Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of pyridine on the heme environment of cytochrome b558 was studied using ESR and optical absorption spectroscopy in relation to the O2(-)-generating activity in the NADPH oxidase system of stimulated pig neutrophils. As the concentration of pyridine increased, the absorption maxima of the alpha- and gamma-bands of cytochrome b558 shifted which correlated with a concomitant decrease in O2(-)-generating activity. In addition, the g = 3.2 signal of cytochrome b558 decreased with the concomitant appearance of a new ESR spectrum that strikingly resembled that of cytochrome P450. The results suggest that pyridine induces a structural modification in the heme environment of cytochrome b558 by shifting the 5th heme ligand (histidine) to a nearby thiolate group without direct binding of pyridine to the heme. The existence of a reactive thiolate near the heme iron was confirmed by pretreatment of blocked cytochrome b558 with p-chloromercuribenzoate, which completely inhibited the formation of the cytochrome P450-like ESR spectrum. The results provide further evidence that a low-spin heme iron of cytochrome b558 with a g-value of 3.2 is essential to the O2(-)-forming reaction of the NADPH oxidase system. From sequence alignments of cytochrome P450 with those of large and small subunits of cytochrome b558, the heme in cytochrome b558 appears to be specifically associated with the large subunit.
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PMID:Modulation of the heme environment of neutrophil cytochrome b558 to a "cytochrome P450-like" structure by pyridine. 785 3

The NADPH oxidase of phagocytes is known to be expressed in Epstein-Barr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% of those found in neutrophils. We have investigated the basis of this low level of expression and find that all four specific components of the NADPH oxidase are expressed in B-lymphocytes, but only p47-phox protein attains levels equivalent with those found in neutrophils. This component was shown to phosphorylate and translocate to the membrane normally on activation. The other cytosolic component, p67-phox, did show a deficit, and by supplementing a B-cell cytosol extract with recombinant p67-phox, this was shown to account for the somewhat reduced activity of B-cell cytosol in a cell-free oxidase system. The cell-free analysis also clearly located the major deficiency in superoxide-generating capacity of B-lymphocytes to the membrane. Western blotting of membrane proteins revealed major reductions in the amount of cytochrome b558. Analysis of the levels of mRNA for both subunits of cytochrome b558, however, showed levels greater than expected. Significantly more mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiated HL60 cells, which are capable of producing large amounts of superoxide. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primarily due to a post-transcriptionally determined block to the accumulation of cytochrome b558.
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PMID:Low NADPH oxidase activity in Epstein-Barr-virus-immortalized B-lymphocytes is due to a post-transcriptional block in expression of cytochrome b558. 786 1

The human neutrophil NADPH oxidase-associated H+ channel acts as a charge compensator for the electrogenic generation of superoxide (O2-.). The expression of the channel activity was found to increase in parallel with that of the stimulatable generation of O2-. in differentiated HL60 cells. HL60 cells induced to differentiate in the presence of succinyl acetone (a inhibitor of heme synthesis) were unable to generate O2-., failed to express p22-phox but retained H+ channel activity. EBV transformed B lymphocyte cell lines from normal and CGD patients lacking expression of either p47-phox or p67-phox all expressed unaltered channel activity; however, the activity was completely absent in the lymphocyte cell line lacking gp91-phox. CHO cells and undifferentiated HL60 cells transfected with gp91-phox cDNA expressed H+ channel activity correlating with the expression of gp91-phox. We therefore conclude that the large subunit of the NADPH oxidase cytochrome b (gp91-phox) is the arachidonate activable H+ channel of human neutrophils.
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PMID:The arachidonate-activable, NADPH oxidase-associated H+ channel. Evidence that gp91-phox functions as an essential part of the channel. 789 Jul 22

Chronic granulomatous disease (CGD) results from defects in the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, central to which is the membrane-bound cytochrome b-245. The cytochrome is composed of two protein subunits, the larger (gp91-phox) being deficient in X-linked CGD. In this study, we have analyzed expression of the cytochrome subunits in B-cell lines from two autosomal CGD patients for whom the disease is caused by deficiency of p22-phox, the smaller subunit. We report the presence of a 65-kD precursor of gp91-phox in the membrane fraction of both p22-phox-deficient cell lines, corresponding to the core protein with N-linked carbohydrate side chains in the high mannose form. Expression of p22-phox in these cells resulted in functional correction of NADPH oxidase. In addition, gp91-phox in the reconstituted cells was processed to its terminally glycosylated form. These data suggest that the association of the 65-kD gp91-phox precursor with p22-phox is a prerequisite for processing of the carbohydrate side chains to the complex form in the Golgi. The detection of this precursor will enable characterization of mutations disrupting the subunit interaction (either naturally occurring or derived by in vitro mutagenesis) and so aid in structure-function analysis of cytochrome b-245. Reconstitution of p22-phox-deficient cells shows the potential of gene therapy for this autosomal form of CGD.
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PMID:p22-phox-deficient chronic granulomatous disease: reconstitution by retrovirus-mediated expression and identification of a biosynthetic intermediate of gp91-phox. 791 88

The NADPH oxidase responsible for generation of superoxide anion and related microbicidal oxidants by phagocytes is assembled from at least five distinct proteins. Two are cytosolic components (p47-phox and p67-phox) that contain Src homology 3 (SH3) domains and associate with a transmembrane cytochrome b558 upon activation. We show here that the SH3 domains of p47-phox bind to proline-rich sequences in p47-phox itself and the p22-phox subunit of cytochrome b558. Binding of the p47-phox SH3 domains to p22-phox was abolished by a mutation in one proline-rich sequence (Pro156-->Gln) noted in a distinct form of chronic granulomatous disease and was inhibited by a short proline-rich synthetic peptide corresponding to residues 149-162 of p22-phox. Expression of mutated p22-phox did not restore oxidase activity to p22-phox-deficient B cells and did not enable p22-phox-dependent translocation of p47-phox to membranes in phorbol ester-stimulated cells. We also show that the cytosolic oxidase components associate with one another through the C-terminal SH3 domain of p67-phox and a proline-rich C-terminal sequence in p47-phox. These SH3 target sites conform to consensus features deduced from SH3 binding sites in other systems. We propose a model in which the oxidase complex assembles through a mechanism involving SH3 domains of both cytosolic proteins and cognate proline-rich targets in other oxidase components.
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PMID:Assembly of the phagocyte NADPH oxidase: binding of Src homology 3 domains to proline-rich targets. 793 8

We have restudied two kindreds that formed the basis of the original report of autosomal recessive chronic granulomatous disease (CGD) associated with leukocyte glutathione peroxidase deficiency. Case 1 from the original study and the surviving brother of the originally reported case 2 both have severe CGD, with no detectable respiratory burst activity in purified intact neutrophils. However, their leukocytes exhibit normal glutathione peroxidase enzyme activity and gene expression. Examination of phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase components known to be defective in CGD reveals no detectable cytochrome b558 nor any membrane activity in a cell-free NADPH oxidase assay system. Molecular analysis of the genes encoding cytochrome b558 subunits shows, in case 1, a C-->T substitution at nucleotide 688 of the gene encoding the gp91-phox subunit of cytochrome b558, resulting in a termination signal in place of Arginine-226. Levels of gp91-phox mRNA are markedly decreased despite normal levels of gene transcription, indicating a post-transcriptional effect of the nonsense mutation on mRNA processing or stability. The X-linked form of CGD developed in this cytogenetically normal female due to the uniform inactivation of the normal X chromosome in her granulocytes, indicated by the expression in her granulocyte mRNA of only one allele of a glucose-6-phosphate dehydrogenase polymorphisms for which she is heterozygous in genomic DNA. Case 2 (of the present study) has distinct mutations in each allele of the p22-phox gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Chronic granulomatous disease and glutathione peroxidase deficiency, revisited. 794 43

In previous studies, we showed that interleukin-4 (IL-4) suppressed porcine (p) macrophage superoxide production and that the mechanism of suppression involved down-regulation of the superoxide-generating enzyme NADPH oxidase heavy-chain 91-kDa subunit mRNA (gp91-phox) expression. In order to examine the effect of IL-4 on expression of the gene encoding the porcine NADPH oxidase light-chain 22-kDa subunit (p22-phox), we cloned the p22-phox cDNA from a macrophage library. The p22-phox cDNA is 786 bp in length and contains a 576-bp open reading frame which predicts a primary translation product of 192 amino acids (aa). Comparison of the porcine and human 22-phox cDNAs showed a high degree of similarity between the two species in their nucleotide (85%) and deduced aa (83%) sequences. as well as in their hydropathy profiles. Notable features, including a high proline content and an iron-coordinating His94, are conserved in both the porcine and human 22-Phox. A single species of mRNA of about 1 kb was detected in macrophages. The mRNA levels remained unchanged in cells treated with lipopolysaccharide (LPS) or with IL-4 at various concentrations from 0-50 ng/ml. Prolonged treatment with LPS or IL-4 did not enhance the effect of these substances on p22-phox mRNA expression. The effect of IL-4 on p22-phox mRNA expression was also compared with another immunosuppressive cytokine, transforming growth factor-beta 1 (TGF beta 1). No change in mRNA expression was found in the cells with or without TGF beta 1 treatment. The results indicated that the heavy and light chains of NADPH oxidase are independently regulated by IL-4 in macrophages.
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PMID:Cloning and expression of the gene encoding the porcine NADPH oxidase light-chain subunit (p22-phox). 795 70

Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to data on cultured human fibroblasts have primarily concerned the identification of cytochrome b558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47-phox and p67-phox. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47-phox, p67-phox and p22-phox. Expression of the gp91-phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47-phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/microgram membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/microgram membrane protein). These data indicate that the expression of p47-phox and p67-phox by human fibroblasts may contribute to the cells' generation of superoxide.
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PMID:The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts. 798 96

When the neutrophil NADPH oxidase is activated to generate superoxide, the cytosolic components, p47phox, p67phox, and the GTP-binding protein Rac, become stably associated with the plasma membrane. The translocation of p47phox and p67phox is dependent on the presence in the membrane of the flavocytochrome b558, itself composed of two subunits, gp91phox and p22phox. In this study we have quantitated Rac1 and Rac2 in human neutrophils and show that > 96% of the Rac protein is Rac2 and that chronic granulomatous disease (CGD) neutrophils contain normal levels of the protein. We used a cell-free translocation system as well as intact normal and CGD neutrophils to determine whether the translocation of Rac2 is dependent upon the presence of the other oxidase components. When cell-free reactions contained any combination of normal, p47phox-deficient or p67phox-deficient cytosol and normal or flavocytochrome b558-deficient membranes, the GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-dependent association of Rac2 with the re-isolated membranes was not significantly different from the control mixture of normal membranes and cytosol. In intact CGD neutrophils lacking p47phox or p67phox and stimulated with phorbol myristate acetate the translocation of Rac2 was also normal, and we conclude that Rac translocation can occur independently of the cytosolic NADPH oxidase components. In contrast, in the absence of flavocytochrome b558 from intact X-chromosome linked CGD neutrophils, Rac2 translocation was reduced to only 25% of the control value. On the basis of these observations we propose that while Rac2 can bind to a site distinct from either gp91phox or p22phox, it depends upon an interaction with the flavocytochrome b558 for maximal stability in the membrane.
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PMID:Rac translocates independently of the neutrophil NADPH oxidase components p47phox and p67phox. Evidence for its interaction with flavocytochrome b558. 798 99

Professional phagocytes, neutrophils, possess a unique membrane-associated NADPH oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O2-. Oxidase activation involves the assembly, in the plasma membrane, of membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The oxidase system consists of two plasma membrane-bound components; low-potential cytochrome b558, which is composed of two subunits of 22 kDa and 91 kDa, and a flavoprotein related to the electron transport between NADPH and heme-binding domains of the oxidase. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome b558 (flavocytochrome b558). At least two cytosolic components, 67 kDa protein and a phosphorylated 47 kDa protein, are known to translocate to the plasma membrane, ensuring assembly of an active O2(-)-generating NADPH oxidase system. More recently, the membrane (Raps) and cytosolic (Racs) GTP-binding proteins have been established as essential to oxidase assembly. It is the purpose of this review to focus on recent data concerning the regulatory mechanisms which lead to organization and activation of the neutrophil NADPH oxidase system.
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PMID:Activation factors of neutrophil NADPH oxidase complex. 801 44


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