Assay of extracellular metallothionein.
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Animals↗
Biomedical subjects
R K Mehra
Publications and source records attributed to R K Mehra.
Selective and tandem amplification of a member of the metallothionein gene family in Candida glabrata.
Metallothioneins constitute a multigene family in the yeast Candida glabrata. Two genes, designated metallothionein-I (MT-I) and one member of the metallothionein-II family (MT-II), were cloned and sequenced previously (Mehra, R. K., Garey, J. R., Butt, T. R., Gray, W. R., and Winge, D. R. (1989) J. Biol. Chem. 264, 19747-19753). Southern analysis of the genomic DNA samples from different wild-type isolates indicated that the MT-I gene was always present as a single copy but multiple (3-9) and tandemly arranged copies of one MT-II gene were present in different strains. Strains of C. glabrata highly resistant to copper salts were obtained by repeated culturing of wild-type isolates in medium containing increasing concentrations of copper sulfate. These strains showed further stable chromosomal amplification (greater than 30 copies) of the MT-II gene. The MT-I gene remained as a single copy. Amplified copies of the MT-II gene were always arranged tandemly. One of the copper-resistant strains acquired more copies of the MT-II gene by apparent duplication of the chromosome carrying this gene. The size of the amplification unit was 1.25 kilobases. The principal MT-I and -II genes of C. glabrata were shown to map to different chromosomes by electrophoretic karyotypic analysis. The length of chromosome carrying MT-II gene increased appreciably in strains exhibiting the highest amplification of this gene. Northern analysis showed increased basal levels of MT-II mRNA in strains having highly amplified MT-II locus.
Amino Acid Sequence↗
Host defenses against copper toxicity.
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Animals↗
Fractionation of milk proteins.
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Animals↗
Candida glabrata metallothioneins. Cloning and sequence of the genes and characterization of proteins.
Southern blot analysis has identified several metallothionein gene sequences in a human pathogenic yeast Candida glabrata. Two of these genes encoding proteins designated MT-I and MT-II have been cloned and sequenced. No introns were found in either of the genes. The complete primary structure of MT-II was also determined by protein sequencing methods. As isolated, MT-I and MT-II consist of 62 and 51 amino acids, respectively. The only residues predicted from the nucleotide sequence but not present in the isolated protein are the amino-terminal methionines in each sequence. MT-I contains 18 cysteines, 14 of which are present as Cys-X-Cys motifs and two additional cysteines in a Cys-X-X-Cys sequence. The sequence of MT-II contains 16 cysteinyl residues, 14 of which are in Cys-X-Cys sequences. Fluorescence spectroscopy indicates the presence of Cu(I)-thiolate bonds in both proteins. The binding stoichiometries are 11-12 for MT-I and 10 for MT-II. Under certain nutritional conditions, a truncated form of MT-II was also produced. Northern analysis of the total cellular RNA from copper-treated cells showed that both MT-I and MT-II genes are regulated by this metal ion in a concentration-dependent fashion. The concentrations of MT-II mRNA appeared to be higher than that of MT-I mRNA at all concentrations of copper sulfate tested. Both genes are inducible by silver but not by cadmium salts. Cadmium ions, however, are effective in reducing the control levels of both MT-I and MT-II mRNAs.
Amino Acid Sequence↗
Characterization of purine hydroxylase I from Aspergillus nidulans.
Purine hydroxylase I from Aspergillus nidulans was purified 850-fold. The purified preparations exhibited the spectral and catalytic properties, including broad specificity for oxidizing and reducing substrates, typical of molybdenum/flavin/iron-sulphur-containing hydroxylases (oxotransferases).
Aspergillus nidulans↗
Isolation, purification and characterization of low molecular weight 110mAg-binding proteins from the rat and goat testis.
Low molecular weight 110mAg-binding proteins have been purified from the goat and rat testis using a combination of acetone fractionation, gel filtration and ion-exchange chromatography. Both the rat and goat 110mAg-binding proteins existed in three isoforms, although the goat isoforms carried higher net negative charge than the corresponding isoforms of the rat. The average molecular weight of the goat isoforms was 8000 daltons, whereas that of the rat isoforms was 6300. The goat 110mAg-binding protein (a mixture of all the isoforms) was stable when heated at 85 degrees C for 15 min. The isoform II of the rat 110mAg-binding protein showed little absorbance at 280 nm, although the corresponding isoform of the goat protein did show significant absorbance at the same wavelength.
Animals↗
Effect of mutation of cysteinyl residues in yeast Cu-metallothionein.
Metallothioneins have been isolated from Saccharomyces cerevisiae CUP1 mutants generated by Wright et al. (Wright, C. F., Hamer, D. H., and McKenney, K. (1986) Nucleic Acids Res. 14, 8489-8499). In the mutant metallothioneins, pairs of cysteinyl residues have been converted to seryl residues. The mutant proteins differ only in the positions of the double substitutions; each mutant molecule contains 10 cysteinyl residues. Each mutant protein lacks the first 8 residues at the amino terminus from the decoded gene sequence of the CUP1 locus. Mutant molecules consist of 53 residues analogous to the wild-type metallothionein and are designated 9/11, 24/26, 36/38, and 49/50 (in reference to the sequence positions of the Cys----Ser conversions). The properties of the mutant metallothioneins are vastly different, and host cells harboring the different plasmid-encoded mutant molecules show marked differences in sensitivity to CuSO4. Growth inhibition was observed at CuSO4 concentrations up to mM in cells containing the 9/11, 24/26, and 36/38 molecules, but not for cells containing protein 49/50. A CuSO4 concentration of 5 mM was required to inhibit the growth of yeast containing either 49/50 or the wild-type metallothionein. In the purified proteins the copper binding stoichiometry of each molecule, except protein 24/26, was nearly 8 mol eq. Protein 24/26 bound 5.5 copper ions/molecule. The Cu(I) chelator bathocuproine disulfonate reacted with over 50% of the copper ions in proteins 9/11, 24/26, and 36/38, but less than 10% of the copper ions in proteins 49/50 and wild-type metallothionein were reactive. The thiolates in 9/11, 24/26, and 36/38 were also more reactive in a disulfide exchange reaction with dithiodipyridine compared with the sulfhydryls in 49/50 and the wild-type molecules. The four mutant copper proteins are luminescent and exhibit a similar quantum yield. The cluster structures contributing to the particular electronic transitions are markedly more sensitive to oxygen in proteins 9/11, 24/26, and 36/38 compared with 49/50 and the wild-type molecules. The air-sensitive proteins exhibit a tertiary fold not recognized by polyclonal antibodies directed to a conformational epitope on yeast Cu-metallothionein. Protein 49/50 cross-reacts with the antibody in a concentration-dependent fashion similar to the wild-type protein. Mutation of 2 cysteinyl residues in the carboxyl portion of metallothionein does not significantly alter properties of the molecule, whereas mutation of several cysteines in the amino-terminal portion of the molecule yields a different conformation.
Carrier Proteins↗
Studies on the gamma-glutamyl Cu-binding peptide from Schizosaccharomyces pombe.
The gamma-glutamyl peptide induced in Schizosaccharomyces pombe in response to metal stress has been purified following exposure of the organism to cadmium and copper salts. Induction of the peptide enables S. pombe to proliferate in media containing high concentrations of cadmium and copper. Two Cd-gamma-Glu peptide complexes are produced which differ in the content of acid-labile sulfur. One Cu-gamma-Glu peptide complex is induced, and it lacks acid-labile sulfur in the metal-binding cluster. The peptides are composed of repeating dipeptide units of gamma-Glu-Cys with a carboxyl-terminal glycine with heterogeneity observed in the repeat unit n. The number of repeats averages 3.2 and 3.8 for the Cd-peptides I and II and 3.6 for the Cu-peptide, in the case of the Cu-complex peptides with n values from 2 to 4 were separated by reverse phase high pressure liquid chromatography. The Cu-gamma-Glu peptide complex is oligomeric, but the exact number of peptide units per complex is not known. The copper binding stoichiometry averages 2.3 g atoms of Cu/mol of peptide, whereas Cd-peptides I and II average 1.8 and 2.7 mol eq of Cd(II)/peptide unit. The pH of half-dissociation of Cu ions from the gamma-Glu peptide is near 1.3, whereas pH values of 4 and 5.4 are sufficient for half-displacement of Cd ions from the sulfide-containing and -lacking peptides II and I, respectively. In the Cu-peptide complex copper is bound as Cu(I) as the complex exhibits luminescence characteristic of Cu(I)-S chelation. The luminescence emission peaks at 619 nm with a corrected excitation peak centered at 290 nm. The luminescence of the Cu-complex indicates the clustering of Cu(I) ions within a solvent-inaccessible complex. The complex is air-labile as the luminescence emission is gradually lost upon air exposure.
Amino Acids↗
Cu(I) binding to the Schizosaccharomyces pombe gamma-glutamyl peptides varying in chain lengths.
The metal-gamma-glutamyl peptide complex of Schizosaccharomyces pombe is an oligomer of peptides of the general structure (gamma-Glu-Cys)n-Gly with n defining the number of dipeptide repeats. The complexes induced with either cadmium or copper salts are heterogeneous with respect to the number of repeat units or n. Peptides isolated from two preparations of the Cd-gamma-Glu complex by reverse-phase HPLC at low pH were of an n range of 2 to 6 with n3 and n4 peptides being predominant. In addition to peptides of the mentioned structure, peptides of n3 and n4 without the terminal Gly were isolated. These n3 and n4 desGly peptides were present in an abundance of about 10-20% of the concentration of the parent peptide. Peptides of unique n were studied in Cu(I) reconstitution experiments in an attempt to understand the significance of the peptide length heterogeneity in the oligomeric metal-thiolate cluster. Cu-gamma-Glu complexes were formed with each peptide as determined by the characteristic 260-nm shoulder in the ultraviolet absorption spectrum and luminescence indicative of Cu(I)-thiolate coordination in a solvent-inaccessible environment. Cluster formation also occurs with desGly peptides, so the carboxyl-terminal Gly is not critical for cluster formation. Maximal Cu binding stoichiometry with n3 and n4 peptides was markedly less than the maximal Cu(I) stoichiometry of a peptide mixture or the native complex. Cu ions in complexes formed with unique n peptides were more reactive with bathocuproine than Cu ions in complexes with a peptide n mixture. The results suggest that metal-peptide complexes consisting of peptides differing in n probably exist and not all metal-peptide complexes have the same n peptide constituents.
Binding Sites↗
Metal-specific synthesis of two metallothioneins and gamma-glutamyl peptides in Candida glabrata.
Cellular resistance to heavy metal cytotoxicity in most species is mediated by the binding of metal ions either to a cysteine-rich polypeptide in the metallothionein family or to short cysteine-containing gamma-glutamyl peptides. One of these metal binding systems has been found in most organisms studied. However, the yeast Candida (Torulopsis) glabrata expresses both metallothionein and the gamma-glutamyl peptides for metal detoxification, and each system is regulated in a metal-specific manner. Exposure of C. glabrata to copper salts stimulates formation of two metallothionein-like polypeptides with a cysteine content of 30 mol% and the repeated sequence Cys-Xaa-Cys. The cells synthesize gamma-glutamyl peptides upon exposure to cadmium salts. Penta- and tetrapeptides that form a cadmium-thiolate cluster in a peptide oligomer containing labile sulfur are synthesized.
Amino Acid Sequence↗
Metallothionein in blood, bile and urine.
The development of radioimmunoassays for rat metallothionein-I (MT-I) has revealed that this protein is present in many extracellular fluids. Plasma concentrations are low in normal animals but may be substantially increased in animals with elevated tissue MT levels. MT-I is also excreted in varying amounts into urine and bile, and there is evidence for the presence of degradation products of MT-I, detectable by radioimmunoassay, in these fluids. Investigation of the secretion of MT can provide valuable information on the turnover of the protein in tissues and lead to the development of novel diagnostic tests for the assessment of trace metal status.
Animals↗
Induction of synthesis and degradation of metallothionein-I in the tissues of rats injected with zinc.
A radioimmunoassay specific for metallothionein-I (MT-I) has been used for the estimation of this protein in various tissues of the rat following a single intraperitoneal injection of ZnSO4 (2 mg Zn/kg body weight). Quantitative information has also been obtained about the rates of clearance of MT-I from selected tissues following Zn injection. Wide variations were observed in the basal levels of the protein in different tissues of the rat. The levels of MT-I (micrograms/g fresh weight) were high in kidneys (16.9) and testes (14.3), whereas liver (2.4), pancreas (2.2), intestinal mucosa (2.6) and spleen (0.53) contained only small amounts of the protein. MT-I levels in the liver and pancreas increased several fold following Zn injection. The liver had the highest MT-I levels (23.1) at 12 h post-injection, whereas pancreas recorded the highest level (21.6) at 4 h post-injection. The intestinal mucosa registered only a small increase in MT-I; kidneys, spleen and testes were almost unaffected. Concentrations of MT-I returned to the basal levels at 72 h post-injection in all the tissues studied. The biological half-lives of MT-I in the liver, pancreas and mucosa were estimated to be 13.7, 9.8 and 15.0 h respectively.
Animals↗
Effects of dietary copper supplementation of rats on the occurrence of metallothionein-I in liver and its secretion into blood, bile and urine.
The appearance and excretion of metallothionein-I (MT-I) was studied in rats given a diet containing 1000 mg of Cu/kg for several weeks. No significant increase in MT-I concentrations in liver, plasma or bile was detected in rats with liver copper concentrations less than 600 micrograms of Cu/g fresh wt. Above this concentration, liver MT-I concentrations increased in proportion to the increase in hepatic copper content. Plasma and bile MT-I concentrations were directly related to those in the liver and were about 10 times those in normal rats. Urinary MT-I concentration also increased 10-fold within 1 week. Fractionation of bile and urine on Sephadex G-50 revealed the presence of monomeric MT-I and a range of possible degradation products of the isoprotein.
Animals↗
Studies on the metabolism of rat liver copper-metallothionein.
The degradation of purified 35S-labelled rat liver isometallothioneins (MT) by lysosomal extracts was studied. Zn-MT-I was more readily hydrolysed than Zn-MT-II, but no significant degradation of the Cu-containing metallothioneins could be detected, even after 24 h incubation. The susceptibility of MT to degradation in vitro may be related to the strength of the metal-thiolate bonds. However, the turnover rates of cytosolic MT in vivo, as established by pulse-labelling techniques, are apparently subject to different controls. The half-lives of MT-I and -II in the liver cytosol of Cu2+-injected rats were only 15.4 +/- 1.5 and 18.2 +/- 1.1 h respectively. Approx. 25% of the total liver MT was present in particulate fractions (probably in lysosomes) of the liver and had a half-life of 25.1 +/- 4.1 h.
Animals↗
Species differences in the occurrence of copper-metallothionein in the particulate fractions of the liver of copper-loaded animals.
Large amounts of Cu-metallothionein were obtained by 2-mercaptoethanol and sodium dodecyl sulphate extractions of the particulate fractions of the liver of pigs given high-Cu2+ diets or rats injected with Cu2+. Three isoproteins were purified from pig liver and characterized on the basis of their physicochemical properties, metal content and amino acid composition. No such pool of Cu-metallothionein was present in the liver of Cu2+-loaded sheep or of rats given Cu2+-supplemented diets.
Amino Acids↗
Metallothionein-I in the plasma and liver of neonatal rats.
The concentrations of metallothionein-I in the plasma and liver of neonatal rats were measured by radioimmunoassay. Plasma concentrations of the protein in male and female 4-day-old rats were 350 and 740 ng/ml respectively, and declined rapidly to only 3.5 ng/ml at 32 days of age. Concentrations in liver were also high in the newborn rats (200 micrograms/g), and declined from 12 days of age onwards.
Animals↗
Purification and properties of purine hydroxylase II from Aspergillus nidulans.
Purine hydroxylase II from Aspergillus nidulans has been purified to near homogeneity. The enzyme has a pI of 5.7, a molecular weight of 300,000, and two subunits with molecular weight of 153,000 each. The enzyme contains 2 FAD, 2 molybdenum atoms, and 4 (2 Fe-2S) iron-sulfur centers per molecule and exhibits broad specificity for reducing and oxidizing substrates. Among the more notable characteristics are the ability to oxidize hypoxanthine and nicotinic acid but not xanthine and virtually complete inactivity with oxygen. Moreover, while the enzyme is inactivated by borate and methanol, it is very resistant to cyanide and arsenite and it not inactivated by allopurinol. At infinite concentrations of reducing and oxidizing substrates, the Km for hypoxanthine was 119 microM, for nicotinic acid was 136 microM, and for NAD+ was 525 microM.
Alcohol Oxidoreductases↗