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1.
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Energy metabolism and lipid peroxidation of human erythrocytes as a function of increased oxidative stress. 总被引:5,自引:0,他引:5
B Tavazzi D Di Pierro A M Amorini G Fazzina M Tuttobene B Giardina G Lazzarino 《European journal of biochemistry》2000,267(3):684-689
To study the influence of oxidative stress on energy metabolism and lipid peroxidation in erythrocytes, cells were incubated with increasing concentrations (0.5-10 mM) of hydrogen peroxide for 1 h at 37 degrees C and the main substances of energy metabolism (ATP, AMP, GTP and IMP) and one index of lipid peroxidation (malondialdehyde) were determined by HPLC on cell extracts. Using the same incubation conditions, the activity of AMP-deaminase was also determined. Under nonhaemolysing conditions (at up to 4 mM H2O2), oxidative stress produced, starting from 1 mM H2O2, progressive ATP depletion and a net decrease in the intracellular sum of adenine nucleotides (ATP + ADP + AMP), which were not paralleled by AMP formation. Concomitantly, the IMP level increased by up to 20-fold with respect to the value determined in control erythrocytes, when cells were challenged with the highest nonhaemolysing H2O2 concentration (4 mM). Efflux of inosine, hypoxanthine, xanthine and uric acid towards the extracellular medium was observed. The metabolic imbalance of erythrocytes following oxidative stress was due to a dramatic and unexpected activation of AMP-deaminase (a twofold increase of activity with respect to controls) that was already evident at the lowest dose of H2O2 used; this enzymatic activity increased with increasing H2O2 in the medium, and reached its maximum at 4 mM H2O2-treated erythrocytes (10-fold higher activity than controls). Generation of malondialdehyde was strictly related to the dose of H2O2, being detectable at the lowest H2O2 concentration and increasing without appreciable haemolysis up to 4 mM H2O2. Besides demonstrating a close relationship between lipid peroxidation and haemolysis, these data suggest that glycolytic enzymes are moderately affected by oxygen radical action and strongly indicate, in the change of AMP-deaminase activity, a highly sensitive enzymatic site responsible for a profound modification of erythrocyte energy metabolism during oxidative stress. 相似文献
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A Bellelli M Brunori S G Condò B Giardina 《The Journal of biological chemistry》1987,262(6):2624-2629
The properties of human hemoglobin reacted with 2-nor-2-formylpyridoxal 5'-phosphate, a bifunctional derivative of pyridoxal 5'-phosphate, have been investigated both from an equilibrium and kinetic point of view. The experimental data, interpreted in terms of the two-state allosteric model, indicate that a perturbed R state is characteristic of this modified low ligand affinity hemoglobin. In flash photolysis experiments, a quickly reacting component is always observed, in spite of the lack of dissociation into free dimers; this kinetic behavior is thought to reflect the presence of functionally independent alpha beta dimers, still connected by the flexible cross-link but forming an open hemoglobin tetramer. Two possible models for the interpretation of the kinetics of CO and/or haptoglobin binding are presented and discussed. 相似文献
5.
G De Sanctis G Falcioni B Giardina F Ascoli M Brunori 《Journal of molecular biology》1986,188(1):73-76
A domain of 108 amino acid residues (32 to 139), obtained by digestion of horse heart apomyoglobin with clostripain, was found to bind protoheme in a 1 to 1 molar ratio. This domain is 33 amino acid residues larger than the protein segment encoded by the central exon in seal myoglobin. Flash photolysis experiments have shown that reconstituted "mini-myoglobin" is very similar to myoglobin in the combination reaction with carbon monoxide and with oxygen, and in the oxygen replacement reaction by carbon monoxide. These experiments provide for the first time direct evidence for the presence of a structural and functional domain, closely corresponding to the segment encoded by the central exon of the myoglobin gene, which contains the information for binding the natural heme and for maintaining the native folding typical of a respiratory protein. 相似文献
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S. Caretto M. C. Giardina C. Nicolodi D. Mariotti 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1994,88(5):520-524
Daucus carota L. cell lines stably resistant to the herbicide chlorsulfuron (CS) have been isolated according to a stepwise selection. Studies carried out during different selection steps show that the specific activity of the target enzyme acetohydroxyacid synthase (AHAS) increases along with CS resistance. Southern hybridization analysis performed with aBrassica napus AHAS probe in a CS highly-resistant cell line reveals the presence of a greatly amplifiedEcoRI fragment of genomic DNA. This indicates that AHAS overproduction induced by stepwise selection is due to gene amplification. Regenerants from some resistant cell lines maintained the CS-resistant trait at the whole plant level. 相似文献
8.
R. Vagnozzi G. Lazzarino B. Tavazzi D. Di Pierro P. Siragusa R. Giuffré B. Giardina 《Biological trace element research》1995,47(1-3):241-246
Short-term incomplete cerebral ischemia was induced in the rat by bilaterally clamping for 5 min the common carotid arteries;
subsequent reperfusion of 10 min was obtained by removing carotid occlusion. At the end of ischemia or reperfusion, animals
were sacrificed by decapitation. A control group was represented by sham-operated rats. Peripheral venous blood samples were
withdrawn from the femoral vein from rats subjected to cerebral reperfusion 5 min before ischemia, at the end of ischemia,
and 10 min after reperfusion. A highly sensitive HPLC method for the direct determination of malondialdehyde, oxypurines,
and nucleosides was used on 200 μL of brain tissue and plasma extracts. Incomplete cerebral ischemia induced the, appearance
of a significant amout of tissue malondialdehyde (undetectable in control animals) and a decrease of ascorbic acid. A further
6.6-fold increase of malondialdehyde and a 18.5% decrease of ascorbic acid occurred after 10 min of reperfusion. Plasma malondialdehyde,
which was present in minimal amount before ischemia, significantly increased after 5 min of ischemia, being strikingly augmented
after 10 min of reperfusion. A similar trend was observed for oxypurines and nucleosides. From these data, it can be affirmed
that tissue concentrations of malondialdehyde and ascorbic acid, and plasma levels of malondialdehyde, oxypurines, and nucleosides,
reflect both the oxygen radical-mediated tissue injury and the depression of energy metabolism thus representing early biochemical
markers of short-term incomplete brain ischemia, and reperfusion in the rat. 相似文献
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S H Orkin J P Sexton T C Cheng S C Goff P J Giardina J I Lee H H Kazazian 《Nucleic acids research》1983,11(14):4727-4734