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)

The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559.
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PMID:The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559. 184 35

A soluble protein containing very weak NADPH-dependent nitroblue tetrazolium reductase activity was partially purified from the cytosol of dormant human neutrophils by DEAE-5PW ion exchange chromatography. This preparation of cytosolic reductase exhibited three nitroblue tetrazolium-reducing bands with approximate molecular masses of 95, 45, and 40 kDa on non-denaturing gel electrophoresis in the presence of 35 mM n-octyl-glucoside, and two major bands with apparent masses of 45 and 40 kDa along with a few variable minor bands on SDS-polyacrylamide gel electrophoresis. The 45 kDa protein is susceptible to endogenous proteases and is rapidly converted to proteolysis products at 36 degrees C. The partially purified cytosolic protein(s) provided a concentration-dependent activation of NADPH oxidase in the cell-free system composed of the membrane, arachidonate and magnesium ion. In addition, polyclonal antibodies raised against rabbit hepatic NADPH:cytochrome P-450 reductase [EC 1.6.99.1] showed positive immunological reactivity toward cytosolic 45 kDa protein and also caused 30 to 40% inhibition of superoxide anion production in the cell-free system.
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PMID:Cytosolic components to activate neutrophilic NADPH oxidase in a cell-free system. 196 55

After phorbol 12-myristate 13-acetate (PMA) stimulation the increase of NADPH:nitroblue tetrazolium reductase activity in the plasma membrane almost corresponded with the stimulated activity of respiratory burst oxidase. Solubilization of plasma membranes from PMA-activated neutrophils with n-octyl glucoside resulted in high recoveries of the two enzymatic activities. When solubilized plasma membrane was subjected to non-denaturing polyacrylamide gel electrophoresis in the presence of 35 mM n-octyl glucoside, we could see three major bands stained with NADPH-dependent nitroblue reductase activity giving molecular masses of approx. 95, 45 and 40 kDa, respectively. Activity was specific for NADPH but not for NADH. These bands also stained weakly in the plasma membranes obtained from resting cells. The activities for NADPH oxidase and nitroblue tetrazolium reductase were found to elute as a very similar protein peak on an anion-exchange HPLC, at about 0.32 M KCl. This elution peak also contains 45 and 40 kDa proteins showing NADPH:nitroblue tetrazolium reductase activity.
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PMID:NADPH: nitroblue tetrazolium reductase found in plasma membrane of human neutrophil. 211 29

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.
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PMID:Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid. 215 45

The superoxide generating NADPH oxidase was studied in an SDS-activated cell-free system. This system requires the participation of both membranal and cytosolic components. Cytosol derived from elicited peritoneal guinea pig macrophages was fractionated by several nucleotide affinity chromatography procedures. Various such fractionations led to the separation of two distinct factors, both of which are necessary for the activation and/or activity of the superoxide-forming NADPH oxidase. One factor (sigma 2), bound to octyl, 2',5'-ADP-, 5'-ATP-, 5'-GTP-agarose and carboxymethyl-Sepharose but did not bind to hexyl, 5'-AMP-, 5'-ADP- and 5'-GDP-agarose. The other factor (sigma 1) did not bind to any of the above matrices. Subsequent elution of sigma 2 from 2',5'-ADP-agarose was effected by ATP, GTP and NADPH but not by NADH. Elution from GTP-agarose was by ATP and GTP but not by NADPH. Elution from ATP-agarose was by ATP, GTP and also, albeit weakly, by NADPH. The above results suggest that sigma 2 contains a site which recognizes the phosphate group at the ribose 2' position in adenosine, and a site that recognizes purine nucleotide triphosphates.
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PMID:Nucleotide binding properties of cytosolic components required for expression of activity of the superoxide generating NADPH oxidase. 215 58

The superoxide (O2-)-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). O2- production requires the cooperation of membrane-associated and cytosolic components. The membrane component can be solubilized by octyl glucoside yielding a highly active oxidase preparation. High performance gel filtration of the solubilized oxidase on Superose 12 in the presence of 40 mM octyl glucoside leads to the total loss of enzymatic activity. This can be restored in previously inactive eluate fractions by "reconstitution" with N-ethylmaleimide or heat (60 degrees C)-inactivated total solubilized membrane. Oxidase activity, that becomes evident upon reconstitution, is eluted from Superose 12 with molecules in the Mr range of 300,000-71,000. The material with reconstitutive capacity is completely dissociated from the oxidase, eluting with molecules in the Mr range of 71,000-11,000. The Superose 12 elution profile of the material responsible for reconstitution coincides with that of membrane-derived phospholipid. Also, the reconstitutive capacity of total solubilized membrane and that of the Mr 71,000-11,000 region of the Superose eluate are recovered in a chloroform extract prepared by the method of Bligh and Dyer. It is concluded that loss of oxidase activity by gel filtration at a high octyl glucoside concentration is the consequence of delipidation. NADPH oxidase activity, revealed by reconstitution of Superose 12 fractions with exogenous phospholipid, correlates closely with the elution profile of cytochrome b559. Reconstitution of activity of delipidated oxidase can also be achieved with natural non-macrophage phospholipids and with synthetic phospholipids. Reconstitution of NADPH oxidase activity by lipids is governed by the following rules: (a) phospholipids are effective; lysophospholipids and neutral lipids are not; (b) phospholipids with polar heads represented by choline, ethanolamine, and serine, as well as cardiolipin, are effective; phosphatidylinositol is much less active; (c) phospholipids with unsaturated fatty acid residues are capable of reconstitution while saturated acyl residues do not confer activity; this specificity appears not to be related to the transition temperature of the phospholipids.
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PMID:Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme. 254 2

The superoxide-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, most notably SDS. Activation requires the cooperation of membrane-associated and cytosolic components. We now report that at least two cytosolic factors are required for SDS-elicited activation of NADPH oxidase of guinea pig macrophages. Treatment of cytosol with ammonium sulfate at 37% saturation led to the partition of the two factors in the supernatant and precipitate fractions (termed components sigma 1 and sigma 2, respectively). Although each fraction by itself was inactive, recombining them resulted in complete recovery of the original ability of native cytosol to support SDS-elicited superoxide production by octyl-glucoside solubilized macrophage membranes. Both components are proteins, as shown by their susceptibility to trypsin and proteinase K, and were inactivated by heating at 60 degrees C. sigma 2, but not sigma 1, was inactivated by treatment with the covalent sulfhydryl reagent N-ethylmaleimide. On high-performance gel filtration, sigma 1 was found to have a molecular mass of 30 to 52 kDa, whereas sigma 2 eluted with molecules of 150 to 440 kDa. Component sigma 1 was partially purified from the ammonium sulfate supernatant fraction of cytosol by hydrophobic interaction chromatography followed by gel filtration. A material behaving like sigma 1 was also found to be present in the cytosol of guinea pig thymus cells, lymph node lymphocytes and brain and of the mouse myeloma cell line MOPC 315. However, sigma 2 appears to be strictly phagocyte specific. The molecular characteristics of sigma 1 components from nonphagocytic cells were similar to those of macrophage sigma 1, as shown by their presence in the supernatant, after treatment of cytosol with ammonium sulfate at 37% saturation, a molecular mass close to 30 to 52 kDa and a similar behavior on hydrophobic interaction chromatography. These findings raise the possibility that cytosolic component sigma 1 might be the bearer of a cellular function, more general than the one suggested by its role in the activation of NADPH oxidase of phagocytes.
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PMID:Activation of the superoxide-forming NADPH oxidase of macrophages requires two cytosolic components--one of them is also present in certain nonphagocytic cells. 255 80

Sodium dodecyl sulfate (SDS) was shown to elicit NADPH-dependent superoxide (O2-) production by a cell-free system derived from sonically disrupted resting guinea pig macrophages (Bromberg, Y., and Pick, E. (1985) J. Biol. Chem. 260, 13539-13545). O2- production was absolutely dependent on the cooperation between a membrane-associated component, sedimenting with the 48,000 X g pellet and a cytosolic factor, nonsedimentable at 265,000 X g. The present report describes the solubilization and characterization of the membrane-associated component of the SDS-activable O2(-)-forming NADPH oxidase (operationally termed pi). Treatment of the 48,000 X g pellet with 30 mM octyl glucoside resulted in complete transfer of pi to the soluble fraction. The solubilized pellet produced an average of 0.92 mumol of O2-/mg of protein/min upon reduction of octyl glucoside content below the critical micellar concentration and in the presence of cytosol, 100 microM SDS, and 0.2 mM NADPH. The activity of solubilized pellet-cytosol combinations was also expressed as NADPH-dependent, azide-resistant oxygen consumption and hydrogen peroxide production. pi was inactivated by the sulfhydryl reagent p-chloromercuribenzoate. Solubilized pellet contained spectroscopically detectable cytochrome b559 (225.6 +/- 15.0 pmol/mg mg protein). Both pi and cytochrome b559 were bound by Cibacron Blue Sepharose and could be eluted by a gradient of octyl glucoside (0-30 mM) in the presence of 1 M KCl. On high performance gel filtration on Superose 12, both pi and cytochrome b559 eluted in the excluded volume; when 25 mM octyl glucoside was present in the elution buffer, pi was partially dissociated from cytochrome b559. Sequential purification of pi on Blue Sepharose followed by gel filtration on Superose 12 in the presence of 25 mM octyl glucoside lead to complete resolution of pi from cytochrome b559 (pi was found in the Mr = 28,000 - 11,000 range while the bulk of cytochrome b559 eluted in the Mr = 113,000 - 71,000 range). We propose that pi is distinct from cytochrome b559 and represents a membrane-associated component in an amphiphile-activated electron transport chain from NADPH to oxygen.
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PMID:Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Characterization of the membrane-associated component. 282 96

Anionic amphiphiles such as long chain unsaturated fatty acids and SDS were shown to activate the superoxide (O2-) producing NADPH oxidase in a cell-free system derived from sonically disrupted phagocytes (macrophages and granulocytes). O2- production required the cooperation of a membrane associated component sedimenting at 48,000 X g (pi) and a cytosolic factor (sigma). The purpose of the present investigation was to find out whether components pi and sigma were also present in non-phagocytic cells that do not produce O2- when stimulated. It was found that the 48,000 X g pellets of guinea pig lymph node and thymus cell sonicates contained significant amounts of component pi, as shown by their ability to support SDS-elicited NADPH-dependent O2- production when supplemented with macrophage cytosol. Lymph node and thymus pi could be extracted from the membrane by 30 mM octyl glucoside, just as its macrophage-derived equivalent. Combining lymph node and thymus 48,000 X g pellet with autologous cytosol did not yield an active enzyme preparation. Also, cytosol from lymph node and thymus cells could not cooperate with macrophage 48,000 X g pellet, indicating that component sigma was lacking in lymphoid cells. Neither pi nor sigma could be detected in guinea pig kidney, the mouse myeloma cell line MOPC 315 and the canine cell line Cf2Th. The 48,000 X g pellet of all nonphagocytic cells examined contained a b-cytochrome that resembled, by its spectral characteristics, the cytochrome b559 thought to be characteristic of phagocytes. In macrophages, cytochrome b559 represented 80% of b-cytochrome content of the 48,000 X g pellet, whereas in non-phagocytic cells, the equivalent material represented only 50 to 60%. There was no correlation between the presence and quantity of the cytochrome b559-like chromophore in the 48,000 X g pellet of a particular cell type and its ability to cooperate with macrophage cytosol in SDS-elicited O2- production.
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PMID:Certain lymphoid cells contain the membrane-associated component of the phagocyte-specific NADPH oxidase. 283 Dec 70

Both 1,2-diacyl- and 1-O-alkyl-2-acyl-sn-glycerols are released during stimulation of human polymorphonuclear leukocytes (PMNL). 1,2-Diacylglycerols have received intense interest as intracellular "second messengers" due to their ability to activate protein kinase C (Ca2+ phospholipid-dependent enzyme). However, little is known about bioactivities of the alkylacylglycerols. This study compared the ability of 1,2-diacyl- and 1-O-alkyl-2-acylglycerols to modulate the respiratory burst of stimulated PMNL, a response which depends on the activation of an NADPH oxidase to generate bactericidal species of reduced oxygen. Direct stimulation by N-formyl-Met-Leu-Phe caused an abrupt release of H2O2 which ceased within 2.5 min. Preincubation with diacylglycerols (1-oleoyl-2-acetylglycerol,5-30 microM, and 1,2-dioctanoylglycerol,2-5 microM) caused a decrease in lag time, 3-fold increase in initial rate of H2O2 release, and marked prolongation of the response to N-formyl-Met-Leu-Phe (features characteristic of a priming effect). Preincubation with alkylacylglycerols (1-O-delta 9-octadecenyl-2-acetylglycerol, 5-30 microM, and 1-O-octyl-2-octanoylglycerol, 20-50 microM) primed initiation (shortened lag time and increased velocity) but, in contrast to diacylglycerols, did not alter duration of H2O2 release. While low concentrations of diacylglycerols (5-30 microM) primed PMNL, higher concentrations (greater than or equal to 70 microM) stimulated the cells directly. In contrast, higher (70-100 microM) concentrations of alkylacylglycerols did not prime the responses but, in fact, inhibited priming (especially of duration) induced by diacylglycerol. The high concentrations of alkylacylglycerol also inhibited direct stimulation induced by high concentrations of diacylglycerol. Direct stimulation by high concentrations of diacylglycerol probably involves activation of protein kinase C, whereas alkylacylglycerol was found to inhibit activation of protein kinase C by diacylglycerol in vitro. Thus, diacylglycerols are complete priming agonists, altering both rate and duration of the response. In contrast, alkylacylglycerols may have biphasic, concentration-related effects in modulation of functions of PMNL. At low concentrations, they may facilitate initiation of functional events; however, as their concentration increases, they may serve to terminate responses. The distinct priming effects of these diglycerides also reveal that priming can involve at least two distinct events: 1) initiation and 2) prolongation.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Selective priming of rate and duration of the respiratory burst of neutrophils by 1,2-diacyl and 1-O-alkyl-2-acyl diglycerides. Possible relation to effects on protein kinase C. 319 43


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