DOCSLIB.ORG

On Functional Role of Cytochrome B5 II. NADH-Linked Ascorbate Radical Reductase Activity in Microsomes

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
On Functional Role of Cytochrome B5 II. NADH-Linked Ascorbate Radical Reductase Activity in Microsomes J. Biochcm., 69, 325-330 (1971, On Functional Role of Cytochrome b5 II. NADH-linked Ascorbate Radical Reductase Activity in Microsomes Toshihiko HARA* and Shigeki MINAhAMI* *The Biochemistry Laboratory , National Nakano Chest Hospital, Tokyo and **the Department of Biochemistry, Kyushu University School of Medicine, Fukuoka Received for publication, Mat 28. 1970 1) :Microsomes catalyze the reduction of ascorbate radical by NADH . The enzyme which functions in this reaction is identified as the microsomal NADH-cytochrome c reductase, the electron transfer system involving cytochrome b5. Reduced cytochrome h5, whether bound to or isolated from the microsomal membrane, reacts rapidly with the ascorbate radical to form ascorbate. With intact microsomes , the cytochrome is maintained in the fully reduced state in the presence of NADH. This condition favors the electron transfer from NADH to ascorbate radical as catalyzed by micro somes. 2) The non-ionic detergent, Emalgen 810, which inhibits the reduction of micro somal bound cytochrome b5 by NADH, also inhibits the NADH-ascorbate radical reductase activity of microsomes. 3) Cytochrome b5 is reduced by ascorbate very slowly but not reduced by ascorbate radical. Under aerobic condition, the cytochrome appears to be only partially reduced by ascorbate due to the autoxidation of the reduced cytochrome. 4) Microsomes with NADH generating system prevent the oxidation of ascorbate. This effect is inhibited by Emalgen 810, the inhibitor of NADH-cytochrome b, reduc tase system of microsomes. Role of NADH-cytochrome b5 reductase system to main tain intracellular level of ascorbate is discussed. It has been demonstrated that oxidation of of an ascorbate oxidizing enzyme in micro NADH in microsomes is activated by catalytic somes. Formation of ascorbate radical as an amount of ascorbate which cannot be replaced intermediate form was indicated by Z amazaki et by dehydroascorbate (1, -2). For the inter al. (1) in their study on plant ascorbate oxidase pretation of such reaction, a "radical-like" [EC 1. 10. 3. 3]. Staudinger ct al. ( ~)_)by using semidehydroascorbate was proposed: the ex the enzyme for generating ascorbate radical, istence of which has been confirmed by direct have demonstrated that ascorbate radical may measurement in electron spin resonance spec be the true electron acceptor from NADH in trometry (3 ). microsomes and that microsomes contain an The generation of ascorbate radical in enzyme system which transfer electron from microsomes needs to postulate the existence NADH to ascorbate radical. On the other Vol. 69, No. 2, 1971 325 326 T. HARA and S. MINAKAMI hand, it has been demonstrated that oxidation the partially reduced cytochrome b5 was autox of ascorbate to dehydroascorbate by ascorbate idized under aerobic condition following a oxidase is inhibited if both NADH and micro first order reaction. The rate constant was somes are present (5), about 0.3 min-1 at 20°C in 0.1M phosphate We are interested in the NADH-ascorbate buffer, pH 7.2. When NADH was added to radical reductase activity in microsomes and microsomes excessively, the NADH oxidation have demonstrated that microsomal NADH proceeded more rapidly than it could be ascribed cytochrome b5 reductase system may function to the autoxidation of cytochrome b5. Under as the catalyst for reducing ascorbate radical such a condition, the increase in the reoxi by NADH. dation rate of cytochrome b5 will accelerate the oxidation velocity of NADH, as observed in the following experiments. The remaining MATERIALS AND METHODS portion of the NADH oxidation may be due to Preparation-Microsomes were prepared as the cyclic reduction-oxidation of cytochrome described previously (6). Ascorbate oxidase P-450. was purified from the cortex of Japanese cu Effect of Non-ionic Detergent on NADH cumbers by the method of Tokuyama ct al. Oxidation by Microsomes-Previously, we have (7 ). The preparation contained copper in the reported that the electron transfer from NADH amount of 1.1 ,ƒÊg/mg protein, having an en to cytochrome b5 in intact microsomes is in zymatic activity of 1,200 units/mg protein by hibited by the non-ionic detergent, Emalgen expressing its unit according to Lovett-Janison 810 ((5). On the other hand, the detergent and Nelson (8). Initial velocity of oxygen showed an inhibitory effect of NADPH oxidase uptake in the presence of 1 mm ascorbate and without showing significant inhibition of NADH 1 unit/ml of ascorbate oxidase was 0.38 mm/ oxidation. This observation may be explained min at 20°C in 0.1 M phosphate buffer, pH 7.0, as follows. The oxidation of NADH is mainly saturated with air. due to the reaction of cytochrome b5 where Analytical Methods-Oxygen uptake was the reoxidation rate of reduced cytochrome b5 measured by a Clark oxygen electrode. The is an important determinant for the overall activity of ascorbate oxidase was estimated as NADH oxidation rate. In this case, the inhi described by Sack et al. (9). The copper bition of intramicrosomal electron transfer by content of the enzyme was measured with the detergent does not necessarily reflect on bathocuproin reagent (10 ). Ascorbate was the overall oxidation rate. For the oxidation determined by the reduction of 2, 6-dichloro of NADPH, the contribution of P-450 system phenol indophenol and subsequent extraction is most probable, in which the rate deter with amyl alcohol. mining step in the sequence is the step to Other spectrophotometrical and chemical reduce P-450. As the reduction of P-450 by analyses were performed in the same way as NADPH is inhibited by the detergent (un described previously (G). published observation), it may depress the rate of NADPH oxidation. The characteristics of the detergent discussed above were used for RESULTS the study of NADH-ascorbate radical reductase Relation between Oxidation of NADH and in microsomes. Oxidation of cytochrome b5 in Microsomes Kinetics of NADH-Ascorbate Radical Re Oxidation of NADH by liver microsomes is ductase Activity of Microsemes in the Presence mediated by cyclic oxidoreduction of cyto and Absence of the Detergent-NADH oxidase chrome b5 or cytochrome P-450. When micro activity of microsomes was activated by the somes were treated with a small amount of presence of ascorbate and this activation was NADH less than necessary to reduce cyto far more pronounced on addition of ascor chrome b5 completely, NADH reduced only the bate oxidase (Fig. 1), as originally observed stoichiometric amount of cytochrome b5. Then by Staudinger et al. (1 ). Under our J. Biochem. ON FUNCTIONAL ROLE OF CYTOCHROME b5 327 Fig. 1. Oxidation of NADH and NADPH by micro Fig. 2. Oxidation of NADH and NADPH by micro somes. The assay mixture contained in 3 ml: 3_mg somes in the presence of Emalgen 810. 0.8`c Emalgen of microsomal protein, 10 mm Amytal, 0.1 M phos 810 was previously added to the assay mixture. The phate buffer, pH 7.2. conditions were the same as in Fig. 1. experimental conditions, the ratio of the ac tivation was more than 25 fold when both ascorbate and ascorbate oxidase were added. On the other hand, the NADPH oxidase ac tivity of microsomes was only slightly activated by ascorbate radical. These results indicate that the ascorbate-dependent (or ascorbate radical-dependent) reduced nicotinamide co enzyme oxidase activity of microsomes is more specific for NADH. The effect of Emalgen 810 on the ascor bate radical-dependent NADH (or NADPH) oxidase activity of microsomes is shown in Fig. 2. By the addition of ascorbate in the Fig, 3. Effect of ascorbate and ascorbate oxidase on presence of the detergent, the NADH oxidase microsomal cytochrome b;, reduced by NADH. The activity was enhanced in the same manner as assay mixture contained in 3 ml: 2.4 mg of micro observed without the detergent. This suggests somal protein, 0.1 M phosphate buffer pH 7.2. that the ascorbate oxidase activity intrinsic to microsomes is not affected by the deter (Fig. 3). In the presence of ascorbate, the re gent. When ascorbate oxidase was further added oxidation of cytochrome b5 was accelerated in to the system, the oxidation of both NADH agreement with the observation by Blume et and NADPH increased, although the activa al. (11), and further addition of ascorbate tions were not so large as observed in the oxidase caused more activation. This obser system without the detergent. Under the vation indicates that the activating effect of condition when the reduction of cytochrome ascorbate on NADH oxidase activity of micro b5 was inhibited by the detergent, the reduc somes can be ascribed to the activating effect tion rate of cytochrome b5 became rate-limit of ascorbate radical on the oxidation of cyto ing in the overall oxidative sequence of chrome b5. The prior additions of ascorbate NADH. and ascorbate oxidase followed by NADH ad Effect of Ascorbate Radical on Microsomal dition did not change the autoxidation rate. Bound Cytochrome b5-The addition of ascor When microsomes were treated with bate or subsequent addition of ascorbate oxidase NADPH aerobically, cytochrome b5 was par to microsomes changed the rate of reoxidation tially reduced to the point where the rates of of cytochrome b5 which was reduced NADH reduction and reoxidation were in balance. Vol. 69, No. 2, 1971 328 T. HARA and S. MINAKAMI When ascorbate was added to microsomes cytochrome b5. This observation also indicates under this condition, the steady state level of that the oxidation of cytochrome b5 by ascor reduced cytochrome b5 fell to a lower level. bate radical, as shown in intact microsomes, This indicates an increase in the reoxidation is due to the direct electron transfer between rate of the cytochrome by the presence of them. Furthermore, the effect of ascorbate ascorbate. to activate the oxidation of microsomal bound Similar phenomenon could be observed cytochrome b5 and to increase the NADH with a use of NADH as a reductant in the oxidizing activity of microsomes, which can presence of Emalgen 810, when the reduction be observed even without adding ascorbate rate of cytochrome b5 was depressed by the oxidase exogenously, seems to have occurred detergent to be comparable to the autoxidation by the cooperation of some endogenous ascor rate of the cytochrome.
Load more

Recommended publications
  • Elevated Hydrogen Peroxide and Decreased Catalase and Glutathione
    Sullivan-Gunn and Lewandowski BMC Geriatrics 2013, 13:104 http://www.biomedcentral.com/1471-2318/13/104 RESEARCH ARTICLE Open Access Elevated hydrogen peroxide and decreased catalase and glutathione peroxidase protection are associated with aging sarcopenia Melanie J Sullivan-Gunn1 and Paul A Lewandowski2* Abstract Background: Sarcopenia is the progressive loss of skeletal muscle that contributes to the decline in physical function during aging. A higher level of oxidative stress has been implicated in aging sarcopenia. The current study aims to determine if the higher level of oxidative stress is a result of increased superoxide (O2‾ ) production by the NADPH oxidase (NOX) enzyme or decrease in endogenous antioxidant enzyme protection. Methods: Female Balb/c mice were assigned to 4 age groups; 6, 12, 18 and 24 months. Body weight and animal survival rates were recorded over the course of the study. Skeletal muscle tissues were collected and used to measure NOX subunit mRNA, O2‾ levels and antioxidant enzymes. Results: Key subunit components of NOX expression were elevated in skeletal muscle at 18 months, when sarcopenia was first evident. Increased superoxide dismutase 1 (SOD1) activity suggests an increase in O2‾ dismutation and this was further supported by elevated levels of hydrogen peroxide (H2O2) and decline in catalase and glutathione peroxidase (GPx) antioxidant protection in skeletal muscle at this time. NOX expression was also higher in skeletal muscle at 24 months, however this was coupled with elevated levels of O2‾ and a decline in SOD1 activity, compared to 6 and 12 months but was not associated with further loss of muscle mass.
    [Show full text]
  • ARS-2014-6136-Ver9-Huang 6P 973..984
    ANTIOXIDANTS & REDOX SIGNALING Volume 23, Number 12, 2015 DOI: 10.1089/ars.2014.6136 ORIGINAL RESEARCH COMMUNICATION Redox Regulation of Pro-IL-1b Processing May Contribute to the Increased Severity of Serum-Induced Arthritis in NOX2-Deficient Mice Ya-Fang Huang,1 Pei-Chi Lo,1 Chia-Liang Yen,2 Peter Andrija Nigrovic,3,4 Wen-Chen Chao,1,5 Wei-Zhi Wang,1 George Chengkang Hsu,1 Yau-Sheng Tsai,1 and Chi-Chang Shieh1,6 Abstract Aims: To elucidate the role of reactive oxygen species (ROS) in arthritis and to identify targets of arthritis treatment in conditions with different levels of oxidant stress. Results: Through establishing an arthritis model by injecting arthritogenic serum into wild-type and NADPH oxidase 2 (NOX2)-deficient mice, we found that arthritis had a neutrophilic infiltrate and was more severe in Ncf1 - / - mice, a mouse strain lacking the expression of the NCF1/p47phox component of NOX2. The levels of interleukin-1b (IL-1b) and IL-6 in inflamed joints were higher in Ncf1 - / - than in controls. Antagonists of tumor necrosis factor-a (TNFa) and IL-1b were equally effective in suppressing arthritis in wild-type mice, while IL-1b blockade was more effective than TNFa blockade in Ncf1 - / - mice. A treatment of caspase inhibitor and the combination treatment of a caspase inhibitor and a cathepsin inhibitor, but not a cathepsin inhibitor alone, suppressed arthritic severity in the wild-type mice, while a treatment of cathepsin inhibitor and the combination treatment of a caspase inhibitor and a cathepsin inhibitor, but not a caspase inhibitor alone, were effective in treating Ncf1 - / - mice.
    [Show full text]
  • Cytochrome P450 Enzymes but Not NADPH Oxidases Are the Source of the MARK NADPH-Dependent Lucigenin Chemiluminescence in Membrane Assays
    Free Radical Biology and Medicine 102 (2017) 57–66 Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Cytochrome P450 enzymes but not NADPH oxidases are the source of the MARK NADPH-dependent lucigenin chemiluminescence in membrane assays Flávia Rezendea, Kim-Kristin Priora, Oliver Löwea, Ilka Wittigb, Valentina Streckerb, Franziska Molla, Valeska Helfingera, Frank Schnütgenc, Nina Kurrlec, Frank Wempec, Maria Waltera, Sven Zukunftd, Bert Luckd, Ingrid Flemingd, Norbert Weissmanne, ⁎ ⁎ Ralf P. Brandesa, , Katrin Schrödera, a Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany b Functional Proteomics, SFB 815 Core Unit, Goethe-Universität, Frankfurt, Germany c Institute for Molecular Hematology, Goethe-University, Frankfurt, Germany d Institute for Vascular Signaling, Goethe-University, Frankfurt, Germany e University of Giessen, Lung Center, Giessen, Germany ARTICLE INFO ABSTRACT Keywords: Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant NADPH oxidase challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized Nox detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous Lucigenin studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox Chemiluminescence activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse Superoxide (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells Reactive oxygen species Membrane assays overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases.
    [Show full text]
  • The Relationship of NADPH Oxidases and Heme Peroxidases: Fallin' In
    MINI REVIEW published: 05 March 2019 doi: 10.3389/fimmu.2019.00394 The Relationship of NADPH Oxidases and Heme Peroxidases: Fallin’ in and Out Gábor Sirokmány 1,2* and Miklós Geiszt 1,2* 1 Department of Physiology, Faculty of Medicine, Semmelweis University, Budapest, Hungary, 2 “Momentum” Peroxidase Enzyme Research Group of the Semmelweis University and the Hungarian Academy of Sciences, Budapest, Hungary Peroxidase enzymes can oxidize a multitude of substrates in diverse biological processes. According to the latest phylogenetic analysis, there are four major heme peroxidase superfamilies. In this review, we focus on certain members of the cyclooxygenase-peroxidase superfamily (also labeled as animal heme peroxidases) and their connection to specific NADPH oxidase enzymes which provide H2O2 for the Edited by: Gabor Csanyi, one- and two-electron oxidation of various peroxidase substrates. The family of NADPH Augusta University, United States oxidases is a group of enzymes dedicated to the production of superoxide and hydrogen Reviewed by: peroxide. There is a handful of known and important physiological functions where Tohru Fukai, Augusta University, United States one of the seven known human NADPH oxidases plays an essential role. In most of Patrick Pagano, these functions NADPH oxidases provide H2O2 for specific heme peroxidases and University of Pittsburgh, United States the concerted action of the two enzymes is indispensable for the accomplishment *Correspondence: of the biological function. We discuss human and other metazoan examples of such Gábor Sirokmány sirokmany.gabor@ cooperation between oxidases and peroxidases and analyze the biological importance med.semmelweis-univ.hu of their functional interaction. We also review those oxidases and peroxidases where this Miklós Geiszt geiszt.miklos@ kind of partnership has not been identified yet.
    [Show full text]
  • Role of NADPH Oxidase in Arsenic-Induced Reactive Oxygen Species Formation and Cytotoxicity in Myeloid Leukemia Cells
    Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells Wen-Chien Chou*†‡, Chunfa Jie§, Andrew A. Kenedy¶, Richard J. Jonesʈ, Michael A. Trush¶, and Chi V. Dang*†¶ʈ** *Program of Human Genetics and Molecular Biology, †Department of Medicine, §McKusick–Nathans Institute of Genetic Medicine, and ʈSidney Kimmel Comprehensive Cancer Center, School of Medicine, ¶Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205 Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved January 21, 2004 (received for review October 16, 2003) Arsenic has played a key medicinal role against a variety of associated and cytosolic subunits, can be stimulated by phorbol ailments for several millennia, but during the past century its myristate acetate (PMA) through protein kinase C-mediated prominence has been displaced by modern therapeutics. Recently, phosphorylation of the p47PHOX subunit (17, 18). This complex attention has been drawn to arsenic by its dramatic clinical efficacy is responsible for the production of superoxide anion (respira- against acute promyelocytic leukemia. Although toxic reactive tory burst) of professional phagocytes encountering microbial oxygen species (ROS) induced in cancer cells exposed to arsenic pathogens, and its importance in host immunity is underscored could mediate cancer cell death, how arsenic induces ROS remains by the immunocompromised congenital disease, chronic gran- undefined. Through the use of gene expression profiling, interfer- ulomatous disease (CGD), which results from mutations in one ence RNA, and genetically engineered cells, we report here that of the subunits of NADPH oxidase (19, 20).
    [Show full text]
  • Trans-Plasma Membrane Electron Transport System in the Myelin Membrane
    Wilfrid Laurier University Scholars Commons @ Laurier Theses and Dissertations (Comprehensive) 2015 Characterization of the Trans-plasma Membrane Electron Transport System in the Myelin Membrane Afshan Sohail Wilfrid Laurier University, [email protected] Follow this and additional works at: https://scholars.wlu.ca/etd Part of the Biochemistry Commons, Molecular and Cellular Neuroscience Commons, and the Molecular Biology Commons Recommended Citation Sohail, Afshan, "Characterization of the Trans-plasma Membrane Electron Transport System in the Myelin Membrane" (2015). Theses and Dissertations (Comprehensive). 1694. https://scholars.wlu.ca/etd/1694 This Thesis is brought to you for free and open access by Scholars Commons @ Laurier. It has been accepted for inclusion in Theses and Dissertations (Comprehensive) by an authorized administrator of Scholars Commons @ Laurier. For more information, please contact [email protected]. Characterization of the Trans-plasma Membrane Electron Transport System in the Myelin Membrane By Afshan Sohail THESIS Submitted to the Department of Chemistry Faculty of Science In partial fulfillment of the requirements for Master of Science in Chemistry Wilfrid Laurier University 2014 © Afshan Sohail 2014 Abstract Myelination is the key feature of evolution in the nervous system of vertebrates. Myelin is the protrusion of glial cells. More specifically, "oligodendrocytes" in the central nervous system (CNS), and "Schwann" cells in the peripheral nervous system (PNS) form myelin membranes. Myelin remarkably, enhances the propagation of nerve impulses. However, myelin restricts the access of extracellular metabolites to the axons. A pathology called "demyelination" is associated with myelin. The myelin sheath is not only an insulator, but it is itself metabolically active. In this study it is hypothesized that the ratio of NAD(P)+/NAD(P)H and the glycolytic pathway of the myelin sheath is maintained via trans-plasma membrane electron transport system (t-PMET).
    [Show full text]
  • Microsomal Reductase Activity in Patients with Thyroid Neoplasms Elena V
    Microsomal reductase activity in patients with thyroid neoplasms Elena V. Proskurnina1, Maria V. Fedorova2, Madina M. Sozarukova3, Aleksandr E. Mitichkin4, Igor V. Panteleev4, Evgeny V. Svetlov4 1 Research Centre for Medical Genetics, Moscow, Russia 2 Faculty of Fundamental Medicine, Lomonosov Moscow State University, Moscow, Russia 3 Kurnakov Institute of General and Inorganic Chemistry of the Russian Academy of Sciences, Moscow, Russia 4 Inozemtsev City Clinical Hospital of Moscow Department of Health, Moscow, Russia Corresponding Author: Elena V. Proskurnina, [email protected] Abstract Objective: Cytochrome b5 reductase (CYB5R) and cytochrome P450 reductase (CYPOR) play an important role in cell metabolism; however, their role in thyroid hormonogenesis and carcinogenesis has not been studied. The activity of CYB5R correlates with metastasizing in breast cancer, but there are no similar studies for CYB5R and CYPOR for thyroid cancer. The aim was to assess the activity of CYB5R and CYPOR in thyroid tissues in benign and malignant thyroid neoplasms. Methods: 36 patients with thyroid neoplasms participated in the study. The control euthyroid goiter group included 10 patients; the thyrotoxic nodular goiter group included 14 patients; the papillary thyroid cancer T1-2N0-1M0 (PTC) group included 12 patients. The activity of CYB5R and CYPOR was assessed with lucigenin-enhanced chemiluminescence stimulated by NADH and NADPH, respectively. Results: The activity of CYB5R and CYPOR increased several times in thyrotoxicosis and approximately by an order of magnitude in some cases of PTC, but the activity change of CYPOR was more pronounced compared to CYB5R. For the PTC group, the subgroups with low and high activity of microsomal reductases were detected.
    [Show full text]
  • Crystal Structures and Atomic Model of NADPH Oxidase
    Crystal structures and atomic model of NADPH oxidase Francesca Magnania,1,2, Simone Nencia,1, Elisa Millana Fananasa, Marta Ceccona, Elvira Romerob, Marco W. Fraaijeb, and Andrea Mattevia,2 aDepartment of Biology and Biotechnology “L. Spallanzani,” University of Pavia, 27100 Pavia, Italy; and bMolecular Enzymology Group, University of Groningen, 9747 AG Groningen, The Netherlands Edited by Carl F. Nathan, Weill Medical College of Cornell University, New York, NY, and approved May 16, 2017 (received for review February 9, 2017) NADPH oxidases (NOXs) are the only enzymes exclusively dedicated TM binds two hemes (1, 2, 13). The enzyme catalytic cycle entails to reactive oxygen species (ROS) generation. Dysregulation of these a series of steps, which sequentially transfer electrons from cyto- polytopic membrane proteins impacts the redox signaling cascades solic NADPH to an oxygen-reducing center located on the that control cell proliferation and death. We describe the atomic extracytoplasmic side of the membrane (hereafter referred to as crystal structures of the catalytic flavin adenine dinucleotide (FAD)- the “outer side”). Thus, a distinctive feature of NOXs is that and heme-binding domains of Cylindrospermum stagnale NOX5. NADPH oxidation and ROS production take place on the op- The two domains form the core subunit that is common to all seven posite sides of the membrane (1, 2). The main obstacle to the members of the NOX family. The domain structures were then structural and mechanistic investigation of NOX’s catalysis and docked in silico to provide a generic model for the NOX family. A regulation has been the difficulty encountered with obtaining well- linear arrangement of cofactors (NADPH, FAD, and two membrane- behaved proteins in sufficient amounts.
    [Show full text]
  • Activity in Human Neutrophils DPI Selectively Inhibits Intracellular
    DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils Alicia Buck, Felix P. Sanchez Klose, Vignesh Venkatakrishnan, Arsham Khamzeh, Claes Dahlgren, Karin Christenson and Johan Bylund Downloaded from ImmunoHorizons 2019, 3 (10) 488-497 doi: https://doi.org/10.4049/immunohorizons.1900062 http://www.immunohorizons.org/content/3/10/488 This information is current as of October 2, 2021. http://www.immunohorizons.org/ Supplementary http://www.immunohorizons.org/content/suppl/2019/10/18/3.10.488.DCSup Material plemental References This article cites 33 articles, 12 of which you can access for free at: http://www.immunohorizons.org/content/3/10/488.full#ref-list-1 Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: by guest on October 2, 2021 http://www.immunohorizons.org/alerts ImmunoHorizons is an open access journal published by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. ISSN 2573-7732. RESEARCH ARTICLE Innate Immunity DPI Selectively Inhibits Intracellular NADPH Oxidase Activity in Human Neutrophils Alicia Buck,* Felix P. Sanchez Klose,* Vignesh Venkatakrishnan,† Arsham Khamzeh,* Claes Dahlgren,† Karin Christenson,* and Johan Bylund* *Department of Oral Microbiology and Immunology, Institute of Odontology, Sahlgrenska Academy at University of Gothenburg, 40530 Downloaded from Gothenburg, Sweden; and †Department of Rheumatology and Inflammation Research, Institute of Medicine, Sahlgrenska Academy at University of Gothenburg, 40530 Gothenburg, Sweden ABSTRACT http://www.immunohorizons.org/ Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation.
    [Show full text]
  • Cytochrome P450 Enzymes but Not NADPH Oxidases Are the Source of the NADPH-Dependent Lucigenin Chemiluminescence in Membrane Assays Crossmark
    Free Radical Biology and Medicine 102 (2017) 57–66 Contents lists available at ScienceDirect Free Radical Biology and Medicine journal homepage: www.elsevier.com/locate/freeradbiomed Cytochrome P450 enzymes but not NADPH oxidases are the source of the NADPH-dependent lucigenin chemiluminescence in membrane assays crossmark Flávia Rezendea, Kim-Kristin Priora, Oliver Löwea, Ilka Wittigb, Valentina Streckerb, Franziska Molla, Valeska Helfingera, Frank Schnütgenc, Nina Kurrlec, Frank Wempec, Maria Waltera, Sven Zukunftd, Bert Luckd, Ingrid Flemingd, Norbert Weissmanne, ⁎ ⁎ Ralf P. Brandesa, , Katrin Schrödera, a Institute for Cardiovascular Physiology, Goethe-University, Frankfurt, Germany b Functional Proteomics, SFB 815 Core Unit, Goethe-Universität, Frankfurt, Germany c Institute for Molecular Hematology, Goethe-University, Frankfurt, Germany d Institute for Vascular Signaling, Goethe-University, Frankfurt, Germany e University of Giessen, Lung Center, Giessen, Germany ARTICLE INFO ABSTRACT Keywords: Measuring NADPH oxidase (Nox)-derived reactive oxygen species (ROS) in living tissues and cells is a constant NADPH oxidase challenge. All probes available display limitations regarding sensitivity, specificity or demand highly specialized Nox detection techniques. In search for a presumably easy, versatile, sensitive and specific technique, numerous Lucigenin studies have used NADPH-stimulated assays in membrane fractions which have been suggested to reflect Nox Chemiluminescence activity. However, we previously found an unaltered activity with these assays in triple Nox knockout mouse Superoxide (Nox1-Nox2-Nox4-/-) tissue and cells compared to wild type. Moreover, the high ROS production of intact cells Reactive oxygen species Membrane assays overexpressing Nox enzymes could not be recapitulated in NADPH-stimulated membrane assays. Thus, the signal obtained in these assays has to derive from a source other than NADPH oxidases.
    [Show full text]
  • Mycobacterium Tuberculosis
    Correction MICROBIOLOGY Correction for “Mycobacterium tuberculosis is protected from NADPH oxidase and LC3-associated phagocytosis by the LCP protein CpsA,” by Stefan Köster, Sandeep Upadhyay, Pallavi Chandra, Kadamba Papavinasasundaram, Guozhe Yang, Amir Hassan, Steven J. Grigsby, Ekansh Mittal, Heidi S. Park, Victoria Jones, Fong-Fu Hsu, Mary Jackson, Christopher M. Sassetti, and Jennifer A. Philips, which was first published September 27, 2017; 10.1073/pnas.1707792114 (Proc Natl Acad Sci USA 114: E8711–E8720). The authors note that the following statement should be added to the Acknowledgments: “The Biomedical Mass Spec- trometry Resource of Washington University is supported by NIH Grants P41-GM103422, P60-DK-20579, and P30-DK56341.” Published under the PNAS license. www.pnas.org/cgi/doi/10.1073/pnas.1718266114 E9752 | PNAS | November 7, 2017 | vol. 114 | no. 45 www.pnas.org Downloaded by guest on September 28, 2021 Mycobacterium tuberculosis is protected from NADPH PNAS PLUS oxidase and LC3-associated phagocytosis by the LCP protein CpsA Stefan Köstera,1, Sandeep Upadhyayb,c,1, Pallavi Chandrab,c, Kadamba Papavinasasundaramd, Guozhe Yangb,c, Amir Hassanb,c, Steven J. Grigsbyb,c, Ekansh Mittalb,c, Heidi S. Parka, Victoria Jonese, Fong-Fu Hsuf, Mary Jacksone, Christopher M. Sassettid, and Jennifer A. Philipsb,c,2 aDivision of Infectious Diseases, Department of Medicine, New York University School of Medicine, New York, NY 10016; bDivision of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St.
    [Show full text]
  • Modulation of CYP2C9 Activity and Hydrogen Peroxide Production by Cytochrome B5 Javier Gómez‑Tabales1, Elena García‑Martín1*, José A
    www.nature.com/scientificreports OPEN Modulation of CYP2C9 activity and hydrogen peroxide production by cytochrome b5 Javier Gómez‑Tabales1, Elena García‑Martín1*, José A. G. Agúndez1 & Carlos Gutierrez‑Merino2* Cytochromes P450 (CYP) play a major role in drug detoxifcation, and cytochrome b5 (cyt b5) stimulates the catalytic cycle of mono‑oxygenation and detoxifcation reactions. Collateral reactions of this catalytic cycle can lead to a signifcant production of toxic reactive oxygen species (ROS). One of the most abundant CYP isoforms in the human liver is CYP2C9, which catalyzes the metabolic degradation of several drugs including nonsteroidal anti‑infammatory drugs. We studied modulation by microsomal membrane‑bound and soluble cyt b5 of the hydroxylation of salicylic acid to gentisic acid and ROS release by CYP2C9 activity in human liver microsomes (HLMs) and by CYP2C9 baculosomes. CYP2C9 accounts for nearly 75% of salicylic acid hydroxylation in HLMs at concentrations reached after usual aspirin doses. The anti‑cyt b5 antibody SC9513 largely inhibits the rate of salicylic acid hydroxylation by CYP2C9 in HLMs and CYP2C9 baculosomes, increasing the KM approximately threefold. Besides, soluble human recombinant cyt b5 stimulates the Vmax nearly twofold while it decreases nearly threefold the Km value in CYP2C9 baculosomes. Regarding NADPH‑ dependent ROS production, soluble recombinant cyt b5 is a potent inhibitor both in HLMs and in CYP2C9 baculosomes, with inhibition constants of 1.04 ± 0.25 and 0.53 ± 0.06 µM cyt b5, respectively. This study indicates that variability in cyt b5 might be a major factor underlying interindividual variability in the metabolism of CYP2C9 substrates. Abbreviations ASA Acetylsalicylic acid a.u.
    [Show full text]