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1.
A cytosolic superoxide dismutase (SOD) was purified and characterized from a fast-growing Mycobacterium sp. strain JC1 DSM 3803 grown on methanol. The native molecular weight of the purified SOD was estimated to be 48 kDa. SDS-PAGE
revealed a subunit of 23 kDa, indicating that the enzyme is a homodimer. The enzyme activity was inhibited by H2O2 and azide. The purified SOD contained 1.12 and 0.56 g-atom of Mn and Fe per mol of enzyme, respectively, suggesting that
it may be a Fe/Mn cambialistic SOD. The apo-SOD reconstitution study revealed that Mn salts were more specific than Fe salts
in the SOD activity. The gene encoding the SOD was identified from the JC1 cosmid genomic library by PCR screening protocol.
The cloned gene, sodA, had an open reading frame (ORF) of 624 nt, encoding a protein with a calculated molecular weight of 22,930 Da and pi of
5.33. The deduced SodA sequence exhibited 97.6% identity with that of Mycobacterium fortuitum Mn-SOD and clustered with other mycobacterial Mn-SODs. A webtool analysis on the basis of SOD sequence and structure homologies
predicted the SOD as a tetrameric Mn-SOD, suggesting that the protein is a dimeric Mn-SOD having tetramer-specific sequence
and structure characteristics. 相似文献
2.
《Free radical research》2013,47(6):519-531
A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms. 相似文献
3.
Xiaojing Xu Yijun Zhou Shanjun Wei Dongtao Ren Min Yang Huahu Bu Mingming Kang Junli Wang Jinchao Feng 《Molecules and cells》2009,27(4):423-428
Superoxide dismutases (SODs) constitute the first line of cellular defense against oxidative stress in plants. SODs generally
occur in three different forms with Cu/Zn, Fe, or Mn as prosthetic metals. We cloned the full-length cDNA of the Thellungiella halophila Cu/Zn-SOD gene ThCSD using degenerate RT-PCR and rapid amplification of cDNA ends (RACE). Sequence analysis indicated that the ThCSD gene (GenBank accession number EF405867) had an open reading frame of 456 bp. The deduced 152-amino acid polypeptide had
a predicted molecular weight of 15.1 kDa, an estimated pI of 5.4, and a putative Cu/Zn-binding site. Recombinant ThCSD protein
was expressed in Escherichia coli and assayed for SOD enzymatic activity in a native polyacrylamide gel. The SOD activity of ThCSD was inactivated by potassium
cyanide and hydrogen peroxide but not by sodium azide, confirming that ThCSD is a Cu/Zn-SOD. Northern blotting demonstrated
that ThCSD is expressed in roots, stems, and leaves. ThCSD mRNA levels increased by about 30-fold when plants were treated with sodium chloride (NaCl), abscisic acid (ABA), and indole-acetic
acid (IAA) and by about 50-fold when treated with UVB light. These results indicate that ThCSD is involved in physiological
pathways activated by a variety of environmental conditions.
These authors contributed equally to this work. 相似文献
4.
Screening the expression characteristics of several miRNAs in G93A‐SOD1 transgenic mouse: altered expression of miRNA‐124 is associated with astrocyte differentiation by targeting Sox2 and Sox9
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Fenghua Zhou Caixia Zhang Yingjun Guan Yanchun Chen Qiang Lu Linlin Jie Hailing Gao Hongmei Du Haoyun Zhang Yongxin Liu Xin Wang 《Journal of neurochemistry》2018,145(1):51-67
5.
Rachenko E. I. Rikhvanov E. G. Varakina N. N. Rusaleva T. M. Borovskii G. B. Voinikov V. K. 《Russian Journal of Plant Physiology》2004,51(2):198-202
The action mechanism of the mitochondrial inhibitor sodium azide on thermotolerance in Saccharomyces cerevisiae was studied. At ambient growth temperature, pretreatment with sodium azide was shown to improve the thermotolerance of parent cells and the hsp104 mutant. Treating with the inhibitor during a mild heat shock suppressed the development of induced thermotolerance due to the inhibition of heat shock protein (Hsp104) synthesis. Treating with the inhibitor immediately before lethal heat shock produced a variety of effects on thermotolerance depending on whether the yeast metabolism was oxidative or fermentative. The conclusions are: (1) the protective effect of sodium azide on the thermotolerance of S. cerevisiae cells grown on glucose-containing medium is not related to Hsp104 functioning, and (2) the mechanisms of basic and induced thermotolerance differ considerably. 相似文献
6.
Methionine biosynthesis in Saccharomyces cerevisiae: mutations at the regulatory locus ETH2. I. Genetic data 总被引:4,自引:0,他引:4
Monique Masselot Huguette de Robichon-Szulmajster 《Molecular & general genetics : MGG》1974,135(4):339-348
Summary Evidence has been obtained that sodium azide is an inhibitor of cell division in wild-type and aziA strains of Salmonella typhimurium. The bacteria grown in media containing sodium azide and glucose formed long filaments. It has been found that sodium azide had a stronger inhibitory effect on DNA synthesis than on cell mass increase. When filaments produced by azide action were transferred to azide-free medium very rapid increase in DNA content was observed during the first 45 min. After this time, when relative DNA content was increased the rate of DNA synthesis was reduced and cell divisions reappeared.Inhibitory effect of azide on DNA biosynthesis in vitro was observed with toluenized cells of S. typhimurium. Only ATP-dependent radioactive dTMP incorporation into DNA was affected by sodium azide. It had no effect on the incorporation in the absence of ATP.Mutant aziC was isolated in S. typhimurium by scoring for clones with normal cell division in the presence of sodium azide. Azide had much less effect on DNA biosynthesis in vivo and in vitro in aziC cells as compared with isogenic controls.This work was supported by the Polish Academy of Sciences within the project 09.3.1., and by the U.S. Public Health Service, grant No. 05-032-1. 相似文献
7.
A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis> 总被引:3,自引:0,他引:3
Yu P 《Applied microbiology and biotechnology》2007,74(1):93-98
The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme
SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The
SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column
chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity. 相似文献
8.
Unique characteristics of superoxide dismutase of a strictly anaerobic archaebacterium Methanobacterium thermoautotrophicum 总被引:3,自引:0,他引:3
The superoxide dismutase (SOD) gene of Methanobacterium thermoautotrophicum (Takao, M., Oikawa, A., and Yasui, A. (1990) Arch. Biochem. Biophys. 283, 210-216), a strictly anaerobic archaebacterium, was expressed in Escherichia coli. The gene product accounted for more than 30% of the host's soluble protein. The purified protein was an active iron-containing tetrameric SOD with specific activity similar to known manganese-containing SODs (MnSODs) of aerobic archaebacteria. Although M. thermoautotrophicum SOD is an iron-containing SOD (FeSOD), it resembles MnSODs in amino acid sequence as judged by criteria distinguishing FeSODs from MnSODs. Moreover, M. thermoautotrophicum SOD is resistant to azide and hydrogen peroxide as MnSODs are, suggesting that its evolution is distinct from known eubacterial FeSODs. 相似文献
9.
Akihide Takeuchi Osamu Miyaishi Kazutoshi Kiuchi Ken‐ichi Isobe 《Developmental neurobiology》2000,45(1):39-46
In previous studies, we have demonstrated that damaged neurons within a boundary area around necrosis fall into delayed cell death due to the cytotoxic effect of microglial nitric oxide (NO), and are finally eliminated by activated microglia. In contrast, neurons in a narrow surrounding region nearby this boundary area remain alive even though they may encounter cytotoxic NO. To investigate the mechanism by which neurons tolerate this oxidative stress, we examined the in vitro and in vivo expression levels of superoxide dismutase (SOD) under pathological conditions. Results from our in situ hybridization and immunohistochemical studies showed up‐regulation of Cu/Zn‐SOD only in neurons outside the boundary area, whereas up‐regulation of Mn‐SOD was detected in both neurons and glial cells in the same region. In vitro experiments using rat PC12 pheochromocytoma and C6 glioma cell lines showed that induction of both Cu/Zn‐ and Mn‐SOD mRNA could only be detected in PC12 cells after treatment with NO donors, while a slight induction of Mn‐SOD mRNA alone could be seen in C6 glioma cells. The mechanism of resistance toward oxidative stress therefore appears to be quite different between neuronal and glial cells. It is assumed that these two types of SOD might play a critical role in protecting neurons from NO cytotoxicity in vivo, and the inability of SOD induction in damaged neurons seems to cause their selective elimination after focal brain injury. © 2000 John Wiley & Sons, Inc. J Neurobiol 45: 39–46, 2000 相似文献
10.
Rikhvanov E. G. Rachenko E. I. Varakina N. N. Rusaleva T. M. Borovskii G. B. Voinikov V. K. 《Russian Journal of Genetics》2004,40(4):341-347
Heat shock protein Hsp104 of Saccharomyces cerevisiae functions as a protector of cells against heat stress. When yeast are grown in media containing nonfermentable carbon sources, the constitutive level of this protein increases, which suggests an association between the expression of Hsp104 and yeast energy metabolism. In this work, it is shown that distortions in the function of mitochondria appearing as a result of mutation petite or after exposure of cells to the mitochondrial inhibitor sodium azide reduce the induction of Hsp104 synthesis during heat shock. Since the addition of sodium azide suppressed the formation of induced thermotolerance in the parent type and in mutant hsp104,the expression of gene HSP104 and other stress genes during heat shock is apparently regulated by mitochondria. 相似文献
11.
The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H2O2. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of
D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian 相似文献
12.
Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat+. The Alr0998 protein could be immunodetected in AnKat+ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat+ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat+ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H2O2 caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO2 fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat+ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase. 相似文献
13.
Background
The M-like protein, also known as SzP, is expressed on the surface of Streptococcus equi subsp. zooepidemicus (S. zooepidemicus). Previous studies demonstrated that SzP is similar to M protein of group A Streptococcus in the structure and characteristics of antiphagocytosis. The M protein is an adhesin that can bind to the host cells, however it is not known whether the SzP of S. zooepidemicus also functions as an adhesin. We conducted an investigation to determine SzP as an adhesin, and one SzP epitope was identified to be responsible for mediating binding to HEp-2 cells. 相似文献14.
Rikhvanov E. G. Varakina N. N. Rusaleva T. M. Rachenko E. I. Voinikov V. K. 《Microbiology》2002,71(6):662-665
The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucose-grown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances the damage inflicted on the cells by heat shock. 相似文献
15.
Mtm1p is essential for the posttranslational activation of manganese-containing superoxide dismutase (SOD2) in Saccharomyces cerevisiae; however, whether the same holds true for Arabidopsis thaliana is unknown. In this study, by using the yeast mtm1 mutant complementation method, we identified a putative MTM gene (AtMTM1, At4g27940) that is necessary for SOD2 activation. Further, analysis of SOD activity revealed that an SOD2 defect is rescued
in the yeast mutant Y07288 harboring the AtMTM1 gene. Related mRNA-level analysis showed the AtMTM1 gene is induced by paraquat but not by hydrogen peroxide, which indicates that this gene is related to the superoxide scavenger
SOD. In addition, an AtMTM1::GFP fusion construct was transiently expressed in the protoplasts, and it was localized to the
mitochondria. Furthermore, sequence deletion analysis of AtMTM1 revealed that the code region (amino acid (aa) 60–198) of Mtm1p plays an important role in localization of the protein to
the mitochondria. Regulation of AtMTM1 gene expression was analyzed using a fusion construct of the 1,766 bp AtMTM1 promoter and the GUS (β-glucuronidase) reporter gene. The screen identified GUS reporter gene expression in the developing
cotyledons, leaves, roots, stems, and flowers but not in the siliques. Our results suggest that AtMTM1 encodes a mitochondrial
protein that may be playing an important role in activation of MnSOD1 in Arabidopsis. 相似文献
16.
17.
18.
Characterization of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) activity in mouse peritoneal cavity cells
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Dhébora Albuquerque Dias Bruna de Barros Penteado Lucas Derbocio dos Santos Pedro Mendes dos Santos Carla Cardozo Pinto Arruda Maria Rosa Chitolina Schetinger Daniela Bitencourt Rosa Leal Jeandre Augusto dos Santos Jaques 《Cell biochemistry and function》2017,35(7):358-363
This study aimed to characterize the activity of ectonucleoside triphosphate diphosphohydrolase (E‐NTPDase; EC 3.6.1.5) in peritoneal cavity cells from BALB/c mice. E‐NTPDase was activated in the presence of both calcium (1.5mM) and magnesium (1.5mM) ions. However, the activity was higher in the presence of Ca2+. A pH of 8.5 and temperature of 37°C were the optimum conditions for catalysis. The apparent Km values were 0.51mM and 0.66mM for the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP), respectively. The Vmax values were 136.4 and 120.8 nmol Pi/min/mg of protein for ATPase and ADPase activity, respectively. Nucleotide hydrolysis was inhibited in the presence of sodium azide (20mM, ATP: P < .05; ADP: P < .001), sodium fluoride (20mM; ATP and ADP: P < .001), and suramin (0.3mM; ATP: P < .01; ADP: P < .05), which is a known profile for NTPDase inhibition. Although all of the diphosphate and triphosphate nucleotides that were tested were hydrolyzed, enzyme activity was increased when adenine nucleotides were used as substrates. Finally, we stress that knowledge of the E‐NTPDase catalytic biochemical properties in mouse peritoneal cavity cells is indispensable for properly determining its activity, as well as to fully understand the immune response profile in both healthy and sick cells. 相似文献
19.
Growth of Scenedesmus species and strains, grown for 28 days in mineral BBM medium in batch-cultures, displayed sigmoidal kinetics that comprised
a lag, exponential and declining growth phases. Total SOD activity in these autotrophically cultured organisms, which oscillated
within 0.6 – 1.4 Umg protein−1, was rather species-specific and only to some extent depended on the growth phase. Contrary, three S. obliquus strains: wild type 276-6, mutant with blocked PS I (strain 56.80) and mutant with blocked PS II (strain 57.80), cultured
for 7 days on BBM medium supplemented with bacto-tryptone and yeast extract (BBM+) turned out to be time-dependent and to
have several times higher total SOD activity than one obtained for Scenedesmus grown autotrophically. Regardless of the media composition, the phase of growth and studied organism, dominant isoforms of
total SOD were together determined Fe- and Mn-SOD.
Profiles of SOD isoforms, obtained after PAGE analysis of all autotrophically and exponentially growing organisms, revealed
that one Mn-SOD and one Cu/Zn-SOD bands located on gels at the same position whereas location of three bands of Fe-SOD depended
on the strain. This suggests the presence of two different groups of Fe-SODs in analyzed organisms. Identical SOD profiles
found in two S. armatus strains (276-4a and 276-4d) and S. subspicatus correspond well with their taxonomic position. The SOD profile of S. armatus B1-76 distinctly differed from two other S. armatus strains but was identical to S. microspinal B1-76 and S. quadricauda G-15 despite the fact that there were significant growth rate differences between these three species. SODs profiles of S. acutus 437 and S. obliguus 453 were species-specific. In S. obliquus strains cultured on BBM+ medium, there are four SOD bands: one slightly visible band of Mn-SOD, two intensive bands of Fe-SOD
and one band of Cu/Zn-SOD. The above finding suggests that antioxidant response of algae kept in batch-cultures differs according
to medium composition and the SOD activity mainly restricted to chloroplasts. 相似文献
20.
A recent study has shown that in the budding tunicate Polyandrocarpa misakiensis, the mitochondrial respiratory chain (MRC) dramatically attenuates the gene activity during senescence. In this study, we examined the possible involvement of superoxide dismutase (SOD) in the attenuation of gene expression of cytochrome c oxidase subunit 1 (COX1) in aged zooids. By RT‐PCR and in situ hybridization, Cu/Zn‐SOD (SOD1) was found to be expressed in most cells and tissues of buds and juvenile zooids but showed a conspicuous decline in senescent adult zooids, except in the gonad tissue in which the cytoplasm of juvenile oocytes was stained heavily. This expression pattern of SOD1 was similar to that of COX1. In contrast to SOD1, Mn‐SOD (SOD2) was expressed constitutively in both somatic and germline tissues of buds, juvenile zooids, and senescent adult zooids. Knockdown of SOD1 by RNAi diminished the gene activity of not only SOD1 but also of COX1. The resultant zooids had transient deficiencies in growth and budding, and they recovered from these deficiencies approximately 1 month later. Our results indicate that in P. misakiensis, SOD1 is a senescence‐associated nuclear gene and that the experimental decline in SOD1 gene expression accompanies the attenuation of MRC gene activity. Although it is uncertain how SOD1 is downregulated during tunicate senescence, the decreased SOD1 activity could be one of the main causes of MRC gene attenuation during normal senescence. 相似文献