A chemiluminescent method for measurement of activated oxygen forms in biological fluids and homogenates

A chemiluminescent method for measuring the concentration of activated oxygen species (O₂² and H₂O₂) is described. Its main features are: high sensitivity (10^?9 M H₂O₂), its applicability to systems with high optical absorbance in the visible spectral region, a wide linear dynamic range, and the possibility for recording the kinetics of the processes, in which activated oxygen species are involved.     

蚕豆叶片发育与衰老过程中超氧物歧化酶活性与丙二醛含量变化

蚕豆植株叶片随茎节自上而下表现出明显的发育与衰老顺序,可作为衰老特征的是叶绿素和蛋白质含量明显下降。蚕豆叶中SOD活性主要定位于12 000× g离心后所得的上清液和叶绿体组分。衰老叶片的SOD总活性和叶绿体组分的相对活性都有所下降,SOD同工酶谱也发生了改变。O_2~ 产生速率随叶龄增大而稍上升;而MDA含量在叶片外观表现枯黄衰老征兆前就急剧上升。可能因为衰老叶片过氧化氢酶活性大幅度下降与SOD之间的不平衡,致使O_2~ 代谢中间产物累积而引起膜的损伤.     

水稻不育花药中H_2O_2的积累与膜脂过氧化的加剧

水稻7017、二九矮细胞质雄性不育系及其保持系花药的POD,CAT和SOD活性研究的结果表明,单核早期时不育及可育花药的酶活性差异不明显,单核晚期、二核及三核期的不育花药显著低于可育花药。在不育花药中缺少两条Cu-Zn SOD同工酶带,而且O_2~ 产生效率为可育的4.1～5.5倍,并有H_2O_2和MDA的积累。不育花药中H_2O_2的积累和膜脂过氧化的加剧可能与花粉败育有关。     

Oxidant inhibition of epithelial active sodium transport

The purpose of this study was to quantify the effects of extracellularly generated partially reduced oxygen species on active sodium (NA⁺) transport across the ventral toad skin, a well-studied epithelium. Sections of skin from decapitated toads were mounted in an Ussing chamber, bathed on both sides with electrolyte solution containing 500 μM xanthine and bubbled continuously with room air. The tissues were short-circuited, and short circuit current (Isc) and tissue resistance (R_t were monitored continuously with an automatic voltage clamp apparatus. Fifteen mU/ml of xanthine oxidase (XO), either purchased from Calbiochem or purified from cream, were instilled in either the apical (mucosal) or basolateral (serosal) baths at t = 0 and T = 10 min. Hydrogen peroxide (H₂O₂) concentrations increased to 200 μM within the first 20 min and then decreased, reaching a value of 40 μM by 60 min. Mean [H₂O₂] was 90 μM. Instillation of XO in the apical bath resulted in a large decrease in Isc and an increase in R_t, their values being 43% and 160% of their corresponding controls 85 min after the first instillation. Addition of superoxide dismutase and catalase completely prevented these changes. Instillation of XO in the basolateral bath had no effect. Similar physiological responses were obtained using the Calbiochem XO or the purified XO, which contained no measurable protease activity. It was concluded that extracellularly generated partially reduced oxygen species may interfere with active Na⁺ transport by possibly damaging apical Na⁺ channel proteins.     

Vanadate-stimulated oxidation of NAD(P)H

Vanadate stimulates the oxidation of NAD(P)H by biological membranes because such membranes contain NAD(P)H oxidases which are capable of reducing dioxygen to O₂⁻ and because vanadate catalyzes the oxidation of NAD(P)H by O₂⁻, by a free radical chain mechanism. Dihydropyridines, such as reduced nicotinamide mononucleotide (NMNH), which are not substrates for membrane-associated NAD(P)H oxidases, are not oxidized by membranes plus vanadate unless NAD(P)H is present to serve as a source of O₂⁻. When [NMNH] greatly exceeds [NAD(P)H], in such reaction mixtures, one can observe the oxidation of many molecules of NMNH per NAD(P)H consumed. This reflects the chain length of the free radical chain mechanism. We have discussed the mechanism and significance of this process and have tried to clarify the pertinent but confusing literature.     

Chemiluminescent oxidation of ribose catalyzed by horseradish peroxidase in presence of hydrogen peroxide

The reaction of ribose with horseradish peroxidase in the presence of H₂O₂ is accompanied by light emission. The detection of horseradish peroxidase Compound II (FeO²⁺) indicates that the enzyme participates in a normal peroxidatic cycle. Hydrogen peroxide converts horseradish peroxidase into Compound I (FeO³⁺) which in turn is converted into Compound II by abstracting a hydrogen atom from ribose forming a ribosyl radical. In aerated solutions oxygen rapidly adds to the ribosyl radical. Based on the spectral characteristics and the enhancement of the chemiluminescence by chlorophyll-a, xanthene dyes, D₂O and DABCO, it is suggested that the excited species, apparently triplet carbonyls and ¹O₂, are formed from the bimolecular decay of the peroxyl radicals via the Russell mechanism.     

Potentiation by the Tetraphenylboron Anion of the Effects of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine and Its Pyridinium Metabolite

The 1-methyl-4-phenylpyridinium species (MPP+) is the four-electron oxidation product of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and is widely assumed to be the actual neurotoxic species responsible for the MPTP-induced destruction of dopaminergic neurons. MPTP is oxidized by the enzyme monoamine oxidase-B to a dihydropyridinium intermediate which is oxidized further to MPP+, an effective inhibitor of the oxidation of the Complex I substrates glutamate/malate in isolated mitochondrial preparations. In the present study, the tetraphenylboron anion (TPB) greatly potentiated the inhibitory effects of MPP+ and other selected pyridinium species on glutamate/malate respiration in isolated mouse liver mitochondria. At 10 microM TPB, the potentiation ranged from approximately 50-fold to greater than 1,000-fold for the several pyridinium species tested. In other experiments, TPB greatly enhanced the accumulation of [3H]MPP+ by isolated mitochondrial preparations. This facilitation by TPB of MPP+ accumulation into mitochondria explains, at least in part, the potentiation by TPB of the above-mentioned inhibition of mitochondrial respiration. Moreover, TPB addition increased the amount of lactate formed during the incubation of mouse neostriatal tissue slices with MPTP and other tetrahydropyridines. The administration of TPB also potentiated the dopaminergic neurotoxicity of MPTP in male Swiss-Webster mice. All of these observations, taken together, are consistent with the premise that the inhibitory effect of MPP+ on mitochondrial respiration within dopaminergic neurons is the ultimate mechanism to explain MPTP-induced neurotoxicity.     

Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells

Summary In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, _DBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID_50,DBDS,MCD (0.5±0.1 m) for the H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is in agreement with the ID_50,Cl ^–,_MCD (0.94±0.07 m) for H₂-DIDS inhibition of MCD cell Cl^– flux, thus relating _DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl^– exchange by a factor of two, from _Cl=0.30±0.02 sec to 0.56±0.06 sec (30mm NaHCO₃). The ID_50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003±0.001 m, so that binding is about an order of magnitude tighter than that for inhibition of rbc K⁺ flux (K _I,K ⁺,_rbc=0.017 m). These experiments indicate that the Na⁺,K^–-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells, modulates DBDS binding kinetics with a physiological ID_50,DBDS,MCD (0.076±0.005 m); 2 m CE also more than doubles the Cl^– exchange time constant from 0.20±0.04 sec to 0.50±0.08 sec (30mm NaHCO₃). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.     

Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL-60 and U 937 cells

The development of cytochrome b558 (Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL-60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFN gamma. O2- release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin-amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol-amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2- production is closely related to Cyt b but not to MPO specific contents. In differentiated HL-60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFN gamma to the RA treatment. However, marked differences in the O2- production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA-stimulated O2- production is rather low ie 100 and 20 times less in granulocytic HL-60 and monocyto-macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte-macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2- production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFN gamma and/or with RA.