• Journal of Microbiology and Biotechnology
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• v.27 no.5
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• pp.983-989
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• 2017

NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of $NADP^+$ in the affinity chromatography process. In the present study, the rat NPR clone containing a $6{\times}$ Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using $Ni^{2+}$-affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

• Journal of Acupuncture Research
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• v.21 no.5
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• pp.1-12
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• 2004

Objective : This study was to investigate the effect of acupuncture on NADPH-diaphorase and nNOS in the brain stem and cerebellum of spontaneously hypertensive rats. Methods : The experimental groups were divided into four groups : Normal, Choksamni(ST36), Kokchi(LI11), arbitrary group. Thereafter we evaluated changes in NADPH-diaphorase positive neurons histochemically and changes in nNOS neurons immunohistochemically. Results : 1. The optical densities of NADPH-diaphorase positive neurons of all the Choksamni & Kokchi groups were significantly different in SuG, DLPAG, IP, Pr, Gi areas of brain stem and cerebellum as compared to normal & arbitrary groups. In PPTg only Choksamni group was significantly different as compared to normal and arbitrary groups. 2. The optical densities of nNOS-positive neurons of Choksamni & Kokchi groups were significantly different in SuG, DLPAG areas of brain stem as compared to normal group. In IP, Pr only Kokchi group was significantly different as compared to normal group. The optical densities of nNOS-positive neurons of Choksamni & Kokchi groups were significantly different in SuG, DLPAG, PPTg, Pr, Gi areas of brain stem as compared to arbitrary group. In IP, Pr only Kokchi group was significantly different as compared to arbitrary group. 3. The optical densities of nNOS-positive neurons of all the Choksamni & Kokchi groups were not significantly different in cerebellum as compared to normal & arbitrary groups. Conclusions : We found out that acupuncture have effects on NADPH-diaphorase and nNOS in the brain stem and cerebellum of spontaneously hypertensive rats.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.2
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• pp.99-106
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• 2009

Although anti-atherogenic effects of cilostazol have been suggested, its effects on the expression of SR in macrophages are unclear. This study investigated the role of cilostazol on CD36 expression of murine macrophages enhanced by HNE, a byproduct of lipid peroxidation. The stimulation of macrophages with HNE led to an increased expression of CD36, which was significantly attenuated by NAC, an antioxidant. Moreover, the increased production of ROS by HNE was completely abolished by NADPH oxidase inhibitors, DPI and apocynin, as well as by the 5-LO inhibitor, MK886, but not by inhibitors for other oxidases. This suggested that NADPH-oxidase and 5-LO were major sources of ROS induced by HNE. In addition, HNE-enhanced expression of CD36 was reduced by these inhibitors, which indicated a role for NADPH oxidase and 5-LO on CD36 expression. In our present study, cilostazol was a significant inhibitor of ROS production, as well as CD36 expression induced by HNE. An increase in NADPH oxidase activity by HNE was significantly attenuated by cilostazol, however cilostazol had no effect on HNE-enhanced 5-LO activity. Together, these results suggest that cilostazol attenuates HNE-enhanced CD36 expression on murine macrophages thorough inhibition of NADPH oxidase-derived ROS generation.

• Journal of Acupuncture Research
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• v.20 no.6
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• pp.148-159
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• 2003

Objective: This study examines the effects of acupoint combination on NADPH-diaphorase and neuronal nitric oxide synthase(nNOS) in the brainstem of spontaneously hypertensive rats. Methods: The changes of NADPH-d-positive neurons using a histochemical method and the changes of nNOS-positive neurons using an immunohistochemical method were evaluated. The optical densities of NADPH-d-positive neurons and nNOS-positive neurons of the Choksamni(ST36) Umnungchon(SP9) groups were significantly increased in all brainstem areas as compared to the Choksamni and Choksamni Kokchi(LI11) groups and decreased, with the exeption of the nNOS-positive neurons in the superficial gray of superior colliculus, as compared to the normal group. Results: Our results demonstrated that electroacupuncture changes the activity in the NO system in the brainstem of SHR and the acupoint combination is one of the important parameters for this effect.

• BMB Reports
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• v.31 no.3
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• pp.227-232
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• 1998

The leukocyte NADPH oxidase of neutrophils is a membrane-bound enzyme that catalyzes the production of $O_2^-$ from oxygen using NADPH as an electron donor. Dormant in resting neutrophils, the enzyme acquires catalytic activity when the cells are exposed to appropriate stimuli. During activation, the cytosolic oxidase components $p47^{phox}$ and $p67^{phox}$ migrate to the plasma membrane, where they associate with cytochrome $b_{558}$, a membrane-bound flavohemoprotein, to assemble the active oxidase. The oxidase can be activated in a cell-free system; the activating agent usually employed is an anionic amphiphile such as sodium dodecyl sulfate (SDS). Because $p47^{phox}$ can translocate by itself during activation, the conformational change in $p47^{phox}$ may be responsible for the activation of NADPH oxidase. We show here that the treatment of $p47^{phox}$ with SDS leads to an increase in the reactivity of the sutbydryl group of cysteines toward N-ethylmaleimide, indicating that the conformational change occurs when $p47^{phox}$ is exposed to SDS. We propose that this change in conformation results in the appearance of a binding site through which $p47^{phox}$ interacts with cytochrome $b_{558}$during the activation process.

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1395-1402
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• 2013

Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3-hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2'-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2'-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.

• Biomolecules & Therapeutics
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• v.26 no.1
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• pp.29-38
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• 2018

During cancer progression, cancer cells are repeatedly exposed to metabolic stress conditions in a resource-limited environment which they must escape. Increasing evidence indicates the importance of nicotinamide adenine dinucleotide phosphate (NADPH) homeostasis in the survival of cancer cells under metabolic stress conditions, such as metabolic resource limitation and therapeutic intervention. NADPH is essential for scavenging of reactive oxygen species (ROS) mainly derived from oxidative phosphorylation required for ATP generation. Thus, metabolic reprogramming of NADPH homeostasis is an important step in cancer progression as well as in combinational therapeutic approaches. In mammalian, the pentose phosphate pathway (PPP) and one-carbon metabolism are major sources of NADPH production. In this review, we focus on the importance of glucose flux control towards PPP regulated by oncogenic pathways and the potential therein for metabolic targeting as a cancer therapy. We also summarize the role of Snail (Snai1), an important regulator of the epithelial mesenchymal transition (EMT), in controlling glucose flux towards PPP and thus potentiating cancer cell survival under oxidative and metabolic stress.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.5
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• pp.325-329
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• 2010

Vascular NADPH oxidase plays a pivotal role in producing superoxide in endothelial cells and thus acts in the initiation and development of inflammatory cardiovascular diseases such as atherosclerosis. Epigallocatechin-3-gallate (EGCG), the major catechin derived from green tea, has multiple beneficial effects for treating cardiovascular disease but the effect of EGCG on the expression of vascular NADPH oxidase remains unknown. In this study, we investigated the mechanism(s) by which EGCG might inhibit the expression of subunits of NADPH oxidase, namely $p47^{phox}$, $p67^{phox}$ and $p22^{phox}$, induced by angiotensin II (Ang II) in human umbilical vein endothelial cells. Ang II increased the expression levels of $p47^{phox}$, $p67^{phox}$, and $p22^{phox}$, but EGCG counteracted this effect on $p47^{phox}$. Moreover, EGCG did not affect the production of reactive oxygen species induced by Ang II. These data suggest a novel mechanism whereby EGCG might provide direct vascular benefits for treating inflammatory cardiovascular diseases.

• BMB Reports
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• v.38 no.3
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• pp.366-369
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• 2005

NADPH-cytochrome P450 reductase (CPR) transfers electrons from NADPH to cytochrome P450 and also catalyzes the one-electron reduction of many drugs and foreign compounds. Various spectrophotometric assays have been performed to examine electron-accepting properties of CPR and its ability to reduce cytochrome $b_5$, cytochrome c, and ferricyanide. In this report, reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by CPR has been assessed as a method for monitoring CPR activity. The principle advantage of this substance is that the reduction of MTT can be assayed directly in the reaction medium by a continuous spectrophotometric method. The electrons released from NADPH by CPR were transferred to MTT. MTT reduction activity was then assessed spectrophotometrically by measuring the increase of $A_{610}$. MTT reduction followed classical Michaelis-Menten kinetics ($K_m\;=\;20\;{\mu}M$, $k_{cat}\;=\;1,910\;min^{-1}$). This method offers the advantages of a commercially available substrate and short analysis time by a simple measurement of enzymatic activity of CPR.

• Applied Microscopy
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• v.45 no.3
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• pp.135-143
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• 2015

The aim of this study was to investigate effects on joint mobilization in neurochemical changes of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) and nitric oxide synthase (NOS) of the spinal cord neurons after right knee joint immobilization (RKJI) and in ultrastructural changes of the femoral nerves innervating the muscles acting on RKJI. A total of 15 guinea pigs were used and divided into 5 groups. Immunohistochemistry was performed to detect NADPH-d and NOS. NADPH-d and NOS were not expressed in the ventral horn of control and experimental groups, but were expressed or not in the dorsal horn according to the duration of release after RKJI and the presence or absence of joint mobilization. Ultrastructures of the femoral nerves in experimental groups had partial demyelination and condensed clumps in axon. Effects on manipulative therapy after RKJI were confirmed from expression of NADPH-d and NOS in the dorsal horn of the lumbosacral spinal cord. Manipulative therapy was more effective against a long-term immobilization than a short-term immobilization.