Construction and analysis of a semi-quantitative energy profile for the reaction catalyzed by the radical enzyme galactose oxidase

The investigation of the effect of oxidized lipoproteins on platelet activity is important for the understanding of the plague formation under atherosclerosis. In the present work, we examined the influence of low density lipoproteins (LDL) on ADP-induced platelet aggregation in the platelet rich plasma. In was demonstrated that mixing of plasma and LDL was accompanied by the decrease of ADP-induced aggregation parameters as compared to control (mixing with buffer). After 1 h incubation, platelet ADP-aggregation in the sample containing oxidized LDL (oxLDL) exceeded the ADP-aggregation in the control sample. The dependence of the aggregation parameters on the incubation time and on the degree of LDL oxidation were obtained. No difference in the cholesterol and phospholipid content was observed between cells incubated with buffer, native or oxidized LDL. Therefore, the possible oxLDL-induced accumulation of cholesterol in platelet membranes is excluded as a reason for the increased cell aggregation.     

13C- and 15N-labeled peptide substrates as mechanistic probes of oligosaccharyltransferase

The carboxamide moiety that links the carbohydrate and protein moieties in N-linked glycoproteins has been unambiguously determined to arise intact from asparagine by the use of chemically synthesized Bz-[4-13C, 15N]Asn-Leu-Thr-NH2 as an oligosaccharyltransferase (OST) substrate. Bz-[4-13C]Asn-Leu-Thr-NH2 was also synthesized and used to evaluate a proposed mechanism of OST catalysis similar to that of glutamine-dependent amidotransferases using 15NH4OAc as a potential external nucleophile. Analysis of NMR and MS spectra of the isotopically labeled peptides and the resulting biosynthesized glycopeptides indicates that free 15NH3 is not lost from the doubly labeled substrate during catalysis nor can exogenous 15NH3 intercept any of several postulated enzyme-bound species. These results indicate that OST-catalyzed glycosylation does not follow a mechanism involving the transient generation of exchangeable "NH3". Thus, in contrast to several glutamine-dependent amidotransferases, OST catalysis does not lead to transient scission of the asparagine beta-carboxamide C-N bond. Together with previously published results, these data argue against nucleophilic activation of the asparagine beta-carboxamide moiety being the underlying chemical mechanism for OST-catalyzed glycosylation of peptides.     

Imidazolyl carboxylic acids as mechanistic probes of flavocytochrome P-450 BM3

omega-Imidazolyl carboxylic acids (C10-C12) have been used as probes of the active site and catalytic mechanism of the fatty acid hydroxylase P-450 BM3 from Bacillus megaterium. These compounds are the most potent inhibitors of P-450 BM3 yet reported. All are mixed inhibitors, increasing the Km and decreasing the kcat for laurate oxidation. All ligate the P-450 BM3 ferric heme iron, inducing a type II shift in the Soret absorbance band from 419 to 424 nm. Binding to the ferrous form is much weaker. 10-(Imidazolyl)decanoic acid was the best inhibitor (Kic = 0.9 microM, Kiu = 5.7 microM), while 12-(imidazolyl)dodecanoic acid (Kic = 1.35 microM, Kiu = 6.9 microM) was superior to 11-(imidazolyl)undecanoic acid (Kic = 7.5 microM, Kiu = 16 microM). Dissociation constants for binding to oxidized P-450 BM3 heme iron were determined spectrophotometrically as 8 microM (C12 azole) and 27 microM (C11 azole). The binding of 10-(imidazolyl)decanoic acid was too tight for an absolute Kd to be determined spectrophotometrically, but this value is <0.2 microM. The binding of different fatty acids to the enzyme was found to have distinct effects on the Kd for the azoles. Laurate induced tighter binding (Kd for the C12 azole lowered to 4.7 microM), while arachidonate weakened the affinity (Kd increased to 23 microM). Arachidonate diminished the affinity for the C10 azole sufficiently that a Kd could be determined by spectrophotometric titration (11 microM). Affinity for the C12 azole was decreased in active-site-mutants R47G (R47 tethers the fatty acid carboxylate group) and F87Y but increased in mutant F87G-indicating an important role for this residue in determining heme accessibility. The C10 azole binds much more weakly to the spin-state-insensitive F87Y (32. 2 microM), suggesting that the inhibitors may bind preferentially to different conformers of P-450 BM3. NADP+ binding in the reductase also tightened affinity of these inhibitors for P-450 BM3 (Kd for the C12 azole decreased to 2.7 microM), but this effect was not observed for FMN-deficient mutant W574D, suggesting that the interdomain effect of NADP+ on inhibitor binding was mediated via flavin mononucleotide. Resonance Raman spectroscopy indicates that the inhibitors form low-spin complexes with P-450 BM3 and that their binding induces movements of the heme vinyls relative to the ring.     

Cyclopropyl indolequinones: mechanistic probes for bioreductive anticancer drug action

A series of cyclopropyl indolequinones based on structures 5-7 was designed and synthesized to probe the structural features essential for bioreductive cytotoxicity. Ring opening of the cyclopropane ring under radical conditions was demonstrated to be mechanistically feasible, and related to the involvement of such one-electron processes in the cytotoxicity of cyclopropyl indolequinones under hypoxic conditions.     

Mechanochemical mechanism for peptidyl free radical generation by amyloid fibrils

beta-Amyloid peptides (A beta) form the core of Alzheimer's disease (AD) senile plaques, and are implicated in AD neurotoxicity. A beta and some derivatives generate free radicals upon fibrilogenesis. A mechanism for free radical generation is proposed, based upon fibril cross beta-sheet structure: (1) During fibrilogenesis there is a small probability of mispacking of A beta monomers, resulting in abnormal fibril packing. (2) Continued fibrilogenesis traps a packing defect within the beta-sheet. Surrounding beta-sheet resists distortion, and the abnormally packed polypeptide(s) is strained. (3) Thermal processes cause homolytic bond scission and radical production from strained polypeptide through mechanically activated thermal decomposition. (4) Reaction with oxygen produces peroxy radicals, prevents unproductive radical recombination, and promotes observed cross-linking, production of reactive oxygen species and peptide fragmentation. Adiabatic mapping suggested significant strain would be generated by beta-sheet misalignment. The mechanism relates the common structure of fibrils to radical production, and may be relevant to cytotoxicity in prion and other amyloidoses.     

Reduction of the ascorbyl free radical to ascorbate by thioredoxin reductase

Recycling of ascorbic acid from its oxidized forms is required to maintain intracellular stores of the vitamin in most cells. Since the ubiquitous selenoenzyme thioredoxin reductase can recycle dehydroascorbic acid to ascorbate, we investigated the possibility that the enzyme can also reduce the one-electron-oxidized ascorbyl free radical to ascorbate. Purified rat liver thioredoxin reductase catalyzed the disappearance of NADPH in the presence of low micromolar concentrations of the ascorbyl free radical that were generated from ascorbate by ascorbate oxidase, and this effect was markedly stimulated by selenocystine. Dehydroascorbic acid is generated by dismutation of the ascorbyl free radical, and thioredoxin reductase can reduce dehydroascorbic acid to ascorbate. However, control studies showed that the amounts of dehydroascorbic acid generated under the assay conditions used were too low to account for the observed loss of NADPH. Electron paramagnetic resonance spectroscopy directly confirmed that the reductase decreased steady-state ascorbyl free radical concentrations, as expected if thioredoxin reductase reduces the ascorbyl free radical. Dialyzed cytosol from rat liver homogenates also catalyzed NADPH-dependent reduction of the ascorbyl free radical. Specificity for thioredoxin reductase was indicated by loss of activity in dialyzed cytosol prepared from livers of selenium-deficient rats, by inhibition with aurothioglucose at concentrations selective for thioredoxin reductase, and by stimulation with selenocystine. Microsomal fractions prepared from rat liver showed substantial NADH-dependent ascorbyl free radical reduction that was not sensitive to selenium depletion. These results suggest that thioredoxin reductase can function as a cytosolic ascorbyl free radical reductase that may complement cellular ascorbate recycling by membrane-bound NADH-dependent reductases.     

Ascorbyl free radical as a real-time marker of free radical generation in briefly ischemic and reperfused hearts. An electron paramagnetic resonance study

The role of free radicals in myocardial reperfusion injury remains controversial. We have developed a new method using ascorbyl free radical (AFR) as a real-time, quantitative marker of free radical generation during myocardial reperfusion. A total of 35 dogs were studied. Twelve open-chest dogs underwent either 5 minutes (n = 5) or 20 minutes (n = 7) of coronary artery occlusion and 30 minutes of reperfusion. Seven additional animals undergoing 20 minutes of coronary occlusion also received the antioxidant enzymes superoxide dismutase and catalase, beginning 10 minutes before occlusion through the end of reperfusion. Exogenous ascorbate was infused intravenously, and the concentration of AFR in the great cardiac vein was continuously measured by electron paramagnetic resonance spectroscopy. Preocclusion AFR concentration was similar in the three groups. Upon reperfusion, AFR rose significantly in each animal group (P < .05). However, the AFR rise in the 20-minute-occlusion group, 38 +/- 17%, was significantly greater than in the 5-minute-occlusion group, 27 +/- 14% (P < .002). In addition, in the animals that received superoxide dismutase and catalase, the rise in the AFR was markedly attenuated, 13 +/- 6% (P < .002). Two dogs that received ascorbate but did not undergo coronary artery occlusion/reperfusion sequences showed no change in coronary venous AFR signal, indicating the stability of the signal over time. Five dogs received ascorbate while undergoing interventions to alter coronary venous flow: intravenous saline, dobutamine, dipyridamole, and nitroglycerin. Coronary venous AFR changes were minimal despite large coronary flow alterations, indicating that the AFR signal is independent of changes in coronary venous flow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence microscopic observation of catalysis by single or few LDH-1 enzyme molecules

Lactate dehydrogenase (LDH-1) catalyzes the reaction of lactate and nonfluorescent NAD+ to pyruvate, NADH (fluorescence at lambda em = 455 nm, lambda em = 365 nm) and H+. The injection of highly diluted LDH-1 solution into a drop of substrate solution results in the formation of a bubble of enzyme inside the drop of substrate. At the contact surface between the enzyme solution and the substrate, discrete and statistically distributed zones of increasing fluorescence intensity and different size can be observed after enzyme injection. These zones can be interpreted as clouds of NADH around a single or a few enzyme molecules. The kinetics of the NADH formation in every fluorescent zone, and the size of the zone, can be described by a zero order production combined with a diffusion controlled loss of the reaction's product NADH from the reaction zone. From the dilution of the enzyme solution and from statistical analysis one can conclude that only few enzyme molecules in the center of the fluorescent reaction zones catalyze the NADH formation.     

Effect of hydrocortisone on oxygen free radical generation by mononuclear cells

Corticosteroids are known to exert antiinflammatory and immunosuppressive effects. Since reactive oxygen species (ROS) induce tissue damage and inflammation and since mononuclear cells (MNCs) generate ROS, we investigated whether corticosteroids inhibit ROS generation by MNCs when given systemically. A single dose of either 300 mg (n = 8) or 100 mg (n = 6) of hydrocortisone (HC) was injected intravenously into eight and six subjects, respectively. Blood samples were obtained before and sequentially after the injection. Following 300 mg HC, N-formylmethionyl leucyl phenylalanine (fMLP)-induced ROS generation, assayed by measuring chemiluminescence with luminol, decreased significantly at 0.5 hours and reached a nadir at 2 hours (8% of basal, P < .001); thereafter, it gradually recovered, but was still below baseline at 24 hours. Following the dose of 100 mg HC, ROS generation decreased significantly at 1 hour (nadir, 30% of basal; P < .01) and gradually recovered to near basal level at 8 hours. Serum cortisol concentrations were markedly elevated over basal and remained elevated throughout the first 8 hours of the experiment, returning to baseline at 24 hours. This inhibition of ROS generation by HC (and other glucocorticoids) may have a role to play in mediating the antiinflammatory action of corticosteroids.     

Use of cholesterol oxidase for assay of total and free cholesterol in serum by continuous-flow analysis

Idescribe an assay for total cholesterol in serum, with use of the AutoAnalyzer ii (Technicon), in which cholesterol esters are saponified by alkali, cholesterol is held in aqueous micellar solution with a surfactant (Triton X-100) and oxidized by cholesterol oxidase, and the hydrogen peroxide produced is measured by chelation with Ti4+ and xylenol orange. An assay for free cholesterol in serum, based on similar principles, is also described, and the two can be run simultaneously on a dual-channel AutoAnalyzer II. Standard solutions of cholesterol in isopropanol have poorer carryover characteristics than sera, and therefore do not reach the same continuous-flow steady state as sera of equivalent concentrations. Consequent potential calibration errors are avoided by using micellar solutions of cholesterol containing albumin for standardization. The formation of cholesterol peroxide in solutions of cholesterol in isopropanol is demonstrated, and this constitutes another potential source of error in the calibration of enzymic cholesterol assays. In analyzing patients' sera, results of the total cholesterol assay correlate well with those of a mechanized method in which cholesterol esterase and cholesterol oxidase are used; an automated Abell method, calibrated with solutions of cholesterol in isopropanol, gave slightly higher values. Determinations of the ratio of free cholesterol to total cholesterol by our automated cholesterol exidase assays given values that agree well with published results in which digitonin precipitation is used.     

Marchantinquinone, isolated from Reboulia hemisphaerica, as inhibitor of lipid peroxidation and as free radical scavenger

Recent years have witnessed a renewed interest in plants as pharmaceuticals in the Western world. This interest is channeled into the discovery of new biologically-active molecules by the pharmaceutical industry and into the adoption of crude extracts of plants for self-medication by the general public. In both of these areas some attention is being paid to the investigation and use of ethnopharmacology, the traditional use of plants for medicinal purposes by particular cultural groups. Ethnopharmacologic leads have resulted in the introduction of new single molecule drugs but have a greater role to play if crude extracts are accepted for clinical use in the West. The problems confronting such usage are discussed. Considerable benefits for developing countries are possible when the local medicinal plants are subjected to scientific methods of validation of traditional use and quality control. This approach has met with success in some parts of the world but is not always appreciated by national governments and international agencies. Related areas of concern such as conservation of ecology and culture must be integrated with any such program. Plants used in traditional medicine therefore have an important role to play in the maintenance of health in all parts of the world and in the introduction of new treatments.     

Nitroxide free radical clearance in the live rat monitored by radio-frequency CW-EPR and PEDRI

The use of RF (100 to 300 MHz) PEDRI and CW-EPR techniques allows the in vivo study of large animals such as whole rats and rabbits. Recently a PEDRI instrument was modified to also allow CW-EPR spectroscopy with samples of similar size and under the same experimental conditions. In the present study, this CW-EPR and PEDRI apparatus was used to assess the feasibility of the detection of a pyrrolidine nitroxide free radical (2,2,5,5,-tetramethylpyrrolidine-1-oxyl-3-carboxylic acid, PCA) in the abdomen of rats. In particular, we have shown that after the PCA administration (4 mmol kg(-1) b.w.): (i) the PCA EPR linewidth does not show line broadening due to concentration effects; (ii) a similar PCA up-take phase is observed by EPR and PEDRI; and (iii) the PCA half-lives in the whole abdomen of rats measured with the CW-EPR (T1/2=26+/-4 min, mean+/-sd, n=10) and PEDRI (T1/2=29+/-4 min, mean+/-sd, n=4) techniques were not significantly different (p > 0.05). These results show, for the first time, that information about PCA pharmacokinetics obtained by CW-EPR is the same as that from PEDRI under the same experimental conditions.     

溴邻苯三酚红氧化法检测Cu^＋和H2O2反应产生的羟自由基

建立了以Cu^ -H2O^-溴邻苯三酚红体系测定羟自由基的新方法。该方法基于Cu^ 和H2O2反应产生羟自由基;它能使溴邻苯三酚红的颜色发生变化,用分光光度计测定其ΔA值的变化,可间接测定羟自由基的产生量,通过研究,得到最佳实验条件。结果表明,该方法快速,稳定性好,操作简便,可作为一种简易的筛选抗氧化剂的方法。     

A method of one-step enzyme labelling of short oligonucleotide probes for filter hybridisation

Here we describe a method of labelling short oligonucleotide probes with enzyme without purification or chemical modifications. Biotinylated oligonucleotides as short as 10 nt are coupled with streptavidin-conjugated enzyme, hybridised and detected with enzyme-triggered chemiluminescence. The detection of hybridisation signal is linear for two orders of magnitude of target dilution. It is shown to be comparable in sensitivity with standard procedures and with radioactive detection. The method is quick, simple and has potential for automation of large-scale oligo-nucleotide hybridisation and multiplex sequencing.     

Evidence of hydroxyl free radical generation by calcium overload in rat myocardium

Although calcium (Ca2+) is important in cardiac dysfunction and has also been reported as a source of oxidative toxicity, the connection between Ca2+ overload and oxygen free radicals in the myocardium is not clear. We have investigated whether Ca2+ overload generates hydroxyl free radicals in rat ventricle. HPLC with electrochemical detection was used to measure the levels of 2,3- and 2,5-dihydroxybenzoic acid (DHBA) formed when the hydroxyl free radical reacts with salicylate. Ringer's solution containing salicylic acid (0.5 nmol microL-1 min-1) was infused through a microdialysis probe in the region of the left anterior descending coronary artery of the rat ventricle. A positive linear correlation was obtained between Ca2+ and hydroxyl free radical formation trapped as 2,3-DHBA (r2 = 0.976) and 2,5-DHBA (r2 = 0.982) in the myocardial dialysate. The administration of ouabain (1 mg kg-1, i.v.), a Ca2+ elevator, into the femoral vein significantly increased the level of 2,3- and 2,5-DHBA. These results indicate that Ca2+ overload generates hydroxyl free radicals in rat heart.     

Characterization of cytochrome c free radical reactions with peptides by mass spectrometry

The reactions of horse heart cytochrome c, hydrogen peroxide, and the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid with a series of polypeptides were investigated using mass spectrometry. The mass spectra obtained from these reactions revealed that after a free radical has been generated on the heme-containing protein horse heart cytochrome c, it can be transferred to other biomolecules. In addition, the number of free radicals transferred to the target molecule could be determined. Recipient peptides/proteins that contained a tyrosine and/or tryptophan amino acid residue were most susceptible to free radical transfer. Using tandem mass spectrometry, the location of the 3,5-dibromo-4-nitrosobenzenesulfonic acid radical adduct on the nonapeptide RWIILGLNK was unequivocally determined to be at the tryptophan residue. We also demonstrated that the presence of an antioxidant in the reaction mixture not only inhibits free radical formation on horse heart cytochrome c, but also interferes with the transfer of the free radical, once it has been formed on cytochrome c.     

Macrophage and microglial responses to cytokines in vitro: phagocytic activity, proteolytic enzyme release, and free radical production

Our rapidly expanding knowledge of the cause and pathogenesis of Community-acquired pneumonia (CAP) offers new opportunities to prevent this disease. Influenza vaccine is effective for the prevention of respiratory illness, including pneumonia, in the setting of influenza A and B infection. Pneumococcal vaccine is effective for preventing the most common form of bacterial CAP, but it is most effective when administered early in the course of chronic illnesses. Even with the widespread availability and proven efficacy of influenza and pneumococcal vaccines, their use has remained suboptimal. Rimantadine and Amantadine have also been used successfully for prevention of influenza A infection. Further improvement in strategies for the prevention of CAP lies in the development of new and improved vaccines, enhanced environmental control, and general education of physicians and the public, so that new approaches such as hospital-based immunization can be applied.