Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rap1A is a GTP-binding protein of the Ras superfamily that is highly abundant in phagocyte membranes. Although Rap1A copurifies with cytochrome b558, a component of the superoxide-generating NADPH oxidase complex of human phagocytes and B lymphocytes, the involvement of Rap1A in the regulation of the oxidative burst in these cells has not been clearly established. Therefore, we have stably transfected human Epstein-Barr virus-transformed B lymphocytes that possess an activable NADPH oxidase complex with cDNAs for mutants of Rap1A "locked" in a GTP-bound (63E) and GDP-bound (17N) state. Both the 17N and 63E mutants of Rap1A inhibited phorbol ester-stimulated O2-. production by 50 and 80%, respectively, while transfection with cDNA for wild-type Rap1A had no effect on the respiratory burst. No effects of the Rap1A mutants on cell viability, proliferation, expression of cell-surface markers, or phorbol 12-myristate 13-acetate-stimulated interleukin-8 generation were detected. These data demonstrate that Rap1A is a regulator of O2-. formation in intact cells. Furthermore, the inhibitory effect of both GTP- as well as GDP-bound mutants indicates that Rap1A functions in a dynamic cycle as opposed to a unidirectional pathway, as is the case for the other NADPH oxidase regulatory GTP-binding protein, Rac.
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PMID:Activated or dominant inhibitory mutants of Rap1A decrease the oxidative burst of Epstein-Barr virus-transformed human B lymphocytes. 803 26

Chronic granulomatous disease is a serious clinical entity. The disease is caused by the failure of NADPH oxidase in phagocytic leukocytes to generate superoxide, needed for the killing of micro-organisms. The patients need careful management aimed at prevention and aggressive treatment of infections. CGD is a heterogeneous syndrome, both clinically and genetically. This disease is caused by a diversity of mutations, and multiple genes are affected. In fact, in the A22 and X91 subtypes of CGD, in which the alpha subunit and the beta subunit of cytochrome b558 are affected, respectively, the mutations are virtually unique for each CGD family tested. The results of these studies provide a better understanding of the mechanism of action of the various components of the superoxide-generating enzyme. Although treatment of CGD patients has improved considerably over the past 30 years, death caused by overwhelming infections is still a serious threat. Prenatal diagnosis now provides the relatives of a CGD patient with the possibility to choose for first-trimester abortion of an affected fetus. Moreover, genetic correction of the disease is now a goal within reach.
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PMID:The genetic basis of chronic granulomatous disease. 807 Aug 13

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2',5'-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.
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PMID:NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization. 811 Jan 98

The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-bound electron transport chain, consists of cytochrome b558, p47-phox, p67-phox, and p21rac1 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b558 in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b558) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b558. In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b558. We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b558 in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:p21rac does not participate in the early interaction between p47-phox and cytochrome b558 that leads to phagocyte NADPH oxidase activation in vitro. 811 10

The 47-kDa subunit of the NADPH oxidase system (p47-phox) of neutrophils undergoes an association with proteins in the Triton X-100-insoluble fraction upon stimulation of the cells with 4 beta-phorbol 12-myristate 13-acetate. This fraction contains the assembled oxidase that catalyzes the generation of superoxide by stimulated cells. In this paper, we report that the addition of an inhibitor of protein kinases (1-(5-isoquinolinylsulfonyl-2-methylpiperazine) to neutrophils that are already stimulated results in the dissociation of p47-phox from this fraction. Antagonists of type 1 and 2A protein phosphatases (calyculin A, okadaic acid) prevented this phenomenon. In contrast, norokadanone, an inactive analog of okadaic acid, did not affect this response. These observations are correlated with previous studies on the phosphorylation of p47-phox and superoxide release. In addition, we show that protein kinase C (PKC) also undergoes an extensive redistribution to the Triton X-100-insoluble fraction in 4 beta-phorbol 12-myristate 13-acetate-stimulated cells, the extent of which is diminished significantly in neutrophils from chronic granulomatous disease patients who lack either p47-phox or cytochrome b558. These studies strongly indicate that PKC and type 1 and/or 2A protein phosphatases are involved in a continuous phosphorylation reaction that maintains the oxidase in the assembled/active state. Moreover, components of the oxidase may target and facilitate the translocation of PKC to a cellular site in close apposition to the oxidase.
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PMID:Reciprocal interactions between protein kinase C and components of the NADPH oxidase complex may regulate superoxide production by neutrophils stimulated with a phorbol ester. 814 69

A rare subgroup (approx. 5%) of all chronic granulomatous disease (CGD) patients suffers from mutations in the gene encoding the small p22-phox subunit of the flavocytochrome b558 heterodimer, the terminal redox component of the phagocyte NADPH oxidase. A male CGD patient with neutrophil granulocytes devoid of any spectrometrically detectable cytochrome b558 owing to autosomally inherited p22-phox deficiency (phenotype, A22-) is reported. The patient was identified as being compound heterozygous for two independent mutations of his p22-phox alleles. On the maternal allele a single base substitution (A186 to T) was found that predicts a nonconservative replacement of Glu 53 by Val. On his paternal p22-phox allele a G was found to be added to a G stretch between nucleotides G195 and G199 in the cDNA sequence. The resulting frame shift predicts an aberrant open reading frame, 16 amino acids longer than the normal p22-phox polypeptide. Genomic DNA was tested for the presence of the mutant allele by mismatch PCR (polymerase chain reaction). For this purpose, a single base mismatch was introduced at nucleotide position 189, leading to digestion of the normal allele by the restriction enzyme HinfI. The maternal allele was found to be present in 50% of the patient's DNA and in 50% of the DNA from his mother. The same mismatch PCR analysis with control DNA from 35 healthy individuals ruled out the possibility that the single base substitution (A186 to T) represents a common polymorphism. Inheritance of the second allelic mutation (G insertion) was verified by restriction enzyme analysis using BslI [CC(N)7GG] to digest PCR-amplified genomic DNA at the mutation site. PCR in combination with restriction enzyme analysis proved to be a powerful tool for verification of point mutations in the compound heterozygous CGD patient analyzed and may be used for prenatal diagnosis in this family.
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PMID:Identification of allele-specific p22-phox mutations in a compound heterozygous patient with chronic granulomatous disease by mismatch PCR and restriction enzyme analysis. 816 15

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.
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PMID:A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox. 818 43

The formation of microbicidal oxidants by stimulated phagocytes is a major mechanism of host defence against infection and may also cause unwanted damage to host tissues in the setting of inappropriate inflammation. Recently, the molecular basis for oxidant production has been defined by elucidating the structure, biochemistry and regulation of the phagocyte NADPH oxidase, a multicomponent enzyme that uses NADPH to reduce molecular oxygen to superoxide anion which is then converted to hydrogen peroxide. Many of the advances resulted from the study of phagocytes obtained from patients with inherited abnormalities of the NADPH oxidase system, known as the chronic granulomatous diseases of childhood (CGD). These patients are susceptible to life-threatening infections. The NADPH oxidase is a complex enzyme system that has been shown to contain cytosolic and membrane components that assemble at the plasma membrane with cell activation. These components include a membrane NADPH-binding flavoprotein, cytochrome b558, the cytosolic proteins p47phox, p67phox and a small ras-related guanosine triphosphatase or rac protein that confers guanosine triphosphate sensitivity to the NADPH oxidase. Clinically, the NADPH oxidase system can be stimulated with interferon-gamma, resulting in reduced infections in patients with CGD. In addition, the recent incorporation of genes for the components of the NADPH oxidase into retrovirus vectors has resulted in successful transduction of these genes into blood stem cells from CGD patients with correction of the functional defect. This suggests that gene therapy for correction of CGD will be possible in the near future.
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PMID:Delineation of the phagocyte NADPH oxidase through studies of chronic granulomatous diseases of childhood. 818 51

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.
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PMID:Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. 820 90

Cell stimulation of blood phagocytes activates the superoxide-producing NADPH oxidase. Cytochrome b558, one of the two oxidase redox components, comprises a light (alpha) and a heavy glycosylated (beta) subunit. The other redox component, a flavoprotein, is now thought to be the heavy subunit, on the basis of amino acid sequence comparisons and of reconstitution experiments with purified components. We published that pyridoxal-5'-diphospho-5'-adenosine is an inactivating affinity label for the NADPH-binding site of particulate oxidase from activated neutrophils. We have now radiolabeled the inactivated oxidase by reducing with Na[3H]BH4 the Schiff base formed between proteins and the reagent. Upon SDS-PAGE, the NADPH-inhibitable incorporation is found at the same position as the immunodetectable cytochrome heavy subunit, before and after deglycosylation. Membranes from either activated cells of a cytochrome-deficient X-linked granulomatous disease patient or normal resting cells show no incorporation at this position. Our results provide experimental evidence for the existence on the cytochrome b558 heavy chain of an NADPH-binding site which can only be affinity-labeled by PLP-AMP when the oxidase is active. This suggests the occurrence of a conformational change in the cofactor binding site upon enzyme activation.
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PMID:Affinity-labeling of an NADPH-binding site on the heavy subunit of flavocytochrome b558 in particulate NADPH oxidase from activated human neutrophils. 824 Mar 26


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