RESUMEN
Metallothionein (MT) is an ubiquitous heavy metal-binding protein which has been identified in animals, plants, protists, fungi and bacteria. In insects, primary structures of MTs are known only for Drosophila and the collembolan, Orchesella cincta. The MT cDNA from O. cincta encodes a 77 amino acid protein with 19 cysteines. Isolations of the protein itself have demonstrated the presence of two smaller metal-binding peptides, whose amino acid sequences correspond to parts of the cDNA, and which apparently result from cleavage of the native protein. The present study was undertaken to complete the picture of cleavage sites within the MT protein by applying protein isolation techniques in combination with mass spectrometry and N-terminal sequence analysis. Further, recombinant expression allowed us to study the intrinsic stability of the MT and to perform in vitro cleavage studies. The results show that the MT from O. cincta is specifically cleaved at two sites, both after the amino acid sequence Thr-Gln (TQ). One of these sites is located in the N-terminal region and the other in the linker region between two putative metal-binding clusters. When expressed in Escherichia coli, the recombinant O. cincta MT can be isolated in an uncleaved form; however, this protein can be cleaved in vitro by the proteolytic activity of O. cincta. In combination with other studies, the results suggest that the length of the linker region is important for the stability of MT as a two domain metal-binding protein.
Asunto(s)
Cadmio/metabolismo , Metalotioneína/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Animales , Expresión Génica , Insectos/metabolismo , Metalotioneína/genética , Metalotioneína/aislamiento & purificación , Metales/metabolismo , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/aislamiento & purificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/metabolismoRESUMEN
Red chlorophyll catabolite (RCC) reductase (RCCR) and pheophorbide (Pheide) a oxygenase (PaO) catalyse the key reaction of chlorophyll catabolism, porphyrin macrocycle cleavage of Pheide a to a primary fluorescent catabolite (pFCC). RCCR was purified from barley and a partial gene sequence was cloned (pHvRCCR). The gene was expressed at all stages of leaf development and in roots. By comparison with different databases, genomic sequences and expressed sequence tags similar to RCCR were found in phylogenetically diverse species, and activity of RCCR was demonstrated in two of them, Arabidopsis thaliana and Marchantia polymorpha. The gene of A. thaliana (AtRCCR) was employed for molecular cloning, heterologous expression and the production of polyclonal antibodies. With recombinant RCCR, the major product of RCC reduction was pFCC-1, but small quantities of its C1 epimer, pFCC-2, also accumulated. The reaction required reduced ferredoxin and was sensitive to oxygen. AtRCCR encoded a 35 kDa protein which was used for chloroplast import experiments. Upon transport, it was processed to a mature form of 31 kDa. The significance of cloning of RCCR is discussed in respect to the evolution of chlorophyll catabolism and to the cloning of PaO.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/genética , Hordeum/genética , Oxidorreductasas/genética , Proteínas de Plantas/genética , Secuencia de Aminoácidos , Proteínas Reguladoras de la Apoptosis , Arabidopsis/química , Secuencia de Bases , Southern Blotting , Clorofila/metabolismo , Cloroplastos/metabolismo , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Hordeum/química , Datos de Secuencia Molecular , Oxidorreductasas/química , Oxidorreductasas/aislamiento & purificación , Hojas de la Planta/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Electrospray ionization (ESI) mass spectra of both well-characterized and novel metallothioneins (MTs) from various species were recorded to explore their metal-ion-binding modes and stoichiometries. The ESI mass spectra of the zinc- and cadmium-binding MTs showed a single main peak corresponding to metal-to-protein ratios of 4, 6, or 7. These findings combined with data obtained by other methods suggest that these MTs bind zinc or cadmium in a single predominant form and are consistent with the presence of three- and four-metal clusters. An unstable copper-specific MT isoform from Roman snails (Helix pomatia) could be isolated intact and was shown to preferentially bind 12 copper ions. To obtain additional information on the formation and relative stability of metal-thiolate clusters in MTs, a mass spectrometric titration study was conducted. One to seven molar equivalents of zinc or of cadmium were added to metal-free human MT-2 at neutral pH, and the resulting complexes were measured by ESI mass spectrometry. These experiments revealed that the formation of the four-metal cluster and of the thermodynamically less stable three-metal cluster is sequential and largely cooperative for both zinc and cadmium. Minor intermediate forms between metal-free MT, Me4MT, and fully reconstituted Me7MT were also observed. The addition of increasing amounts of cadmium to metal-free blue crab MT-I resulted in prominent peaks whose masses were consistent with apoMT, Cd3MT, and Cd6MT, reflecting the known structure of this MT with two Me3Cys9 centers. In a similar reconstitution experiment performed with Caenorhabditis elegans MT-II, a series of signals corresponding to apoMT and Cd3MT to Cd6MT species were observed.
Asunto(s)
Metalotioneína/química , Metalotioneína/metabolismo , Animales , Cadmio/metabolismo , Cobre/metabolismo , Estabilidad de Medicamentos , Humanos , Técnicas In Vitro , Espectrometría de Masas/métodos , Peso Molecular , Unión Proteica , Zinc/metabolismoRESUMEN
The roundworm Caenorhabditis elegans adapted for survival at high concentrations of Cd(II) expresses two isoforms of metallothionein, CeMT-I and CeMT-II. To characterize one of these proteins CeMT-II was prepared as its Cd containing form by expressing its cDNA heterologously in Escherichia coli. The purified 63-amino-acid protein was identified as the desired product by ion-spray mass spectrometry and was found to resemble in most of its chemical and spectroscopic features the metallothioneins of other animal phyla. The recombinant protein contains a total of 18 cysteine residues and, as documented by electrophoresis and mass spectrometry, binds firmly six Cd ions through the cysteine's side chains. The (113)Cd NMR spectrum features six (113)Cd resonances. Their chemical shift positions between 615 and 675 ppm denote the existence of clusters of tetrahedrally coordinated cadmium thiolate complexes. The metal thiolate coordination dominates also the electronic far-UV absorption spectrum. It is characterized by a massive absorption profile with Cd thiolate shoulders at 255 and 235 nm. Upon replacement of Cd by Zn the profile was blue-shifted by 30 nm.
Asunto(s)
Caenorhabditis elegans/química , Proteínas del Helminto/aislamiento & purificación , Metalotioneína/aislamiento & purificación , Animales , Secuencia de Bases , Caenorhabditis elegans/genética , ADN de Helmintos/genética , ADN Recombinante/genética , Expresión Génica , Genes de Helminto , Proteínas del Helminto/química , Proteínas del Helminto/genética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metalotioneína/química , Metalotioneína/genética , Datos de Secuencia Molecular , Peso Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The induction of metallothionein was studied in the springtail Orchesella cincta (Collembola), a species of insect living in forest soils. Upon dietary exposure to Cd, two Cd-binding, cysteine-rich peptides were isolated from whole-body homogenates, using gel filtration and reversed-phase FPLC. Mass spectrometric analysis revealed that the molecular masses of these peptides were 2989 Da and 4139 Da, respectively. Amino acid sequencing of the 2989-Da peptide resulted in a sequence typical for a metallothionein. Sequencing of the 4139-Da protein was unsuccessful, probably due to N-terminal blockage. Using different PCR techniques (3' and 5' RACE) with (degenerate) primers based on the identified amino acid sequence of the 2989 Da peptide, a metallothionein cDNA was isolated. The sequence of this cDNA potentially codes for a protein of 77 amino acids. The 2989 Da peptide corresponds to the C-terminal part of this protein. The 4139-Da protein is probably encoded by the N-terminal part of this protein. These results suggest that the identified peptides are products of one gene, and that the primary gene product is subject to post-translational processing. The deduced amino acid sequence of the O. cincta metallothionein shows low sequence similarity with metallothioneins from Drosophila. The similarity between O. cincta MT and MTs of invertebrates is not higher than that between O. cincta and vertebrates.
Asunto(s)
Cadmio/farmacología , Sistema Digestivo/metabolismo , Insectos/efectos de los fármacos , Metalotioneína/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Resistencia a Medicamentos , Metalotioneína/aislamiento & purificación , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Suelo/parasitologíaRESUMEN
The basal amounts of metallothionein (MT) and its rates of biosynthesis were compared in resting and proliferating Chang liver (CCl-13) cells. In resting cells the total amounts of the detectable isoforms MT-2 and MT-1e were approx. 1.6x10(6) and 4x10(5) molecules per cell respectively. In exponentially growing cultures the cellular contents of both isoforms increased co-ordinately approx. 4-fold and decreased again to the initial values within 48 h after entering density-mediated growth arrest. As documented for MT-2 its transient accretion was attributable to a 10-fold rise in the rate of biosynthesis of this protein during the growth phase. Measurements of the relative amounts of MT-2 mRNA indicated the occurrence of a more than 50% increase within the first 12 h after subculturing of the cells, followed by a return to basal levels thereafter. These results denote a direct link between the programming of MT synthesis and proliferation and thus attest to a central housekeeping function of the MTs.
Asunto(s)
Hígado/citología , Hígado/metabolismo , Metalotioneína/metabolismo , Northern Blotting , División Celular , Extractos Celulares/análisis , Línea Celular , Cromatografía Líquida de Alta Presión , ADN/biosíntesis , Humanos , Hígado/química , Metalotioneína/biosíntesis , Metalotioneína/aislamiento & purificación , Hibridación de Ácido Nucleico , Proteínas/análisis , ARN/aislamiento & purificaciónRESUMEN
A novel copper-binding metallothionein (MT) has been purified from mantle tissue of the terrestrial snail Helix pomatia using gel-permeation chromatography, ion-exchange chromatography and reverse-phase HPLC. Copper was removed from the thionein by addition of ammonium tetrathiomolybdate. The resulting apothionein (molecular mass 6247 Da) was S-methylated and digested with trypsin, endoproteinase Arg-C and endoproteinase Lys-C. Amino acid sequences of the resulting peptides were determined by collision-induced dissociation tandem MS. The protein is acetylated at its N-terminus, and consists of 64 amino acids, 18 of which are cysteine residues. A comparison with the cadmium-binding MT isolated from the midgut gland of the same species shows an identical arrangement of the cysteines, but an unexpectedly high variability in the other amino acids. The two MT isoforms differ in total length and at 26 positions of their peptide chains. We suggest that the copper-binding MT isoform from the mantle of H. pomatia is responsible for regulatory functions in favour of copper, probably in connection with the metabolism of the copper-bearing protein, haemocyanin.
Asunto(s)
Cobre/metabolismo , Caracoles Helix/química , Metalotioneína/química , Metalotioneína/metabolismo , Secuencia de Aminoácidos , Animales , Apoproteínas/aislamiento & purificación , Apoproteínas/metabolismo , Metalotioneína/aislamiento & purificación , Metalotioneína/fisiología , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , EstereoisomerismoRESUMEN
Partial metal depletion and 113Cd-NMR studies have suggested that the recombinant Cd-containing metallothionein of the sea urchin Strongylocentrotus purpuratus (Cd7-MTA) binds its metal ions in a four-metal (Cd4Cys11) and a three-metal (Cd3Cys9) cluster associated with the N-terminal and C-terminal halves of the protein, respectively [Wang, Y., Mackay, E.A., Zerbe, O., Hess, D., Hunziker, P.E., Vasak, M. & Kägi, J.H.R. (1995) Biochemistry 34,7460-7467]. This partitioning has now been confirmed by bisecting native Cd7-MTA with subtilisin into products bearing only a single metal-thiolate cluster. Their separation by reverse-phase HPLC and on-line electrospray mass spectrometry in combination with sequence analysis revealed selective cleavage of the protein into a set of N-terminal polypeptides containing 37-39 residues with four Cd ions and a set of C-terminal polypeptides containing 24 and 25 residues with three Cd ions. Thus, sea urchin MTA like its mammalian counterparts is made up of two separate cluster-harboring domains. The fragmentation pattern indicated that the sites of cleavage are located in the peptide loop interspaced between the first two metal-bound cysteine residues of the C-terminal domain. Accordingly, with cleavage, one of the putative nine thiolate ligands of the three-metal cluster was lost to the N-terminal fragment. The coordinational consequences of this repartition were reflected in massive chiroptical changes accompanying the cleavage process. While the liberated N-terminal domain retained the CD profile of the four-metal cluster in the parent protein and thereby indicated preservation of its structure, the CD features attributable to the intact three-metal cluster were largely lost on cleavage. The vanished features bear strong resemblance to the large biphasic ellipticity signal at 250 nm which dominates the CD spectrum of native Cd7-MTA, and allow us thus to attribute this signal to excitonic coupling interactions of Cd-thiolate chromophores in the three-metal cluster.
Asunto(s)
Cadmio/química , Metalotioneína/química , Erizos de Mar/química , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Animales , Quelantes/farmacología , Dicroismo Circular , Ácido Edético/farmacología , Espectrometría de Masas , Metalotioneína/efectos de los fármacos , Metalotioneína/genética , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , EspectrofotometríaRESUMEN
A protocol based on a combination of established methods for the characterization and identification of inducible stress proteins in human cell lines is described. A particular protein spot, collected from several micropreparative two-dimensional electrophoresis (2-DE) gels, is concentrated into a new gel prior to simultaneous electrotransfer onto a Cationic Durapore (CD) membrane and onto a polyvinylidene (PVDF) backup membrane. The protein blotted onto the PVDF support is subjected to N-terminal sequence analysis. From the protein bound to the CD membrane the peptide mass profile is obtained by proteolytic digestion of the protein followed by the separation of the resulting peptides by high performance liquid chromatography (HPLC) and their detection by on-line electrospray mass spectrometry (LC/MS). Additional internal sequence information may be obtained by amino acid sequence analysis of peptides collected in the HPLC effluent. The efficiency of this strategy is demonstrated with two proteins extracted from 15 micropreparative 2-DE gels of an extract of a human liver cell line. The peptide mass fingerprinting of a 60 kDa protein with a pI of 5.3 assigned 22 of 37 peptides to the heat shock protein 60 (Hsp 60). The result was confirmed by the N-terminal sequence analysis of the undigested protein and of an internal tryptic fragment. The second sample, a 40 kDa protein with a pI of 4.9, was identified as a processed form of the heat shock cognate 71 kDa protein (Hsc 70).
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Proteínas de Choque Térmico/análisis , Espectrometría de Masas/métodos , Membranas Artificiales , Polivinilos , Secuencia de Aminoácidos , Línea Celular , Cromatografía Líquida de Alta Presión , Humanos , Datos de Secuencia MolecularRESUMEN
The characterization of degradation products of metallothionein (MT) in rat liver is reported. Using gel filtration chromatography on Sephadex G-75 an unknown zinc (Zn)-containing peak was found in livers of rats injected with zinc. It contained at least 13 peptide fractions, designated as LPI-1, LPI-2, LPI-3, LPI-4, LPII-1, LPII-2, LPII-3, LPIII-1, LPIII-2, LPIII-3, LPIII-4, LPIII-5 and LPIII-6. While two of these fractions (LPII-1 and LPIII-4) were identified as the carboxyl-terminal half (alpha-domain) of rat MT-1, LPI-2 and LPI-3 corresponded to the amino-terminal half (beta-domain) of rat MT-2. In contrast, neither the beta-domain of rat MT-1 nor the alpha-domain of rat MT-2 could be recovered. The data may indicate that the two metal clusters of Zn-MT-1 and Zn-MT-2 differ in their stabilities resulting in differences in their susceptibility towards proteolytic degradation.
Asunto(s)
Hígado/metabolismo , Metalotioneína/metabolismo , Zinc/metabolismo , Secuencia de Aminoácidos , Animales , Masculino , Metalotioneína/análisis , Metalotioneína/química , Datos de Secuencia Molecular , Péptidos/análisis , Péptidos/aislamiento & purificación , Ratas , Ratas Sprague-DawleyRESUMEN
1. Two cadmium-binding metallothionein (Mt) isoforms, called Mta and Mtb, were isolated from terrestrial snails (Arianta arbustorum), using various chromatographic techniques, such as gel-permeation chromatography and reversed-phase HPLC. The purified proteins were S-methylated and cleaved by means of different enzymes (trypsin, endoproteinase Glu-C, and endoproteinase Asp-N). Amino acid sequences were determined by automated Edman degradation and collision-induced dissociation (CID) tandem MS. According to their primary structures, both isoforms should be attributed to class-I Mts. 2. The two forms are structurally identical, differing only by one amino acid exchange in position 60 of the peptide chain. Both isoproteins consist of 66 amino acids, 18 of which are cysteine residues. Most of the cysteine residues are arranged in seven Cys-Xaa-Cys motifs. Mta and Mtb possess an N-terminal acetylated-serine residue and contain a short N-terminal motif which shows a high degree of similarity with the N-termini of histones H4 and H2A. 3. A comparison of Mta and Mtb with other invertebrate Mts shows a very high degree of sequence similarity with a cadmium-binding Mt from Helix pomatia, a species that is closely related to Arianta arbustorum. Moreover, Mta and Mtb, as expected, also exhibit structural similarities with Mts from other molluscan species, such as mussels and oysters. It is suggested that Mta and Mtb represent two allelic isoforms, reflecting the genetic polymorphism of Mt in Arianta arbustorum.
Asunto(s)
Metalotioneína/química , Caracoles/química , Secuencia de Aminoácidos , Animales , Contaminación Ambiental , Isomerismo , Espectrometría de Masas , Metalotioneína/aislamiento & purificación , Datos de Secuencia Molecular , Mapeo Peptídico , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We explored the molecular genetics underlying the massive induction of isoMTs by Zn2+ or Cd2+ in metal tolerant rabbit kidney (RK-13) sub-line cells, using band shift assays and Southern blotting analysis. In sub-line cells accommodated to intermediate metal concentrations (100 microM Zn2+; 1-20 microM Cd2+) evidence suggested that the increase in the capacity for isoMT synthesis is brought about by an increased binding activity of the nuclear transcription factors MTF-1 and Sp1. Using quantitative band shift analysis with a mouse MRE-d oligonucleotide probe, the binding of both transcription factors was found to be enhanced two to three times over the binding activity measured in the unexposed parental RK-13 cells. Their increase in binding activity is probably the cause of the overexpression of MT genes and the development of metal tolerance in these cells. In cells tolerant to the highest concentrations of metal the analysis of Southern blot signals revealed MT gene amplification to be the most probable cause of the increased MT production. Thus, in cells of sub-lines growing in the presence of 350 microM Zn2+, two of the isoMT genes were coordinately triplicated and in cells tolerant to 150 microM Cd2+ one isoMT gene was amplified two-fold.
Asunto(s)
Cadmio/metabolismo , Riñón/metabolismo , Metalotioneína/metabolismo , Zinc/metabolismo , Animales , Cadmio/farmacología , Línea Celular , Regulación de la Expresión Génica , Metalotioneína/genética , Conejos , Factores de Transcripción/metabolismo , Zinc/farmacologíaRESUMEN
The mode of metal binding in sea urchin metallothionein (MT) was explored by electronic absorption, chiroptical, NMR, and mass spectroscopic methods. Recombinant sea urchin MT containing 7 equiv of the natural mixture of Cd isotopes was stripped of the metal by exposure to low pH and reconstituted with 113Cd (> 95% enriched). Comparison of the electronic spectroscopic and chiroptical features and the 113Cd NMR spectra of the reconstituted material with those of the native recombinant material indicated that the reconstituted material had regained the native conformation. The shoulder at 250 nm in the electronic absorption spectrum, the biphasic circular dichroism profile centered at 250 nm, and the chemical shift positions (605-695 ppm) of the seven 113Cd NMR resonances all strongly suggested that sea urchin MT like all other well characterized MTs contains clusters made up of tetrahedral Cd-thiolate units. The 113Cd chemical shift correlation spectrum of the reconstituted protein proved the existence of such metal clusters and allowed the unambiguous assignment of some of the metal connectivities. Homonuclear decoupling experiments in which Cd resonances were selectively saturated indicated moreover a partitioning of the metal complement into two separate clusters containing three and four Cd ions. The same proposition was supported by the selective reduction of three 113Cd resonances upon partial metal depletion following exposure of the protein to EDTA. Thus, the three-metal cluster is energetically less stable than the four-metal cluster.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Metalotioneína/química , Secuencia de Aminoácidos , Animales , Cadmio , Ácido Edético , Isótopos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Metalotioneína/biosíntesis , Metalotioneína/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Mapeo Peptídico , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Erizos de Mar , Espectrofotometría , Espectrofotometría Atómica , Subtilisinas , Compuestos de SulfhidriloRESUMEN
Metallothionein from tissues of rabbits exposed to cadmium chloride was separated into seven distinct isoforms by reverse-phase liquid chromatography and their complete amino acid sequences were determined. Five of the seven isometallothioneins showed structural features so far not identified in other mammalian metallothioneins. Thus, two isoproteins contain a polypeptide with a chain length of 62 rather than 61 amino acid residues. Two isoforms are characterized by an additional positive charge and one by the presence of an isopeptide bond between aspartic acid and serine in the N-terminal half of the protein. The isoproteins characterized were identified from different sources: rabbit liver and kidney and a rabbit kidney cell-line (RK-13). In all three, the structural characteristics of the individual isoforms are retained, indicating that in the different tissues the same mechanisms control the synthesis and the stability of the different cadmium-induced isoMTs.
Asunto(s)
Metalotioneína/química , Secuencia de Aminoácidos , Animales , Cadmio/farmacología , Cloruro de Cadmio , Línea Celular , Cloruros/farmacología , Cromatografía Líquida de Alta Presión , Concentración de Iones de Hidrógeno , Riñón/química , Hígado/química , Metalotioneína/aislamiento & purificación , Datos de Secuencia Molecular , Mapeo Peptídico , Conejos , TripsinaRESUMEN
Cadmium-induced metallothioneins from the common sea mussel, Mytilus edulis, were shown to comprise of two groups of isoforms having apparent molecular masses of 10 kDa and 20 kDa. The 10-kDa group was resolved by anion-exchange chromatography into four fractions while the 20-kDa group was resolved into three fractions using this method. After metal removal and S-methylation of the cysteine residues using methyl-p-nitrobenzenesulphonate the complete amino acid sequences were determined. Five isoforms of the 20-kDa group were shown to possess monomeric units consisting of 71 amino acids. These proteins were distinct from the four 72-amino-acid proteins of the 10-kDa group. The FASTA algorithm has been used to compare the degree of similarity between the mussel metallothionein MT-10-IV isoform and other metallothioneins. The mussel MT-10-IV isoform exhibited substantial similarity to other molluscan metallothioneins. Moreover, the mussel metallothionein exhibited more similarity to vertebrate metallothioneins than to those of non-molluscan invertebrates, thus suggesting that the mussel metallothioneins are class I metallothioneins.
Asunto(s)
Bivalvos/química , Metalotioneína/química , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Humanos , Metalotioneína/aislamiento & purificación , Datos de Secuencia Molecular , Mapeo Peptídico , Homología de Secuencia de AminoácidoRESUMEN
A cadmium-binding metallothionein has been purified from metal-exposed Roman snails (Helix pomatia) using gel-permeation, ion-exchange and reverse-phase high-performance liquid chromatography. The S-methylated protein was digested with trypsin and the endoproteinases Asp-N, Glu-C and Arg-C. While most of the resulting peptides could be sequenced by Edman degradation, the intact protein, as well as the N-terminal peptide, proved to be blocked. Analysis by mass spectrometry showed that the N-terminal amino acid was an acetylated serine residue. Snail metallothionein, which is suggested to be involved in the detoxification of cadmium, contains 66 amino acid residues, 18 of which are cysteine residues arranged in seven Cys-Xaa-Cys motifs. The calculated molecular mass of the protein is 6.62 kDa. The primary structure of snail metallothionein reveals a clear relationship with molluscan and vertebrate metallothioneins, but lower similarity with metallothioneins of other invertebrate species. The N-terminal region of the isolated protein proved to be unique among the metallothionein sequences determined so far, showing high degrees of similarity with the N-terminal sequences of histones H2A and H4 which may be important for regulatory functions.
Asunto(s)
Histonas/química , Metalotioneína/química , Homología de Secuencia de Aminoácido , Secuencia de Aminoácidos , Animales , Cadmio/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Caracoles Helix , Humanos , Metalotioneína/aislamiento & purificación , Datos de Secuencia Molecular , Mapeo PeptídicoRESUMEN
The synthesis of metallothionein (MT) was investigated in three different human epithelial cell lines, each derived from one of the embryonic germ layers. The accretion of different isoforms of the protein was monitored using a sensitive neutral-pH h.p.l.c. method. Induction of MT synthesis by zinc ions and dexamethasone revealed differences between the three cell lines, both with respect to the number and the amounts of the different isoMTs formed. Dose-response experiments showed that an increase in dexamethasone concentration enhances MT accretion asymptotically to a limit, whereas within the concentration range explored zinc produces an exponential augmentation.
Asunto(s)
Metalotioneína/biosíntesis , Células Cultivadas , Cromatografía Líquida de Alta Presión , Dexametasona/farmacología , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Células HeLa , Humanos , Hígado/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Metalotioneína/aislamiento & purificación , Zinc/farmacologíaRESUMEN
The effects of increasing concentrations of Zn(II) and Cd(II) on the expression of the four isometallothioneins (isoMTs), namely MT-1a, MT-2a, MT-2d and MT-2e, in rabbit kidney cells (RK-13) and the development of cellular tolerance to these metal ions were studied. The results showed that, whereas in parental cells MT concentration was low and composed nearly exclusively of MT-2a and MT-1a, all four isoMTs increased massively in abundance when the cells were exposed to toxic concentrations of Zn(II) or Cd(II), the relative increase being largest in the two minor isoforms MT-2d and MT-2e. While the response of the four isoMTs to the challenge by Zn(II) or Cd(II) was qualitatively comparable, there were differences in sensitivity and delay time, Cd(II) being the more efficient inducer and much faster in eliciting the onset of isoMT synthesis. An even larger production of isoMTs resulted when RK-13 cells were cultured in the presence of a series of metal concentrations yielding sub-lines of increased metal tolerance. In this instance too, there were marked differences in the response to Cd(II) and Zn(II). Thus, in cells of sub-lines selected for tolerance to moderate concentrations of Cd(II) the kinetic analysis of isoMT accretion gave indications of a saturable induction process while no such evidence was forthcoming for Zn(II). In sub-line cells selected for tolerance to the highest concentrations of Cd(II) or Zn(II) isoMT formation was increased by another order of magnitude, reaching for some isoforms a 100- to 1000-fold augmentation over the amounts measured in cells of the unexposed parental cells. A potentiation of this magnitude goes beyond the range of ordinary regulation of gene expression. It is to be viewed instead as an enlargement of the capacity of isoMT synthesis acquired by a variety of mechanisms in the surviving cells.
Asunto(s)
Cadmio/toxicidad , Riñón/metabolismo , Metalotioneína/biosíntesis , Zinc/toxicidad , Animales , División Celular , Células Cultivadas , Inducción Enzimática/efectos de los fármacos , Riñón/citología , Riñón/efectos de los fármacos , Conejos , Factores de TiempoRESUMEN
Reversed-phase high-performance liquid chromatography coupled with an on-line integrating chromatographic data system was used to separate and quantify the four major isometallothioneins MT-1a, MT-2a, MT-2d, and MT-2e present in metallothionein samples from cultured rabbit kidney cells as prepared by gel-filtration and/or ion-exchange chromatography. The standard curves for each of these isoforms showed closely comparable linear correlations between the amount of protein applied to the column and their integrated absorbance peak area at 220 nm. The lower limit of quantification is 30 pmol, sufficient to assess basal isometallothionein concentrations and to follow their variation upon metal exposure.
Asunto(s)
Riñón/química , Metalotioneína/análisis , Animales , Cadmio/química , Células Cultivadas , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Estudios de Evaluación como Asunto , Riñón/citología , Conejos , Zinc/químicaRESUMEN
The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.