RESUMO
Intense synchrotron radiation produces specific structural and chemical damage to crystalline proteins even at 100 K. Carboxyl groups of acidic residues (Glu, Asp) losing their definition is one of the major effects observed. Here, the susceptibilities to X-ray damage of acidic residues in tetrameric malate dehydrogenase from Haloarcula marismortui are investigated. The marked excess of acidic residues in this halophilic enzyme makes it an ideal target to determine how specific damage to acidic residues is related to their structural and chemical environment. Four conclusions are drawn. (i) Acidic residues interacting with the side-chains of lysine and arginine residues are less affected by radiation damage than those interacting with serine, threonine and tyrosine side-chains. This suggests that residues with higher pK(a) values are more vulnerable to damage than those with a lower pK(a). However, such a correlation was not found when calculated pK(a) values were inspected. (ii) Acidic side-chains located in the enzymatic active site are the most radiation-sensitive ones. (iii) Acidic residues in the internal cavity formed by the four monomers and those involved in crystal contacts appear to be particularly susceptible. (iv) No correlation was found between radiation susceptibility and solvent accessibility.
Assuntos
Aminoácidos/química , Aminoácidos/efeitos da radiação , Cristalografia por Raios X/métodos , Haloarcula marismortui/química , Malato Desidrogenase/química , Malato Desidrogenase/efeitos da radiação , Modelos Químicos , Simulação por Computador , Relação Dose-Resposta à Radiação , Concentração de Íons de Hidrogênio , Malato Desidrogenase/ultraestrutura , Modelos Moleculares , Conformação Proteica/efeitos da radiação , Desnaturação Proteica/efeitos da radiação , Doses de Radiação , Soluções , Solventes/química , Relação Estrutura-Atividade , Raios XRESUMO
Mesembryanthemum crystallinum, a facultative halophyte and C(3)-Crassulacean acid metabolism (CAM) intermediate plant, has become a favoured plant for studying stress response mechanisms during C(3)-CAM shifts. One hour of exposure to excess light (EL) caused inhibition of photosynthetic electron transport in M. crystallinum leaves as indicated by chlorophyll a fluorescence measurements. This was accompanied by an increase in NADP-malic enzyme (ME), one of the key cytosolic enzymes involved in CAM, and by a general increase in superoxide dismutase (SOD) activity. In contrast, NAD-ME activity (the mitochondrial form of ME) was not affected by EL. Exposure to EL and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) treatment of a whole plant in low light induced hydrogen peroxide (H(2)O(2)) and C(3) to CAM transition. In contrast, treatment with 3-3,4-dichlorophenyl-1,1-dimethyl urea (DCMU) has blocked high light-induced H(2)O(2) accumulation and C(3)-CAM transition. Moreover, the abundance of transcripts encoding different SODs, ascorbate peroxidase and SOD activity was differently regulated by DCMU and DBMIB. Results of applying EL or high light, H(2)O(2) and photosynthetic electron transport inhibitors suggest that the redox events in the vicinity of PSII and/or PSI and photo-produced H(2)O(2) play a major role in the regulation of C(3)-CAM transition and photooxidative stress responses in M. crystallinum.
Assuntos
Cloroplastos/enzimologia , Peróxido de Hidrogênio/metabolismo , Mesembryanthemum/enzimologia , Estresse Oxidativo/fisiologia , Cloroplastos/efeitos da radiação , Dibromotimoquinona/farmacologia , Diurona/farmacologia , Transporte de Elétrons/fisiologia , Transporte de Elétrons/efeitos da radiação , Regulação da Expressão Gênica de Plantas/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Mesembryanthemum/citologia , Mesembryanthemum/efeitos da radiação , Oxirredução/efeitos da radiação , Estresse Oxidativo/efeitos da radiação , Estimulação Luminosa , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiação , Superóxido Dismutase/metabolismo , Superóxido Dismutase/efeitos da radiaçãoRESUMO
Chloroplast NADP-dependent malate dehydrogenase is one of the best-studied light-regulated enzymes. In C3 plants, NADP-MDH is a part of the 'malate valve' that controls the export of reducing equivalents in the form of malate to the cytosol. NADP-MDH is completely inactive in the dark and is activated in the light with reduced thioredoxin. Compared with its permanently active NAD-linked counterparts, NADP-MDH exhibits N- and C-terminal sequence extensions, each bearing one regulatory disulphide. Upon reduction of the C-terminal disulphide, the enzyme active site becomes accessible for the substrate. Reduction of the N-terminal disulphide promotes a conformational change advantageous for catalysis. To trace the evolutionary development of this intricate regulation mechanism, we isolated cDNA clones for NADP-MDH from the mossfern Selaginella and from two unicellular green algae. While the NADP-MDH sequence from Selaginella demonstrates the classic cysteine pattern of the higher plant enzyme, the sequences from the green algae are devoid of the N-terminal regulatory disulphide. Phylogenetic analysis of new sequences and of those available in the databases led to the conclusion that the chloroplast NADP-MDH and the cytosolic NAD-dependent form arose via duplication of an ancestral eubacterial gene, which preceded the separation of plant and animal lineages. Redox-sensitive NADP-MDH activity was detected only in the 'green' plant lineage starting from the primitive prasinophytic algae but not in cyanobacteria, Cyanophora paradoxa, red algae and diatoms. The latter organisms therefore appear to utilize mechanisms other than the light-regulated 'malate valve' to remove from plastids excessive electrons produced by photosynthesis.
Assuntos
Clorófitas/genética , Malato Desidrogenase/genética , Plantas/genética , Clorófitas/enzimologia , Cisteína/genética , DNA Complementar/química , DNA Complementar/genética , Evolução Molecular , Regulação Enzimológica da Expressão Gênica , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Malato Desidrogenase (NADP+) , Dados de Sequência Molecular , Filogenia , Plantas/enzimologia , Subunidades Proteicas , Sequências Reguladoras de Ácido Nucleico , Análise de Sequência de DNARESUMO
The induction of nicotinamide adenine dinucleotide phosphate-malic enzyme (NADP-ME) in etiolated maize (Zea mays) seedlings by UV-B and UV-A radiation, and different levels of photosynthetically active radiation (PAR, 400-700 nm) was investigated by measuring changes in activity, protein quantity and RNA levels as a function of intensity and duration of exposure to the different radiations. Under low levels of PAR, exposure to UV-B radiation but not UV-A radiation for 6 to 24 h caused a marked increase in the enzyme levels similar to that observed under high PAR in the absence of UV-B. UV-B treatment of green leaves following a 12-h dark period also caused an increase in NADP-ME expression. Exposure to UV-B radiation for only 5 min resulted in a rapid increase of the enzyme, followed by a more gradual rise with longer exposure up to 6 h. Low levels of red light for 5 min or 6 h were also effective in inducing NADP-ME activity equivalent to that obtained with UV-B radiation. A 5-min exposure to far-red light following UV-B or red light treatment reversed the induction of NADP-ME, and this effect could be eliminated by further treatment with UV-B or red light. These results indicate that physiological levels of UV-B radiation can have a positive effect on the induction of this photosynthetic enzyme. The reducing power and pyruvate generated by the activity of NADP-ME may be used for respiration, in cellular repair processes and as substrates for fatty acid synthesis required for membrane repair.
Assuntos
Malato Desidrogenase/efeitos da radiação , Fotossíntese/efeitos da radiação , Raios Ultravioleta , Zea mays/efeitos da radiação , Indução Enzimática , Malato Desidrogenase/metabolismo , RNA/metabolismo , RNA/efeitos da radiação , Fatores de Tempo , Zea mays/enzimologiaRESUMO
The effects of UV-B radiation on growth, photosynthesis, UV-B-absorbing compounds and NADP-malic enzyme have been examined in different cultivars of Phaseolous vulgaris L. grown under 1 and 12 mM nitrogen. Low nitrogen nutrition reduces chlorophyll and soluble protein contents in the leaves and thus the photosynthesis rate and dry-matter accumulation. Chlorophyll, soluble protein and Rubisco contents and photosynthesis rate are not significantly altered by ambient levels of UV-B radiation (17 microW m-2, 290-320 nm, 4 h/day for one week). Comparative studies show that under high nitrogen, UV-B radiation slightly enhances leaf expansion and dry-matter accumulation in cultivar Pinto, but inhibits these parameters in Vilmorin. These results suggest that the UV-B effect on growth is mediated through leaf expansion, which is particularly sensitive to UV-B, and that Pinto is more tolerant than Vilmorin. The effect of UV-B radiation on UV-B-absorbing compounds and on NADP-malic enzyme (NADP-ME) activity is also examined. Both UV-B radiation and low-nitrogen nutrition enhance the content of UV-B-absorbing compounds, and among the three cultivars used, Pinto exhibits the highest increases and Arroz the lowest. The same trend is observed for the specific activity and content of NADP-ME. On a leaf-area basis, the amount of UV-B-absorbing compounds is highly correlated with the enzyme activity (r2 = 0.83), suggesting that NADP-ME plays a key role in biosynthesis of these compounds. Furthermore, the higher sensitivity of Vilmorin than Pinto to UV-B radiation appears to be related to the activity of NADP-ME and the capacity of the plants to accumulate UV-B-absorbing compounds.
Assuntos
Fabaceae/efeitos da radiação , Plantas Medicinais , Raios Ultravioleta , Dióxido de Carbono , Clorofila , Fabaceae/enzimologia , Fabaceae/crescimento & desenvolvimento , Fabaceae/fisiologia , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Nitrogênio/metabolismo , Fotossíntese/efeitos da radiação , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Rats were exposed to gamma radiation from a 60Co source, receiving 0.25 Gy at weekly intervals. During 2 d before each irradiation, the animals received daily intragastric doses of 26 mg pantothenol or 15 mg beta-carotene per kg body weight. One hour after the third irradiation session, the animals were killed and their livers were analyzed. In animals not supplied with pantothenol, the irradiation resulted in a significant decrease of total liver lipids and a 50% decrease in phospholipids. Liver cholesterol was decreased by about 20%. Irradiation produced lipid peroxidation as expressed by doubling of the amounts of conjugated dienes and ketone dienes and of thiobarbituric acid reactive compounds. The amount of CoA in liver was decreased by 24% and that of reduced glutathione by 40%. The NAD+/NADH ratio was increased by 60% and the activity of NADP-dependent malate dehydrogenase (decarboxylating) was decreased by 26%. The amount of pantothenic acid and its derivatives (expressed as pantolactone-generating compounds) in blood decreased by about 80%. In rats to which pantothenol was administered, the content of pantothenic acid in blood was tripled compared to nonirradiated (control) rats, and all the biochemical parameters measured in liver were the same as in nonirradiated animals.
Assuntos
Raios gama/efeitos adversos , Fígado/efeitos dos fármacos , Fígado/efeitos da radiação , Ácido Pantotênico/análogos & derivados , Animais , Antioxidantes , Colesterol/análise , Colesterol/efeitos da radiação , Coenzima A/análise , Coenzima A/efeitos da radiação , Esquema de Medicação , Feminino , Glutationa/biossíntese , Glutationa/química , Glutationa/efeitos da radiação , Dissulfeto de Glutationa/biossíntese , Dissulfeto de Glutationa/química , Dissulfeto de Glutationa/efeitos da radiação , Intubação Gastrointestinal , Ácido Láctico/análise , Ácido Láctico/efeitos da radiação , Lipídeos/análise , Lipídeos/efeitos da radiação , Fígado/química , Malato Desidrogenase/análise , Malato Desidrogenase/efeitos da radiação , Malato Desidrogenase (NADP+) , NAD/análise , NAD/efeitos da radiação , Ácido Pantotênico/sangue , Ácido Pantotênico/farmacologia , Fosfolipídeos/análise , Fosfolipídeos/efeitos da radiação , Proteínas/química , Ácido Pirúvico/análise , Ácido Pirúvico/efeitos da radiação , Protetores contra Radiação/farmacologia , Ratos , Ratos Endogâmicos , Espécies Reativas de Oxigênio , Substâncias Reativas com Ácido Tiobarbitúrico/análise , Substâncias Reativas com Ácido Tiobarbitúrico/efeitos da radiação , beta Caroteno/administração & dosagem , beta Caroteno/farmacologiaRESUMO
Ultraviolet (UV) radiation is one of the major risk factors of cataract (loss of eye-lens transparency). The influence of UVB radiation (300 nm; 100 microW cm-2) on the activity and apparent kinetic constants (Km and Vmax) of rat lens hexokinase (HK;EC 2.7.1.1), phosphofructokinase (PFK; EC 2.7.1.11), isocitrate dehydrogenase (ICDH; EC 1.1.1.41) and malate dehydrogenase (MDH; EC 1.1.1.37) of energy metabolism has been investigated by irradiating the lens homogenate of three- and 12-month-old rats. In the three-month-old group specific activities of HK and PFK are reduced by 56 and 43%, respectively, and there is no change in ICDH and MDH activities after a 24 h exposure. On the other hand, in the 12-month-old group the decreases are 72, 71, 24 and 16% for HK, PFK, ICDH and MDH, respectively. UVB irradiation increases the apparent Km of HK and PFK (in both age groups), whereas the Km of ICDH and MDH is not altered. While the decrease in Vmax of these enzymes due to UVB exposure is only marginal in three-month-old rats, it is more pronounced (significant) in 12-month-old rats. A similar decrease in enzyme activities of HK and PFK is also observed upon UVB exposure of the intact rat lens. The photoinduced changes in energy metabolism may in turn have a bearing on lens transparency, particularly at an older age.
Assuntos
Envelhecimento , Cristalino/enzimologia , Cristalino/efeitos da radiação , Raios Ultravioleta , Animais , Metabolismo Energético/efeitos da radiação , Hexoquinase/metabolismo , Hexoquinase/efeitos da radiação , Isocitrato Desidrogenase/metabolismo , Isocitrato Desidrogenase/efeitos da radiação , Cinética , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Masculino , Fosfofrutoquinase-1/metabolismo , Fosfofrutoquinase-1/efeitos da radiação , Proteínas/metabolismo , Ratos , Ratos WistarRESUMO
Light-dependent reduction of target disulfides on certain chloroplast enzymes results in a change in activity. We have modeled the tertiary structure of four of these enzymes, namely NADP-linked glyceraldehyde-3-P dehydrogenase, NADP-linked malate dehydrogenase, sedoheptulose bisphosphatase, and fructose bisphosphatase. Models are based on x-ray crystal structures from non-plant species. Each of these enzymes consists of two domains connected by a hinge. Modeling suggests that oxidation of two crucial cysteines to cystine would restrict motion around the hinge in the two dehydrogenases and influence the conformation of the active site. The cysteine residues in the two phosphatases are located in a region known to be sensitive to allosteric modifiers and to be involved in mediating structural changes in mammalian and microbial fructose bisphosphatases. Apparently, the same region is involved in covalent modification of phosphatase activity in the chloroplast.
Assuntos
Cloroplastos/enzimologia , Cisteína , Frutose-Bifosfatase/química , Gliceraldeído-3-Fosfato Desidrogenases/química , Malato Desidrogenase/química , Monoéster Fosfórico Hidrolases/química , Estrutura Terciária de Proteína , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dissulfetos/efeitos da radiação , Fabaceae/enzimologia , Frutose-Bifosfatase/metabolismo , Frutose-Bifosfatase/efeitos da radiação , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/efeitos da radiação , Luz , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Modelos Moleculares , Dados de Sequência Molecular , NADP/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Monoéster Fosfórico Hidrolases/efeitos da radiação , Plantas Medicinais , Homologia de Sequência de Aminoácidos , Suínos , Triticum/enzimologiaRESUMO
Research into dose- and time-dependent alterations of quantitative and qualitative composition of the peritoneal cells has been carried out. Exposure at 3, 5, 7, or 9 Gy reduced the total number of peritoneal cells, increased the activity of the lactate-, malate-, and qlucoso-6-phosphate dehydrogenases, of the acid phosphatase and catepsin D; the amount of the cell protein in adhesing macrophages was also increased. The exposed macrophages showed enhanced phagocytic and cytotoxic activity as evidenced by the increase in luminol- and lucigenin-enhanced chemilumenscence in vitro. The most pronounced changes took place 7-9 days after exposure at 9 Gy.
Assuntos
Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/efeitos da radiação , Doses de Radiação , Fosfatase Ácida/metabolismo , Fosfatase Ácida/efeitos da radiação , Animais , Catepsina D/metabolismo , Catepsina D/efeitos da radiação , Adesão Celular/efeitos da radiação , Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/efeitos da radiação , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/efeitos da radiação , Medições Luminescentes , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Fatores de TempoRESUMO
The influence of external single gamma-irradiation with a dose of 1 Gy on the isoenzyme composition of lactate dehydrogenase, malate dehydrogenase, esterase and acid phosphatase in the cytoplasm of rat brain cells has been investigated. Irradiation was shown to cause differently directed changes in the ratio of the isoenzymes under study at different times after exposure. The isoenzyme spectrum of lactate dehydrogenase and malate dehydrogenase was shown to be normalized on day 30 after irradiation, whereas the isoform composition of esterase and acid phosphatase was not stabilized at that time.
Assuntos
Fosfatase Ácida/efeitos da radiação , Encéfalo/efeitos da radiação , Esterases/efeitos da radiação , Isoenzimas/efeitos da radiação , L-Lactato Desidrogenase/efeitos da radiação , Malato Desidrogenase/efeitos da radiação , Fosfatase Ácida/análise , Animais , Encéfalo/enzimologia , Densitometria , Esterases/análise , Isoenzimas/análise , L-Lactato Desidrogenase/análise , Malato Desidrogenase/análise , Masculino , Doses de Radiação , Ratos , Ratos Wistar , Fatores de TempoRESUMO
A model has been built for the plant NADP-malate dehydrogenase from Zea mays, a key enzyme in photosynthesis, which undergoes light-dependent regulation. The model was based on sequence and presumed structural homology to the known three-dimensional structure of mammalian porcine cytosolic NAD-malate dehydrogenase. A cystine-loop present in an extended C-terminal region of plant NADP-malate dehydrogenases was modelled using molecular mechanics and computer graphical methods, based on the assumption that a disulphide bridge exists in the inactive form of the enzyme between Cys351 and Cys363. The predicted conformation of the intact C-terminal cystine-loop suggests that the extended polypeptide will bind in the active centre and inhibit enzyme activity. Another ionizable cysteine residue in the active site is predicted to control the charge of the catalytic His215 and might be responsible for the uniquely tight binding of the positively charged nicotinamide ring of NADP+ in this and other C4 and C3 plant NADP-malate dehydrogenases.
Assuntos
Malato Desidrogenase/química , Plantas/enzimologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Coenzimas , Luz , Malato Desidrogenase/efeitos da radiação , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência do Ácido Nucleico , Especificidade da Espécie , Suínos , Zea mays/enzimologiaRESUMO
Halophilic malate dehydrogenase (hMDH) from Haloarcula marismortui has been isolated, purified and characterized by biochemical and biophysical solution studies. A stabilization mechanism at extremely high concentrations of salt, based on the formation of co-operative hydrate bonds between the protein and hydrated salt ions, was suggested from thermodynamic analysis of native enzyme solutions. Recently the gene coding for hMDH was isolated and sequenced and an active enzyme cloned (F. Cendrin, J. Chroboczek, G. Zaccai, H. Eisenberg and M. Mevarech, unpublished work). A study of the crystal structure of hMDH in a high-salt physiological medium is in progress (O. Butbul-Dym & J. Sussman, personal communication). Here we discuss in depth implications of these recent developments on our earlier results.
Assuntos
Halobacteriaceae/enzimologia , Malato Desidrogenase/química , Fenômenos Químicos , Físico-Química , Halobacteriaceae/efeitos da radiação , Malato Desidrogenase/efeitos da radiação , Nêutrons , Espalhamento de Radiação , Ultracentrifugação , Difração de Raios XRESUMO
An experiment was performed to isolate the small atypical mitochondria produced during the irradiation of normal mitochondria with an He-Ne laser. Rat liver mitochondria were irradiated with a low-power continuous-wave He-Ne laser (energy dose, 5 J cm-2), followed by isolation using a sucrose gradient. In the irradiated sample, two bands were observed, one corresponding to normal mitochondria and the other to atypical mitochondria. Certain biochemical features of the mitochondria were investigated: mitochondrial enzyme activity and the presence of DNA and RNA were demonstrated. Hybridization experiments carried out with labelled mitochondrial probes, containing the genes for cytochrome oxidase subunit I and 12S rRNA, confirmed the mitochondrial nature of the isolated RNA.
Assuntos
Mitocôndrias Hepáticas/efeitos da radiação , Adenilato Quinase/metabolismo , Adenilato Quinase/efeitos da radiação , Animais , Fracionamento Celular , DNA Mitocondrial/genética , DNA Mitocondrial/efeitos da radiação , Glutamato Desidrogenase/metabolismo , Glutamato Desidrogenase/efeitos da radiação , Hélio , L-Lactato Desidrogenase/metabolismo , L-Lactato Desidrogenase/efeitos da radiação , Lasers , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Masculino , Mitocôndrias Hepáticas/metabolismo , Mitocôndrias Hepáticas/fisiologia , Neônio , RNA/efeitos da radiação , Ratos , Ratos Endogâmicos , Transcrição Gênica/efeitos da radiaçãoRESUMO
The functional molecular mass for ubiquinol-1 oxidation by the Escherichia coli terminal oxidase, cytochrome o, was determined by radiation-inactivation analysis of membranes from a cytochrome d-deficient mutant. The functional molecular mass for ubiquinol-l oxidase activity was found to be 38.3 +/- 2.65 kDa.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Escherichia coli/enzimologia , Quinona Redutases/metabolismo , Membrana Celular/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/química , Cinética , Malato Desidrogenase/metabolismo , Malato Desidrogenase/efeitos da radiação , Peso Molecular , Quinona Redutases/química , Quinona Redutases/efeitos da radiaçãoRESUMO
The mechanism by which the ferredoxin-thioredoxin system activates the target enzyme, NADP-malate dehydrogenase, was investigated by analyzing the sulfhydryl status of individual protein components with [14C]iodoacetate and monobromobimane. The data indicate that ferredoxin-thioredoxin reductase (FTR)--an iron-sulfur enzyme present in oxygenic photosynthetic organisms--is the first member of a thiol chain that links light to enzyme regulation. FTR possesses a catalytically active dithiol group localized on the 13 kDa (similar) subunit, that occurs in all species investigated and accepts reducing equivalents from photoreduced ferredoxin and transfers them stoichiometrically to the disulfide form of thioredoxin m. The reduced thioredoxin m, in turn, reduces NADP-malate dehydrogenase, thereby converting it from an inactive (S-S) to an active (SH) form. The means by which FTR is able to combine electrons (from photoreduced ferredoxin) with protons (from the medium) to reduce its active disulfide group remains to be determined.
Assuntos
Oxirredutases/efeitos da radiação , Tolueno/análogos & derivados , Catálise , Ferredoxinas/análise , Proteínas Ferro-Enxofre , Luz , Malato Desidrogenase/efeitos da radiação , Oxirredução/efeitos da radiação , Oxirredutases/metabolismo , Plantas/enzimologia , Compostos de Sulfidrila/análise , Tiorredoxinas/análise , Tolueno/análiseRESUMO
The accuracy of the radiation-inactivation technique for estimating molecular size was investigated with a range of proteins of known molecular mass. With the use of irradiation with a 16 MeV electron beam, inactivation was examined both in frozen samples at 77 K and in freeze-dried samples at room temperature. The effect of the presence of detergents and chloroplast membrane preparations was also measured. It was demonstrated that proteins added as internal standards, including malate dehydrogenase, glucose-6-phosphate dehydrogenase and cytochrome c, can provide an accurate calibration of molecular size. However, a disadvantage of the technique was that the target size of oligomeric enzymes could be that of either the monomers, dimers or higher oligomers. The detergent Triton X-100 increased the rate of inactivation of the proteins investigated.
Assuntos
Proteínas/efeitos da radiação , Membrana Celular/enzimologia , Membrana Celular/efeitos da radiação , Cloroplastos/efeitos da radiação , Enzimas/efeitos da radiação , Liofilização , Congelamento , Substâncias Macromoleculares , Malato Desidrogenase/efeitos da radiação , Proteínas de Membrana/efeitos da radiação , Peso Molecular , Padrões de ReferênciaRESUMO
Light-grown cultures of Neurospora crassa showed photoregulation of a number of enzymes. Proteases and cytosolic malate dehydrogenase showed an increase in activity. There was a decrease in the activity of mitochondrial malate dehydrogenase, isocitrate dehydrogenase and cytosolic glucose-6P-dehydrogenase, isocitrate dehydrogenase and isocitrate lyase.
Assuntos
Luz , Neurospora crassa/enzimologia , Neurospora/enzimologia , Citosol/enzimologia , Frutose-Bifosfato Aldolase/efeitos da radiação , Glucosefosfato Desidrogenase/efeitos da radiação , Isocitrato Desidrogenase/efeitos da radiação , Isocitrato Liase/efeitos da radiação , Malato Desidrogenase/efeitos da radiação , Mitocôndrias/enzimologia , Neurospora crassa/efeitos da radiaçãoAssuntos
Micro-Ondas , Mitocôndrias Hepáticas/enzimologia , Adenosina Trifosfatases/efeitos da radiação , Adenilato Quinase/efeitos da radiação , Animais , Complexo IV da Cadeia de Transporte de Elétrons/efeitos da radiação , Técnicas In Vitro , Malato Desidrogenase/efeitos da radiação , Masculino , NAD/efeitos da radiação , Doses de Radiação , Ratos , Ratos Endogâmicos , Frações Subcelulares/enzimologiaRESUMO
The inactivation of Coxiella burnetii in suspension or in cultures of guinea pig peritoneal macrophages by ultraviolet (UV) light was studied. The effect of UV treatment on the activity of macrophage organelle marker enzymes and their subsequent equilibration in linear sucrose gradients was also determined. It was shown that UV treatment of 600 mu W/cm2 for 15 s at a distance of 10 cm inactivated C. burnetti, either in suspension (10(8) organisms per ml) or within guinea pig peritoneal macrophages. Similar UV treatment had little effect on the activity or equilibration of macrophage organelle marker enzymes in linear sucrose gradients. However, longer exposure caused considerable inactivation of these enzymes.