Evaluation of relative contributions of two enzymes supposed to metabolise hydrogen peroxide in Paracoccus denitrificans.
(9/4896)
A biosensor exploiting an electrochemically mediated enzyme-catalysed reaction was used to quantify relative contributions of cytoplasmic catalase and periplasmic cytochrome c peroxidase to the overall rate of hydrogen peroxide breakdown in cells of Paracoccus denitrificans. The effects of antimycin (an inhibitor of electron flow to cytochrome c peroxidase), the reaction rate versus substrate concentration profiles for the whole cells and subcellular fractions, and the time courses of oxygen concentration demonstrated a profound decrease in the capacity of cytochrome c peroxidase to reduce H2O2 under in vivo conditions. The reason is suggested to be a competition for available electrons between the enzyme and terminal oxidases metabolising oxygen produced by catalase. (+info)
Role of antioxidant defenses against ethanol-induced damage in cultured rat gastric epithelial cells.
(10/4896)
Reactive oxygen species appears to be involved in the pathogenesis of ethanol-induced gastric mucosal injury in vivo. Because ingested ethanol diffuses into the gastric mucosa, targeting both epithelium and endothelium, in the present study we examined the possible protective effect of antioxidants on ethanol damage in gastric epithelial cells and endothelial cells in vitro. Cytotoxicity by ethanol was quantified by measuring 51Cr release. The effects of impairment of the glutathione redox cycle and of inhibition of cellular catalase were examined. The generation of superoxide was assessed by the reduction in cytochrome c. Ethanol caused a time- and dose-dependent increase in 51Cr release from epithelial cells. Incubation of cells with DL-buthionine-(S,R)-sulfoximine, while reducing glutathione production, dose dependently enhanced ethanol-induced injury. 1,3-Bis(chloroethyl)-nitrosourea, while inhibiting glutathione reductase activity, also sensitized cells to ethanol. In contrast, the inhibition of catalase with 3-amino-1,2, 4-triazole did not alter the susceptibility of epithelial cells to ethanol. Ethanol induced damage to endothelial cells in a similar fashion. In endothelial cells, however, neither impairment of the glutathione cycle nor inhibition of catalase influenced ethanol-induced damage. Epithelial cells, when exposed to ethanol, increased superoxide production as a function of ethanol concentration, whereas endothelial cells did not. The glutathione redox cycle, but not cellular catalase, plays a critical role in protecting epithelial cells against ethanol damage, whereas neither antioxidant seems to play a role in protection of endothelial cells. The distinct difference in antioxidant protection against ethanol appears to depend on the capability of each cell to produce cytotoxic oxygen species in response to ethanol exposure. (+info)
Tretinoin prevents age-related renal changes and stimulates antioxidant defenses in cultured renal mesangial cells.
(11/4896)
Age-related progressive glomerular sclerosis in the rat is associated with increased expression of tumor necrosis factor-beta1 and increased protein content in the renal cortex, enhanced production of H2O2, in both renal glomeruli and mesangial cells (MCs) cultured from them, as well as augmented glomerular oxidative damage. We have previously shown that tretinoin-treated old male Fischer 344 rats have 30% lower protein content in the renal cortex than control old rats. Here, we report that this effect may depend on the inhibition of the expression of tumor necrosis factor-beta1, a matrigenic cytokine, and osteopontin, a protein with cell adhesive and chemotactic properties. In addition, we show that tretinoin prevents the cytotoxicity of H2O2 in cultured human MCs by increasing both the catalase activity and the reduced glutathione content, which are dose- and time-dependent changes. These increases were not dependent on each other: when these effects were previously inhibited with 3-amino-1,2,4-atriazole or L-buthionine-(S, R)-sulfoximine, respectively, tretinoin still induced the increase of the other noninhibited antioxidant defense. An enhanced gene transcription is the most likely mechanism involved in the tretinoin-induced stimulation of MC antioxidant defense systems because 1) preincubation of MCs with actinomycin D or cycloheximide fully abolished it; 2) tretinoin-incubated MCs showed increased levels of catalase mRNA and gamma-glutamyl-cysteine synthetase (catalytic subunit) mRNA, the latter being the rate-limiting step in de novo reduced glutathione synthesis; and 3) the stability of both mRNA was unchanged by tretinoin. These results show one strategy of protecting renal cells from H2O2-mediated injury based on increasing their antioxidant defenses. (+info)
Role of the lateral channel in catalase HPII of Escherichia coli.
(12/4896)
The heme-containing catalase HPII of Escherichia coli consists of a homotetramer in which each subunit contains a core region with the highly conserved catalase tertiary structure, to which are appended N- and C-terminal extensions making it the largest known catalase. HPII does not bind NADPH, a cofactor often found in catalases. In HPII, residues 585-590 of the C-terminal extension protrude into the pocket corresponding to the NADPH binding site in the bovine liver catalase. Despite this difference, residues that define the NADPH pocket in the bovine enzyme appear to be well preserved in HPII. Only two residues that interact ionically with NADPH in the bovine enzyme (Asp212 and His304) differ in HPII (Glu270 and Glu362), but their mutation to the bovine sequence did not promote nucleotide binding. The active-site heme groups are deeply buried inside the molecular structure requiring the movement of substrate and products through long channels. One potential channel is about 30 A in length, approaches the heme active site laterally, and is structurally related to the branched channel associated with the NADPH binding pocket in catalases that bind the dinucleotide. In HPII, the upper branch of this channel is interrupted by the presence of Arg260 ionically bound to Glu270. When Arg260 is replaced by alanine, there is a threefold increase in the catalytic activity of the enzyme. Inhibitors of HPII, including azide, cyanide, various sulfhydryl reagents, and alkylhydroxylamine derivatives, are effective at lower concentration on the Ala260 mutant enzyme compared to the wild-type enzyme. The crystal structure of the Ala260 mutant variant of HPII, determined at 2.3 A resolution, revealed a number of local structural changes resulting in the opening of a second branch in the lateral channel, which appears to be used by inhibitors for access to the active site, either as an inlet channel for substrate or an exhaust channel for reaction products. (+info)
Transient and steady-state kinetics of the oxidation of substituted benzoic acid hydrazides by myeloperoxidase.
(13/4896)
Myeloperoxidase is the most abundant protein in neutrophils and catalyzes the production of hypochlorous acid. This potent oxidant plays a central role in microbial killing and inflammatory tissue damage. 4-Aminobenzoic acid hydrazide (ABAH) is a mechanism-based inhibitor of myeloperoxidase that is oxidized to radical intermediates that cause enzyme inactivation. We have investigated the mechanism by which benzoic acid hydrazides (BAH) are oxidized by myeloperoxidase, and we have determined the features that enable them to inactivate the enzyme. BAHs readily reduced compound I of myeloperoxidase. The rate constants for these reactions ranged from 1 to 3 x 10(6) M-1 s-1 (15 degrees C, pH 7.0) and were relatively insensitive to the substituents on the aromatic ring. Rate constants for reduction of compound II varied between 6.5 x 10(5) M-1 s-1 for ABAH and 1.3 x 10(3) M-1 s-1 for 4-nitrobenzoic acid hydrazide (15 degrees C, pH 7.0). Reduction of both compound I and compound II by BAHs adhered to the Hammett rule, and there were significant correlations with Brown-Okamoto substituent constants. This indicates that the rates of these reactions were simply determined by the ease of oxidation of the substrates and that the incipient free radical carried a positive charge. ABAH was oxidized by myeloperoxidase without added hydrogen peroxide because it underwent auto-oxidation. Although BAHs generally reacted rapidly with compound II, they should be poor peroxidase substrates because the free radicals formed during peroxidation converted myeloperoxidase to compound III. We found that the reduction of ferric myeloperoxidase by BAH radicals was strongly influenced by Hansch's hydrophobicity constants. BAHs containing more hydrophilic substituents were more effective at converting the enzyme to compound III. This implies that BAH radicals must hydrogen bond to residues in the distal heme pocket before they can reduce the ferric enzyme. Inactivation of myeloperoxidase by BAHs was related to how readily they were oxidized, but there was no correlation with their rate constants for reduction of compounds I or II. We propose that BAHs destroy the heme prosthetic groups of the enzyme by reducing a ferrous myeloperoxidase-hydrogen peroxide complex. (+info)
Antioxidant mechanisms in apolipoprotein E deficient mice prior to and following closed head injury.
(14/4896)
Apolipoprotein E deficient mice have distinct memory deficits and neurochemical derangements and their recovery from closed head injury is impaired. In the present study, we examined the possibility that the neuronal derangements of apolipoprotein E deficient mice are associated with oxidative stress, which in turn affects their ability to recover from close head injury. It was found that brain phospholipid levels in apolipoprotein E deficient mice are lower than those of the controls (55+/-15% of control, P<0. 01), that the cholesterol levels of the two mice groups are similar and that the levels of conjugated dienes of the apolipoprotein E deficient mice are higher than those of control mice (132+/-15% of P<0.01). Brains of apolipoprotein E deficient mice had higher Mn-superoxide dismutase (134+/-7%), catalase (122+/-8%) and glutathione reductase (167+/-7%) activities than control (P<0.01), whereas glutathione peroxidase activity and the levels of reduced glutathione and ascorbic acid were similar in the two mouse groups. Closed head injury increased catalase and glutathione peroxidase activities in both mouse groups, whereas glutathione reductase increased only in control mice. The superoxide dismutase activity was unaffected in both groups. These findings suggest that the antioxidative metabolism of apolipoprotein E deficient mice is altered both prior to and following head injury and that antioxidative mechanisms may play a role in mediating the neuronal maintenance and repair derangements of the apolipoprotein E deficient mice. (+info)
Isolation and characterization of the catalase gene from Rhizobium sp. SNU003, a root nodule symbiont of Canavalia lineata.
(15/4896)
A catalase gene from Rhizobium sp. SNU003, a root nodule symbiont of Canavalia lineata, was cloned and its nucleotide sequence was determined. The Rhizobium DNA of about 280 bp was amplified using two PCR primers synthesized from the conserved sequences of the type I catalase gene. The nucleotide sequence of the amplified fragment revealed three regions that were conserved in the catalase, showing it as being part of the catalase gene. A genomic Southern hybridization using this fragment as a probe showed that the 5.5 kb PstI, 1.8 kb EcoRI, and 0.7 kb StyI fragments hybridized strongly with the probe. The Rhizobium genomic library constructed into the EMBL3 vector was screened, and one catalase clone was selected. The nucleotide sequence of the 5.5 kb PstI fragment from the clone revealed an open reading frame of 1455 bp, encoding a polypeptide of 485 amino acids with a molecular mass of 54,958 Da and a pI of 6.54. The predicted amino acid sequence of the catalase is 66.3% identical to that of Bacteroides fragilis, but was only 53.3% identical to the Rhizobium meliloti catalase. (+info)
Identification of aerobically and anaerobically induced genes in Enterococcus faecalis by random arbitrarily primed PCR.
(16/4896)
Enterococci have emerged among the leading causes of nosocomial infection. With the goal of analyzing enterococcal genes differentially expressed in environments related to commensal or environmental colonization and infection sites, we adapted and optimized a method more commonly used in the study of eukaryotic gene expression, random arbitrarily primed PCR (RAP-PCR). The RAP-PCR method was systematically optimized, allowing the technique to be used in a highly reproducible manner with gram-positive bacterial RNA. In the present study, aerobiosis was chosen as a variable for the induction of changes in gene expression by Enterococcus faecalis. Aerobically and anaerobically induced genes were detected and identified to the sequence level, and differential gene expression was confirmed by quantitative, specifically primed RT-PCR. Differentially expressed genes included several sharing identity with those of other organisms related to oxygen metabolism, as well as hypothetical genes lacking identity to known genes. (+info)