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)

Rac, a small molecular weight GTPase in the Ras superfamily, participates in the activation of the multicomponent superoxide-generating NADPH oxidase of human neutrophils. Rac is 30% identical to Ras overall, but is 75% identical within the sequence corresponding to the effector region of Ras, which regulates mitogenesis through interactions with the protein kinase Raf1. We investigated the role of this region in Rac1 using site-directed mutagenesis. In a cell-free semirecombinant NADPH oxidase system, mutants in the 26, 33, 38, and 45 amino acids showed 20-110-fold reduced binding to the oxidase complex as judged by EC50 values and reduced (44-80%) maximal activities in superoxide generation. Only the GTP gamma S-bound form associated, since the GDP-bound form of Rac neither activated alone nor competed with GTP gamma S-Rac. EC50 values for neither p47-phox nor p67-phox were affected when mutant Racs were used in place of Rac. Data indicate direct binding of the Rac effector region to one or more components of the respiratory burst oxidase. Results indicate a general role for conserved effector-equivalent regions in small GTPases in the regulation of protein-protein interactions.
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PMID:Ras effector-homologue region on Rac regulates protein associations in the neutrophil respiratory burst oxidase complex. 794 51

The Rac proteins, Rac1 and Rac2, are essential components of the NADPH oxidase system of phagocytes and regulate the actin assembly associated with membrane ruffling. These functions are controlled by the GTP-bound form of Rac. The biochemical interaction between Rac and its only known GDP-dissociation stimulator (termed smgGDS) was characterized. SmgGDS was able to stimulate the incorporation of guanosine 5'-[gamma-thio]-triphosphate GTP[gamma S] into the RhoA, Rac2, Rac1, Rap1A and CDC42Hs GTP-binding proteins, but the activity was greatest toward RhoA and Rac2. Isoprenoid modification of these proteins was not absolutely required for the interaction with smgGDS. Interestingly, the activity of smgGDS toward Rac1 could not be observed in a [3H]GDP/GTP exchange assay under conditions where it stimulated incorporation of GTP[gamma S] into Rac1. We determined that smgGDS prevented the loss of Rac1 activity during the [3H]GDP/GTP exchange assay by demonstrating the ability of smgGDS to inhibit the loss of Rac1 GTP[gamma S]-binding during incubations at 30 degrees C. This stabilizing effect was exactly counterbalanced by the ability of smgGDS to stimulate the release of [3H]GDP from Rac1, thereby producing no net observable effect in the exchange assay. SmgGDS was able to effectively stimulate the release of GDP but not GTP[gamma S] from Rac1. SmgGDS maintains Rac1 in a nucleotide-free form after release of GDP, indicating that the reaction between Rac1 and smgGDS involves a substituted enzyme mechanism.
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PMID:SmgGDS stabilizes nucleotide-bound and -free forms of the Rac1 GTP-binding protein and stimulates GTP/GDP exchange through a substituted enzyme mechanism. 798 Apr 44

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

The neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotide-binding regulatory (G) protein Rac1, were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Rac1, the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Rac1 in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KCl-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.
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PMID:Activation of the O2(-)-generating NADPH oxidase in a semi-recombinant cell-free system. Assessment of the function of Rac in the activation process. 800 73

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

Recent progress in our understanding of the regulation of the phagocyte NADPH oxidase by the Rac GTP-binding protein(s) has provided the first detailed glimpse into the mechanisms of leukocyte regulation by a small GTP-binding protein. Studies over the past year have indicated that the activity of the NADPH oxidase can be modulated by regulation of the GTP/GDP state of Rac. Proteins exist in leukocytes that are able to modify GTP-binding protein function in this manner, and their activity may be regulated by signals generated upon phagocyte stimulation.
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PMID:Regulation of the human neutrophil NADPH oxidase by the Rac GTP-binding proteins. 802 12

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

The Rac GTP-binding proteins regulate the actin cytoskeleton and the superoxide-forming NADPH oxidase of phagocytic leukocytes. These functions of Rac are determined by the GTP/GDP state of the protein, which can be modulated by GTPase-activating proteins (GAPs). The interaction of Ras with both downstream signaling targets and GAPs is mediated via an "effector" domain (amino acids 30-40). We demonstrate that the effector domain of Rac2 is required for both NADPH oxidase activation and actin assembly, but that mutations in this region do not decrease the responsiveness of Rac to GAPs. In contrast, mutations of residues 12 (Gly-->Val) or 61 (Gln-->Leu) inhibit both intrinsic- and GAP-stimulated GTP hydrolysis by Rac2. A double mutation in which both the effector domain and Q61L were modified restored NADPH oxidase activation and membrane ruffling, while the equivalent effector domain and G12V double mutation did not. The Rac2 Q61L mutant had an increased "affinity" for NADPH oxidase activation and for GAP binding as compared to the wild type or G12V proteins. These experiments suggest that Rac contains at least two "effector" interaction sites, and that changes in binding interactions at one of these sites may influence the function of the other.
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PMID:Differing structural requirements for GTPase-activating protein responsiveness and NADPH oxidase activation by Rac. 808 25

Rho-related proteins are members of the ras superfamily of small GTP-binding proteins. Their function in fibroblasts has been analysed using microinjection of living cells. Rho appears to link plasma membrane receptors to the assembly of focal adhesions and actin stress fibres. The closely related protein rac, on the other hand, links receptors to the polymerization of actin at the plasma membrane to form membrane ruffles and pinocytotic vesicles. In phagocytic cells, rac has been shown to be required for activation of a membrane-bound NADPH oxidase in response to receptor activation. These systems provide the basis for a working model for the mechanism of action of the rho family of small GTPases.
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PMID:Cellular responses regulated by rho-related small GTP-binding proteins. 810 28

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


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