-terminal beta domain with over 5 mol eq Cu(I) bound.

Allosteric Site↗
PubMed1985

Yeast metallothionein. Sequence and metal-binding properties.

The protein product of the CUP1 locus in Cu-resistant Saccharomyces cerevisiae has been purified and characterized. The protein was found to lack the first 8 amino acids predicted by the nucleotide sequence of the gene. The residues removed from the amino-terminal region include 5 hydrophobic residues, two of which are aromatic. The unique amino terminus starting at Gln9 of the putative DNA translation product was observed for metallothionein purified in the presence of various protease inhibitors or from a pep4 mutant yeast strain deficient in vacuolar proteases. The remainder of the primary structure of the protein is equivalent to the decoded DNA sequence, so yeast metallothionein is a 53-residue polypeptide of molecular weight 5655. The isolated protein contained 8 copper ions ligated by 12 cysteines/molecule. Reconstitution studies of the apo-molecule revealed that 8 mol eq of Cu(I) conferred maximal stability against proteolysis and depleted the zinc content of zinc-saturated metallothionein. These assays suggested that the protein has 8 binding sites for Cu(I). Ag(I) ions bound to the protein with the same stoichiometry. Yeast metallothionein was also observed to coordinate Cd(II) and Zn(II) ions in vitro. In studies of direct binding, protection against proteolysis, and metal ion exchange, these divalent ions were found to associate with the protein with a maximal stoichiometry of 4 ions/molecule. Yeast metallothionein thus exhibits two distinct binding configurations for Cu(I) and Cd(II) as does the mammalian protein.

Amino Acid Sequence↗
PubMed1985

Independence of the domains of metallothionein in metal binding.

Mammalian metallothionein is a low molecular weight protein with two metal-binding domains. To determine if metal binding in one domain affects binding in the other, we prepared peptides corresponding to the regions that enfold the two metal-thiolate clusters. Metal reconstitution studies of these peptides revealed stoichiometries of metal binding similar to those observed within the intact molecule. Thus, the alpha domain coordinates 4 Cd(II), 6 Cu(I), or 6 Ag(I) ions regardless of whether the domain is part of the total protein or is studied as a separate peptide. Likewise, the beta domain binds 3 Cd(II), 6 Cu(I), or 6 Ag(I) ions in both the intact protein and as a separate peptide. If cluster B in intact metallothionein is preformed with Cu(I) or Ag(I), cluster A saturates with either 4 mol eq of Cd(II) or 6 mol eq of Ag(I). Similarly, preformation of the A cluster with Cd(II) does not affect the binding of 6 Cu(I) ions in the B cluster. Therefore, the metal-dependent folding of the protein to create one cluster occurs independent of constraints or influences from the other domain. Formation of the protein with a tetrahedrally coordinated metal in one cluster and a trigonally coordinated metal in the other center is possible.

Cadmium↗
PubMed1985

Distinct metal-binding configurations in metallothionein.

In a study of the binding stoichiometry of various metals to rat liver metallothionein, the protein appears to coordinate metals in 2 distinct configurations. Ions of at least 18 different metals were shown to associate with the protein suggesting that there is little specificity in binding. Most metals exhibited saturation binding at 7 mol eq forming M7-metallothionein. These included Bi(III), Cd(II), Co(II), Hg(II), In(III), Ni(II), Pb(II), Sb(III), and Zn(II). Others metals including Os(III), Pd(II), Pt(IV), Re(V), Rh(III), and Tl(III) give a positive indication of binding, but stoichiometries were unclear. Ag(I) and Cu(I) bound in clusters as M12-metallothionein. This binding stoichiometry was determined in 3 ways: (a) by determining the equivalence point in Cu- and Ag-titrated samples where resistance to proteolysis is maximal; (b) by determining the point where Zn ions are completely displaced from Zn7-metallothionein; and (c) by direct binding studies. Ag-reconstituted protein, recovered from gel filtration, had an average Ag content of 11.5 g atoms/mol of protein. A similar stoichiometry for the Cu-protein resulted from displacement of Zn from Zn7-metallothionein by Cu(I). The M12-protein was converted to the M7-protein by displacement of Ag(I) or Cu(I) with 7 mol eq of Hg(II). Whereas the distribution of metals in the 2 domains of M7-metallothionein is M4 alpha and M3 beta, the arrangement in the M12-molecule is probably M6 alpha and M6 beta. We propose that metallothionein ligates Ag(I) and Cu(I) in a trigonal geometry by bridging thiolates. This is in contradistinction to a tetrahedral binding geometry in the M7-protein. Distinct binding configurations may result in different tertiary structures for M7- and M12-proteins which may relate to metabolic specificity of Zn-metallothionein and Cu-metallothionein, respectively.

Animals↗
PubMed1984

Structural characterization of the isoforms of neonatal and adult rat liver metallothionein.

Metallothionein was purified from the livers of adult and neonatal rats. The complete amino acid sequences of isoforms I and II of Cd-induced adult metallothionein were determined by automated Edman degradation of CNBr and tryptic peptides of carboxymethylated proteins. Both isoproteins contain 61 residues, but differ at 12 of those positions. The positions of the 20 cysteinyl residues are invariant with respect to those in other known mammalian metallothioneins. Based on the following criteria, the two soluble and constitutive Zn-metallothionein isoforms from neonatal liver appear identical to their corresponding forms in the adult animal: 1) they behave identically on reverse-phase high-pressure liquid chromatography, anion-exchange chromatography and nondenaturing gel electrophoresis; 2) their amino acid compositions are the same within experimental error; and 3) their amino acid sequences are identical in the first 23 residues, even though isoforms I and II differ in 7 of those positions. It appears highly likely that isoforms from both neonatal and adult rats are encoded by the same genes, and therefore that the large age-dependent concentration differences seen in the liver must be due to variation in gene regulation.

Age Factors↗
PubMed1984

Preferential binding of copper to the beta domain of metallothionein.

Proteolytic studies of rat liver metallothionein reconstituted in vitro with Cu salts revealed that the 2 metal centers fill in an ordered fashion. The B cluster in the NH2-terminal beta domain fills prior to Cu binding in cluster A. This is in contradistinction to cluster formation induced by the binding of Cd or Zn ions in which cluster A is the center of initial binding. The formation of metal cluster B by Cu occurs in a cooperative fashion yielding a saturated cluster with approximately 6 Cu+ ions bound. The B cluster is saturated with Cd or Zn after binding of only 3 metal ions. The preferential binding of Cd and Cu to the alpha and beta domains, respectively, and the tolerance toward proteolysis of these 2 different half saturated molecules permit the isolation of each domain. The metal cluster in each isolated domain can be reversibly formed with predicted stoichiometries of Cd and Cu. The folding of the polypeptide therefore appears to create each cluster independently. The metal binding data suggest that Cu-metallothionein contains 11-12 Cu ions, 6 bound in the beta domain and 5-6 in the alpha domain. In contrast, Cd-metallothionein contains 7 Cd ions, 3 bound to beta and 4 to alpha.

Amino Acids↗
PubMed1984

Crystals of cadmium, zinc metallothionein.

Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space group is P41212(P43212) with unit cell dimensions a = b = 31.0 A and c = 120.0 A, and one molecule in the crystallographic asymmetric unit. The crystals are square bipyramids elongated on the tetragonal c-axis and are grown by repetitive seeding. The crystals are suitable for high resolution structure analysis. Assays of dissolved crystals show that the crystals have the same Cd and Zn content and amino acid composition as the native, as-isolated protein.

Animals↗
PubMed1984

Formation of the metal-thiolate clusters of rat liver metallothionein.

The isoforms of rat liver apo-metallothionein (MT) were reconstituted in vitro with Cd and Zn ions to study the order of binding of the seven metal sites. Reconstitution with seven Cd ions resulted in a metalloprotein similar to induced Cd,Zn-MT by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin and the pH-dependent release of Cd. Proteolytic digestion of MT reconstituted with sub-optimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in cluster A. Upon saturation of the four sites in cluster A, binding occurs in the three metal center, cluster B. Samples reconstituted with one to four Cd or Zn ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain encompassing the four metal center. Samples renatured with five to seven Cd ions per MT molecule showed decreasing quantities of alpha fragment and increasing amounts of nativelike MT. The binding process in each domain is cooperative. Reconstitution of apo-MT with two Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4-alpha cluster. Likewise, when Cd5-renatured MT was digested with subtilisin, 30% of the molecules were identified as Cd7-MT with the remainder as Cd4-alpha fragment.

Animals↗
PubMed1983

Order of metal binding in metallothionein.

Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions to study the order of binding of the 7 metal sites in the two separate metal clusters, one containing four metal ions (cluster A) and the other containing three (cluster B). Reconstitution with 7 Cd ions resulted in a metalloprotein similar to induced Cd,Zn-metallothionein by the criteria of electrophoretic mobility, insensitivity to proteolysis by subtilisin, and the pH-dependent release of Cd. Proteolytic digestion of metallothionein reconstituted with suboptimal quantities of Cd followed by separation of Cd-containing polypeptide fragments by electrophoresis and chromatography revealed metal ion binding initially occurs in the 4-metal center, cluster A. Upon saturation of the 4 sites in cluster A, binding occurs in the 3-metal center, cluster B. Samples reconstituted with 1 to 4 Cd ions per protein molecule, followed by digestion with subtilisin, yielded increasing amounts of a proteolytically stable polypeptide fragment identical with the alpha fragment domain that is known to encompass the 4-metal center. Samples renatured with 5 to 7 Cd ions per metallothionein molecule showed decreasing quantities of alpha fragment and increasing amounts of native-like metallothionein. Similar results were obtained in reconstitution studies with Zn ions. Samples reconstituted with 7 Cd eq followed by incubation with EDTA revealed that cluster B Cd ions were removed initially. The binding process in each domain is cooperative. Reconstitution of apometallothionein with 2 Cd ions followed by proteolysis yields a 50% recovery of saturated Cd4 alpha cluster. Likewise, when Cd5-renatured metallothionein was digested with subtilisin, 30% of the molecules were identified as Cd7 metallothionein with the remainder as Cd4 alpha fragment.

Amino Acids↗
PubMed1983

Single crystals of cadmium, zinc metallothionein.

Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space group is P4(1)2(1)2 (P4(3)2(1)2) with unit cell dimensions a = b = 31.0 A and c = 120.0 A, and one molecule in the crystallographic asymmetric unit. The crystals are square bipyramids elongated on the tetragonal c-axis and are grown by repetitive seeding. The crystals are suitable for high resolution structure analysis. Assays of dissolved crystals show that the crystals have the same Cd and Zn content and amino acid composition as the native, as-isolated protein.

Animals↗
PubMed1983

A method for distinguishing Cu,Zn- and Mn-containing superoxide dismutases.

A procedure has been developed to distinguish between the two forms of eukaryotic superoxide dismutases using a common activity assay. Treatment of cellular fractions with 2% sodium dodecyl sulfate at 37 degrees C for 30 min selectively inactivates the mitochondrial, manganese-containing variant without affecting the cytosolic copper, zinc-superoxide dismutase. After removing excess sodium dodecyl sulfate by precipitation with potassium chloride, the supernate is assayed using the xanthine oxidase-cytochrome c method.

Animals↗
PubMed1983

Antigenicity of metallothionein.

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 1-25 of the molecule competed with 125I-labeled metallothionein as effectively as the native protein. The crossreactivity was unaffected whether the protein was native or denatured. The antigenicity is thus independent of the degree of folding of the protein and, although all antigenic sites depend to some degree on conformation or topography, this favors a sequential (or continuous) rather than a discontinuous nature of the determinants. The two regions in metallothionein that appear to be important in the interaction of the molecule with the antisera include the NH2-terminal acetylated methionine and the cluster of lysines in the sequence from residues 20-25. These regions are homologous in the various vertebrate metallothioneins known to crossreact with rabbit anti-rat metallothionein antiserum. This implies that the induction in rabbits of antiserum to rat metallothionein is an autoimmune phenomenon. The results support the two-cluster model of metallothionein and are compatible with predictions of sites of antigenicity based on regions of high hydrophilicity.

Animals↗
PubMed1982

113Cd NMR study of a metallothionein fragment. Evidence for a two-domain structure.

A 32-residue polypeptide fragment, designated alpha I, of rat liver metallothionein obtained by subtilisin digestion was studied by 113Cd NMR. The amino acid composition of the fragment corresponded to residues 30-61 of the metallothionein primary structure, and it contained 3.4 g atoms of Cd2+/mol of alpha I-fragment. Four 113Cd resonances were observed, three of which had identical chemical shifts to those assigned to the four-metal cluster in human liver metallothionein-2 under the same pH and buffer conditions. The 5-ppm chemical shift difference between the remaining resonance assigned to the four-metal cluster in the intact protein can be explained to result from the removal of the NH2-terminal polypeptide fragment containing the three-metal cluster. These results provide unambiguous evidence for the two-domain structure of metallothionein, containing a separate three- and a four-metal cluster.

Animals↗