Đề tài nghiên cứu nhằm 2 mục tiêu: Tạo ra vector tái tổ hợp chứa đoạn gen mã hóa enzyme NQO1; thiết kế vector biểu hiện tạm thời thành công đoạn gen đó trong tế bào động vật HEK. Mời các bạn cùng tham khảo nội dung chi tiết.

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50-kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH....

In a previous study, the S100A8/A9 protein, a Ca2+- and arachidonic acid-binding protein, abundant in neutrophil cytosol, was found to potentiate the activation of the redox component of the O2– generating oxidase in neutrophils, namely the membrane-bound flavocytochrome b, by the cytosolic phox proteins p67phox, p47phox and Rac ` (Doussiere J., Bouzidi F. and Vignais P.V. (2001) Biochem. Biophys. Res. Commun. 285, 1317–1320). This led us to check by immunoprecipitation and protein fractionation whether the cytosolic phox proteins could bind to S100A8/A9. Following incubation of a cytosolic extract from nonactivated bovine neutrophil with protein A–Sepharose bound to antip67phox antibodies, the...

In the O2– generating flavocytochrome b, the membranebound component of the neutrophil NADPH oxidase, electrons are transported from NADPH to O2 in the following sequence: NADPH fi FAD fi heme b fi O2. Although p-iodonitrotetrazolium (INT) has frequently been used as a probe of the diaphorase activity of the neutrophil flavocytochrome b, the propensity of its radical to interact reversibly with O2 led us to question its specificity. This study was undertaken to reexamine the interaction of INT with the redox components of the neutrophil flavocytochrome b. ...

Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP + by NADH(forward reaction). This reaction is stimulatedby an electrochemical protongradient,Dp, presumably throughan increased release of NADPH. The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) andNADP(H), respectively.

A new NADP(H)-dependent alcohol dehydrogenase (the YCR105Wgene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2–6.4. ADHVII shows a broad sub-strate specificity similar to the recently characterized ADHVI (64% identity), although they show some differ-ences in kinetic properties. ADHVI and ADHVII are the onlymembersof the cinnamyl alcohol dehydrogenase family inyeast. SimultaneousdeletionofADH6andADH7wasnot lethal for the yeast....

H2O2 generation is a limiting step in thyroid hormone bio-synthesis. Biochemical studies have confirmed that H2O2is generated by a thyroid Ca 2+ /NADPH-dependent oxidase. Decreased H2O2 availability may be another mechanism of inhibition of thyroperoxidase activity produced by thio-ureylene compounds, as propylthiouracil (PTU) and methimazole (MMI) are antioxidant agents. Therefore, we analyzed whether PTU or MMI could scavenge H2O2or inhibit thyroidNADPHoxidase activityin vitro.

NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A⁄A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kin-etic mechanism and practical application of PTDH-E175A⁄A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization.

ATP-NAD kinase phosphorylates NAD to produce NADP by using ATP, whereas ATP-NADH kinase phosphorylates both NAD and NADH. Three NAD kinase homologues, namely, ATP-NAD kinase (Utr1p), ATP-NADH kinase (Pos5p) and function-unknown Yel041wp (Yef1p), are found in the yeastSaccharomyces cerevisiae. In this study, Yef1p was identified as an ATP-NADH kinase. The ATP-NADH kinase activity of Utr1p was also confirmed.

Many biosynthetic reactions and bioconversions are limited by low availab-ility of NADPH. With the purpose of increasing the NADPH concentra-tion and⁄or the flux through the pentose phosphate pathway inAspergillus niger, the genes encoding glucose 6-phosphate dehydrogenase (gsdA), 6-phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains.

Ferredoxin–NADP(H) reductases (FNRs) represent a pro-totype of enzymes involved in numerous metabolic path-ways. We found that pea FNR ferricyanide diaphorase activity was inhibited by Zn 2+ (Ki 1.57lM). Dichloro-phenolindophenol diaphorase activity was also inhibited by Zn 2+ (Ki 1.80lM), but the addition of ferrocyanide was required, indicating that the inhibitor is an arrangement of both ions.Escherichia coliFNR was also inhibited by Zn-ferrocyanide, suggesting that inhibition is a consequence of common structural features of these flavoenzymes....

The guanidinium chloride- and urea-induced unfolding of FprA, a mycobacterium NADPH-ferredoxin reductase, was examined in detail using multiple spectroscopic techniques, enzyme activity measurements and size exclusion chromatography. The equilibrium unfolding of FprA by urea is a cooperative process where no stabilization of any partially folded inter-mediate of protein is observed. In comparison, the unfolding of FprA by guanidinium chloride proceeds through intermediates that are stabilized by interaction of protein with guanidinium chloride. ...

The Baeyer–Villiger monooxygenase (BVMO), 4-hydroxy-acetophenone monooxygenase (HAPMO), uses NADPH andO2 to oxidize a variety of aromatic ketones and sulfides. The FAD-containing enzyme has a 700-fold preference for NADPH over NADH. Sequence alignment with other BVMOs, which are all known to be selective for NADPH, revealed three conservedbasic residues,whichcouldaccount for the observed coenzyme specificity. The corresponding residues in HAPMO (Arg339, Lys439 and Arg440) were mutated and the properties of the purified mutant enzymes were studied. ...

Activation of the superoxide-producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1c, which is expressed in the testis and fetal brain.

The mouldHypocrea jecorina(Trichoderma reesei) has two genes coding for enzymes with high similarity to the NADP-dependent glycerol dehy-drogenase. These genes, calledgld1andgld2, were cloned and expressed in a heterologous host. The encoded proteins were purified and their kinetic properties characterized.

The staphylococcal enterotoxins produced byStaphylococcus aureusare associated with pyrogenic response in humans and primates. This study investigates the role of NADPH oxidase and nuclear factor-kappa B (NF-jB) on enterotoxin staphylococcal enterotoxin C1 (SEC1)-induced pyrogenic cytokine production in human peripheral blood mononuclear cells (PBMC).

NADPH oxidases of the Nox family exist in various supergroups of eukaryotes but not in prokaryotes, and play crucial roles in a variety of biological processes, such as host defense, signal transduction, and hor-mone synthesis.

Ferredoxin (flavodoxin)-NADP(H) reductases (FNRs) are ubiquitous flavoenzymes that deliver NADPH or low-potential one-electron donors (ferredoxin, flavodoxin, adrenodoxin) to redox-based metabolic reactions in plastids, mitochondria and bacteria. Plastidic FNRs are quite efficient reductases.

NADPH oxidase is a major source of the superoxide produced in cardio-vascular tissues. Expression of NOX1, a catalytic subunit of NADPH oxi-dase, is induced by various vasoactive factors, including angiotensin II, prostaglandin (PG) F2a and platelet-derived growth factor (PDGF). To clarify the molecular basis of this transcriptional activation, we delineated the promoter region of the NOX1gene.

The N-termini of the NADPH : protochlorophyllide oxidoreductase (POR) proteins A and B from barley and POR from pea were determined by acet-ylation of the proteins and selective isolation of the N-terminal peptides for mass spectrometry de novosequence analysis. We show that the cleav-age sites between the transit peptides and the three mature POR proteins are homologous.