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At least 595 records · Page 33Linked to original sources
PubMed2004

In vivo bioluminescent monitoring of chemical toxicity using heme oxygenase-luciferase transgenic mice.

Transgenic mice expressing the luciferase (luc) gene under the control of the heme oxygenase-1 promoter (Ho1) were used to measure the induction of heme oxygenase in response to known toxicants. Transgenic Ho1-luc expression was visualized in vivo using a low-light imaging system (IVIS). Ho1-luc activation was compared to Ho1-luc expression, HO1 protein levels, standard markers of toxicity, and histology. Male and female Ho1-luc transgenic mice were exposed to acute doses of cadmium chloride (CdCl2, 3.7 mg/kg), doxorubicin (15 mg/kg), and thioacetamide (300 mg/kg). These agents induced the expression of Ho1-luc in the liver and other tissues to varying degrees. The greatest increase in Ho1-luc activity was observed in the liver in response to CdCl2; intermediate responses were observed for doxorubicin and thioacetamide. Induction of the Ho1-luc transgene by these agents was similar to endogenous protein levels of heme oxygenase as assessed by Western blotting, and generally correlated with plasma levels of circulating enzymes reflecting hepatic or general tissue damage. Histopathology confirmed the toxic effects of CdCl2 on liver and kidney; doxorubicin on kidney, liver, and intestine; and thioacetamide on the liver. Tissue damage was much more pronounced than the luciferase expression following thioacetamide treatment when compared with tissue damage and bioluminescence of the other toxicants. Nevertheless, the induction of Ho1-luc expression following exposure to these agents suggests that the Ho1-luc transgenic mouse may prove useful as a model for in vivo screening of compounds that induce luciferase expression as a marker of toxicity.

Alanine Transaminase↗
PubMed2002

Sugars have potential to curtail oxygenase activity of Rubisco.

Sugars play a critical role in regulating overall cellular metabolism and owing to their general compatibility with various cellular events plants invariably show enhanced levels of sugars for maintaining desired osmoticum under osmotic stress. Sugars (sucrose and trehalose) and sugar-alcohols (glycerol, mannitol, inositol, and sorbitol) with the exception of sorbitol lowered oxygenase activity of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) without altering carboxylase activity under unstressed conditions. Most interestingly, these solutes including sorbitol fully curtailed NaCl-induced enhancement in oxygenase activity, even at concentrations as low as 50mM. However, none of these solutes could alleviate NaCl-suppressed carboxylase activity. In summary, our findings demonstrate that one of the most important roles of sugars and sugar-alcohols in plants exposed to salt stress is to curtail oxygenase activity of Rubisco.

Carbohydrates↗
PubMed1999

Heme oxygenase-carbon monoxide signalling pathway in atherosclerosis: anti-atherogenic actions of bilirubin and carbon monoxide?

Atherosclerosis is a major contributor to cardiovascular disease, and genetic disorders of lipoprotein metabolism are recognized risk factors in atherogenesis. The gaseous monoxides nitric oxide (NO) and carbon monoxide (CO), generated within the blood vessel wall, have been identified as important cellular messengers involved in the regulation of vascular smooth muscle tone. Microsomal heme oxygenases degrade heme to biliverdin and CO, and the cytosolic enzyme biliverdin reductase then catalyzes reduction of biliverdin to bilirubin, both powerful chain-breaking antioxidants. Two principal isozymes of heme oxygenase have been identified, a constitutive isoform HO-2 (M(r) approximately 34,000) and an inducible isoform HO-1 (M(r) approximately 32,000), which is expressed at a low basal level in vascular endothelial and smooth muscle cells and is induced by heavy metals, oxidative stress, inflammatory mediators and oxidized low density lipoproteins. Although NO and CO modulate intracellular cGMP levels, platelet aggregation and smooth muscle relaxation, CO has a much lower affinity for soluble guanylyl cyclase than NO. Decreased production or sensitivity to NO in atherosclerosis may be compensated for by an induction of HO-1, with bilirubin acting as a cellular antioxidant and CO as a vasodilator. This review examines the evidence that oxidized low density lipoproteins (LDL), hypoxia and pro-inflammatory cytokines induce HO-1 expression and activity in vascular endothelial and smooth muscle cells, and evaluates the anti-atherogenic potential of the heme oxygenase signalling pathway.

Arteriosclerosis↗
PubMed2003

Carbon monoxide modulates the response of human basophils to FcepsilonRI stimulation through the heme oxygenase pathway.

We report the effects of exogenous and endogenous carbon monoxide (CO) on the immunological activation of human basophils. Hemin (1-100 microM), a heme oxygenase substrate analogue, significantly increased the formation of bilirubin from partially purified human basophils, thus indicating that these cells express heme oxygenase. This effect was reversed by preincubating the cells for 30 min with Zn-protoporphyrin IX (100 microM), a heme oxygenase inhibitor. Hemin (100 microM) also decreased immunoglobulin G anti-Fcepsilon (anti-IgE)-induced activation of basophils, measured by the expression of a membrane granule-associated protein, identified as cluster differentiation protein 63 (CD63), and by histamine release. These effects were reversed by Zn-protoporphyrin IX (100 microM), by oxyhemoglobin (HbO(2)), a CO scavenger (100 microM), and by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), an inhibitor of the soluble guanylyl cyclase (100 microM). Exposure of basophils to exogenous CO (10 microM for 30 min) also decreased their activation, while nitrogen (N(2)) was ineffective. HbO(2) and ODQ reversed the inhibition, reversing both membrane protein CD63 expression and histamine release to basal values. Both hemin and exogenous CO significantly raised cGMP levels in basophils and blunted the rise of calcium levels caused by immunological activation. This study suggests that CO increases cGMP formation, which in turn induces a fall in intracellular Ca(2+) concentration, thereby resulting in the inhibition of human basophil activation.

Antibodies, Anti-Idiotypic↗
PubMed2001

Homocysteine attenuates endothelial haem oxygenase-1 induction by nitric oxide (NO) and hypoxia.

The disrupted metabolism of homocysteine (Hcy) causes hyperhomocysteinemia, a condition associated with the impairment of nitric oxide (NO) bio-availability, tissue hypoxia and increased risk of vascular disease. Here, we examined how Hcy modulates the induction of the stress protein haem oxygenase-1 (HO-1) evoked by NO releasing agents and hypoxia in vascular endothelial cells. We found that Hcy (0.5 mM) markedly reduced the increase in haem oxygenase activity and HO-1 protein expression induced by sodium nitroprusside (SNP, 0.5 mM) but did not affect HO-1 activation mediated by S-nitroso-N-acetyl-penicillamine. Cells pre-treated with Hcy followed by addition of fresh medium containing SNP still exhibited an augmented haem oxygenase activity. Interestingly, high levels of Hcy were also able to abolish hypoxia-mediated HO-1 expression in a concentration-dependent manner. These novel findings indicate that hyperhomocysteinemia interferes with crucial signaling pathways required by cells to respond and adapt to stressful conditions.

Animals↗
PubMed1998

Modulation of the UVA activation of haem oxygenase, collagenase and cyclooxygenase gene expression by epigallocatechin in human skin cells.

We have investigated the modifying effects of epigallocatechin, a major polyphenolic constituent of green tea, on ultraviolet-A-activated gene expression in human fibroblasts and keratinocytes using the stress responsive enzymes: haem oxygenase-1, interstitial collagenase and cyclooxygenase-2. Although epigallocatechin strongly reduced ultraviolet-A-induced haem oxygenase-1 activation in skin-derived 'fibroblasts, the same compound activated collagenase and cyclooxygenase expression. In a keratinocyte cell line, ultraviolet-A-mediated haem oxygenase-1 over-expression was low and epigallocatechin failed to modulate it further. In contrast to the results with fibroblasts, ultraviolet-A activation of cyclooxygenase in keratinocytes was reduced by epigallocatechin. The results indicate that the effect of this green tea polyphenol on cellular stress responses is complex and may involve direct effects on signal transduction as well as changes that may be associated with its antioxidant activity.

Catechin↗
PubMed2000

Mechanism of heme degradation by heme oxygenase.

Heme oxygenase catalyzes the three step-wise oxidation of hemin to alpha-biliverdin, via alpha-meso-hydroxyhemin, verdoheme, and ferric iron-biliverdin complex. This enzyme is a simple protein which does not have any prosthetic groups. However, heme and its two metabolites, alpha-meso-hydroxyhemin and verdoheme, combine with the enzyme and activate oxygen during the heme oxygenase reaction. In the conversion of hemin to alpha-meso-hydroxyhemin, the active species of oxygen is Fe-OOH, which self-hydroxylates heme to form alpha-meso-hydroxyhemin. This step determines the alpha-specificity of the reaction. For the formation of verdoheme and liberation of CO from alpha-meso-hydroxyhemin, oxygen and one reducing equivalent are both required. However, the ferrous iron of the alpha-meso-hydroxyheme is not involved in the oxygen activation and unactivated oxygen is reacted on the 'activated' heme edge of the porphyrin ring. For the conversion of verdoheme to the ferric iron-biliverdin complex, both oxygen and reducing agents are necessary, although the precise mechanism has not been clear. The reduction of iron is required for the release of iron from the ferric iron-biliverdin complex to complete total heme oxygenase reaction.

Electron Spin Resonance Spectroscopy↗
PubMed1999

Structural and mechanistic studies on 2-oxoglutarate-dependent oxygenases and related enzymes.

Mononuclear nonheme-Fe(II)-dependent oxygenases comprise an extended family of oxidising enzymes, of which the 2-oxoglutarate-dependent oxygenases and related enzymes are the largest known subgroup. Recent crystallographic and mechanistic studies have helped to define the overall fold of the 2-oxoglutarate-dependent enzymes and have led to the identification of coordination chemistry closely related to that of other nonheme-Fe(II)-dependent oxygenases, suggesting related mechanisms for dioxygen activation that involve iron-mediated electron transfer.

Amino Acid Oxidoreductases↗
PubMed2003

Design, preparation, and characterization of mixed dimers of inducible nitric oxide synthase oxygenase domains.

A limitation of site-directed mutagenesis of homodimeric proteins is that both subunits will carry the same mutation. We have devised a way to prepare mixed dimers, in which only one chain bears a desired mutation, or each chain can bear a different mutation. Using the inducible nitric oxide oxygenase domain as a model, our strategy focused on the co-expression of two differentially tagged versions of the oxygenase domain, with isolation of the desired mixed dimer in two chromatography steps. We evaluated expression vectors encoding polyhistidine (His(6)), cellulose binding domain, glutathione-S-transferase, and polyglutamate (Glu(7))-tagged versions of the oxygenase domain for satisfactory levels of soluble protein expression and for their ability to form mixed dimers. The combination of His(6)- and Glu(7)-tagged subunits was successful in both respects, and the mixed dimers could be separated from either form of homodimer by sequential immobilized metal affinity chromatography and anion exchange chromatography. The UV-Vis spectrum, substrate binding properties, and enzymatic activity were not altered in the mixed dimer wild-type (His(6)/Glu(7)) compared to the two homodimers (His(6)/His(6) and Glu(7)/Glu(7)). We then characterized a mixed dimer variant in which one chain contained an E371A substitution (which prevents binding of the substrate L-arginine) while the other subunit was left unaltered. This species binds L-arginine and has about one-half the activity of the wild-type homodimer. Mutants known to destabilize the iNOS dimer (E411A, D454A, and W188F) were also investigated; in these cases co-expression with the wild-type subunit did not lead to the formation of stable mixed dimers.

Catalytic Domain↗
PubMed2001

Oxygenases: mechanisms and structural motifs for O(2) activation.

Recent structural and mechanistic analysis of oxygenase enzymes together with the study of biomimetic model reactions have provided new insights into the catalytic mechanisms of oxygenase-catalysed reactions. High-valent iron-oxo intermediates have been implicated in heme- and pterin-dependent mono-oxygenases. Structural motifs have been identified for binding of non-heme iron(II) (His,His,Glu) and iron(III) (His(2)Tyr(2)) in non-heme-dependent dioxygenases, but additional factors influencing the choice of reaction pathway are emerging from model studies.

Models, Chemical↗
PubMed2002

Protein engineering of oxygenases for biocatalysis.

Oxygenase enzymes have seen limited practical applications because of their complexity, poor stabilities, and often low catalytic rates. However, their ability to perform difficult chemistry with high selectivity and specificity has kept oxygenases at the forefront of engineering efforts. Growing understanding of structure-function relationships and improved protein engineering methods are paving the way for applications of oxygenases in chemical synthesis and bioremediation.

Animals↗
PubMed1997

Histochemical localization of heme oxygenase-2 protein and mRNA expression in rat brain.

Heme oxygenase (HO) proteins are members of the HSP30 family and consist of 2 isozymes identified to date, termed HO-1 and HO-2. Separate genes encode the isozymes and protein products which are immunochemically distinct, share less than 50% similarity at the amino acid sequence level. Each form, however, shows greater than 90% similarity among species, including human and the rat (reviewed in ref.). Furthermore, these isozymes function in a well-defined role to carry out oxidation of the heme molecule (Fe-protoporphyrin IX) in concert with NADPH-cytochrome P450 reductase. The oxidation of heme is isomer specific and results in the formation of bile pigments, carbon monoxide, and iron. The heme molecule constitutes the prosthetic moiety of hemoproteins, such as hemoglobin, myoglobin, catalase, soluble guanylate cyclase, cytochrome b5, cytochromes P450 and NO synthase. HO-1 also known as heat shock protein (HSP) 32 is encoded by a gene which is exquisitely stress-responsive and a host of stimuli that mediate oxidative stress cause induction of the protein both in vivo and in vitro. The HO-2 form shows a unique pattern of regulation from that of HO-1. HO-2 is a constitutive protein and its expression is not affected by the inducers of HO-1 tested to date; rather, the only known regulator of HO-2 yet identified is adrenal glucocorticoids. The two isozymes display vast differences in tissue distribution and under normal conditions HO-1 is present in the whole brain at the limit of immunodetection and is discreetly localized in select neuronal populations. HO-1 protein (approximately 32 kDa) and its approximately 1.8 kb transcript are increased, however, in response to stressful stimuli primarily in non-neuronal cell populations. The heme oxygenase system serves in both a catabolic and anabolic capacity in the cell. In the former capacity, it down-regulates cellular heme and hemoprotein levels. And, as such it inactivates the most effective catalyst for formation of free radicals, the heme molecule. In its anabolic role, as noted above, heme oxygenase produces bile pigments, carbon monoxide, and iron, all of which are biologically active: bile pigments function as antioxidants; the carbon monoxide generated by HO activity has been correlated with the generation of cGMP; and iron regulates expression of various genes, including that of HO-1 itself, as well as transferrin receptors, ferritin, and NO synthase. We used rabbit anti-rat HO-2 polyclonal antibody and HO-2 cDNA to localize HO-2 immunoreactive protein and the 1.3- and 1.9 kb homologous transcripts, respectively, in rodent brain as visualized by histochemical staining procedures. These protocols provide the first detailed description of methodologies successfully used to define the pattern of HO-2 expression at the transcriptional and translational levels in the adult rat brain and glucocorticoid-treated newborn rats. The procedures described herein have the virtue of being non-radioactive, as well as applicability to the systemic organs, such as the cardiovascular system and the male reproductive organs. Visualization of cellular HO-2 expression aids in assessment of potential sites of carbon monoxide, iron, and bilirubin production within the nervous system.

Animals↗
PubMed1995

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

Base Sequence↗
PubMed1994

Identification of histidine 25 as the heme ligand in human liver heme oxygenase.

Electronic and resonance Raman spectroscopic studies are reported for the His25Ala mutant of human liver heme oxygenase (HO) and its complex with heme. In the oxidized (ferric) form of the enzyme.substrate complex, the heme is shown to be in a high-spin, five-coordinate state. This is distinct from the same complex in the wild-type enzyme in which the heme is six-coordinate, ligated to a proximal histidine and a water molecule in an environment reminiscent of aquometmyoglobin. The reduced (ferrous) form of the complex of the H25A heme oxygenase mutant has lost the very prominent resonance Raman band at approximately 217 cm-1 seen in the wild-type complex that has been unambiguously assigned to the proximal Fe-N(His) vibrational frequency [Sun et al. (1993) Biochemistry 32, 14151; Takahashi et al. (1994) Biochemistry 33, 1010]. The absence of this band in the spectrum of the mutant protein definitively identifies His 25 as the proximal ligand of the heme substrate. Furthermore, this ferrous heme-H25A HO complex exists as an equilibrium mixture between a five-coordinate, high-spin species and a four-coordinate, intermediate-spin species. Although the H25A mutant protein shows no heme oxygenase activity, the heme is competent to bind carbon monoxide. Studies of the CO adduct of the H25A HO complex show v(CO) and v(Fe-CO) frequencies at 1960 and 529 cm-1, respectively, that are characteristic of a hydrophobic carbon monoxide binding site on a heme with a weak proximal ligand.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Monoxide↗
PubMed2002

Models of the low-spin iron(III) hydroperoxide intermediate of heme oxygenase: magnetic resonance evidence for thermodynamic stabilization of the d(xy) electronic state at ambient temperatures.

The (13)C pulsed ENDOR and NMR study of [meso-(13)C-TPPFe(OCH(3))(OO(t)Bu)](-) performed in this work shows that although the unpaired electron in low-spin ferrihemes containing a ROO(-) ligand resides in a d(pi) orbital at 8 K, the d(xy) electron configuration is favored at physiological temperatures. The variable temperature NMR spectra indicate a dynamic situation in which a heme with a d(pi) electron configuration and planar porphyrinate ring is in equilibrium with a d(xy) electron configuration that has a ruffled porphyrin ring. Because of the similarity in the EPR spectra of the hydroperoxide complexes of heme oxygenase, cytochrome P450, and the model heme complex reported herein, it is possible that these two electron configurations and ring conformations may also exist in equilibrium in the enzymatic systems. The ruffled porphyrinate ring would aid the attack of the terminal oxygen of the hydroperoxide intermediate of heme oxygenase (HO) on the meso-carbon, and the large spin density at the meso-carbons of a d(xy) electron configuration heme suggests the possibility of a radical mechanism for HO. The dynamic equilibrium between the ruffled (d(xy)) and planar (d(pi)) conformers observed in the model complexes also suggests that a flexible heme binding cavity may be an important structural motif for heme oxygenase activity.

Carbon Isotopes↗
PubMed2003

The hydroxide complex of Pseudomonas aeruginosa heme oxygenase as a model of the low-spin iron(III) hydroperoxide intermediate in heme catabolism: 13C NMR spectroscopic studies suggest the active participation of the heme in macrocycle hydroxylation.

13C NMR spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase (Fe(III)-OH), where OH(-) has been used as a model of the OOH(-) ligand to gain insights regarding the elusive ferric hydroperoxide (Fe(III)-OOH) intermediate in heme catabolism at ambient temperatures. Analysis of the heme core carbon resonances revealed that the coordination of hydroxide in the distal site of the enzyme results in the formation of at least three populations of Fe(III)-OH complexes with distinct electronic configurations and nonplanar ring distortions that are in slow exchange relative to the NMR time scale. The most abundant population exhibits a spin crossover between S = (1)/(2) and S = (3)/(2) spin states, and the two less abundant populations exhibit pure, S = (3)/(2) and S = (1)/(2), (d(xy)())(1) electronic configurations. We propose that the highly organized network of water molecules in the distal pocket of heme oxygenase, by virtue of donating a hydrogen bond to the coordinated hydroxide ligand, lowers its ligand field strength, thereby increasing the field strength of the porphyrin (equatorial) ligand, which results in nonplanar deformations of the macrocycle. This tendency to deform from planarity, which is imparted by the ligand field strength of the coordinated OH(-), is likely reinforced by the flexibility of the distal pocket in HO. These findings suggest that if the ligand field strength of the coordinated OOH(-) in heme oxygenase is modulated in a similar manner, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.

Biomimetic Materials↗
PubMed2003

Heme oxygenase activity and some indices of antioxidant protection in rat liver and kidney in glycerol model of rhabdomyolysis.

Activity of heme oxygenase, superoxide dismutase, and catalase, the content of reduced glutathione and total heme in the liver and kidneys, and serum absorption spectrum in the Soret band were studied in rats with glycerol-induced rhabdomyolysis. Glycerol increased the content of heme-containing metabolites in the serum and the total heme content in the liver and kidneys, and decreased the content of reduced glutathione and catalase activity in the examined organs. Superoxide dismutase activity increased in the liver and decreased in the kidneys. Heme oxygenase activity increased in the liver and kidneys 2 and 6 h postinjection, respectively. The effects of heme delivered to the liver and kidneys from the vascular bed on the antioxidant defense and heme oxygenase activity were studied.

Animals↗
PubMed1991

The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells.

Chronic hypoxia increases the expression of a set of stress proteins (oxygen regulated proteins or ORPs) which is implicated in the development of drug resistance and radiation sensitivity in tumour cells. Five major ORPs have been documented, and two, ORP 80 and ORP 100, are considered to be identical to the glucose regulated stress proteins GRP78 and GRP94, respectively. We report here that ORP 33 is a form of the heme catabolic enzyme, heme oxygenase, using evidence obtained from northern blotting, two-dimensional polyacrylamide gel electrophoresis and western analysis. Heme oxygenase is believed to be an important component of the cellular response to oxidative stress. The significance of heme oxygenase as a hypoxia-induced stress protein is discussed.

Aerobiosis↗