Gene/Protein
Disease
Symptom
Drug
Enzyme
Compound
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Gene/Protein
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Target Concepts:
Gene/Protein
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Enzyme
Compound
Query: EC:1.6.3.1 (
NADPH oxidase
)
11,281
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
The enzyme
4-hydroxyphenylacetate
,
NAD(P)H:oxygen oxidoreductase
(1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when
4-hydroxyphenylacetate
(4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of
4-HPA
. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity.
4-HPA
1-hydrocylase was inhibited by KCl, which was uncompetitive with
4-HPA
. Values of Ki determined for inhibitors competitive with
4-HPA
were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM
4-HPA
, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-
4-hydroxyphenylacetic acid
. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of
4-HPA
that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.
...
PMID:Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. 23 37
Exposure to airborne pollutants such as tobacco smoke is associated with increased activation of inflammatory-immune processes and is thought to contribute to the incidence of respiratory tract disease. We hypothezised that cigarette smoke (CS) could synergize with activated inflammatory/immune cells to cause oxidative injury or result in the formation of unique reactive oxidants. Isolated human neutrophils were exposed to gas-phase CS, and the production of nitrating and chlorinating oxidants following neutrophil stimulation was monitored using the substrate
4-hydroxyphenylacetate
(HPA). Stimulation of neutrophils in the presence of CS resulted in a reduced oxidation and chlorination of HPA, suggesting inhibition of
NADPH oxidase
or myeloperoxidase (MPO), the two major enzymes involved in inflammatory oxidant formation. Peroxidase assays demonstrated that neutrophil MPO activity was not significantly affected after CS-exposure, leaving the
NADPH oxidase
as a likely target. The inhibition of neutrophil oxidant formation was found to coincide with depletion of cellular GSH, and a similar modification of critical cysteine residues, such as those in
NADPH oxidase
components, might be involved in reduced respiratory burst activity. As alpha,beta-unsaturated aldehydes such as acrolein have been implicated in thiol modifications by CS, we exposed neutrophils to acrolein prior to stimulation, and observed inhibition of
NADPH oxidase
activation in relation to GSH depletion. Additionally, translocation of the cytosolic components of
NADPH oxidase
to the membrane, a necessary requirement for enzyme activation, was inhibited. Protein adducts of acrolein (or related aldehydes) could be detected in several neutrophil proteins, including
NADPH oxidase
components, following neutrophil exposure to either CS or acrolein. Alterations in neutrophil function by exposure to (environmental) tobacco smoke may affect inflammatory/infectious conditions and thereby contribute to tobacco-related disease.
...
PMID:Cigarette smoke impairs neutrophil respiratory burst activation by aldehyde-induced thiol modifications. 1124 41
Mammalian spermatozoa must undergo a preparation period known as capacitation to become capable of fertilizing oocytes. Controlled amounts of reactive oxygen species (ROS), such as superoxide anion (O2.-) and hydrogen peroxide (H2O2) have been shown essential for capacitation and acrosome reaction. The presence of an oxidase in the sperm plasma membrane has been suggested. The objective of the present study was to provide evidence for the production of O2.- by capacitating cryopreserved bovine spermatozoa. Percentages of capacitation and acrosome reaction were determined by the chlortetracycline assay. The effect of several nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors on capacitation was also studied. H2O2 production was determined by the fluorometric assay using the
p-hydroxyphenylacetic acid
-horseradish peroxidase system. Superoxide dismutase (SOD) activity was determined spectrophotometrically at 480 nm. Heparin-dependent capacitation was inhibited by all
NADPH oxidase
inhibitors tested (p < 0.05). Significant levels of H2O2 were produced during capacitation with heparin; such production was inhibited by diphenyleneiodonium, one of the
NADPH oxidase
inhibitors. The addition of catalase to the incubation medium failed to modify the capacitation rate; inhibition was only observed when SOD was present (p < 0.05). Endogenous SOD activity was diminished during heparin-dependent capacitation (p < 0.05). Similar levels of acrosome reaction induced by lysophosphatidylcholine were obtained in both heparin and O2.--dependent capacitation. Overall results suggest the participation of a sperm oxidase in bovine sperm capacitation. H2O2, generated by O2.- dismutation, failed to participate in capacitation, although this ROS may have been able to decrease endogenous SOD activity. Exogenous O2.- promotes physiological capacitation in cryopreserved bovine sperm, thus allowing the acquisition of fertilizing capacity.
...
PMID:Participation of superoxide anion in the capacitation of cryopreserved bovine sperm. 1264 29
The production of reactive oxygen species, including H
2
O
2
, is a process that can be used in signaling, cell death, or immune response. To quantify oxidative stress in cells, a fluorescence technique has been modified from a previously described method to measure H
2
O
2
release from cells (1-5). This assay takes advantage of H
2
O
2
-mediated oxidation of horseradish peroxidase (HRP) to Complex I, which, in turn, oxidizes
p-hydroxyphenylacetic acid
(pHPA) to a stable, fluorescent pHPA dimer (2,2'-dihydroxy-biphenyl-5,5' diacetate [(pHPA)
2
]). The H
2
O
2
-dependent HRP-mediated oxidation of pHPA is a sensitive and specific assay for quantifying H
2
O
2
release from cells. This assay can measure H
2
O
2
release from whole cells, mitochondria, or the
NADPH oxidase
.
...
PMID:Determination of H
2
O
2
Generation by pHPA Assay. 2828 Jul 52
