Molecular characterization of cu/Zn SOD gene in Asian clam Corbicula fluminea and mRNA expression and enzymatic activity modulation induced by metals
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Corbicula fluminea
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Superoxide dismutase is an important antioxidant enzyme in animals and plays an important role to remove excess free radicals which achieved mainly through the copper-zinc superoxide dismutase and manganese superoxide dismutase. Copper may directly or indirectly affect the copper-zinc superoxide dismutase activity, while the change of reactive oxygen species induced by copper deficiency may regulate manganese superoxide dismutase expression through the transcription factor NF-κB and AP-1.
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Abstract In order to determine the primary structure of banana shrimp, Penaeus merguiensis , vitellogenin (Vg), we previously purified vitellin (Vt) from the ovaries of vitellogenic females, and chemically analyzed the N‐terminal amino acid sequence of its 78 kDa subunit. In this study, a cDNA from this species encoding Vg was cloned based on the N‐terminal amino acid sequence of the major 78 kDa subunit of Vt and conserved sequences of Vg/Vt from other crustacean species. The complete nucleotide sequence of Vg cDNA was achieved by RT‐PCR and 5′ and 3′ rapid amplification of cDNA ends (RACE) approaches. The full‐length Vg cDNA consisted of 7,961 nucleotides. The open reading frame of this cDNA encoding a precursor peptide was comprised of 2,586 amino acid residues, with a putative processing site, R‐X‐K/R‐R, recognized by subtilisin‐like endoproteases. The deduced amino acid sequence was obtained from the Vg cDNA and its amino acid composition showed a high similarity to that of purified Vt. The deduced primary structure, of P . merguiensis Vg was 91.4% identical to the Vg of Penaeus semisulcatus and was also related to the Vg sequences of six other crustacean species with identities that ranged from 86.9% to 36.6%. In addition, the amino acid sequences corresponding to the signal peptide, N‐terminal region and C‐terminal region of P . merguiensis Vg were almost identical to the same sequences of the seven other reported crustacean species. Results from RT‐PCR analysis showed that Vg mRNA expression was present in both the ovary and hepatopancreas of vitellogenic females but was not detected in other tissues including muscle, heart, and intestine of females or in the hepatopancreas of mature males. These results indicate that the Vg gene may be expressed only by mature P . merguiensis females and that both the ovary and hepatopancreas are possible sites for Vg synthesis in this species of shrimp. Mol. Reprod. Dev. © 2006 Wiley‐Liss, Inc.
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Abstract: In some families with amyotrophic lateral sclerosis (ALS), the disease is linked to mutations in the gene encoding CuZn‐superoxide dismutase. The mutant CuZn‐superoxide dismutases appear to cause motor neuron degeneration by a toxic property, suggested to be linked to an altered reactivity of the active‐site Cu ions. Asp 90 Ala mutant CuZn‐superoxide dismutase was isolated from six patients with ALS, allowing properties of the mutant enzyme synthesized and conditioned in patients with ALS to be examined. The molecular mass of the Asp 90 Ala mutant CuZn‐superoxide dismutase was 45 Da lower than that of the wild‐type enzyme, as expected from the amino acid exchange. The mobility after sodium dodecyl sulfate‐polyacrylamide gel electrophoresis was markedly increased, however, suggesting altered properties of the polypeptide. The mutant CuZn‐superoxide dismutase showed a minimal reduction in stability but did not differ significantly from the wild‐type enzyme in enzymic activity, in content and affinity for active‐site Cu ions and in the propensity to catalyze formation of hydroxyl radicals. Our findings suggest that the deleterious effect of mutant CuZn‐superoxide dismutases on motor neurons in ALS is not related to altered reactivity of active‐site Cu ions, resulting in increased oxidant stress. Attention should therefore also be directed at other mechanisms and properties of the mutant polypeptides and their degradation products.
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Superoxide Dismutase(SOD)(EC 1.15.1.1)is a metalloenzyme that is found in almost all organisms and catalyzes the dismutation of superoxide anion radical to hydrogen peroxide and molecular oxygen.Three unique and highly compartmentalized mammalian SOD have been biochemically and molecularly characterized to date:Cu,Zn superoxide dismutase(CuZnSOD,SOD1), MnSOD(Manganese Superoxide Dismutase,SOD2)and EC-SOD(Extracellular Superoxide Dismutase,SOD3).Cu,Zn superoxide dismutase(CuZnSOD,SOD1)is a copper and zinc-containing homodimer that is found almost exclusively in intracellular cytoplasmic spaces.CuZnSOD is widely distributed and comprises about 90% of the total SOD.Cytoplasmic and periplasmic SOD exists as dimers, whereas chloroplastic and extracellular enzymes exist as tetramers.Structure supports independent functional evolution in prokaryotes and eukaryotes.CuZnSOD are thought to protect the brain,lungs,and other tissues from oxidative stress.This paper reviewed the gene, molecular and chemical structure and biological function of CuZnSOD.
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Preparations of liver large granule fraction from control and copper-injected rats were treated with digitonin (0.01-0.16 mg/mg protein) and supernatants rich in lysosomal matrix and in mitochondrial intermembrane space were obtained. After copper injection the superoxide dismutase activity in all supernatants was significantly increased. The granular CuZn-superoxide dismutase in the two animal groups was localized in lysosomes only. The cytosolic and lysosomal CuZn-superoxide dismutase in control preparations showed an equal electrophoretic pattern (two peaks with Rf = 0.42 and 0.47). After copper injection three new electrophoretic peaks of the lysosomal CuZn-superoxide dismutase activity (with Rf = 0.37, 0.52 and 0.62 respectively) appeared. The increased heterogeneity of the granular CuZn-superoxide dismutase activity after copper injection is explained by the oxidative degradation of the CuZn-superoxide dismutase in lysosomes.
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A mixture of tetradecamer oligodeoxyribonucleotides complementary to the codons specifying the carboxyl-terminal sequence, Ile-His-Pro-Phe-His, of angiotensin was chemically synthesized as two pools and used for the isolation of a cDNA clone specific for angiotensinogen from a cDNA bank of rat liver mRNA sequences. The two pools (oligo 1 and oligo 2), each containing 24 oligodeoxyribonucleotides, were first used as primers to initiate reverse transcription of rat liver mRNA. One of the pools (oligo 1) was found to prime a specific 32P-labeled cDNA of approximately 160 nucleotides that contained the anticoding sequence corresponding exactly to the amino acid sequence of rat angiotensin. This cDNA, in turn, was used to rescreen cDNA clones that were isolated by initially selecting the rat liver cDNA bank by hybridization with the oligo 1 mixture. One clone thus obtained, designated pRag16, was subjected to nucleotide sequence analysis and verified to contain a nearly full-length cDNA sequence coding for rat angiotensinogen precursor. The deduced amino acid sequence indicates that the precursor molecular consists of angiotensinogen of 453 amino acid residues and a putative signal peptide of 24 amino acid residues. The predicted molecular weight and amino acid composition of angiotensinogen agree well with those determined by using the purified protein. An angiotensin moiety is located at the amino-terminal part of angiotensinogen, preceded directly by the signal peptide and followed by a large carboxyl-terminal sequence that contains two internally homologous sequences and three potential glycosylation sites.
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A human Colony Stimulating Factor-1 (CSF-1 or Macrophage-CSF) encoding cDNA was previously isolated from the pancreatic tumor cell line MIA-PACA-2 (I). This cDNA encodes a protein of 26 Kd molecular weight. To isolate a murine cDNA homologous to human M-CSF, a probe from the coding region of the human cDNA was prepared by nick-translation. A recotnbinant lambda phage library made from the mRNA of the murine fibroblast cell line. L929, was screened with the human M-CSF cDNA probe The cDNA isolated from the murine library has an open reading frame of 1656 nucleotides which encodes a protein of 552 amino acids. The first 32 amino acids encode a putative signal peptide which when cleaved yields a mature protein beginning with the amino acid sequence: Lys-Glu-Val-Ser. This deduced sequence is identical to the amino terminal residues reported for M-CSF as purified from L-cell supernatant? (2). The mature protein deduced from the cDNA sequence has a molecular weight of 57.261 d. All ten of the cysteine residues are found in approximately the amino-terminal half of the molecule. There are three potential relinked glycosylation sites (Asn-X-Thr/Ser) located at residues 154, 172, and 378. From the first amino acid encoded by the cDNA to residue 180 and from residue 476 to the cartxwy-terminal residue, approximately 80% homology is found between the murine and human cDNA's at both the nucleotide and amino acid levels. For comparison the deduced human M-CSF protein sequence is aligned below that of the murine sequence. The open reading frame in the murine cDNA encodes an additional 295 amino acids which are not found in the deduced amino-acid sequence of human M-CSF (I). However, higher molecular weight forms of human M-CSF mRNA have also been identified by Northern analysis of MIA-PACA-2 mRNA using the 26 Kd encoding cDNA as a probe (3). Furthermore, the murine M-CSF encoding cDNA we isolated was incorporated into an animal cell expression vector under SV40 early promoter transcrlptional control. When the vector was transfected Into the simian cell line, COS-7, murine M-CSF biological activity, as measured on mouse bone marrow cells, was observed in the transfected-culture supernalants
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Previous studies have demonstrated a 50% increase in pulmonary superoxide dismutase (SOD) activity in oxygen-adapted rats and have suggested that SOD plays a significant role in the development of "tolerance". To further study these events, the cuprozinc SOD was purified from rat liver and found to be similar to previously purified cuprozinc superoxide dismutases. A rabbit antisera to rat cuprozinc SOD was produced and used to perform antibody titrations of SOD in the lungs of rats exposed to 85% O2 for 5 days. The absolute amount of cuprozinc SOD increased 41% by antibody titration which accounted for most, if not all, of the 48% increase demonstrated in total SOD activity. Spectrophotometric assays at pH 7.8 and 10.0 of pure rat cuprozinc SOD and crude lung homogenates suggest that there is also a manganese SOD present, but the role of that SOD in the development of oxygen tolerance has not been established.
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