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At least 811 records · Page 45Linked to original sources
PubMed1993

Domains of rat heme oxygenase-2: the amino terminus and histidine 151 are required for heme oxidation.

Cleavage of heme b (Fe-protoporphyrin IX) at the alpha-meso carbon bridge is catalyzed by heme oxygenase isozymes, HO-1 and HO-2, to form biliverdin IX alpha. Currently, we have examined the requirement for the amino terminus and the hydrophobic carboxy terminus of rat HO-2 expressed in Escherichia coli for heme degradation activity and have assessed the importance of His 151 for this activity. His 151 is in the longest span of amino acids (24 residues) which are present, with only a single conservative substitution, in seven cloned heme oxygenases including the apparent single isozyme in chicken. We show His 151 is essential for cleavage of heme, as substitution of alanine for this residue by site-directed mutagenesis resulted in expression of an inactive protein with immunoreactivity toward antibody to rat HO-2. A cDNA construct in which nucleotides encoding the 33 N-terminal amino acid residues were deleted, when expressed, produced a protein of predicted size and immunoreactivity with antibody to HO-2 but also devoid of heme degrading activity. The presence of additional residues at this terminus, for the most part, accounts for the larger size of HO-2 compared to HO-1. Conversely, the hydrophobic region at the carboxy terminus did not appear to be essential for heme degradation. A construct in which the sequence encoding the primarily hydrophobic amino acids of the carboxy terminus was replaced by a sequence encoding predominantly hydrophilic residues expressed a protein which retained full capability to convert heme to biliverdin. Further, the construct with a hydrophilic carboxy terminus was not appreciably associated with bacterial membranes, suggesting that the carboxy terminus in the wild-type protein serves as a membrane anchor for this enzyme.

Amino Acid Sequence↗
PubMed1997

Microsomal alcohol oxygenase: purification and characterization of a cytochrome P450 responsible for oxidation of 7-hydroxy-delta 8-tetrahydrocannabinol to 7-oxo-delta 8-tetrahydrocannabinol in guinea pig liver.

Guinea pig hepatic enzyme, microsomal alcohol oxygenase, was able to oxidize both 7 alpha- and 7 beta-hydroxy-delta 8-tetrahydrocannabinol (7 alpha- and 7 beta-hydroxy-delta 8-THC) to 7-oxo-delta 8-THC. A cytochrome P450, named P450GPF-B, which mediates this oxidative metabolism was purified from hepatic microsomes of untreated female guinea pigs. The purified enzyme showed a single protein band of molecular mass 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal amino acid sequence of P450GPF-B is highly homologous with those of several cytochrome P450s belonging to the CYP3A subfamily. 18O derived from atmospheric oxygen was incorporated into 31 and 6%, respectively, of 7-oxo-delta 8-THC formed from 7 alpha- and 7 beta-hydroxy-delta 8-THC when the substrates were incubated with P450GPF-B under 18O2. The antibody against P450GPF-B significantly suppressed the oxidative activities of 7 alpha- and 7 beta-hydroxy-delta 8-THC to 7-oxo-delta 8-THC in hepatic microsomes of guinea pig. These results indicate that P450GPF-B is a major enzyme responsible for the hepatic microsomal alcohol oxygenase activities in the guinea pig.

Amino Acid Sequence↗
PubMed1998

Semisynthetic flavocytochromes based on cytochrome P450 2B4: reductase and oxygenase activities.

A synthetic flavocytochrome with the reductase and oxygenase activities was obtained by covalent binding of riboflavin to cytochrome P450 2B4. The reactions catalyzed by the newly synthesized flavocytochromes were studied. Formation of carbon monoxide complex with the reduced form of hemoprotein led to 60-80% inhibition of oxygenase reactions, indicating the leading role of reduced heme iron in generating active oxygen species by flavocytochromes.

Animals↗
PubMed1995

Differential control of the metal-mediated activation of the human heme oxygenase-1 and metallothionein IIA genes.

Heme oxygenase-1, an essential enzyme in heme catabolism, and metallothionein IIA, a small metal-binding protein with clusters of cysteins, are remarkably induced in HeLa cells following the treatment with cadmium or zinc. Both proteins are considered to be involved in the defense system against metal toxicity. Here we showed by transient expression assays that the cadmium-responsive element (CdRE) of the human heme oxygenase-1 gene is not responsive to zinc, whereas a metal-regulatory element (MRE) of the human metallothionein IIA gene is able to respond to either cadmium or zinc. The CdRE is recognized by a certain nuclear protein(s) which is however unable to bind to an MRE of the metallothionein IIA gene. These results suggest that the metal-selective activation of each gene promoter is mediated by a separate mechanism.

Base Sequence↗
PubMed1996

The heme-responsive element of the mouse heme oxygenase-1 gene is an extended AP-1 binding site that resembles the recognition sequences for MAF and NF-E2 transcription factors.

Jun and Fos (AP-1) transcription factors were recently proposed to mediate induction of the mouse heme oxygenase-1 gene by different agents including heme and cadmium. In this report we show that the AP-1 binding sequence, TGAGTCA, is necessary but insufficient for gene activation in response to heme or cadmium. The minimal heme response element was identified as an extended AP-1 binding site, (T/C)GCTGAGTCA. In addition to the AP-1 heptad, this element also contains an interdigitated antioxidant response element, GCnnnGTCA. Specific antioxidant response elements from the NAD(P)H:quinone oxidoreductase-1 and the glutathione S-transferase Ya subunit genes were in fact responsive to heme but not all sequences that conform to the consensus antioxidant response element were activated by this agent. The heme response element resembles the consensus binding sites for the product of the maf oncogene and for the transcription factor NF-E2. The potential role of these and related transcription factors and the implication of the composite heme response element in heme oxygenase-1 gene regulation are discussed.

Animals↗
PubMed1999

Induction of haem oxygenase contributes to the synthesis of pro-inflammatory cytokines in re-oxygenated rat macrophages: role of cGMP.

Macrophage activation and the resulting inflammatory response may be a major component of tissue injury upon hypoxia and re-oxygenation. Activation of the haem oxygenase (HO)/carbon monoxide (CO) pathway may be an important regulator of the inflammatory response, through production of cyclic 3', 5'-monophosphate (cGMP). We have assessed whether HO contributes to the increased production of the pro-inflammatory cytokines TNF-alpha and IL-6 in re-oxygenated rat peritoneal macrophages.Hypoxia/re-oxygenation markedly increased levels of HO-1 mRNA and cGMP. The increase in cGMP was reduced by the HO-1 inhibitor tin-protoporphyrin (SnPP-9) given during re-oxygenation. Hypoxia and re-oxygenation also increased IL-6 and TNF-alpha mRNA expression, as well as IL-6 and TNF-alpha concentrations in the cell supernatant. These increases were nullified by SnPP-9 and by Methylene Blue, an inhibitor of guanylate cyclase, but were not affected by L-NNA, an inhibitor of NO synthesis. The inhibitory effect of SnPP on the synthesis of cytokines was reversed by co-administration of the stable analogue of cGMP, 8-Br-cGMP. Our results indicate that activation of haem oxygenase and of the CO/cGMP pathway is a major stimulus for the synthesis and release of pro-inflammatory cytokines in re-oxygenated macrophages. This pathway may play a central role in pathological situations in which local tissue hypoxia/re-oxygenation triggers a systemic inflammatory response, for example in patients with shock.

Animals↗
PubMed1997

The nitric oxide donor SIN-1 protects endothelial cells from tumor necrosis factor-alpha-mediated cytotoxicity: possible role for cyclic GMP and heme oxygenase.

In cultured endothelial cells, incubation with TNF-alpha (50 ng/ml) for 72 h markedly reduced viability of endothelial cells. A 6-h pre-incubation with the nitric oxide (NO) donor linsidomine (SIN-1, 10-150 microM) protected endothelial cells in a concentration-dependent manner and increased viability by up to 59% of control. The unmetabolized parent compound molsidomine and the NO-free metabolite of SIN-1 3-morpholinoiminoacetonitrile (SIN-1C) were without cytoprotective effect. Cytoprotection by SIN-1 was completely abolished by the NO scavenger 2-phenyl-4,4,5,5, -tetramethylimidazoline-1-oxyl-3-oxide (PTIO, 30 microM). A cytoprotective effect comparable to SIN-1 was observed when preincubating the cells with dibutyryl cyclic GMP (10-100 microM). Moreover, no protection by SIN-1 occurred in the presence of cycloheximide (1 microM) or 1H--1,2,4-oxadiazole-4, 3-a-quinoxalin-1-one (ODQ, 0.1 microM), a selective inhibitor of soluble guanylyl cyclase. Tin protoporphyrin-IX (SnPP, 25 microM), an inhibitor of heme oxygenase, was found to attenuate SIN-1-induced cytoprotection. Our results demonstrate that SIN-1 produces a long-term endothelial protection against cellular injury by TNF-alpha, presumably via a cyclic GMP-dependent pathway leading to up-regulation of protective proteins such as heme oxygenase.

Acetonitriles↗
PubMed1975

The involvement of superoxide anions in the autoxidation of various cofactors of cysteamine-oxygenase.

The reduction of tetranitroblue tetrazolium with cysteamine, mediated by a number of dyes, elemental sulphur, elemental selenium and selenide, under aerobic conditions, was inhibited to various extent upon addition of superoxide dismutase. A strict parallelism between the ability to produce O2- ions and the property of those compounds to act as cofactors for cysteamine-oxygenase, to yield hypotaurine, has been observed. Based on the fact that the autoxidation of cysteamine also gives rise to O2- formation, though to a minor extent, we propose a mechanism for cysteamine-oxygenase action. This mechanism was derived from the data obtained in the model system studied.

Animals↗
PubMed2000

Elevated basal activity of tryptophan oxygenase in alcohol-preferring C57BL mice.

Tryptophan oxygenase activity in alcohol-preferring C57Bl mice and control CBA and DBA/2 mice was studied under nonstressful conditions and after glucocorticoid-induced stress. Elevated basal tryptophan oxygenase activity in C57Bl mice is probably responsible for reduced brain content of tryptophan and serotonin associated with alcohol preference.

Alcoholism↗
PubMed1987

Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate. II. Anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase.

The localization of anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was studied by determining the enzyme activities in subcellular fractions obtained by differential centrifugation of the mycelia of Streptomyces aureofaciens after lysozyme treatment. Glucose-6-phosphate dehydrogenase was a typical cytoplasmic enzyme both in the low- and high-production strain. Anhydrotetracycline oxygenase was found in the membrane fraction of the low-production strain. In the high-production strain, it was detected in several fractions, the highest activity being found in cytoplasm. The presence of 10 microM benzyl thiocyanate in the culture medium significantly changed the distribution of the latter enzyme in both strains. The redistribution of the enzymes is discussed with respect to tetracycline over-production.

Cell Fractionation↗
PubMedDate not supplied

Modulatory influence of tin-protoporphyrin on gossypol-induced alterations of heme oxygenase activity in male Wistar rats.

Gossypol--a male contraceptive is toxic and causes anorexia, reduction in body weight, hypokalemia etc. It prevents liberation of oxygen from oxyhemoglobin and has hemolytic effect on erythrocytes and leads to microcytic hypochromic anemia. SnPP has been shown to either competitively suppress or to significantly ameliorate a variety of naturally occuring or experimentally induced forms of jaundice in animals and man by inhibiting heme degradation. In this paper novel tissue-dependent response to differential dosing regimen of gossypol and gossypol in association with Sn-protoporphyrin (SnPP) is described. Gossypol was found to be a stimulator of heme oxygenase activity in the liver and kidney to varying degrees. This tissue response contrasted with that of the spleen, where gossypol decreased the activity of the enzyme. The increase in enzymatic activity was accompanied by a decline in the total microsomal protein content on gossypol administration. The gossypol mediated an increase of heme oxygenase activity, elevated bilirubin levels leading to hyperbilirubinemia. The stimulatory effect of gossypol was counteracted to a considerable extent when SnPP was simultaneously administered. Hence, we envision the importance of combined rather than single exposures in defining the realms of toxicology of these and other related drugs. We further envisage the existence of important gossypol-heme interactions in the regulation of heme metabolism.

Animals↗
PubMed2004

Characterization of a heme oxygenase of Clostridium tetani and its possible role in oxygen tolerance.

In order to colonize mammalian wounds, the anaerobic bacterium Clostridium tetani must presumably cope with temporary oxic conditions. Therefore, the recently decoded genome sequence was searched for genes which could confer oxygen tolerance. A few identified systems such as superoxide dismutases and peroxidases are probably responsible for this protection against toxic oxygen species. Another system was detected, a heme oxygenase which could have a role in establishing or maintaining an anoxic microenvironment in the process of wound colonization. The hemT gene encoding the heme oxygenase is expressed in C. tetani, as shown by reverse transcription-PCR. When overexpressed in Escherichia coli, the enzyme converts heme to biliverdin under strict oxic conditions.

Biliverdine↗
PubMed2005

Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta).

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a beta-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting beta-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4'-beta-ionone ring oxygenase.

Blotting, Southern↗
PubMed2002

Formation of benzoquinol moiety in cornoside by salidroside mono-oxygenase, a cytochrome P450 enzyme, from Abeliophyllum distichumcell suspension cultures.

A microsomal fraction prepared from Abeliophyllum distichumNakai (Oleaceae) cell suspension cultures oxidized salidroside, a glucoside of 4-hydroxyphenylethyl alcohol, to cornoside possessing a unique benzoquinol ring. The enzyme named salidroside mono-oxygenase required NADPH as the only cofactor, and molecular oxygen. The reaction was strongly inhibited by CO as well as several cytochrome P450 inhibitors, such as cytochrome c and miconazole, indicating the involvement of a cytochrome P450 enzyme. Salidroside mono-oxygenase accepted salidroside as the only substrate, but did not oxidize 4-hydroxyphenylethyl alcohol, the salidroside aglucone, and 4-hydroxybenzoic acid. The optimum pH of the reaction was 7.5, and apparent K(m) values for salidroside and NADPH were 44 micro M and 33 micro M, respectively. The benzoquinol ring formation mechanism is discussed in comparison to the mechanism for ipso substitution of 4-hydroxybenzoate by active oxygen species followed by elimination leading to hydroquinone.

Benzoquinones↗
PubMed2004

Targeted site-directed mutagenesis of a heme oxygenase locus by gene replacement in the moss Ceratodon purpureus.

The moss Physcomitrella patens is so far the only plant species in which it is possible for nuclear genes to be modified by homologous recombination at a reasonably efficiency. Here we describe the use of homologous recombination for another moss, Ceratodon purpureus. Our approach is based on the repair of the ptr116 mutant allele. In this mutant, codon 31 of the heme oxygenase gene CpHO1 is mutated to a stop codon. Heme oxygenase is necessary for the conversion of heme to biliverdin, the precursor of the phytochrome chromophore. Thus, in ptr116 the phytochrome-mediated responses of phototropism, chlorophyll accumulation and branching are lost. Protoplast transformation with DNA encoding the wild-type protein resulted in a rescue of 0.8% of regenerated protoplasts. In about half of the analyzed lines, formation of CpHO1 concatemers was observed at the CpHO1 locus, whereas in the other half, the mutant CpHO1 gene was replaced by a single DNA copy. This gene repair led to the exchange of single bases, and thus provides the first demonstration of efficient site-directed mutagenesis in a plant nuclear genome. Our studies also revealed an effective mechanism for gene inactivation in Ceratodon. When wild-type protoplasts were transformed with intact or modified CpHO1 genes, approximately 40% of regenerated protoplasts showed the ptr phenotype.

Amino Acid Sequence↗
PubMed2007

Heme oxygenase-1 expression in premature and mature neonates during the first week of life.

Newborns are exposed to mechanical and oxidative stress during labor and to relative hyperoxia thereafter during the course of adaptation to the extrauterine conditions. Part of the adaptation mechanism is the rapid degradation of fetal hemoglobin and the oxidation of its heme moiety by heme oxygenases (HOs). Heme oxygenase-1 enzyme (HO-1) is the inducible isoform, which is induced by and protective against oxidative stress. We hypothesized that HO-1 may play a role in the physiological adaptation of newborns. We therefore measured the HO-1 mRNA expression with cRT-PCR during the first week after birth in healthy mature and premature newborns. We found that HO-1 was induced until day 2 or 3 after birth, but its level had dropped below the birth HO-1 mRNA level by the end of the first week. HO-1 levels and inducibility were similar in mature newborns and premature newborns. The fact that HO-1 was inducible even in gestation week 26 suggests that HO-1 plays an important role in the early adaptation processes.

Gene Expression↗
PubMed2004

Heme oxygenase-2 products activate IKCa: role of CO and iron in guinea pig portal vein smooth muscle cells.

Hemin (10 microM) and carbon monoxide (CO) increased iberiotoxin-blockable IKCa in portal vein smooth muscle cells. CO-induced IKCa activation was abolished by 10 microM ODQ, 10 microM cyclopiazonic acid and 1 microM KT5823. The hemin-induced effect on IKCa was abolished by pretreatment with Sn-protoporphyrin IX, a heme oxygenase inhibitor and Fe2+ chelator but was insensitive to inhibitors of soluble guanylate cyclase (GC) and cGMP-dependent protein kinase (PKG). There was no effect of hemin on IKCa in the presence of 3 microM dithiotreitol into the bath or 3 mM glutathione into the pipette solution. Superoxide dismutase (1000 U/ml) or catalase (3000 U/ml) added into the pipette solution also abolished the effect of hemin on IKCa in this tissue. Additionally, 10 microM hemin could not influence IKCa in Ca2+-free external solution or in the presence of 30 microM SKF 95356. It was concluded that CO increases IKCa via its "conventional" signaling pathway, which involves soluble GC and PKG activation and subsequent stimulation of sarcoplasmic reticulum Ca2+ pump activity resulting in Ca2+-dependent activation of IKCa due to the accumulation of Ca2+ into the space near the plasma membrane. On the other hand, internally produced CO could not yield the same IKCa increase, while Fe2+ derived from heme oxygenase 2-dependent degradation of hemin in portal vein smooth muscle cells gives rise to reactive oxygen species namely hydroxyl and superoxide radicals. Both radicals are responsible for the SKF 95356-sensitive non-selective cation channel activation, the Ca2+ influx and the subsequent increase of Ca2+ concentration near the plasma membrane that augments the KCa channel activity.

Animals↗
PubMed2005

Differential regulation of chlorophyll a oxygenase genes in rice.

Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase. We have identified two genes (OsCAO1 and OsCAO2) from the rice genome that are highly homologous to previously studied chlorophyll a oxygenase (CAO) genes. They are positioned in tandem, probably resulting from recent gene duplications. The proteins they encode contain two conserved functional motifs - the Rieske Fe-sulfur coordinating center and a non-heme mononuclear Fe-binding site. OsCAO1 is induced by light and is preferentially expressed in photosynthetic tissues. Its mRNA level decreases when plants are grown in the dark. In contrast, OsCAO2 mRNA levels are higher under dark conditions, and its expression is down-regulated by exposure to light. To elucidate the physiological function of the CAO genes, we have isolated knockout mutant lines tagged by T-DNA or Tos17. Mutant plants containing a T-DNA insertion in the first intron of the OsCAO1 gene have pale green leaves, indicating chlorophyll b deficiency. We have also isolated a pale green mutant with a Tos17 insertion in that OsCAO1 gene. In contrast, OsCAO2 knockout mutant leaves do not differ significantly from the wild type. These results suggest that OsCAO1 plays a major role in chlorophyll b biosynthesis, and that OsCAO2 may function in the dark.

Chlorophyll↗