Separation of highly fluorescent proteins by SDS-PAGE in Acroporidae corals.

This study characterized the spectral properties of Acropora tenuis, A. nasuta, A. secale, and A. aspera, all of which showed strong colorful fluorescence under ultraviolet light-A (black light). The emission maxima of fluorescence from the intact corals were 517, 482, 484, and 514 nm in A. tenuis, A. nasuta, A. secale, and A. aspera, respectively. Using a soluble fraction of cell-free extract of the corals, we applied a method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate each fluorescent protein component contained in the corals. Green fluorescent bands were detected in all Acropora examined, although their apparent molecular mass and relative content were different. A. aspera had two orange bands in addition to the green one. The major excitation and emission peaks of the orange fluorescence bands were almost identical (476 and 478 nm), however, they were discernible by their spectral profiles and molecular masses. Some biochemical properties of the highly fluorescent proteins of Acropora are described and implications of the results are discussed.

Inhibitory effects of nitric oxide on oxidative phosphorylation in plant mitochondria.

Plant nitrate reductase (NR) produces nitric oxide (NO) when nitrite is provided as the substrate in the presence of NADH [H. Yamasaki and Y. Sakihama (2000) FEBS Lett. 468, 89-92]. Using a NR-dependent NO producing system, we investigated the effects of NO on the energy transduction system in plant mitochondria isolated from mung bean (Vigna radiata). Plant mitochondria are known to possess two respiratory electron transport pathways-the cytochrome and alternative pathways. When the alternative pathway was inhibited by n-propyl gallate, the addition of NR strongly suppressed respiratory O(2) consumption driven by the cytochrome pathway. In contrast, the alternative pathway measured in the presence of antimycin A was not affected by NO. The extent of the steady-state membrane potential (Deltapsi) generated by respiratory electron transport rapidly declined in response to NO production. The addition of bovine hemoglobin, a quencher of NO, resulted in the recovery of Deltapsi to the uninhibited level. Consistent with its inhibition of Deltapsi, NO produced by NR strongly suppressed ATP synthesis in the mitochondria. These results provide substantial evidence to confirm that the plant alternative pathway is resistant to NO and support the idea that the alternative pathway may lower respiration-dependent production of active oxygens under conditions where NO is overproduced.

Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species.

We examined the ability of plant nitrate reductase (NR) to produce nitric oxide (NO) using in vitro assays. Electrochemical and fluorometric measurements both showed that NO is produced by corn NR in the presence of nitrite and NADH at pH 7. The NO production was inhibited by sodium azide, a known inhibitor for NR. During the reaction, absorbance of 2',7'-dichlorodihydrofluorescein increased markedly. This change was completely suppressed by sodium azide, glutathione or depletion of oxygen. We conclude that plant NR produces both NO and its toxic derivative, peroxynitrite, under aerobic conditions when nitrite is provided as the substrate for NR.

Reduction of phenoxyl radicals mediated by monodehydroascorbate reductase.

Monodehydroascorbate (MDA) reductase catalyzes the reduction of MDA, the only organic radical substrate for the enzyme reported so far. Here, we show that cucumber MDA reductase is also capable of reducing phenoxyl radicals which are generated by horseradish peroxidase (HRP) with H2O2. The addition of MDA reductase plus NADH suppressed the HRP/H2O2 dependent oxidation of quercetin, accompanied by the oxidation of NADH. The quenching of the quercetin radical by MDA reductase plus NADH was confirmed by ESR. MDA reductase with NADH also suppressed the HRP/H2O2 dependent oxidation of hydroxycinnamates, including ferulic acid, coniferyl alcohol, and chlorogenic acid. Thus, the phenoxyl radicals of plant phenols can be reduced to their respective parent phenols by MDA reductase via a mechanism similar to the reduction of MDA.

Flavonoid-Peroxidase Reaction as a Detoxification Mechanism of Plant Cells against H2O2.

Recent studies have revealed that dietary flavonoids are potent radical scavengers, acting in a manner similar to ascorbate and [alpha]-tocopherol. However, it is still not clear whether flavonoids have a similar antioxidative function in plants. We examined the possibility that flavonoids could function as stress protectants in plant cells by scavenging H2O2. Two major flavonoids, quercetin and kaempferol glycosides, were isolated from leaves of the tropical tree Schefflera arboricola Hayata. Both glycosides and aglycones of isolated flavonols were oxidized by H2O2 in the presence of horse-radish peroxidase and/or in a soluble fraction of S. arboricola leaf extract. The rates of oxidation were in the order quercetin > kaempferol > quercetin glycoside >> kaempferol glycoside. Judging from the effects of inhibitors such as KCN, p-chloromercuribenzoate, and 3-amino-1H-1,2,4-triazole, we conclude that guaiacol peroxidase in the soluble fraction catalyzes H2O2-dependent oxidation of flavonols. In the flavonol-guaiacol peroxidase reaction, ascorbate had the potential to regenerate flavonols by reducing the oxidized product. These results provide further evidence that the flavonoid-peroxidase reaction can function as a mechanism for H2O2 scavenging in plants.

Bleaching of the red anthocyanin induced by superoxide radical.

Red anthocyanin prepared from petals of Hibiscus rosa-sinensis L. was photobleached in the EDTA-riboflavin system. The rate of bleaching monitored at 565 nm depended on the light intensity and EDTA concentrations. Anaerobic conditions and/or addition of superoxide dismutase prevented the bleaching of anthocyanin, whereas mannitol and catalase did not. A similar bleaching was observed under dark conditions in the xanthine-xanthine oxidase system. The results indicate that anthocyanin is bleached by the nonenzymatic reaction with the superoxide radical and suggest that the pigment can function as an antioxidant. The antioxidative efficiency of cyanidin to superoxide was 10-fold higher than that of cyanidin-3-sophoroside as a Hibiscus anthocyanin.