Single crystals have been grown of Cd,Zn metallothionein isoform I1 from rat liver. The space gro... more Single crystals have been grown of Cd,Zn metallothionein isoform I1 from rat liver. The space group is P41212 (P43212) with unit cell dimensions a = 2, = 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. Metallothionein is a low molecular weight protein with the ability to bind a variety of metals, commonly Zn, Cd, and Cu (1). The protein was initially isolated and characterized from mammalian kidney and liver (2-4). Primary structures are known for human (5) , equine (6) , mouse (7) , crab (8), and Neurospora (9) metallothioneins. These sequences are strongly homologous, contain 57-61 residues, are rich in cysteine, lysine and serine, and are devoid in aromatic amino acids. The mammalian proteins contain 20 conserved cysteine residues. Coordination of the metals by cysteine is tetrahedral (I). '13Cd NMR' of rabbit liver Cd,Zn metallothionein demonstrates that the metals are arranged in polynuclear clusters (10). Using homonuclear decoupling, the 'I3Cd NMR spectra
Mammalian metallothionein is a low molecular weight protein with two metal-binding domains. To de... more 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.
The crystal structure of Cd,Zn metallothionein isoform II from rat liver has been determined usin... more The crystal structure of Cd,Zn metallothionein isoform II from rat liver has been determined using the anomalous scattering data from five Cd in the native protein. The structure of a 4Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by an iterative solvent leveling and map inversion procedure. The structure is folded into two domains. The N-terminal domain (beta) of residues 1-29 enfolds a three-metal cluster of 1 Cd and 2 Zn coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The C-terminal domain (alpha) of residues 30-61 enfolds a 4Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, diameter 15-20 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. The Cd1Zn2(cys)9 cluster is homologous with a 12 atom fragment of the Cd4(cys)11 cluster.
Copper-sulfur (CuS) multinuclear clusters exist in biological macromolecules that function in a v... more Copper-sulfur (CuS) multinuclear clusters exist in biological macromolecules that function in a variety of cellular processes ranging from copper ion buffering, signal transduction to copper ion storage. The sulfur ligands are typically provided exclusively by cysteinyl thiolates and proteins and peptides containing CuS clusters have an abundance of cysteine residues. A common sequence motif in these polypeptides is Cys-Xaa-Cys or Cys-Xaa-Xaa-Cys in which Xaa represents any other amino acid.
Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions... more 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 ' The designated terms used are: clusters A and B, the 4-and 3metal centers, respectively; a, the COOH-terminal domain of metallothionein which encompasses cluster A. ~~ 13063 This is an Open Access article under the CC BY license.
Metallothionein, which accumulates in livers of rats which have received subcutaneous injections ... more Metallothionein, which accumulates in livers of rats which have received subcutaneous injections of cadmium, has been purified to homogeneity by the use of Sephadex chromatography, acetone fractionation and chromatography on diethyl aminoethyl (DEAE)-cellulose. Anion exchange chromatography of Cd-thionein yielded two components differing in amino acid composition. Both forms of purified Cd-thionein displayed such characteristic features as high cysteine content and lack of aromatic amino acids. A molecular weight of 10,200 and a typical metal content of five atoms of cadmium and two atoms of zinc per protein molecule were common features of the two forms. The low molecular weight proteins which are induced in rats exposed to zinc, mercury or silver have been purified by the same procedure as was used for Cd-thionein. In each case the thionein was resolved into what appeared to be the same two fractions on DEAE-cellulose. The two forms of each metalloprotein exhibited mobilities identical to those of the corresponding Cd-thionein on polyacrylamide-gel electrophoresis. The amino acid compositions of the more anionic forms of Hg-thionein and Znthionein were quite similar to that of the corresponding Cd-thionein. Thus, the identity of the proteins induced in rats by zinc, mercury and silver with the previously known metallothionein induced by cadmium has been established.
Metal-binding domains are comprised of a metal ion (or metal ions) that has its coordination site... more Metal-binding domains are comprised of a metal ion (or metal ions) that has its coordination sites saturated by ligands which come from a short stretch of amino acid sequence. Recently, it has been discovered that several classes of proteins involved in nucleic acid binding and gene regulation may contain metal-binding domains. Two approaches to studying these systems will be discussed. The f i t involves analysis of amino acid sequences and includes methods for identifying potential metal-binding domain forming sequences and attempts to predict three-dimensional structures of metal-binding domains based on the structures of other metalloproteins and of small inorganic complexes. Using these methods an attractive model for the "zinc finger" metal-binding domains from Transcription Factor IIIA and related proteins has been developed. The second approach involves the preparation of peptides which correspond to individual metal-binding domains. The peptides can be characterized with regard to their metal-binding and structural properties using a variety of chemical and spectroscopic techniques. This approach has been applied to the "zinc finger" domains among other systems.
The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma vi... more The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.
Archives of Biochemistry and Biophysics, Jun 1, 1978
Liver metallothionein can be induced by the injection of a variety of metal ions, including Cd, H... more Liver metallothionein can be induced by the injection of a variety of metal ions, including Cd, Hg, Ag, Au, and Zn, into rats. The protein so induced contains bound zinc in addition to the other metal ion used for induction. In Cd-induced metailothionein Zn ~ § was found to occupy two of the eight possible metal-binding sites in thionein prepared from animals killed at least 10 h after the injection of cadmium. Thion'ein isolated at earlier times contained little or no zinc. The amount of thionein formed is proportional to the dose of Cd, but the hepatic uptake of Cd precedes the mobilization of Zn by several hours. This Cdinduced mobilization of Zn is most significant in the liver. The origin of the mobilized Zn is primarily from the excretory pathway, with the diet also being a contributing factor. In mice and rats, the increased hepatic Zn arising from Cd injection can largely be accounted for by a decrease in fecal Zn. This effect of Cd-induced Zn mobilization is also observed in rats and mice made zinc deficient. The mechanism of Zn mobilization is not resolved, but may involve both an effect of cadmium on excretion and the retention of zinc in the liver caused by an elevated level of thionein. The results presented suggest a physiological role for thionein in zinc metabolism.
Proper isolation and purification techniques for metal-lothionein (MT) and related proteins in no... more Proper isolation and purification techniques for metal-lothionein (MT) and related proteins in nonmammalian species are essential to the study of these molecules at all levels of biological organization. There are a number of basic approaches to preparing these proteins from biological tissues that have both positive and negative aspects depending upon the subsequent scientific questions under investigation. A general flow diagram for the isolation of MT and related proteins is given in Figure 1. The following discussion will attempt to examine each of the steps in this general isolation procedure and point out both the advantages and known disadvantages in each procedure. If tissues are to be stored frozen, this should be done at-70°C. Inhibition of endogenous tissue proteases during homogenization procedures is essential to preventing degradation of MT and related proteins. A number of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) or a2-macroglobulin have been commonly used to inhibit protein degradation with varying degrees of success. PMSF dissolved in ethanol and added to a final concentration of 0.5 to 1.0 nM is usually effective in preventing proteolysis by serine proteases. Since PMSF hydrolyzes quickly, it should be added directly to sample. In addition, utilization of reducing agents such as ,B-mercaptoethanol (P-ME) or dithioth-reitol (DTT) to prevent copper oxidation of protein SH groups may be helpful. In the case of p-ME 5 to 20 mM final concentration is sufficient, whereas only 0.5 to 1.0 mM DTT is necessary (1) because of its higher reducing potential. While addition of p-ME or DTT and use of nitrogen-gassed buffers appears to be essential for proper study of copper-containing proteins (2,3), these agents may alter the normal intracellular metal-binding proffles for metals such as Cd, Zn, or Hg because of their ability to form complexes with metal ions. The relative importance ofthis knowledge depends upon which scientific questions are being asked in the system under study. Centrifugation Following homogenization, a centrifugation step is commonly used to remove particulate matter such as unbroken cells and various organelles prior to initiation of chro-matographic procedures. One potential problem of interest in this step concerns the finding in rainbow trout liver (4) that endogenous lipids in the livers of this fish species are capable of trapping some metal-binding proteins and thus produce an artificial metal distribution during subsequent chromatographic steps. The solution to this problem simply involves centrifuging the homog-enate for 2 hr at 100,000g to get maximal …
Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space gro... more 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.
The role of copper metallothionein (CuMT) in copper metabolism and metalloenqme activation is poo... more The role of copper metallothionein (CuMT) in copper metabolism and metalloenqme activation is poorly understood. We have chosen marine crusmceans, in which a direct correlation exists between levels of Cu(I)MT ad Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-25 1, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homurus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclearcopper sites, which subsequently slowly return to their native stereochemical configuration. Fiiy, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.
Transition Metal Ion Uptake in Yeasts and Filamentous Fungi * Hydroxamates and Polycarboxylates a... more Transition Metal Ion Uptake in Yeasts and Filamentous Fungi * Hydroxamates and Polycarboxylates as Iron Transport Agents (Siderophores) in Fungi * Enzymology of Siderophore Biosynthesis in Fungi * Molecular Biology of Iron Transport in Fungi * Reductive Iron Assimilation in Saccharomyces cerevisiae * Iron Storage in Fungi * Manganese: Function and Transport in Fungi * Uptake of Zinc by Fungi * Accumulation of Radionuclides in Fungi * Metal Ion Resistance and the Role of Metallothionein in Yeast * Cadystin (Phytochelatin) in Fungi * Molecular Genetic Analysis of Cadmium Tolerance in Schizosaccharomyces pombe * Superoxide Dismutases in Saccharomyces Cerevisiae * Evolution and Biological Roles of Fungal Superoxide Dismutases Ferrochelatase of Saccharomyces cerevisiae * Heme-Mediated Gene Regulation in Saccharomyces cerevisiae.
The human chorionic gonadotrophin (hCG) preparation (ca. 3000 IU/mg) from pooled pregnancy urine ... more The human chorionic gonadotrophin (hCG) preparation (ca. 3000 IU/mg) from pooled pregnancy urine has been fractionated using anion-exchange and gel exclusion chromatography. The various fractions, many of which were heterogeneous, were assayed using radioimmunoassay (RIA) and a testicular radio-receptor assay (RRA). Three fractions were obtained with potencies between 9000 and 20 000 IU/mg; five fractions were characterized by potencies between 1000 and 6000 IU/mg; and four fractions exhibited low potencies (< 500 IU/mg). A linear relationship between RRA and RIA potency (P) was found to exist for the various fractions: ln(PRRA) = 1.14 ln(PRIA) \p=n-\1.67. The major fractions, which exhibited various potencies, were further characterized by amino acid and carbohydrate analysis. Urine was collected from an individual at various times of gestation, and the hCG fraction was concentrated by benzoic acid and ethanol precipitation. Ion-exchange chromatography of the hCG fraction yielded multiple immunoreactive components throughout pregnancy: thus the observed heterogeneity in the commercial preparation does not appear to result from processing pooled urine from numerous donors. Also, heterogeneity was observed in an individual donor whose urine was not treated with the benzoic acid and alcohol fractionation scheme. It seems indisputable that individual urinary hCG is heterogenous, and this does not appear to reflect isolation artefacts.
Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-1L,, ... more Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-1L,, an amplified locus, and MT-11b, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CotSO 4 required to inhibit the growth by 50~o (ICso) of a C. glabrata strain harboring an amplified MT'Ha locus and a singie-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The ICso decreased to approx. 1 mM when the amplified MT-II., locus was deleted. The disruption of the MT-IIb gene decreased the ICso further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-H genes was attributable to MT-I; no evidence was found for the production of (~'EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-II., revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-Ilb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IL., gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.
Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of c... more Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTZZb is more highly inducible than MTIIa and cells harboring a single MTIZb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between-503 and-292 upstream of the MT coding sequences. Expression of M T I is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.
Single crystals have been grown of Cd,Zn metallothionein isoform I1 from rat liver. The space gro... more Single crystals have been grown of Cd,Zn metallothionein isoform I1 from rat liver. The space group is P41212 (P43212) with unit cell dimensions a = 2, = 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. Metallothionein is a low molecular weight protein with the ability to bind a variety of metals, commonly Zn, Cd, and Cu (1). The protein was initially isolated and characterized from mammalian kidney and liver (2-4). Primary structures are known for human (5) , equine (6) , mouse (7) , crab (8), and Neurospora (9) metallothioneins. These sequences are strongly homologous, contain 57-61 residues, are rich in cysteine, lysine and serine, and are devoid in aromatic amino acids. The mammalian proteins contain 20 conserved cysteine residues. Coordination of the metals by cysteine is tetrahedral (I). '13Cd NMR' of rabbit liver Cd,Zn metallothionein demonstrates that the metals are arranged in polynuclear clusters (10). Using homonuclear decoupling, the 'I3Cd NMR spectra
Mammalian metallothionein is a low molecular weight protein with two metal-binding domains. To de... more 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.
The crystal structure of Cd,Zn metallothionein isoform II from rat liver has been determined usin... more The crystal structure of Cd,Zn metallothionein isoform II from rat liver has been determined using the anomalous scattering data from five Cd in the native protein. The structure of a 4Cd cluster was solved by direct methods. A 2.3 A resolution electron density map was calculated by an iterative solvent leveling and map inversion procedure. The structure is folded into two domains. The N-terminal domain (beta) of residues 1-29 enfolds a three-metal cluster of 1 Cd and 2 Zn coordinated by six terminal cysteine thiolate ligands and three bridging cysteine thiolates. The C-terminal domain (alpha) of residues 30-61 enfolds a 4Cd cluster coordinated by six terminal and five bridging cysteine thiolates. All seven metal sites have tetrahedral coordination geometry. The domains are roughly spherical, diameter 15-20 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. The Cd1Zn2(cys)9 cluster is homologous with a 12 atom fragment of the Cd4(cys)11 cluster.
Copper-sulfur (CuS) multinuclear clusters exist in biological macromolecules that function in a v... more Copper-sulfur (CuS) multinuclear clusters exist in biological macromolecules that function in a variety of cellular processes ranging from copper ion buffering, signal transduction to copper ion storage. The sulfur ligands are typically provided exclusively by cysteinyl thiolates and proteins and peptides containing CuS clusters have an abundance of cysteine residues. A common sequence motif in these polypeptides is Cys-Xaa-Cys or Cys-Xaa-Xaa-Cys in which Xaa represents any other amino acid.
Purified isoforms of rat liver apometallothionein were reconstituted in vitro with Cd and Zn ions... more 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 ' The designated terms used are: clusters A and B, the 4-and 3metal centers, respectively; a, the COOH-terminal domain of metallothionein which encompasses cluster A. ~~ 13063 This is an Open Access article under the CC BY license.
Metallothionein, which accumulates in livers of rats which have received subcutaneous injections ... more Metallothionein, which accumulates in livers of rats which have received subcutaneous injections of cadmium, has been purified to homogeneity by the use of Sephadex chromatography, acetone fractionation and chromatography on diethyl aminoethyl (DEAE)-cellulose. Anion exchange chromatography of Cd-thionein yielded two components differing in amino acid composition. Both forms of purified Cd-thionein displayed such characteristic features as high cysteine content and lack of aromatic amino acids. A molecular weight of 10,200 and a typical metal content of five atoms of cadmium and two atoms of zinc per protein molecule were common features of the two forms. The low molecular weight proteins which are induced in rats exposed to zinc, mercury or silver have been purified by the same procedure as was used for Cd-thionein. In each case the thionein was resolved into what appeared to be the same two fractions on DEAE-cellulose. The two forms of each metalloprotein exhibited mobilities identical to those of the corresponding Cd-thionein on polyacrylamide-gel electrophoresis. The amino acid compositions of the more anionic forms of Hg-thionein and Znthionein were quite similar to that of the corresponding Cd-thionein. Thus, the identity of the proteins induced in rats by zinc, mercury and silver with the previously known metallothionein induced by cadmium has been established.
Metal-binding domains are comprised of a metal ion (or metal ions) that has its coordination site... more Metal-binding domains are comprised of a metal ion (or metal ions) that has its coordination sites saturated by ligands which come from a short stretch of amino acid sequence. Recently, it has been discovered that several classes of proteins involved in nucleic acid binding and gene regulation may contain metal-binding domains. Two approaches to studying these systems will be discussed. The f i t involves analysis of amino acid sequences and includes methods for identifying potential metal-binding domain forming sequences and attempts to predict three-dimensional structures of metal-binding domains based on the structures of other metalloproteins and of small inorganic complexes. Using these methods an attractive model for the "zinc finger" metal-binding domains from Transcription Factor IIIA and related proteins has been developed. The second approach involves the preparation of peptides which correspond to individual metal-binding domains. The peptides can be characterized with regard to their metal-binding and structural properties using a variety of chemical and spectroscopic techniques. This approach has been applied to the "zinc finger" domains among other systems.
The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma vi... more The oncogenic E7 proteins of human papilloma virus (HPV 16) and of cottontail rabbit papilloma virus (CRPV) have been purified from an expression system in Escherichia coli. The proteins as purified from E. coli contain one tightly bound Zn(II) ion per molecule. The metal site shows facile exchange with either Cd(II) or Cu(I). The HPV 16 E7 maximally bound one Cd(II) or two Cu(I) ions, while the CRPV E7 bound two Cd(II) or three Cu(I) ions. The Cd(II) and Cu(I) E7 molecules exhibited optical transitions in the ultraviolet suggestive of metal:thiolate coordination. E7 proteins from HPV 16 and CRPV contain 7 and 8 cysteines/molecule, respectively. Reaction of the E7 proteins with the sulfhydryl reagent, dithiodipyridine, revealed that all the cysteinyl sulfurs are present in the reduced thiol state. Cu(I)-E7 molecules are luminescent with maximal emission at 570 nm. The observed emission at room temperature is indicative of metal coordination within a compact protein environment shielded from solvent interactions. The emission maxima occurs at the same wavelength (570 nm) as Cu(I)-cysteinyl sulfur clusters in Cu(I)-metallothioneins. The single Zn(II) atom in each protein can be removed from E7 in the presence of EDTA. The resulting apoE7 molecules remain soluble and can be partially reconstituted with Cd(II) to regain the ultraviolet charge transfer transitions.
Archives of Biochemistry and Biophysics, Jun 1, 1978
Liver metallothionein can be induced by the injection of a variety of metal ions, including Cd, H... more Liver metallothionein can be induced by the injection of a variety of metal ions, including Cd, Hg, Ag, Au, and Zn, into rats. The protein so induced contains bound zinc in addition to the other metal ion used for induction. In Cd-induced metailothionein Zn ~ § was found to occupy two of the eight possible metal-binding sites in thionein prepared from animals killed at least 10 h after the injection of cadmium. Thion'ein isolated at earlier times contained little or no zinc. The amount of thionein formed is proportional to the dose of Cd, but the hepatic uptake of Cd precedes the mobilization of Zn by several hours. This Cdinduced mobilization of Zn is most significant in the liver. The origin of the mobilized Zn is primarily from the excretory pathway, with the diet also being a contributing factor. In mice and rats, the increased hepatic Zn arising from Cd injection can largely be accounted for by a decrease in fecal Zn. This effect of Cd-induced Zn mobilization is also observed in rats and mice made zinc deficient. The mechanism of Zn mobilization is not resolved, but may involve both an effect of cadmium on excretion and the retention of zinc in the liver caused by an elevated level of thionein. The results presented suggest a physiological role for thionein in zinc metabolism.
Proper isolation and purification techniques for metal-lothionein (MT) and related proteins in no... more Proper isolation and purification techniques for metal-lothionein (MT) and related proteins in nonmammalian species are essential to the study of these molecules at all levels of biological organization. There are a number of basic approaches to preparing these proteins from biological tissues that have both positive and negative aspects depending upon the subsequent scientific questions under investigation. A general flow diagram for the isolation of MT and related proteins is given in Figure 1. The following discussion will attempt to examine each of the steps in this general isolation procedure and point out both the advantages and known disadvantages in each procedure. If tissues are to be stored frozen, this should be done at-70°C. Inhibition of endogenous tissue proteases during homogenization procedures is essential to preventing degradation of MT and related proteins. A number of protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF) or a2-macroglobulin have been commonly used to inhibit protein degradation with varying degrees of success. PMSF dissolved in ethanol and added to a final concentration of 0.5 to 1.0 nM is usually effective in preventing proteolysis by serine proteases. Since PMSF hydrolyzes quickly, it should be added directly to sample. In addition, utilization of reducing agents such as ,B-mercaptoethanol (P-ME) or dithioth-reitol (DTT) to prevent copper oxidation of protein SH groups may be helpful. In the case of p-ME 5 to 20 mM final concentration is sufficient, whereas only 0.5 to 1.0 mM DTT is necessary (1) because of its higher reducing potential. While addition of p-ME or DTT and use of nitrogen-gassed buffers appears to be essential for proper study of copper-containing proteins (2,3), these agents may alter the normal intracellular metal-binding proffles for metals such as Cd, Zn, or Hg because of their ability to form complexes with metal ions. The relative importance ofthis knowledge depends upon which scientific questions are being asked in the system under study. Centrifugation Following homogenization, a centrifugation step is commonly used to remove particulate matter such as unbroken cells and various organelles prior to initiation of chro-matographic procedures. One potential problem of interest in this step concerns the finding in rainbow trout liver (4) that endogenous lipids in the livers of this fish species are capable of trapping some metal-binding proteins and thus produce an artificial metal distribution during subsequent chromatographic steps. The solution to this problem simply involves centrifuging the homog-enate for 2 hr at 100,000g to get maximal …
Single crystals have been grown of Cd,Zn metallothionein isoform II from rat liver. The space gro... more 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.
The role of copper metallothionein (CuMT) in copper metabolism and metalloenqme activation is poo... more The role of copper metallothionein (CuMT) in copper metabolism and metalloenqme activation is poorly understood. We have chosen marine crusmceans, in which a direct correlation exists between levels of Cu(I)MT ad Cu(I)-hemocyanin during the molt cycle (Engel and Brouwer, Biol. Bull. 173, 239-25 1, 1987) as unique model systems to study the involvement of MTs in metalloprotein activation and degradation. We have isolated three low-molecular weight, cysteine-rich copper proteins from the American lobster Homurus americanus, which we designate as CuMT-1, CuMT-2, and CuMT-3, respectively. As a first attempt to fully characterize these proteins, we have determined the sequence of the first 56 amino acids of CuMT-1. The results show this protein to belong to the class I MTs, i.e., related in primary structure to equine renal MT. CuMT-1 cannot transfer its copper to copper-depleted apohemocyanin. CuMT-2 belongs to the same class of MTs as CuMT-1, but CuMT-3 does not. The latter can reactivate lobster hemocyanin containing reduced amounts of Cu(I). Spectroscopic studies show that Cu(I) transfer from CuMT-3 to apohemocyanin initially results in the formation of distorted binuclearcopper sites, which subsequently slowly return to their native stereochemical configuration. Fiiy, we present evidence that shows that the class I MTs in marine crustacea are involved in the sequestration of elevated levels of heavy-metal ions. These observations strongly suggest that the different forms of MT have different biological functions.
Transition Metal Ion Uptake in Yeasts and Filamentous Fungi * Hydroxamates and Polycarboxylates a... more Transition Metal Ion Uptake in Yeasts and Filamentous Fungi * Hydroxamates and Polycarboxylates as Iron Transport Agents (Siderophores) in Fungi * Enzymology of Siderophore Biosynthesis in Fungi * Molecular Biology of Iron Transport in Fungi * Reductive Iron Assimilation in Saccharomyces cerevisiae * Iron Storage in Fungi * Manganese: Function and Transport in Fungi * Uptake of Zinc by Fungi * Accumulation of Radionuclides in Fungi * Metal Ion Resistance and the Role of Metallothionein in Yeast * Cadystin (Phytochelatin) in Fungi * Molecular Genetic Analysis of Cadmium Tolerance in Schizosaccharomyces pombe * Superoxide Dismutases in Saccharomyces Cerevisiae * Evolution and Biological Roles of Fungal Superoxide Dismutases Ferrochelatase of Saccharomyces cerevisiae * Heme-Mediated Gene Regulation in Saccharomyces cerevisiae.
The human chorionic gonadotrophin (hCG) preparation (ca. 3000 IU/mg) from pooled pregnancy urine ... more The human chorionic gonadotrophin (hCG) preparation (ca. 3000 IU/mg) from pooled pregnancy urine has been fractionated using anion-exchange and gel exclusion chromatography. The various fractions, many of which were heterogeneous, were assayed using radioimmunoassay (RIA) and a testicular radio-receptor assay (RRA). Three fractions were obtained with potencies between 9000 and 20 000 IU/mg; five fractions were characterized by potencies between 1000 and 6000 IU/mg; and four fractions exhibited low potencies (< 500 IU/mg). A linear relationship between RRA and RIA potency (P) was found to exist for the various fractions: ln(PRRA) = 1.14 ln(PRIA) \p=n-\1.67. The major fractions, which exhibited various potencies, were further characterized by amino acid and carbohydrate analysis. Urine was collected from an individual at various times of gestation, and the hCG fraction was concentrated by benzoic acid and ethanol precipitation. Ion-exchange chromatography of the hCG fraction yielded multiple immunoreactive components throughout pregnancy: thus the observed heterogeneity in the commercial preparation does not appear to result from processing pooled urine from numerous donors. Also, heterogeneity was observed in an individual donor whose urine was not treated with the benzoic acid and alcohol fractionation scheme. It seems indisputable that individual urinary hCG is heterogenous, and this does not appear to reflect isolation artefacts.
Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-1L,, ... more Candida glabrata harbors multiple genes encoding metallothionein (MT). We have disrupted MT-1L,, an amplified locus, and MT-11b, a single-copy gene, to determine the roles of various MT genes in CuSO4 resistance in C. glabrata. The concentration of CotSO 4 required to inhibit the growth by 50~o (ICso) of a C. glabrata strain harboring an amplified MT'Ha locus and a singie-copy MT-IIb and MT-I genes was 7 mM in a synthetic complete medium. The ICso decreased to approx. 1 mM when the amplified MT-II., locus was deleted. The disruption of the MT-IIb gene decreased the ICso further to 0.1 mM. The CuSO4 resistance in a strain lacking both of the MT-H genes was attributable to MT-I; no evidence was found for the production of (~'EC)nG isopeptides. The comparison of the nucleotide sequence of MT-IIb to that of MT-II., revealed the same coding sequence with differences in the 5' region. However, substantial differences were found in the 3' region. MT-Ilb was expressed since we were able to purify the protein from the strain that had an intact MT-IIb gene, but a deleted MT-IL., gene. In addition, CuSO4 resistance was provided by MT-IIb. Northern analysis of the total RNA from varied C. glabrata strains indicated no significant changes in the expression of MT-I in the presence or absence of the MT-II genes.
Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of c... more Candida glabrata strains and a stable plasmid were developed that were suitable for analysis of copper-inducible expression from promoters of the three metallothionein (MT) genes. The two homologous MTII genes, MTIIa and MTIIb, encode the same polypeptide but are differentially induced by copper salts. MTZZb is more highly inducible than MTIIa and cells harboring a single MTIZb exhibit a greater resistance to copper salts compared to cells harboring a single MTIIa. The differential copper inducibility was mapped to sequences between-503 and-292 upstream of the MT coding sequences. Expression of M T I is highly Cu-regulated, but this MT gene confers much less resistance than MTII genes.
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Papers by Dennis Winge