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PubMed1987

Regulation of xanthine oxidase activity and immunologically detectable protein in rats in response to dietary protein and iron.

To investigate the mechanism for changes in xanthine oxidase activity in response to dietary protein and iron, we fed rats diets containing 50, 20 or 5% casein with either normal iron (35 mg Fe/kg diet) or low iron (5 mg Fe/kg diet). Xanthine oxidase activity changed in liver and intestinal mucosa in response to protein and iron, but immunologically detectable xanthine oxidase protein did not change. When total liver RNA isolated from these rats was translated by a rabbit reticulocyte lysate, we found no difference in the amount of xanthine oxidase that was translated. These results demonstrated that the changes in xanthine oxidase activity were not accompanied by changes in the amount of protein. Since xanthine oxidase can exist in an inactive desulfo form, we asked if xanthine oxidase activity was changed by the content of sulfur-containing amino acids in the diet. Xanthine oxidase activity in intestinal mucosa of the rats fed the 5% casein + methionine diet was significantly greater than that of the rats fed the 5% casein diet alone. These findings suggest that xanthine oxidase activity may be regulated by interconversion of active and inactive desulfo enzyme.

Animals↗
PubMed2005

The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2.

The Ca2+- and arachidonic acid-binding S100A8/A9 protein complex was recently identified by in vitro studies as a novel partner of the phagocyte NADPH oxidase. The present study demonstrated its functional relevance by the impaired oxidase activity in neutrophil-like NB4 cells, after specific blockage of S100A9 expression, and bone marrow polymorphonuclear neutrophils from S100A9-/- mice. The impaired oxidase activation could also be mimicked in a cell-free system by pretreatment of neutrophil cytosol with an S100A9-specific antibody. Further analyses gave insights into the molecular mechanisms by which S100A8/A9 promoted NADPH oxidase activation. In vitro analysis of oxidase activation as well as protein-protein interaction studies revealed that S100A8 is the privileged interaction partner for the NADPH oxidase complex since it bound to p67phox and Rac, whereas S100A9 did interact with neither p67phox nor p47phox. Moreover, S100A8/A9 transferred the cofactor arachidonic acid to NADPH oxidase as shown by the impotence of a mutant S100A8/A9 complex unable to bind arachidonic acid to enhance NADPH oxidase activity. It is concluded that S100A8/A9 plays an important role in phagocyte NADPH oxidase activation.

Animals↗
PubMed2005

Clinically relevant osmolar stress inhibits priming-induced PMN NADPH oxidase subunit translocation.

BACKGROUND: The plasma membrane NADPH oxidase is responsible for the external generation of superoxide by neutrophils (polymorphonucleocytes [PMNs]). The oxidase is a multicomponent enzyme, active only when all subunits are translocated to and assembled at the membrane. We have recently demonstrated that platelet-activating factor (PAF) priming of PMNs translocates the cytosolic p67 subunit to the membrane position. Osmolar stress attenuates PAF priming of the oxidase. Consequently, we hypothesized that clinically relevant osmolar stress inhibits PAF priming-induced p67 translocation. METHODS: Isolated human PMNs were incubated at 37 degrees C for 5 minutes in buffer or 180 mmol/L hypertonic saline (HTS) followed by 3 minutes of incubation with or without 2 mumol/L PAF (resting, PAF, HTS, and HTS-PAF). Digital microscopy was used to determine p67 location in whole PMNs. Subcellular fractions were prepared and membrane translocation of p67 determined by protein electrophoresis. Resting cytosol fractions were immunodepleted of p67 and NADPH oxidase activity measured using p67-deficient sodium dodecyl sulfate cell-free oxidase assays: resting, PAF, or HTS-PAF membrane (1 mug) was combined with immunodepleted resting cytosol (25 mug). RESULTS: By all methodologies, PAF stimulated translocation of p67 to the PMN membrane and this translocation was prevented by osmolar stress (HTS-PAF). In cell-free oxidase assays, the membrane content of p67 after PAF stimulation was increased sufficiently to induce oxidase activity, whereas resting and HTS-PAF membrane did not (0.1 +/- 0.02, 0.23 +/- 0.04, and 0.14 +/- 0.04, respectively, p < 0.01) (resting versus HTS-PAF, no difference). CONCLUSION: PAF priming of the PMN oxidase involves translocation of p67 to the plasma membrane. Clinically relevant osmolar stress with hypertonic saline prevents this PAF-induced translocation of the p67 oxidase subunit. This finding provides new insight into the mechanisms responsible for osmolar control of PMN functional responses.

Cell Membrane↗
PubMed1992

Differential tissue-specific expression and induction of cytochrome P450IVA1 and acyl-CoA oxidase.

We have examined the tissue-specific expression and inducibility of acyl-CoA oxidase and cytochrome P450IVA1 (P450IVA1) RNA in rats. Groups of three rats were dosed daily by gavage with methylclofenapate at 25 mg/kg in 5 ml/kg corn oil for nine weeks, or were administered a vehicle control. P450IVA1 and acyl-CoA oxidase RNA were detected using an RNase protection assay. Similar levels of acyl-CoA oxidase RNA were present in control liver and kidney, but the level of this RNA in lung, muscle and testis was 6-11%, and in pancreas was 0.13%, of that in liver. Treatment of rats with methylclofenapate led to an 11-fold induction of acyl-CoA oxidase RNA in liver and also produced a significant induction of this RNA in kidney, lung, muscle and testis of 1.7-fold, 1.3-fold, 2-fold and 1.7-fold, respectively. Acyl-CoA oxidase RNA was not induced in pancreas. P450IVA1 RNA was present in control liver and also in kidney of control rats at 28% of the level in liver. In contrast to acyl-CoA oxidase RNA, P450IVA1 RNA was not detected in lung, pancreas or testis. Methylclofenapate treatment of rats led to an 18-fold induction of P450IVA1 RNA in liver, and a sevenfold induction in kidney. Induction of P450IVA1 was not detected in any of the other tissues examined. Quantification of the relative amounts of acyl-CoA oxidase and P450IVA1 RNA in control liver revealed that acyl-CoA oxidase RNA was present in a 17.5-fold molar excess over P450IVA1 RNA. Western blotting with an anti-P450IVA IgG revealed two bands of similar apparent molecular mass in liver and kidney microsomes, but not in microsomes from the testis of control rats. Methylclofenapate treatment of rats caused an increase in the intensity of these bands in microsomes from liver, but no induction was obvious in kidney. Immunocytochemical staining for both the microsomal P450IVA and peroxisomal acyl-CoA oxidase proteins was restricted to the proximal convoluted tubule in the kidney cortex, with staining being most intense in the S3 region.

Acyl-CoA Oxidase↗
PubMed2004

Homocysteine activates NADH/NADPH oxidase through ceramide-stimulated Rac GTPase activity in rat mesangial cells.

BACKGROUND: We recently demonstrated that homocysteine (Hcys) increases superoxide (O2-) production via NADH/NADPH oxidase in renal mesangial cells. This O2- production contributes to increased expression of tissue inhibitor of metalloproteinase (TIMP-1) and consequent deposition of collagen in response to Hcys. However, the mechanism by which Hcys activates NADH/NADPH oxidase remains unknown. Given that ceramide is an intracellular activator of this oxidase in several cell types, the present study tests the hypothesis that Hcys activates NADH/NADPH oxidase through a ceramide-mediated signaling pathway in rat mesangial (MG) cells, resulting in O2- production. METHODS: Rat MG cells were incubated with L-homocysteine (L-Hcys) to determine the mechanism by which Hcys activates NADH/NADPH oxidase. Thin layer chromatography (TLC), Western blot analysis, Rac GTPase activity pull down assay, and NADH/NADPH oxidase activity measurements were performed. RESULTS: TLC analysis demonstrated that L-Hcys increased de novo production of ceramide in MG cells. L-Hcys and increased ceramide did not alter the amount of NADH/NADPH oxidase subunit p47phox and p67phox in both membrane and cytosolic fractions from MG cells. However, L-Hcys or ceramide markedly increased the level of GTP-bound Rac, which was accompanied by enhanced activity of NADH/NADPH oxidase. These Hcys or ceramide-induced actions were substantially blocked by a Rac GTPase inhibitor, GDPbetaS, and a de novo ceramide synthesis inhibitor, fumonisin B1 (FB1). CONCLUSION: These results indicate that Hcys activates NADH/NADPH oxidase by stimulating de novo ceramide synthesis, and subsequently enhancing Rac GTPase activity in rat MG cells. This ceramide-Rac GTPase signaling pathway may mediate Hcys-induced oxidative stress in these glomerular cells.

Animals↗
PubMed2003

Temperature dependence of NADPH oxidase in human eosinophils.

The phagocyte NADPH oxidase helps kill pathogens by producing superoxide anion, O2-. This enzyme is electrogenic because it translocates electrons across the membrane, generating an electron current, Ie. Using the permeabilized patch voltage-clamp technique, we studied the temperature dependence of Ie in human eosinophils stimulated by phorbol myristate acetate (PMA) from room temperature to >37 degrees C. For comparison, NADPH oxidase activity was assessed by cytochrome c reduction. The intrinsic temperature dependence of the assembled, functioning NADPH oxidase complex measured during rapid temperature increases to 37 degrees C was surprisingly weak: the Arrhenius activation energy Ea was only 14 kcal mol(-1) (Q10, 2.2). In contrast, steady-state NADPH oxidase activity was strongly temperature dependent at 20-30 degrees C, with Ea 25.1 kcal mol(-1) (Q10, 4.2). The maximum Ie measured at 34 degrees C was -30.5 pA. Above 30 degrees C, the temperature dependence of both Ie and O2- production was less pronounced. Above 37 degrees C, Ie was inhibited reversibly. After rapid temperature increases, a secondary increase in Ie ensued, suggesting that high temperature promotes assembly of additional NADPH oxidase complexes. Evidently, about twice as many NADPH oxidase complexes are active near 37 degrees C than at 20 degrees C. Thus, the higher Q10 of steady-state Ie reflects both increased activity of each NADPH oxidase complex and preferential assembly of NADPH oxidase complexes at high temperature. In summary, NADPH oxidase activity in intact human eosinophils is maximal precisely at 37 degrees C.

Adult↗
PubMed1996

In vitro analysis of the role of glucose oxidase from Talaromyces flavus in biocontrol of the plant pathogen Verticillium dahliae.

Culture filtrates from Talaromyces flavus grown on glucose contained high levels of glucose oxidase activity, while culture filtrates from T. flavus grown on xylan contained negligible glucose oxidase activity. Culture filtrates from T-flavus grown on both media contained complex protein profiles. However, only culture filtrates from T. flavus grown on glucose inhibited germination of microsclerotia of Verticillium dahliae in in vitro inhibition assays. A polyclonal antiserum preparation, pABGO-1, raised against purified glucose oxidase from T. flavus was highly specific for glucose oxidase. Only one protein band in culture filtrates (from glucose medium), migrating at 71 kDa, was detected in Western blots (immunoblots) with this antiserum. This band comigrated with purified glucose oxidase. No bands were detected in culture filtrates from the xylan medium. Glucose oxidase was removed via immunoprecipitation from culture filtrates of T. flavus grown in glucose medium, resulting in filtrates which no longer inhibited in vitro microsclerotial germination. When glucose oxidase-depleted filtrates were amended with purified glucose oxidase from T. flavus, the ability to kill microsclerotia in vitro was restored to original levels. We conclude that glucose oxidase is the only protein in culture filtrates of T. flavus responsible for inhibition of germination of microsclerotia of V. dahliae.

Animals↗
PubMed2000

Regulation of lysyl oxidase by interferon-gamma in rat aortic smooth muscle cells.

Lysyl oxidase is an essential catalyst for the cross-linking of extracellular collagen and elastin. Abnormalities in lysyl oxidase activity may contribute to the pathogenesis of arterial diseases characterized by abnormal matrix remodeling. This study tested the hypothesis that interferon (IFN)-gamma, a proinflammatory cytokine present in aortic aneurysm and arteriosclerotic plaque rupture, downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells. Steady-state lysyl oxidase mRNA levels decreased in a concentration- and time-dependent manner to 30% of control levels after 24 hours of treatment with IFN-gamma. Cell layer lysyl oxidase activity decreased in parallel with the observed changes in steady-state mRNA. Nuclear runoff studies suggested that transcriptional regulation was responsible for at least 40% of the observed downregulation. mRNA decay studies suggested that IFN-gamma also decreased lysyl oxidase mRNA half-life from 9 to 6 hours. Downregulation of lysyl oxidase by IFN-gamma did not appear to require new protein synthesis. This study documents that IFN-gamma downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms. If similar regulation occurs in vivo, it is possible that IFN-gamma-mediated changes in lysyl oxidase may contribute to arterial diseases characterized by abnormal extracellular matrix.

Animals↗
PubMed2000

Myocardial ischemia/reperfusion injury in NADPH oxidase-deficient mice.

Previous studies have suggested that oxygen-derived free radicals are involved in the pathophysiology of myocardial ischemia/reperfusion (MI/R) injury. Specifically, neutrophils have been shown to mediate postischemic ventricular arrhythmias and myocardial necrosis. We hypothesized that MI/R injury would be reduced in the absence (-/-) of NADPH oxidase. Heterozygous control mice (n=23) and NADPH oxidase(-/-) mice (n=24) were subjected to 30 minutes of coronary artery occlusion and 24 hours of reperfusion. Myocardial area at risk per left ventricle was similar in heterozygous control hearts (55+/-3%) and NADPH oxidase(-/-) hearts (61+/-4%). Contrary to our hypothesis, the size of infarct area at risk was similar in the heterozygous control mice (42+/-4%) and NADPH oxidase(-/-) mice (34+/-5%) (P=not significant). In addition, echocardiographic examination of both groups revealed that left ventricle fractional shortening was similar in NADPH oxidase(-/-) mice (n=8; 27+/-2.5%) and heterozygous control mice (n=10; 23.3+/-3. 3%) after MI/R. Superoxide production, as detected by cytochrome c reduction, was significantly impaired (P<0.01) in NADPH oxidase(-/-) mice (n=6) compared with heterozygous mice (n=7) (0.04+/-0.03 versus 2.2+/-0.08 nmol O(2).min(-1).10(6) cells(-1)). Intravital microscopy of the inflamed mesenteric microcirculation demonstrated that leukocyte rolling and adhesion were unaffected by the absence of NADPH oxidase. Oyster glycogen-stimulated neutrophil transmigration into the peritoneum was also similar in both the heterozygous control mice and NADPH oxidase(-/-) mice (P:=not significant). These findings suggest that NADPH oxidase does not contribute to the development of myocardial injury and dysfunction after MI/R.

Animals↗
PubMed1988

Coregulation of NADPH oxidase activation and phosphorylation of a 48-kD protein(s) by a cytosolic factor defective in autosomal recessive chronic granulomatous disease.

The mechanisms regulating activation of the respiratory burst enzyme, NADPH oxidase, of human neutrophils (PMN) are not yet understood, but protein phosphorylation may play a role. We have utilized a defect in a cytosolic factor required for NADPH oxidase activation observed in two patients with the autosomal recessive form of chronic granulomatous disease (CGD) to examine the role of protein phosphorylation in activation of NADPH oxidase in a cell-free system. NADPH oxidase could be activated by SDS in reconstitution mixtures of cytosolic and membrane subcellular fractions from normal PMN, and SDS also enhanced phosphorylation of at least 16 cytosolic and 14 membrane-associated proteins. However, subcellular fractions from CGD PMN plus SDS expressed little NADPH oxidase activity, and phosphorylation of a 48-kD protein(s) was selectively defective. The membrane fraction from CGD cells could be activated for NADPH oxidase when mixed with normal cytosol and phosphorylation of the 48-kD protein(s) was restored. In contrast, the membrane fraction from normal cells expressed almost no NADPH oxidase activity when mixed with CGD cytosol, and phosphorylation of the 48-kD protein(s) was again markedly decreased. Protein kinase C (PKC) activity in PMN from the two patients appeared to be normal, suggesting that a deficiency of PKC is not the cause of the defective 48-kD protein phosphorylation and that the cytosolic factor is not PKC. These results demonstrate that the cytosolic factor required for activation of NADPH oxidase also regulates phosphorylation of a specific protein, or family of proteins, at 48 kD. Although the nature of this protein(s) is still unknown, it may be related to the functional and phosphorylation defects present in CGD PMN and to the activation of NADPH oxidase in the cell-free system.

Autoradiography↗
PubMed1992

Host defense activity in various hosts. Human neutrophil NADPH oxidase activity.

The superoxide generation of neutrophil NADPH oxidase from healthy subjects, patients with respiratory infections, and patients receiving effective therapy with antibiotics or steroids was investigated. In young healthy nonsmokers the mean oxidase activity of neutrophils in women was significantly lower than that in men. In healthy women the mean oxidase activity was significantly lower in young nonsmokers than in young smokers or the elderly. In young nonsmokers, oxidase activity significantly increased during respiratory infections; however, in elderly nonsmokers, no significant increase in oxidase activity was observed during respiratory infections. The mean oxidase activity in patients receiving steroids was very low. In in vitro experiments using cell-free activation systems of NADPH oxidase, steroids were found to injure the membrane-bound components of the oxidase enzyme. These results suggest that decreased superoxide generation in patients receiving steroids may result from steroid-induced damage in the membrane-bound components of the NADPH oxidase system. The inhibitory effect of steroids on superoxide production may reduce bactericidal action of neutrophils, ie, one defense mechanism of the body against many kinds of pathogens. Therefore, long-term therapy with steroids in the elderly should be avoided at all costs.

Adult↗
PubMed1996

Uric acid, xanthine oxidase and other risk factors of hypertension in normotensive subjects.

Uric acid produced by xanthine oxidase (also a source of superoxide radicals) has been known to increase in hypertensive patients. In this study we evaluated the possible involvement of uric acid and xanthine oxidase in the pathogenesis of hypertension by examining their association with mean arterial pressure (MAP) and factors related to blood pressure. These factors include age, quetelet index (weight/height2), cholesterol, creatinine, calcium (Ca), magnesium (Mg), sodium (Na), potassium (K) and urea. Fifty Two (male-19, female-33) normal healthy individuals were studied. Correlation studies of demographic variables showed that age was positively correlated with MAP [r = 0.309, p = 0.026] and cholesterol [r = 0.503, p = 0.000] while quetelet index was positively correlated with age [r = 422, p = 0.000] MAP [r = 0.331, p = 0.016] and xanthine oxidase [r = 0.331, p = 0.016]. MAP was positively correlated with uric acid [r = 0.511, p = 0.000], cholesterol [r = 0.492, p = 0.000] and xanthine oxidase enzyme activity [r = 0.388, p = 0.004] and negatively correlated with plasma calcium [r = 0.603, p = 0.000]. Correlation studies of measured parameters with uric acid and xanthine oxidase showed that uric acid was positively correlated with creatinine [r = 0.627, p = 0.000], plasma magnesium [r 0.442, p = 0.001] and negatively correlated with plasma calcium [r = 0.546, p = 0.000] while xanthine oxidase was negatively correlated with plasma calcium [r = -0.404, p = 0.003] and plasma sodium [r = -0.288, p = 0.038]. Stepwise multiple regression with MAP as dependent variable showed that 65% of total variability of blood pressure can be accounted for by plasma calcium, cholesterol, creatinine, plasma K, plasma Na, uric acid and xanthine oxidase in order of increasing R2 [xanthine oxidase: T-value = 3.26, R2 = 0.653]. It can be concluded that in normotensive subjects, uric acid and xanthine oxidase have significant association with blood pressure and thus are one of the many factors which are involved in the cause or effect of hypertension.

Adult↗
PubMed1980

Free and membrane-bound xanthine oxidase in bovine milk during cooling and heating.

The effect of cold storage (5 C, 24 h) and heat treatment (60 C, 5 min) of milk on activities of free and membrane-bound xanthine oxidase has been studied. Both treatments enhanced total xanthine oxidase activity in milk. Activity of membrane-bound xanthine oxidase increased and free xanthine oxidase decreased in buttermilk while it increased in skim milk on cold storage. Heat of milk increased free and membrane-bound xanthine oxidase activities in both buttermilk and skim milk. The state of xanthine oxidase activity in skim milk from reconstituted milk, which was prepared by mixing xanthine oxidase inactivated skim milk and fresh cream, showed that only the free enzyme migrated from the cream phase to skim milk on cold storage. Very little xanthine oxidase activity was detectable in skim milk on heat treatment of the reconstituted milk sample. The overall increased activity of xanthine oxidase in milk during cold storage or heat treatment may not be due to the release of fat globule membrane enzyme to skim milk.

Animals↗
PubMed1992

Studies on the relationship between xanthine oxidase and histamine release during intestinal ischemia-reperfusion.

Recent studies have demonstrated a connection between xanthine oxidase-generated reactive oxygen intermediates and histamine release during ischemia-reperfusion. In the present work, the effect of modulation of the endogenous histamine level on the xanthine oxidase activity was examined during the reperfusion of a canine ileal segment following a 2 hr of complete ischemia. The xanthine oxidase activity and the plasma histamine level peaked simultaneously at the beginning of reperfusion, reaching mean values of 14.9 nmol/ml/min and 12.1 nmol/l, respectively. Pretreatment with aminoguanidine, a blocker of diamine oxidase (histaminase), resulted in significantly higher levels of histamine during reperfusion, but this elevation was not accompanied by a further increase in xanthine oxidase activity. Pretreatment with the mast cell stabilizer cromolyn significantly diminished the rise in plasma histamine level, with an unchanging activity of xanthine oxidase. No significant alteration could be observed in the postocclusive activity of xanthine oxidase following the intra-arterial administration of 0.5, 1, or 5 nmol of histamine during the last 10 min of the ischemic period. These data suggest that the amount of histamine liberated during reperfusion does not result in a further increase in the xanthine oxidase activity. The release of histamine is not a cause, but rather an effect of the elevated activity of intestinal xanthine oxidase.

Animals↗
PubMed2006

[Recombinant Aspergillus niger glucose oxidase expressed in Trichoderma reesei].

It was expected that recombinant Aspergillus niger glucose oxidase could be expressed in Trichoderma reesei with stable activity. T. reesei CBHI promoter--CBHI ss. gene--A. niger glucose oxidase gene--T. reesei CBHI terminator--A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator--pUC19 (pCBHGOD) vector was constructed in E. coli DH5alpha by PCR application and gene cloning methods. T. reesei QM9414 protoplast was transformed by T. reesei CBHI promoter-CBHI ss. Gene--A. niger glucose oxidase gene--T. reesei CBHI terminator-A. nidulans gpd promoter--E. coli Hygromycin B phosphotransferase gene--A. nidulans trpC terminator linear DNA fragment (CBHGOD fragment) that was made by digestion of pCBHGOD with Kpn I. T. reesei mutant clone with homologous recombinant A. niger glucose oxidase gene was selected by PCR method. Recombinant glucose oxidase was produced by mutant T. reesei strain under induction of wheat straw for 5 days. Recombinant glucose oxidase molecular mass was showed the same as native A. niger glucose oxidase standard from Sigma company by Western blot analysis. Recombinant glucose oxidase activity was 25u/mL in medium. The yield was 0.5 g/L in comparison with Sigma company glucose oxidase standard. There was no recombinant GOD degradation during Trichoderma reesei cultivation that was showed in Western blot analysis. Trichoderma reesei has capability to be a new recombinant host for Aspergillus niger GOD production.

Aspergillus niger↗
PubMed1989

Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme.

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.

Ammonium Sulfate↗
PubMed1989

Use of an affinity label to probe the function of the NADPH binding component of the respiratory burst oxidase of human neutrophils.

The respiratory burst oxidase of neutrophils can be activated in a cell-free system in which solubilized membranes, cytosol, and Mg2+ are required and in which sodium dodecyl sulfate is used to convert the dormant oxidase to an active form. The 2',3'-dialdehyde analog of NADPH was used as an affinity label for the cytosolic NADPH binding component of the respiratory burst oxidase from human neutrophils. When treated with this affinity label in the presence of sodium cyanoborohydride to reduce Schiff bases, neutrophil cytosol was shown to lose at least 90% of its activity in the cell-free system. In contrast to normal cytosol, treated cytosol had lost its ability to abolish the lag time required for activation of the oxidase, suggesting that the treated cytosol was no longer able to participate in the rate-limiting activation step. Furthermore, the treated cytosol had lost its ability to convert the oxidase from a form with a high Km to a form with a low Km for NADPH. The ability of dialdehyde-treated cytosol to activate the oxidase could be restored by untreated cytosol with a concentration dependence suggesting that only one kinetically active component of the oxidase was inhibited by treatment with the NADPH analog. Like the dialdehyde-treated cytosol, cytosols from patients with chronic granulomatous disease caused by a deficiency in a cytosolic Mr = 47,000 protein (pp47) fail to participate in the rate-limiting activation step (Curnutte, J. T., Scott, P. J., and Babior, B. M. (1989) J. Clin. Invest. 83, 1236-1240). These chronic granulomatous disease cytosols were nevertheless able to restore limited activity to the dialdehyde-inactivated cytosol in a cell-free activation system. These results are consistent with a model in which (a) the NADPH binding subunit of the oxidase exists in a very slowly dissociating complex with one or more additional cytosolic components, including pp47, and (b) the NADPH binding component of the oxidase controls the affinity of the enzyme for NADPH, either directly or through the binding of additional cytosolic factors.

Affinity Labels↗
PubMed1985

Ontogenetic development of the monoamine oxidase activity and of the metabolism of biogenic monoamines in rat brain.

The article examines the ontogenetic development of the monoamine oxidase activity and of the metabolism of the dopamine, noradrenaline and serotonin in the brain of newborn, 10-day-, 20-day- and 2-month-old rats. Monoamine oxidase activity is determined using three substrates: tyramine, serotonin and beta-phenylethylamine. Monoamine oxidase A (substrate serotonin) and the total monoamine oxidase activity (substrate tyramine) are found to manifest identical development, their activity increasing quickly after birth, to reach constant values after the 10th day. The general course of the development during the first ten postnatal days shows that the post partum increase in the total monoamine oxidase activity in rat brain is predominantly due to monoamine oxidase A. Monoamine oxidase B (substrate beta-phenylethylamine) develops after the 10th postnatal day. Evidently monoamine oxidase A plays a decisive role for controlling the level of the biogenic monoamines in the young organism during the first days of the ontogenesis. Investigation of the changes in the content of dopamine, noradrenaline and serotonin in the age groups of the experimental animals chosen reveals a rapid increase in the dopamine and noradrenaline levels even during the first ten days of the ontogenesis. The increased total monoamine oxidase activity and the increased dopamine content correspond to its increased turnover rate during ontogenesis. The turnover rate of noradrenaline remains unchanged between the 10th and 20th postnatal days. The increasing serotonin level between the 20th and 60th post partum days corresponds to its increased turnover rate.

Aging↗