Ca(v)1.2 calcium channel is glutathionylated during oxidative stress in guinea pig and ischemic human heart

Glutathionylation as a posttranslational modification of proteins is becoming increasingly recognized, but its role in many diseases has not been demonstrated. Oxidative stress and alterations in calcium homeostasis are associated with the development of cardiac hypertrophy. Because the cardiac L-type Ca²⁺ channel can be persistently activated after exposure to H₂O₂, the aim of this study was to determine whether alterations in channel function were associated with glutathionylation of the α_1C subunit (Ca_v1.2) channel protein. Immunoblot analysis indicated that Ca_v1.2 protein is significantly glutathionylated after exposure to H₂O₂ and glutathione in vitro and after ischemia-reperfusion injury. L-type Ca²⁺ channel macroscopic current and intracellular calcium were significantly increased in myocytes after exposure to oxidized glutathione and reversed by glutaredoxin. The increase in current correlated with an increase in open probability of the channel assessed as changes in single-channel activity after exposing the human long N-terminal Ca_v1.2 to H₂O₂ or oxidized glutathione. We also demonstrate that the Ca_v1.2 channel is significantly glutathionylated in ischemic human heart. We conclude that oxidative stress is associated with an increase in glutathionylation of the Ca_v1.2 channel protein. We suggest that the associated constitutive activity contributes to the development of pathology in ischemic heart disease.     

Glutathione – linking cell proliferation to oxidative stress

SignificanceThe multifaceted functions of reduced glutathione (gamma-glutamyl–cysteinyl–glycine; GSH) continue to fascinate plants and animal scientists, not least because of the dynamic relationships between GSH and reactive oxygen species (ROS) that underpin reduction/oxidation (redox) regulation and signalling. Here we consider the respective roles of ROS and GSH in the regulation of plant growth, with a particular focus on regulation of the plant cell cycle. Glutathione is discussed not only as a crucial low molecular weight redox buffer that shields nuclear processes against oxidative challenge but also a flexible regulator of genetic and epigenetic functions.Recent advancesThe intracellular compartmentalization of GSH during the cell cycle is remarkably consistent in plants and animals. Moreover, measurements of in vivo glutathione redox potentials reveal that the cellular environment is much more reducing than predicted from GSH/GSSG ratios measured in tissue extracts. The redox potential of the cytosol and nuclei of non-dividing plant cells is about −300 mV. This relatively low redox potential maintained even in cells experiencing oxidative stress by a number of mechanisms including vacuolar sequestration of GSSG. We propose that regulated ROS production linked to glutathione-mediated signalling events are the hallmark of viable cells within a changing and challenging environment.Critical issuesThe concept that the cell cycle in animals is subject to redox controls is well established but little is known about how ROS and GSH regulate this process in plants. However, it is increasingly likely that redox controls exist in plants, although possibly through different pathways. Moreover, redox-regulated proteins that function in cell cycle checkpoints remain to be identified in plants. While GSH-responsive genes have now been identified, the mechanisms that mediate and regulate protein glutathionylation in plants remain poorly defined.Future directionsThe nuclear GSH pool provides an appropriate redox environment for essential nuclear functions. Future work will focus on how this essential thiol interacts with the nuclear thioredoxin system and nitric oxide to regulate genetic and epigenetic mechanisms. The characterization of redox-regulated cell cycle proteins in plants, and the elucidation of mechanisms that facilitate GSH accumulation in the nucleus are keep steps to unravelling the complexities of nuclear redox controls.     

Glutathionylation of chikungunya nsP2 protein affects protease activity

BackgroundChikungunya fever is an emerging disease caused by the chikungunya virus and is now being spread worldwide by the mosquito Aedes albopictus. The infection can cause a persistent severe joint pain and recent reports link high levels of viremia to neuropathologies and fatalities. The viral protein nsP2 is a multifunctional enzyme that plays several critical roles in virus replication. Virus infection induces oxidative stress in host cells which the virus utilizes to aid viral propagation. Cellular oxidative stress also triggers glutathionylation which is a post-translational protein modification that can modulate physiological roles of affected proteins.MethodsThe nsP2 protease is necessary for processing of the virus nonstructural polyprotein generated during replication. We use the recombinant nsP2 protein to measure protease activity before and after glutathionylation. Mass spectrometry allowed the identification of the glutathione-modified cysteines. Using immunoblots, we show that the glutathionylation of nsP2 occurs in virus-infected cells.ResultsWe show that in virus-infected cells, the chikungunya nsP2 can be glutathionylated and we show this modification can impact on the protease activity. We also identify 6 cysteine residues that are glutathionylated of the 20 cysteines in the protein.ConclusionsThe virus-induced oxidative stress causes modification of viral proteins which appears to modulate virus protein function.General significanceViruses generate oxidative stress to regulate and hijack host cell systems and this environment also appears to modulate virus protein function. This may be a general target for intervention in viral pathogenesis.     

Cytoprotective effects of 12-oxo phytodienoic acid,a plant-derived oxylipin jasmonate,on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.MethodsThe cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H₂O₂) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).ResultsAmong the jasmonates, only OPDA suppressed H₂O₂-induced cytotoxicity. OPDA pretreatment also inhibited the H₂O₂-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.     

Protein kinase A activation by retinoic acid in the nuclei of HL60 cells

BackgroundActivation of protein kinase A (PKA) occurs during retinoic acid (RA)-induced granulocytic differentiation of human promyelocytic leukemia HL60 cells. It is known that the RIIα regulatory subunit of PKA, is modified by RA (retinoylated) in the early stages of differentiation. We have investigated the effects of RA on PKA during cell differentiation in order to understand the potential significance of this process in the retinoylation of RIIα subunits.MethodsImmunoblotting, immunoprecipitation, confocal microscopy, PCR, and PKA activity assays were employed for characterizing the effects of RA on PKA.ResultsWe found that RA induces intracellular mobility of RIIα and the activation of PKA in HL60 cells. Increases in RIIα levels were observed in RA-treated HL60 cells. RA treatment altered intracellular localization of the PKA subunits, RIIα and Cα, and increased their protein levels in the nuclei as detected by both immunoblotting and immunostaining analyses. Coincident with the increase in nuclear Cα, RA-treated HL60 cells showed increases in both the protein phosphorylation activity of PKA and the levels of phosphorylated proteins in nuclear fractions as compared to control cells. In addition, RIIα protein was stabilized in RA-treated HL60 cells as compared to control cells.ConclusionsThese results suggest that RA stabilizes RIIα protein and activates PKA in the nucleus, with a resultant increase in the phosphorylation of nuclear proteins.General significanceOur evidence suggests that retinoylation of PKA might contribute to its stabilization and activation and that this could potentially participate in RA's ability to induce granulocytic differentiation of HL60 cells.     

Redox Proteomics of the Inflammatory Secretome Identifies a Common Set of Redoxins and Other Glutathionylated Proteins Released in Inflammation,Influenza Virus Infection and Oxidative Stress

Protein cysteines can form transient disulfides with glutathione (GSH), resulting in the production of glutathionylated proteins, and this process is regarded as a mechanism by which the redox state of the cell can regulate protein function. Most studies on redox regulation of immunity have focused on intracellular proteins. In this study we have used redox proteomics to identify those proteins released in glutathionylated form by macrophages stimulated with lipopolysaccharide (LPS) after pre-loading the cells with biotinylated GSH. Of the several proteins identified in the redox secretome, we have selected a number for validation. Proteomic analysis indicated that LPS stimulated the release of peroxiredoxin (PRDX) 1, PRDX2, vimentin (VIM), profilin1 (PFN1) and thioredoxin 1 (TXN1). For PRDX1 and TXN1, we were able to confirm that the released protein is glutathionylated. PRDX1, PRDX2 and TXN1 were also released by the human pulmonary epithelial cell line, A549, infected with influenza virus. The release of the proteins identified was inhibited by the anti-inflammatory glucocorticoid, dexamethasone (DEX), which also inhibited tumor necrosis factor (TNF)-α release, and by thiol antioxidants (N-butanoyl GSH derivative, GSH-C4, and N-acetylcysteine (NAC), which did not affect TNF-α production. The proteins identified could be useful as biomarkers of oxidative stress associated with inflammation, and further studies will be required to investigate if the extracellular forms of these proteins has immunoregulatory functions.     

Intracellular glutathione regulates Andrographolide-induced cytotoxicity on hepatoma Hep3B cells

Abstract

Andrographolide (ANDRO), a diterpenoid lactone isolated from the traditional herbal plant Andrographis paniculata, was reported to induce apoptosis in hepatoma Hep3B cells in our previous study (Ji LL, Liu TY, Liu J, Chen Y, Wang ZT. Andrographolide inhibits human hepatoma-derived Hep3B cells growth through the activation of c-Jun N-terminal kinase. Planta Med 2007; 73: 1397–1401). The present investigation was carried out to observe whether cellular reduced glutathione (GSH) plays important roles in ANDRO-induced apoptosis. ANDRO initially increased intracellular GSH levels which then decreased later, while inhibition of cellular GSH synthesis by L-Buthionine-(S,R)-sulfoximine (BSO) augmented ANDRO-induced cytotoxicity and apoptosis in Hep3B cells. On the other hand, the thiol antioxidant dithiothreitol (DTT) rescued ANDRO-depleted cellular GSH, and abrogated ANDRO-induced cytotoxicity and apoptosis. Furthermore, BSO pretreatment augmented ANDRO-decreased expression of antioxidant protein thioredoxin 1 (Trx1), while DTT reversed this decrease. Further results showed that ANDRO increased the activity of the GSH-related antioxidant enzyme glutathione peroxidase (GPx) and the production of intracellular reactive oxygen species (ROS). Taken together, this study demonstrates that the intracellular redox system plays important roles in regulating the cytotoxicity of ANDRO on hepatoma Hep3B cells.     

On-chip Escherichia coli culture, purification, and detection of expressed proteins

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST–GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.M. Kim and S. Y. Lee contributed equally to this work.     

Identification of proteins undergoing glutathionylation in oxidatively stressed hepatocytes and hepatoma cells

Protein glutathionylation is a post-translational modification consisting of the formation of a mixed disulfide between protein cysteines and glutathione (GSH). To identify proteins undergoing glutathionylation in primary rat hepatocytes and in human HepG2 hepatoma cells, we radiolabeled the intracellular GSH pool with L-[(35)S] cysteine. Cells were then exposed to oxidative stress. Proteins were separated by two-dimensional gel electrophoresis under nonreducing conditions, and glutathionylated proteins were located by autoradiography and identified by mass spectrometry after tryptic digestion. Several proteins previously not known to undergo glutathionylation were thus recognized. Among the identified proteins some are the same or belong to the same functional class as those we have already identified in a previous paper on T cell blasts (actin, nucleophosmin, phosphogluconolactonase, myosin, profilin, cyclophilin A, stress 70 protein, ubiquitin in HepG2 cells and actin, peroxiredoxin 5, cytochrome C oxidase, heat shock cognate 70 in hepatocytes) while others are newly recognized (Ran specific GTPase activating protein, histidine triad nucleotide binding protein 2 in HepG2 cells and enoyl CoA hydratase in hepatocytes). The technique described proved equally applicable to a variety of cell types.     

Overexpression of a small GTP-binding protein Ran1 in Arabidopsis leads to promoted elongation growth and enhanced disease resistance against P. syringae DC3000

Plants resist infection through an innate immune response, which is usually associated with slowing of growth. The molecular mechanisms underlying the trade-off between plant growth and defense remain unclear. The present study reveals that growth/defense trade-offs mediated by gibberellin (GA) and salicylic acid (SA) signaling pathways are uncoupled during constitutive overexpression of transgenic AtRAN1 and AtRAN1_Q72L (active, GTP-locked form) Arabidopsis plants. It is well known that the small GTP-binding protein Ran (a Ras-related nuclear protein) functions in the nucleus–cytoplasmic transport of proteins. Although there is considerable evidence indicating that nuclear–cytoplasmic partitioning of specific proteins can participate in hormone signaling, the role of Ran-dependent nuclear transport in hormone signaling is not yet fully understood. In this report, we used a combination of genetic and molecular methods to reveal whether AtRAN1 is involved in both GA and SA signaling pathways. Constitutively overexpressed AtRAN1 promoted both elongation growth and the disease resistance response, whereas overexpression of AtRAN1_Q72L in the atran2atran3 double mutant background clearly inhibited elongation growth and the defense response. Furthermore, we found that AtRAN1 coordinated plant growth and defense by promoting the stability of the DELLA protein RGA in the nucleus and by modulating NPR1 nuclear localization. Interestingly, genetically modified rice (Oryza sativa) overexpressing AtRAN1 exhibited increased plant height and yield per plant. Altogether, the ability to achieve growth/defense trade-offs through AtRAN1 overexpression provides an approach to maximizing crop yield to meet rising global food demands.     

单核细胞增多性李斯特菌谷胱甘肽还原酶GR的生物学特性

[目的]本文旨在构建单核细胞增多性李斯特菌谷胱甘肽还原酶(glutathione reductase,GR)基因lmo1433(gr)缺失株,并研究GR在细菌生长和运动过程中发挥的作用及与谷氧还蛋白(glutaredoxin,Grx)系统间的调控关系,探究GR参与细菌抗氧化应激和致病力的生物学功能,为阐明氧化还原蛋白介...     

Proteomic Interactions in the Mouse Vitreous-Retina Complex

Purpose

Human vitreoretinal diseases are due to presumed abnormal mechanical interactions between the vitreous and retina, and translational models are limited. This study determined whether nonstructural proteins and potential retinal biomarkers were expressed by the normal mouse vitreous and retina.

Methods

Vitreous and retina samples from mice were collected by evisceration and analyzed by liquid chromatography-tandem mass spectrometry. Identified proteins were further analyzed for differential expression and functional interactions using bioinformatic software.

Results

We identified 1,680 unique proteins in the retina and 675 unique proteins in the vitreous. Unbiased clustering identified protein pathways that distinguish retina from vitreous including oxidative phosphorylation and neurofilament cytoskeletal remodeling, whereas the vitreous expressed oxidative stress and innate immunology pathways. Some intracellular protein pathways were found in both retina and vitreous, such as glycolysis and gluconeogenesis and neuronal signaling, suggesting proteins might be shuttled between the retina and vitreous. We also identified human disease biomarkers represented in the mouse vitreous and retina, including carbonic anhydrase-2 and 3, crystallins, macrophage inhibitory factor, glutathione peroxidase, peroxiredoxins, S100 precursors, and von Willebrand factor.

Conclusions

Our analysis suggests the vitreous expresses nonstructural proteins that functionally interact with the retina to manage oxidative stress, immune reactions, and intracellular proteins may be exchanged between the retina and vitreous. This novel proteomic dataset can be used for investigating human vitreoretinopathies in mouse models. Validation of vitreoretinal biomarkers for human ocular diseases will provide a critical tool for diagnostics and an avenue for therapeutics.     

The Notch ligand Delta-like 1 integrates inputs from TGFbeta/Activin and Wnt pathways

Unlike the well-characterized nuclear function of the Notch intracellular domain, it has been difficult to identify a nuclear role for the ligands of Notch. Here we provide evidence for the nuclear function of the Notch ligand Delta-like 1 in colon cancer (CC) cells exposed to butyrate. We demonstrate that the intracellular domain of Delta-like 1 (Dll1icd) augments the activity of Wnt signaling-dependent reporters and that of the promoter of the connective tissue growth factor (CTGF) gene. Data suggest that Dll1icd upregulates CTGF promoter activity through both direct and indirect mechanisms. The direct mechanism is supported by co-immunoprecipitation of endogenous Smad2/3 proteins and Dll1 and by chromatin immunoprecipitation analyses that revealed the occupancy of Dll1icd on CTGF promoter sequences containing a Smad binding element. The indirect upregulation of CTGF expression by Dll1 is likely due to the ability of Dll1icd to increase Wnt signaling, a pathway that targets CTGF. CTGF expression is induced in butyrate-treated CC cells and results from clonal growth assays support a role for CTGF in the cell growth-suppressive role of butyrate. In conclusion, integration of the Notch, Wnt, and TGFbeta/Activin signaling pathways is in part mediated by the interactions of Dll1 with Smad2/3 and Tcf4.     

Nucleophilic Distribution of Metallothionein in Human Tumor Cells

Metallothionein (MT), a major zinc-binding intracellular protein thiol, has been associated with cytoprotection from heavy metals, antineoplastic drugs, mutagens, and cellular oxidants. Despite its small mass (7 kDa), nuclear partitioning of MT has been observed in both normal and malignant tissues. The factors controlling MT sequestration are unknown. Thus, we examined the regulation of MT subcellular distribution in human cancer cell lines that exhibit prominent nuclear MT. The nuclear disposition of MT was unaltered during cell cycle passage in synchronized cells. MT redistributed to the cytoplasm when cells were exposed to reduced temperature. Cytoplasmic redistribution was also seen in DU-145 and HPC36M prostatic cancer cells after ATP depletion, but not in PC3-MA2 and SCC25/CP cells. Pretreatment with 10 μMCdCl₂did not significantly alter MT distribution but did render all cells sensitive to cytoplasmic redistribution after either reduced temperature or ATP depletion. Thus, nuclear retention of MT is energy requiring and this ability of MT to accumulate in subcellular compartments against its concentration gradient may be important in the capacity of MT to supply Zn or other metals to target sites within the cell.     

Expression and purification of a biologically active basic fibroblast growth factor fusion protein

Basic fibroblast growth factor (bFGF) is a potent mitogen of many cell types and plays an important role in angiogenesis. To help identify proteins that bind to bFGF and mediate its intracellular transport and signaling, we overexpressed and purified a bFGF fusion protein in Escherichia coli. The fusion protein consists of bFGF fused to the C-terminus of glutathione S-transferase (GST). The GST-bFGF fusion protein was purified using SP-Sepharose and glutathione-Sepharose affinity chromatography. The ability of the purified GST-bFGF to stimulate the growth of human umbilical vein endothelial cells (HUVECs) was equivalent to that of purified recombinant 18 kDa bFGF.     

SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expression

Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.     

siRNA下调20S蛋白酶体β5亚基抑制立氏立克次体细胞内增殖

[目的]探究宿主20S蛋白酶体β5亚基(proteasome 20S subunit beta 5,PSMB5)对革兰氏阴性专性胞内寄生菌立氏立克次体胞内生长繁殖的影响.[方法]利用小干扰RNA转染Vero和THP-1宿主细胞,设置未转染细胞为空白对照组、无义小干扰RNA转染组为阴性对照组、PSMB5特异性小干扰RNA...     

Changes in glutathione-dependent redox status and mitochondrial energetic strategies are part of the adaptive response during the filamentation process in Candida albicans

Candida albicans is an opportunist pathogen responsible for a large spectrum of infections, from superficial mycosis to systemic diseases called candidiasis. Its ability to grow in various morphological forms, such as unicellular budding yeast, filamentous pseudohyphae and hyphae, contributes to its survival in the diverse microenvironments it encounters in the host. During infection in vivo, C. albicans is faced with high levels of reactive oxygen species (ROS) generated by phagocytes, and the thiol-dependent redox status of the cells reflects their levels of oxidative stress. We investigated the role of glutathione during the transition between the yeast and hyphal forms of the pathogen, in relation to possible changes in mitochondrial bioenergetic pathways. Using various growth media and selective mutations affecting the filamentation process, we showed that C. albicans filamentation was always associated with a depletion of intracellular glutathione levels. Moreover, the induction of hypha formation resulted in general changes in thiol metabolism, including the oxidation of cell surface − SH groups and glutathione excretion. Metabolic adaptation involved tricarboxylic acid (TCA) cycle activation, acceleration of mitochondrial respiration and a redistribution of electron transfer pathways, with an increase in the contribution of the alternative oxidase and rotenone-insensitive dehydrogenase. Changes in redox status and apparent oxidative stress may be necessary to the shift to adaptive metabolic pathways, ensuring normal mitochondrial function and adenosine triphosphate (ATP) levels. The consumption of intracellular glutathione levels during the filamentation process may thus be the price paid by C. albicans for survival in the conditions encountered in the host.     

Modulation of NADP+-dependent isocitrate dehydrogenase in aging

Abstract

NADPH is an important cofactor in many biosynthesis pathways and the regeneration of reduced glutathione, critically important in cellular defense against oxidative damage. It is mainly produced by glucose-6-phosphate dehydrogenase, malic enzyme, and NADP⁺-specific isocitrate dehydrogenases (ICDHs). Here, we investigated age-related changes in ICDH activity and protein expression in IMR-90 human diploid fibroblast cells and tissues from Fischer 344 rats. We found that in IMR-90 cells the activity of cytosolic ICDH (IDPc) gradually increased with age up to the 46–48 population doubling level (PDL) and then gradually decreased at later PDL. 2′,7′-Dichloro-fluorescein fluorescence which reflects intracellular ROS generation was increased with aging in IMR-90 cells. In ad libitum-fed rats, we noted age-related, tissue-specific modulations of IDPc and mitochondrial ICDH (IDPm) activities and protein expression in the liver, kidney and testes. In contrast, ICDH activities and protein expression were not significantly modulated in diet-restricted rats. These data suggest that modulation of ICDH is an age-dependent and a tissue-specific phenomenon.     

Antioxidant Enzymes of Larvae of the Cabbage Looper Moth,Trzchoplusza Ni: Subcellular Distribution and Activities of Superoxide Dismutase,Catalase and Glutathione Reductase

In the mid-fifth instar larvae of the cabbage looper moth, Trichoplusia ni, the subcellular distribution of total superoxide dismutase was as follows: 3.05 units (70.0%), 0.97 units (22.3%), and 0.33 units (7.6%) mg^?1 protein in the mitochondrial, cytosolic and nuclear fractions, respectively. No superoxide dismutase activity was detected in the microsomal fraction. Catalase activity was unusually high and as follows: 283.4 units (47.3%), 150.1 units (25.1%). 142.3 units (23.8%), and 22.9 units (3.8%) mg^?1 protein in the mitochondrial, cytosolic, microsomal (containing peroxisomes), and nuclear fractions. No glutathione peroxidase activity was found, but appreciable glutathione reductase activity was detected with broad subcellular distribution as follows: 3.86 units (36.1%), 3.68 units (34.0%). 2.46 units (23.0%). and 0.70 units (6.5%) mg^?1 protein in the nuclear, mitochondrial, and cytosolic fractions, respectively. The unusually wide intracellular distribution of catalase in this phytophagous insect is apparently an evolutionary adaptation to the absence of glutathione peroxidase; hence, lack of a glutathione peroxidase-glutathione reductase role in alleviating stress from lipid peroxidation. Catalase working sequentially to superoxide dismutase, may nearly completely prevent the formation of the lipid peroxidizing OH radical from all intracellular compartments by the destruction of H₂O₂ which together with O^?₂ is a precursor of OH.