小川 久光

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 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.

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.

Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O-2(-) concentration upon incubation with all-trans -retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA-induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K-d value of 6.7 nm.

Undifferentiated human promyelocytic leukemia HL-60 cells show little or no superoxide production, but generate a very low O-2(-) concentration upon incubation with all-trans -retinoic acid (ATRA). Its production reaches a maximum within 20 h, and thereafter is maintained at an almost constant level. The differentiated cells show phorbol 12-myristate 13-acetate (PMA)-stimulated NADPH oxidase activity consistent with the amount of gp91phox (phagocytic oxidase) expressed in the plasma membrane. Three isoforms of p21-activated serine/threonine kinases, PAK68, PAK65 and PAK62, were found in both cytosolic and membrane fractions, and their contents were significantly increased during induced differentiation. The amount of Rac identified in the two fractions was also markedly enhanced by ATRA-induced differentiation. In contrast, neither PAK nor Rac was seen in the plasma membrane of undifferentiated HL-60 or human neutrophil, but they were abundant in the cytoplasmic fraction. Binding of Rac with PAK isoforms was shown in the membrane upon induced differentiation of HL-60 cells. Direct binding of purified Rac1 to PAK68 was quantified using a fluorescent analog of GTP (methylanthraniloyl guanosine-5'-[beta,gamma-imido]triphosphate) bound to Rac as a reporter group. Rac1 bound to PAK68 with a 1 : 1 stoichiometry and with a K-d value of 6.7 nm.

The Caenorhabditis elegans unc-13, unc-18, and unc-64 genes are required for normal synaptic transmission. The UNC-18 protein binds to the unc-64 gene product C. elegans syntaxin (Ce syntaxin). However, it is not clear how this protein complex is regulated. We show that UNC-13 transiently interacts with the UNC-18-Ce syntaxin complex, resulting in rapid displacement of UNC-18 from the complex. Genetic and biochemical evidence is presented that UNC-13 contributes to the modulation of the interaction between UNC-18 and Ce syntaxin.

Native human cytosolic alanine aminotransferase (EC 2.6.1.2) was found to be a homodimer consisting of two 55 kDa subunits. The human enzyme was more cationic and more susceptible to heat inactivation and heavy metal-inhibition than its rat homologue. Isoelectric focussing separated three human isoforms and four rat isoforms of cALT that differed in their isoelectric points. Two subtypes of the enzyme which differed in apparent molecular weight on sodium dodecylsulfate polyacrylamide gel electrophoresis were detected in rat, the liver type and the muscular type. This microheterogeneity was not found for human cytosolic alanine aminotransferase. Immunohistochemical studies revealed that the rat liver enzyme was exclusively localized in periportal hepatocytes, consistent with its role in gluconeogenesis. In human hepatocytes the plasma membrane was intensely stained, implicating an intracellular localization near the plasma membrane.

Uridine monophosphate (UMP) synthase is a bifunctional enzyme catalyzing the last two steps of de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase (OPRT) and orotidine-5'-monophosphate decarboxylase (ODC). Loss of either enzymatic activity results in hereditary erotic aciduria, a rare autosomal recessive disorder characterized by retarded growth, anemia, and excessive urinary excretion of erotic acid. We have isolated the UMP synthase chromosomal gene from a lambda EMBL-3 human genomic library and report a single-copy gene spanning similar to 15 kb. The UMP synthase genomic structure encodes six exons ranging; in size from 115 bp to 672 bp, and all splicing junctions adhere to the canonical GT/AG rule. Cognate promoter elements implicated in glucocorticoid- and cAMP-mediated regulation as well as in liver-, myeloid-, and lymphocyte-specific expression are located within the 5' flanking sequence. Molecular investigation of UMP synthase deficiency in a Japanese erotic aciduria patient revealed mutations R96G (A-to-G transition; nt 286) and G429R (G-to-C transversion; nt 1285) in one allele and V109G (T-to-G transversion; nt 326) in the other allele. Expression of human UMP synthase cDNAs containing these mutations in pyrimidine auxotrophic Escherichia coli and in recombinant baculovirus-infected Sf21 cells demonstrates impaired activity presumably associated with the urinary erotic acid substrate accumulations observed in vivo. We further establish the identity of two polymorphism, G213A (v=.26) and 440Gpoly (v =.27) located in exons 3 and 6, respectively, which did not significantly compromise either OPRT or ODC function.

It was found that cytochrome P-450(arom) purified from human placenta microsomes is glycosylated, and the sugar chain was cleaved with endoglycosidase H (Endo H). The core glycosylation of P-450(arom) was examined with two heterologous expression systems, cultured insect cells and in vitro translation system. The P-450(arom) protein expressed in the insect cells was glycosylated, and the sugar chain was sensitive to Endo H. It was also glycosylated when translated with the wheat germ cell-free system in the presence of rough microsomal membrane, and the sugar chain could be removed by Endo H treatment. Since the P-450(arom) molecule has two potential glycosylation sites (Asn-12 and Asn-180), we replaced each of the 2 asparagine residues with alanine by site-directed mutagenesis and examined the glycosylation of the two mutant proteins in the cell-free system. The core glycosylation did not occur when the Asn-12 residue was mutated, whereas the mutant protein with modified Asn-180 residue was glycosylated. These results demonstrated that the potential glycosylation site (Asn-12) in the N-terminal portion of P-450(arom) is the site of glycosylation. We conclude that the N terminus of P-450(arom) is translocated across the endoplasmic reticulum membrane to be glycosylated at the luminal side.