Katsuko Kakinuma

Development of novel fluorescence probes that can reliably detect reactive oxygen species and distinguish specific species.

We designed and synthesized 2-[6-(4′-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF) and 2- [6-(4′-amino)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (APF) as novel fluorescence probes to detect selectively highly reactive oxygen species (hROS) such as hydroxyl radical (⋅OH) and reactive intermediates of peroxidase. Although HPF and APF themselves scarcely fluoresced, APF selectively and dose-dependently afforded a strongly fluorescent compound, fluorescein, upon reaction with hROS and hypochlorite (−OCl), but not other reactive oxygen species (ROS). HPF similarly afforded fluorescein upon reaction with hROS only. Therefore, not only can hROS be differentiated from hydrogen peroxide (H2O2), nitric oxide (NO), and superoxide (O 2 ⨪ ) by using HPF or APF alone, but −OCl can also be specifically detected by using HPF and APF together. Furthermore, we applied HPF and APF to living cells and found that HPF and APF were resistant to light-induced autoxidation, unlike 2′,7′-dichlorodihydrofluorescein, and for the first time we could visualize −OCl generated in stimulated neutrophils. HPF and APF should be useful as tools to study the roles of hROS and−OCl in many biological and chemical applications.

PHYSIOLOGICAL PRODUCTION OF SINGLET MOLECULAR OXYGEN IN THE MYELOPEROXIDASE-H2O2-CHLORIDE SYSTEM

The putative role of singlet oxygen (1O2) in the respiratory burst of neutrophils has remained elusive due to the lack of reliable means to study its quantitative production. To measure 1O2 directly from biological or chemical reactions in the near infrared region, we have developed a highly sensitive detection system which employs two InGaAs/InP pin photodiodes incorporated with a dual charge integrating amplifier circuit. Using this detection system, we detected light emission derived from a myeloperoxidase (MPO)‐mediated reaction in physiological conditions: pH 7.4, 1–30 nM MPO, 10–100 μM H2O2 and 100–130 mM Cl− in place of Br− without the use of deuterium oxide. The MPO‐H2O2‐Cl− system exhibited a single emission peak at 1.27 μm with a spectral distribution identical to that of delta singlet oxygen. Our results suggest physiological production of 1O2 in the MPO‐H2O2‐Cl− system at an intravacuolar neutral pH. The MPO‐mediated generation of 1O2, which may have an important role in host defense mechanisms, is discussed in connection with previous results.

Fused p47phox and p67phox truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components.

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47 phox , p67 phox , and Rac with the membrane-associated flavocytochrome b 558. Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529–7538). To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1–286) and p67N-(1–210) were constructed. In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K m for NADPH. O⨪2 generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b 558 relipidated with phosphatidylinositol- or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b 558 in the active complex.

Superoxide anion hyperproduction by neutrophils in a case of myelodysplastic syndrome. Association with sweet's syndrome and interstitial pneumonia

The complication of Sweets syndrome and interstitial pneumonia occurred in a patient with myelodysplastic syndrome. Superoxide anion production by the patients neutrophils was considerably higher than that by neutrophils obtained from normal controls after stimulation with opsonized zymosan, phorbol myristate acetate, or myristic acid. Prednisone, which a potent inhibitor of superoxide anion production by neutrophils, dramatically improved the skin and pulmonary lesions, suggesting that they were parts of the same clinical spectrum associated with the superoxide anion hyperproduction.

Effect of saturated and unsaturated fatty acids on the oxidative metabolism of human neutrophils. The role of calcium ion in the extracellular medium

The ability of fatty acids to stimulate the generation of superoxide anion (O2-) by human neutrophils was investigated with respect to their Krafft points. Saturated (myristic acid) and unsaturated (elaidic and oleic acid) induced a marked O2- generation and release from human neutrophils at pH 7.4 in the absence of Ca2+, while 0.3 mM Ca2+ inhibited both myristic acid and elaidic acid-induced O2- release. [14C]Myristic acid association with neutrophils was reduced by addition of Ca2+, whereas oleic acid association was not affected. When the pH of the reaction mixture was lowered to 6.4, 0.6 mM Ca2+ did not inhibit the O2- generation by human neutrophils. These results indicate that the inhibitory effect of Ca2+ on the fatty acid-induced O2- generation might be due to the ionic interaction between the carboxyl group of the fatty acid and Ca2+. Furthermore, 11-methyltridecanoic acid, a branched isomer of myristic acid, which showed the low Krafft point even in the presence of Ca2+, stimulated O2- generation by human neutrophils not only in the absence but also in the presence of 0.6 mM Ca2+. The effect of Ca2+ on the fatty acid-induced O2- generation by neutrophils was discussed with reference to its possible relationship to the Krafft point.

Electron transfer reactions in the NADPH oxidase system of neutrophils - involvement of an NADPH-cytochrome c reductase in the oxidase system

Membrane-bound NADPH oxidase of pig blood neutrophils was solubilized with heptylthioglucoside in a high yield. The solubilized preparation from myristate-stimulated cells (sample S) showed high O2- generating activity, and the preparation from resting cells (sample R) had no activity, but the two samples had equal amounts of flavins and cytochrome b-558 (cyt b-558). The electron transfer reactions to exogenous cytochrome c (cyt c) or cyt b-558 in samples S and R were examined. Under anaerobic conditions, NADPH-dependent cyt c reductase activity appeared higher in sample S than in sample R, and the addition of FMN and FAD greatly enhanced the reductase activity of sample S, but not that of sample R. No marked difference between the reductase activities of samples S and R was seen with NADH. Photoreduction of the NADPH oxidase system was examined in the absence of NADPH under anaerobic conditions by monitoring the reduction rates of exogenous cyt c using a flashlight with cut-off filters between 400 and 500 nm. Cyt c reduction was much higher in sample S than in sample R on photoexcitation at about 450 nm. Photoreduction was carried out with a band-pass filter for selective irradiation at 450 nm. Marked reduction of exogenous cyt c was observed only in sample S: the small reduction of cyt c by sample R was independent of the light wavelength and was equal to the blank level. In contrast, no difference in the reduction of cyt b-558 by the two samples was found by either NADPH or photoreduction. Under aerobic conditions, no direct reduction of either cyt c or cyt b-558 was observed. These results suggest that an NADPH-cyt c reductase (a membrane-bound flavoprotein) is involved in the NADPH oxidase system of stimulated neutrophils.

Inhibitory effect of Cibacron Blue F3GA on the O2− generating enzyme of guinea pig polymorphonuclear leukocytes

Abstract Cibacron Blue F3GA(CB-F3GA), which interacts with nucleotide-requiring enzymes, was used to determine the cellular location of the NADPH-binding site of the O 2 − generating enzyme in polymorphonuclear leukocytes(PMN). A fraction of plasma membranes, in which the O 2 − generating enzyme is located, was prepared from myristate(MA)-activated PMN by Percoll density gradient centrifugation. CB-F3GA was found to be a competitive inhibitor of the membrane-bound NADPH dependent O 2 − generating enzyme; the Ki value of CB-F3GA was about 0.8 μM. The dye did not inhibit O 2 − generation and release from PMN activated with MA or phorbol myristate acetate(PMA). These results suggest that the NADPH binding site of the O 2 − generating enzyme is located on the inner surface of the plasma membrane. The low Ki value of this enzyme for the dye suggests that the NADPH binding site has a dinucleotide fold.

A novel mutation at a probable heme-binding ligand in neutrophil cytochrome b558 in atypical X-linked chronic granulomatous disease

A membrane-bound cytochrome b558, a heterodimer consisting of gp91-phox and p22-phox, is a critical component of the superoxide (O2-)-generating reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes. Chronic granulomatous disease (CGD) is characterized by recurrent bacterial infection caused by a defect of the oxidase. Both subunits are absent from phagocytes in typical X-linked recessive CGD patients who are primarily defective in gp91-phox. We report here an atypical case of X-linked CGD in which neutrophils showed a complete absence of O2–-forming NADPH oxidase activity, but a small amount (about 10% of control) of both subunits was detected by immunoblot analysis. Spectrophotometric studies of the neutrophils with a recently developed sensitive method gave no evidence for the heme spectrum in the cytochrome b558 of this CGD. Reverse transcription/polymerase chain reaction and sequence analysis revealed a C to T transition replacing histidine at amino acid position 101 (His101) by tyrosine in gp91-phox. These results provide evidence that His101 of gp91-phox is the one of the heme-binding ligands of cytochrome b558.

Two cytosolic components of the neutrophil NADPH oxidase, P47-PHOX and P67-PHOX, are not flavoproteins

Two cytosolic proteins, p47-phox and p67-phox, have been shown to be essential components of the NADPH-dependent oxidase of human neutrophils, although the specific role of each of these proteins in the multicomponent electron transport complex is undetermined. The superoxide-generating activity of this oxidase can be reproduced in a cell-free system, combining cytosol and membranes from unstimulated neutrophils in the presence of fatty acid and NADPH. In the present studies, cytosol was treated with myristic acid, arachidonic acid, or sodium dodecyl sulfate in the absence of membranes and the resultant precipitate collected by centrifugation and analyzed. Both p47-phox and p67-phox precipitated in the presence of fatty acid. However, neither FAD nor FMN was localized in the precipitates, even though substantial amounts of p47-phox and p67-phox precipitated. These results suggest that neither p47-phox nor p67-phox is a flavoprotein and that neither, therefore, is the oxidase component which accepts electrons from NADPH.

Essential requirement of magnesium ion for optimal activity of the NADPH oxidase of guinea pig polymorphonuclear leukocytes

The NADPH-dependent O2-(H2O2)-forming oxidase-rich plasma membranes were purified from myristate (MA)-activated polymorphonuclear leukocytes using a Percoll-density gradient method. The specific activity of the enzyme in the plasma membrane fraction was twelve times higher than that in the cells. Studies on the effect of divalent cations and chelators on the O2- and H2O2 generating activity of the oxidase showed that Mg2+, but not Ca2+, enhanced the activity significantly. Zn2+, on the other hand, was slightly inhibitory to the oxidase activity. EDTA markedly inhibited the oxidase activity whereas EGTA enhanced it. The optimal oxidase activity was seen in the presence of mumolar concentrations of Mg2+ and reached a maximum at Mg2+ concentrations of 40-50 microM. The addition of Mg2+ resulted in a decrease in the apparent Km of the oxidase for NADPH from 40 microM to 25 microM and an increase in apparent Vmax by 1.5 times. These results suggest that Mg2+ enhances both NADPH binding and catalytic activities of the oxidase.