Hisamitsu Ogawa

NADPH oxidase 4 (Nox4) is constitutively active, While Nox2 requires the cytosolic regulatory Subunits p47(phox) and p67(phox) and activated Rac With activation by phorbol 12-myristate 13-acetate (PMA). This Study Was undertaken to identify the domain oil Nox4 that confers constitutive activity Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production With a K-m For NADPH of 55 +/- 10 mu M. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of similar to 200 mol of H2O2 min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox-2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H2O2 production in the absence Of cytosolic Subunits. Ill contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression Of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electrom transferase activities. Nox I DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction Of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide(470min(-1)), and other electron acceptors (artificial dyes and cytochrom b(5)). Rates were similar to those observed for H2O2 production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and Was seen with NADPH but not NADH. These results Indicate that the Nox4 DH domain exists in all Intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium or NADP+. The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover Of Superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1 (Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L) between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox 1 activity, but NOXO1N (1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox] activation and their interactions might be responsible for regulating the O(2)(-)- producing activity in Caco-2 cells.

In order to understand the molecular mechanism of development during early embryogenesis in diapause and non-diapause of the silkworm, mRNA from diapause and non-diapause eggs was compared using the differential display technique. We cloned the full length of a cDNA encoding a novel RNA helicase-like (RHL) protein by the RACE method using a cDNA fragment which was one of the specific cDNAs in the non-diapause eggs. A BLAST search using the predicted amino acid sequence of RHL revealed a low homology (21-25% identity of its partial length) with that of the DEAD-box RNA helicase. Gene expression of the RHL gene of the diapause and non-diapause eggs was investigated by RT-PCR until 60 h after oviposition. Amplified RHL cDNA was observed through all the stages in the non-diapause eggs, while in the diapause eggs, cDNA was found in eggs 0-12h after oviposition but disappeared 24-60h after oviposition. When the diapause eggs were activated by HCl treatment after chilling at 4 degrees C for 6 days from 48h after oviposition (artificial diapause termination), cDNA was observed from 12h after HCl treatment. We also investigated the immunohistochemical distribution and localization of RHL in non-diapause eggs using anti-recombinant His-tag RHL antiserum. RHL was distributed in blastoderm cells and yolk cells and was localized in the nucleus and the cytosol of yolk cells. These data suggest that RHL has an important role in the early embryo of the silkworm. (c) 2006 Elsevier Ltd. All rights reserved.

A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 muM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% alpha-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 muM, respectively, while the K-m value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 muM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1: 1 but neither p67N nor Rac alone showed significant binding.

A series of truncated forms of His(6)-tagged gp91phox were expressed, solubilized, and purified in the presence of 30 muM FAD. The truncated gp91phox with the longest sequence in the C-terminal region (221-570) (gp91C) showed the highest activity (turnover rate, 0.92) for NADPH diaphorase in the presence of either 0.3% Triton X-100 or 0.5% Genapol X-80. Activity was not inhibited by superoxide dismutase but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium. The flavinated gp91C contained approximately 0.9 mol of FAD/mol of protein (MW 46 kDa) and 12% alpha-helix content. In the absence of p47phox, p67phox showed considerable activation of gp91C in the presence of Rac. Carboxyl-terminal truncated p67phox (1-210) (p67N), which is the minimal active fragment, was fused with Rac or Q61LRac. The fusion protein p67N-Rac (or p67N-Q61LRac) showed a 2-fold higher stimulatory effect on NBT reductase activity of gp91C than the combination of the individual cytosolic p67N and Rac proteins. In contrast, Rac-p67N, a fusion with the opposite orientation, showed a smaller significant effect on the enzyme activity. The EC50 values for p67phox, p67N, p67N-Rac, and Rac-p67N were 8.00. 4.35, 2.56, and 15.2 muM, respectively, while the K-m value for NADPH in the presence and absence of the cytosolic components was almost the same (40-55 muM). In the presence of Rac, p67N or p67phox bound to gp91C with a molar ratio of approximately 1: 1 but neither p67N nor Rac alone showed significant binding.