Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Cadmium-mediated inhibition of testicular heme oxygenase activity: the role of NADPH-cytochrome c (P-450) reductase

The concerted activity of two microsomal enzymes, heme oxygenase and NADPH-cytochrome c (P-450) reductase, is required for isomer-specific oxidation of heme molecule; heme oxygenase is commonly believed to be rate limiting in this activity. In this report, we provide evidence strongly suggesting the rate-limiting role of the reductase in oxidation of heme molecule in rat testis. In the testis and the liver of rats treated with Cd (20 mumol/kg, sc, 24 h) heme oxygenase activity, assessed by the formation of bilirubin, was decreased by 50% and increased by 7-fold, respectively. In these animals, the reductase activity was decreased by nearly 75% in the testis, but remained unchanged in the liver. Similarly, the reductase activity in the liver was not altered when heme oxygenase activity was increased by 20-fold in response to bromobenzene treatment. Addition of purified testicular reductase preparation (purified over 4000-fold), or hepatic reductase, to the testicular microsomes of Cd-treated rats obliterated the Cd-mediated inhibition of heme oxygenase activity. The chromatographic separation of heme oxygenase and the reductase of the testicular microsomal fractions revealed that the reductase activity was markedly decreased (75%) while the heme oxygenase activity, when assessed in the presence of exogenous reductase, was not affected by in vivo Cd treatment. In vitro, the membrane-bound reductase preparation obtained from the testis was more sensitive to the inhibitory effect of Cd than the liver preparation. However, the purified reductase preparations from the testis and the liver exhibited a similar degree of sensitivity to Cd. Based on the molar ratio of heme oxygenase to the reductase in the microsomal membranes of the liver and the testis it appeared that the testicular heme oxygenase, which is predominantly HO-2 isoform, interacts with the reductase less effectively than HO-1; in the induced liver, heme oxygenase is predominantly the HO-1 isoform. It is suggested that due to the low abundance of NADPH-cytochrome c (P-450) reductase and the apparently lower affinity of the enzyme for HO-2, the reductase exerts a regulatory action on heme oxygenase activity in the testis.     

Detection of two heme oxygenase isoforms in the human testis

This study shows heme oxygenase multiplicity is common to rat and human tissues. The isozymes in man and rat, however, are heterogenous proteins that share certain characteristics. Two forms of heme oxygenase, HO-1 and HO-2, were identified in human testis. HO-2 form was the prevalent form. Human and rat HO-1 differed in chromatographic behavior and molecular weight; human HO-1 was a larger molecule (35,400 vs 30,000). The two forms, however, were similar in that immunochemically human HO-1 exhibited reactivity toward antibody to rat HO-1. Human and rat HO-2 also were dissimilar in chromatographic behavior and showed only a weak immunological cross-reactivity. Human and rat HO-1 were essentially the same size. As in rat organs, the microsomal cytochrome P-450 content in human testis was reciprocal to heme oxygenase activity.     

Resolution of the rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1)

In the present study we report on the detection of a distinct pattern of heme oxygenase isoform composition in the rat brain. In this organ only the noninducible form of heme oxygenase, HO-2, could be clearly detected. This pattern of composition distinguishes the brain from other organs tested to date, namely the liver, testis, and spleen. The rat brain microsomal fraction displayed a rather impressive rate of heme oxygenase activity. This fraction also exhibited a rate of NADPH-cytochrome P-450 reductase activity that was sufficient to fully support the oxygenase activity. The brain microsomal fraction was solubilized and subjected to ion-exchange chromatography on DEAE-Sephacel. The chromatographic elution pattern of heme oxygenase activity was compared with those of the liver and testis. In the brain only one peak of heme oxygenase activity was detected. The peak exhibited an elution profile similar to that of HO-2 of the liver and the testis. The presence of an activity peak was not detected in the elution profile at the region where the inducible isoform of heme oxygenase, HO-1, was expected. Cross-reactivity was observed between the solubilized brain microsomal fraction and antiserum to the testis HO-2 when subjected to Ouchterlony double diffusion immunoanalysis. A reaction was not observed when antiserum to liver HO-1 was employed. The presence of HO-2 in the brain microsomal preparation was also established by Western immunoblotting analysis. A protein having a mobility that was identical to the purified testicular HO-2 (Mr 36,000) was present in the brain microsomal preparation when probed with antiserum to HO-2. However, our attempts to demonstrate the presence of HO-1 in the brain microsomal preparation by a similar technique, but using antiserum to HO-1, were not successful. It is proposed that HO-2 is responsible for the bulk, if not all, of the brain microsomal heme oxygenase activity. It is further proposed that tissue-specific regulatory mechanisms are responsible for both the refractory response of the brain heme oxygenase to known metallic inducers and the absence of a detectable amount of the HO-1 isoform.     

Evidence suggesting that the two forms of heme oxygenase are products of different genes

Recently, we have reported on the presence of two forms of heme oxygenase in rat liver and testis microsomes, referred to as HO-1 and HO-2 (M. D. Maines, G. M. Trakshel, and R. K. Kutty (1986) J. Biol. Chem. 261, 411-419; G. M. Trakshel, R. K. Kutty, and M. D. Maines (1986) J. Biol. Chem. 261, 11131-11137). Although the two forms differed in several biochemical properties, we could not ascertain whether they represented two isozymes or whether they were isoforms of heme oxygenase. In the present study, we provide evidence suggesting that the two forms are isozymes and represent different gene products. We also provide data suggesting that HO-1 is the commonly known heme oxygenase form. The molecular weight and immunochemical properties of HO-1 and HO-2 did not vary depending on the tissue source examined, i.e. liver and testis. Major differences, however, were noted in the amino acid composition of the two forms including the presence of 3 cysteine/cystine residues in HO-2 only. Using antibody to HO-2, four testis clones and two liver clones were isolated, and one liver and one testis clone were sequenced. Both clones revealed a 274-base-pair insert, and the sequence of both inserts was the same. The validity of assignment was confirmed by matching a 14-amino-acid peptide obtained from purified HO-2 with the sequence. Approximately 43% amino acid homology was detected between the HO-2 insert and the published amino acid sequence of heme oxygenase. However, amino acid homology search revealed the presence of two regions of homology: one 22-mer sequence with only one unmatched amino acid, and one 10-mer sequence with one unmatched amino acid. Heme oxygenase appeared to be the HO-1 form, an assignment based on its amino acid sequence matching the sequence of 2 peptides obtained from purified HO-1 and the immunochemical properties of the cobalt-, hematin-, and bromobenzene-induced rat liver enzyme. The secondary structure prediction analysis revealed an area of 100% structural homology with only 72% sequence homology. We predict this region may represent the catalytic site of the enzyme.     

Retinoic acid can enhance the stimulation by thyroid hormone of heme oxygenase activity in the liver of thyroidectomized rats.

The effects of retinoic acid (RA) (50 micrograms/100 g body wt. per day) on hepatic heme oxygenase activity, delta-aminolevulinate synthase (ALAS) activity and on cytochrome P-450 content were determined in thyroidectomized rats treated with T3 (10 micrograms/100 g body wt. per day) or diluent. RA, when administered for 3 days, failed to influence significantly the activity of either heme oxygenase or ALAS, however, the retinoid depleted hepatic cytochrome P-450 content by 17% (P less than 0.01) and microsomal heme content by 47% (P less than 0.001). T3 administration enhanced heme oxygenase activity by 72% (P less than 0.001) and ALAS activity by 251% (P less than 0.001) above levels in diluent treated controls and depleted cytochrome P-450 levels by 55% (P less than 0.001) and heme levels by 75% (P less than 0.001). When RA and T3 were administered together, the retinoid markedly enhanced the T3 stimulation of heme oxygenase activity; 173% above controls (P less than 0.001), and 61% above T3 alone (P less than 0.001). However, RA failed to influence the effect of T3 on ALAS activity or cytochrome P-450 depletion. The results indicate that RA can influence the levels of hepatic cytochrome P-450 and can modulate the stimulation of heme oxygenase activity by thyroid hormone in vivo.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

Increased rat testicular heme oxygenase activity associated with depressed microsomal heme and cytochrome P-450 levels after repeated administration of human chorionic gonadotropin

Repeated administration of human chorionic gonadotropin to rats results in a maximal depression of testicular microsomal heme and cytochrome P-450 levels at 24 h, followed by increases that plateau at pretreatment levels by day six. Associated with the depressed levels of microsomal heme and cytochrome P-450 is an increase of testicular microsomal heme oxygenase activity at 12-24 h. Testicular mitochondrial delta-aminolevulinic acid synthase activity was increased at 24 h, and remained elevated throughout the 9-day treatment period. Pretreatment with 1,4,6-androstatrien-3,17-dione, an aromatase inhibitor, failed to prevent the depression of testicular microsomal heme or cytochrome P-450 or increased heme oxygenase activity caused by repeated administration of human chorionic gonadotropin, and administration of estradiol benzoate failed to alter testicular microsomal heme oxygenase activity suggesting that these parameters were not related to altered testicular estrogen content caused by increased aromatase activity. These results suggest that increased testicular heme oxygenase activity is associated with decreased microsomal heme and cytochrome P-450 content during human chorionic gonadotropin-induced desensitization.     

Effect of cis-platinum on kidney cytochrome P-450 and heme metabolism: evidence for the regulatory role of the pituitary hormones

A novel action of the gonadotropic hormones of the adenohypophysis on the regulation of kidney heme metabolism and cytochrome P-450 concentrations is described. The treatment of rats with cis-platinum for 7 days caused a greater than twofold increase in the microsomal cytochrome P-450 and heme concentrations in the kidney. The sodium dodecyl sulfate-gel electrophoresis of the microsomal preparation revealed increased levels of both apocytochrome P-450 and heme in the molecular weight region corresponding to cytochrome P-450. In hypophysectomized rats, similar increases in heme and the cytochrome contents in the kidney were observed. Conversely, the treatment of rats with human chorionic gonadotropin (hCG) fully reversed the effect of cis-platinum on heme and cytochrome P-450 concentrations. The cellular basis of increases in concentrations of heme and the hemoprotein was explored by measuring the incorporation of [14C]glycine-labeled hemoglobin heme into the kidney microsomal heme fractions. In comparison with the control rats, the specific 14C activity of heme in microsomal fraction was not increased. Moreover, the effect of cis-platinum on kidney cytochrome P-450 appeared to be unrelated to alterations in the activities of the rate-limiting enzymes of heme biosynthesis and degradation pathways, delta-aminolevulinate synthetase, and heme oxygenase, respectively. On the other hand, ferrochelatase activity and the concentration of total porphyrins in the kidney were profoundly altered by cis-platinum treatment; a twofold increase in ferrochelatase activity and a marked reduction (40%) in the total porphyrin concentration were observed. Also, the activities of uroporphyrinogen-I synthetase and delta-aminolevulinate dehydratase were decreased in cis-platinum-treated animals. The latter effects reflect a direct inhibitory action of cis-platinum. It appears that the cis-platinum-mediated increase in the microsomal heme concentrations involves an accelerated rate of heme production as a consequence of increased ferrochelatase activity. This, in turn, could increase the production of cytochrome P-450. It is suggested that the anterior pituitary hormones control the concentration of the cytochrome P-450 in the kidney, and this process may be interrupted by cis-platinum.     

The murine Ah locus. Comparison of the complete cytochrome P1-450 and P3-450 cDNA nucleotide and amino acid sequences

Mouse cytochrome P1-450 and P3-450 are most closely associated with induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1) and acetanilide 4-hydroxylase activity, respectively. Full-length cDNA clones of P1-450 and P3-450 were generated from mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mouse liver. P1-450 cDNA is 2620 nucleotides in length and has a coding region (base 110 to 1,675) that produces a protein with 521 residues (Mr = 58,914). P3-450 cDNA is 1,894 nucleotides in length and yields a protein with 513 residues (Mr = 58,183). P1-450 mRNA is the first reported example in mouse in which UAG is used as the termination codon. P1-450 and P3-450, both induced by polycyclic hydrocarbons and regulated by the Ah receptor, exhibit overall nucleotide and protein homology of 68, and 73%, respectively. Segments of high homology, interspersed with regions of low homology, support the hypothesis of gene conversion or unequal crossing over as possible mechanisms for divergence of these two genes. Mouse P1-450 and P3-450 cDNAs were compared with previously published data on rat P-450e cDNA and rabbit form 2 protein, corresponding to two P-450 genes from the "phenobarbital inducible" P-450 gene subfamily. Nucleotide homology between a member of either gene subfamily is about 30%, and protein homology is about 15%, suggesting that the Ah locus-associated P-450 gene subfamily diverged from the phenobarbital inducible P-450 subfamily more than 200 million years ago. An N-terminal and a C-terminal cysteinyl fragment corresponding to the regions around P1-450 Cys-158 and Cys-458, respectively, are the only two cysteinyl peptides conserved among all four proteins compared. Because of greater homology in the C-terminal conserved cysteinyl fragment between the two gene subfamilies and a greater hydrophobic pocket in the C-terminal conserved cysteinyl fragment, the data favor this cysteine as the more likely candidate for the thiolate ligand to the heme iron in the P-450 enzyme active-site.     

Heme oxygenase-1 induction in skeletal muscle cells: hemin and sodium nitroprusside are regulators in vitro

The heat shock protein heme oxygenase-1 (HO-1)is regulated by a variety of physiological and pharmacological factors.In skeletal muscle tissue, HO-1 has been shown to be induced only byexercise and electrical stimulation in vivo. Both hemin and sodiumnitroprusside (SNP) are potent inducers of HO-1 in other tissues. Inthis study, we examined the effects of these two agents on HO-1induction in L6.G8 rat skeletal myoblast cells. Hemin and SNP increasedcellular heme oxygenase activity in both a time- andconcentration-dependent manner. Increases in the HO-1 mRNA level andprotein expression accompanied changes in heme oxygenase activity. Theability of SNP to induce HO-1 in L6.G8 cells was reduced bycoincubation with hydroxocobalamin, a known nitric oxide (NO)scavenger, suggesting that NO itself may be involved in HO-1 genestimulation. These results indicate that HO-1 expression is sensitiveto both hemin and SNP in skeletal myoblast cells and may indicate animportant regulatory mechanism of heme catabolism in skeletal muscletissue.

     

Curcumin, an antioxidant and anti-inflammatory agent, induces heme oxygenase-1 and protects endothelial cells against oxidative stress

Curcumin, a widely used spice and coloring agent in food, has been shown to possess potent antioxidant, antitumor promoting and anti-inflammatory properties in vitro and in vivo. The mechanism(s) of such pleiotropic action by this yellow pigment is unknown; whether induction of distinct antioxidant genes contributes to the beneficial activities mediated by curcumin remains to be investigated. In the present study we examined the effect of curcumin on endothelial heme oxygenase-1 (HO-1 or HSP32), an inducible stress protein that degrades heme to the vasoactive molecule carbon monoxide and the antioxidant biliverdin. Exposure of bovine aortic endothelial cells to curcumin (5-15 microM) resulted in both a concentration- and time-dependent increase in HO-1 mRNA, protein expression and heme oxygenase activity. Hypoxia (18 h) also caused a significant (P < 0.05) increase in heme oxygenase activity which was markedly potentiated by the presence of low concentrations of curcumin (5 microM). Interestingly, prolonged incubation (18 h) with curcumin in normoxic or hypoxic conditions resulted in enhanced cellular resistance to oxidative damage; this cytoprotective effect was considerably attenuated by tin protoporphyrin IX, an inhibitor of heme oxygenase activity. In contrast, exposure of cells to curcumin for a period of time insufficient to up-regulate HO-1 (1.5 h) did not prevent oxidant-mediated injury. These data indicate that curcumin is a potent inducer of HO-1 in vascular endothelial cells and that increased heme oxygenase activity is an important component in curcumin-mediated cytoprotection against oxidative stress.     

Heme oxygenase-1 mRNA levels,metallothionein mRNA levels,lipid peroxidation and microsomal CYP1A activities in rats treated with 3,3-dichlorobenzidine and some other inducers of P450

The effect of 3,3-dichlorobenzidine (DCB), a potent inducer of CYP1A, on the levels of heme oxygenase-1 mRNA and metallothionein mRNAs was examined in the kidney, liver and lung of rats administered a single ip dose (157 μmol/kg) of the compound. DCB treatment increased heme oxygenase-I mRNA abundance in the kidney significantly from barely detectable levels in untreated animals; the maximum increase in the liver and lung was 24-fold and 4-fold, respectively. Hepatic microsomal heme oxygenase activity was also induced by DCB. In contrast with DCB, 2 other P450 inducers, β-naphthoflavone (β-NF) and phenobarbital did not elevate tissue HO-1 rnRNA levels. DCB pretreatment also elevated metallothionein mRNA levels in the kidney, liver and lung, with the effect in the lung being the least pronounced. In contrast with HO-1 mRNA, metallothionein mRNA was increased by the other P450 inducers examined. In vivo lipid peroxidation and in vitro NADPH-dependent microsomal lipid peroxidation were increased in the liver of DCB-treated rats but not in those of phenobarbital- or β-naphthoflavone-treated rats. Treatment with DCB or β-NF did not alter total hepatic microsomal P450 content, as measured spectrophotometrically, but induced the activity of CYP1A2. In contrast, the activity of CYP1A1 was induced to a lesser extent by DCB than by β-NF. The data show that DCB induces HO-1 as weD as P450 1A, confirm stimulation of lipid peroxidation by the compound, and suggest oxidative stress as a mechanism of HO-1 induction by the compound.     

Activity of key enzymes of heme metabolism and cytochrome P-450 content in the rat liver in experimental rhabdomyolysis and hemolytic anemia

The 5-aminolevulinate synthase, heme oxygenase, tryptophan-2,3-dioxygenase activities, the content of total heme and cytochrome P-450 content in the rat liver and absorption spectrum of blood serum in Soret region under glycerol model of rhabdomiolisis and hemolytic anemia caused by single phenylhydrazine injection have been investigated. The glycerol injection caused a considerable accumulation of heme-containing products in the serum and the increase of the total heme content, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as the increase of the 5-aminolevulinate synthase and heme oxygenase activities in the liver during the first hours of its action and the decrease of cytochrome P-450 content in 24 h. Administration of phenylhydrazine lead to the increasing of hemolysis products content in blood serum too, although it was less expressed. The phenylhydrazine injection caused the increase of activities of 5-aminolevulinate synthase, holoenzyme, total activity and heme saturation of tryptophan-2,3-dioxygenase, as well as decrease of cytochrome P-450 content in the rat liver in 2 h. The increase of the total heme content and heme oxygenase activity has been observed in 24 h. The effect of heme arrival from the blood stream, as well as a direct influence of glycerol and phenylhydrazine on the investigated parameters are discussed.     

Spectral characterization of diarylpropane oxygenase, a novel peroxide-dependent, lignin-degrading heme enzyme

Diarylpropane oxygenase, an H2O2-dependent lignin-degrading enzyme from the basidiomycete fungus Phanerochaete chrysosporium, catalyzes the oxygenation of various lignin model compounds with incorporation of a single atom of dioxygen (O2). Diarylpropane oxygenase is also capable of oxidizing some alcohols to aldehydes and/or ketones. This enzyme (Mr = 41,000) contains a single iron protoporphyrin IX prosthetic group. Previous studies revealed that the Soret maximum of the ferrous-CO complex of diarylpropane oxygenase is at approximately 420 nm, as in ferrous-CO myoglobin (Mb), and not like the approximately 450 nm absorption of the CO complex of the ubiquitous heme monooxygenase, cytochrome P-450. This spectral difference between two functionally similar heme enzymes is of interest. To elucidate the structural requirements for heme iron-based oxygenase reactions, we have compared the electronic absorption, EPR, and resonance Raman (RR) spectral properties of diarylpropane oxygenase with those of other heme proteins and enzymes of known axial ligation. The absorption spectra of native (ferric), cyano, and ferrous diarylpropane oxygenase closely resemble those of the analogous myoglobin complexes. The EPR g values of native diarylpropane oxygenase, 5.83 and 1.99, also agree well with those of aquometMb. The RR spectra of ferric diarylpropane oxygenase have their spin- and oxidation-state marker bands at frequencies analogous to those of aquometMb and indicate a high-spin, hexacoordinate ferric iron. The RR spectra of ferrous diarylpropane oxygenase have frequencies analogous to those of deoxy-Mb that suggest a high-spin, pentacoordinate Fe(II) in the reduced form. The RR spectra of both ferric and ferrous diarylpropane oxygenase are less similar to those of horseradish peroxidase, catalase, or cytochrome c peroxidase and are clearly distinct from those of P-450. These observations suggest that the fifth ligand to the heme iron of diarylpropane oxygenase is a neutral histidine and that the iron environment must resemble that of the oxygen transport protein, myoglobin, rather than that of the peroxidases, catalase, or P-450. Given the functional similarity between diarylpropane oxygenase and P-450, this work implies that the mechanism of oxygen insertion for the two systems is different.     

Selective induction of heme oxygenase-1 isozyme in rat testis by human chorionic gonadotropin

A radioimmunoassay was developed to assess the response of testicular HO-1 to agents known to increase the microsomal heme oxygenase activity. Treatment of rats with human chorionic gonadotropin (hCG) increased the microsomal heme oxygenase activity in rat testis. The following data suggest that the increase was specific to the HO-1 isozyme: (a) The elution profile of heme oxygenase activity from a DEAE-Sephacel column showed an increase in the HO-1 peak, but not in the HO-2 peak, (b) the Western immunoblot of the testis microsomes showed an increase in HO-1 protein, and (c) the amount of HO-1 protein that was present in the microsomes, when measured by radioimmunoassay, was doubled. Using radioimmunoassay, it was shown that other agents known to increase the testicular heme oxygenase, sodium arsenate and sodium arsenite, also increased the microsomal content of HO-1. An inhibitor of the testicular microsomal heme oxygenase activity, cadmium, also increased the microsomal HO-1 protein. The findings suggest that inducibility of HO-1 extends to tissues other than the liver, in this instance, the testis, and further support the possibility that HO-1 is the only inducible form of heme oxygenase.     

Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2) 相似文献   

Protein expressed by the ho2 gene of the cyanobacterium Synechocystis sp. PCC 6803 is a true heme oxygenase. Properties of the heme and enzyme complex

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform-1, Syn HO-1, has been characterized, while isoform-2, Syn HO-2, has not. In this study, a full-length ho2 gene was cloned using synthetic DNA and Syn HO-2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO-1, the purified Syn HO-2 bound hemin stoichiometrically to form a heme-enzyme complex and degraded heme to biliverdin IXalpha, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO-2 was found to be comparable to that of Syn HO-1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO-2 converted heme to verdoheme. This shows that during the conversion of hemin to alpha-meso-hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin-Syn HO-2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin-Syn HO-1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa-coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO-2 is different from that of Syn HO-1.     

Purification and characterization of the major constitutive form of testicular heme oxygenase. The noninducible isoform

Recently we reported on the presence of two isoforms of heme oxygenase in rat liver microsomes, referred to as HO-1 and HO-2, and that only HO-1 is inducible (Maines, M. D., Trakshel, G. M., and Kutty, R. K. (1986) J. Biol. Chem. 261, 411-419). Presently we report on the detection of two isoforms of the enzyme in rat testis and purification to near homogeneity of the noninducible isoform, HO-2. A comparative characterization of the liver HO-1 and the testicular HO-2 is also provided. The relative abundance of the isoforms in the two organs was dissimilar. In the testis, the predominant form was HO-2, and only minute amounts of HO-1 were detected. In the liver, however, a 1:2 ratio of HO-1 to HO-2 was noted. The activity of HO-2 in both organs was refractory to cadmium, an inducer of the hepatic HO-1. Under nondenaturing electrophoresis conditions, HO-2 showed a higher mobility than HO-1; on a sodium dodecyl sulfate-polyacrylamide gel, HO-2 displayed a higher monomeric Mr. The apparent Mr values for HO-2 and HO-1 were 36,000 and 30,000, respectively. The isoforms differed in immunochemical properties. Antiserum to the liver HO-1 did not recognize the testicular HO-2 when examined by double immunodiffusion or by Western immunoblotting. HO-2 was more sensitive to heat inactivation than HO-1. When exposed at 65 degrees C (10 min), 70% of HO-1 activity was retained; however, nearly 80% of HO-2 activity was lost. The apparent Km values for heme for HO-1 and HO-2 were 0.24 and 0.40 microM, respectively. HO-1 and HO-2 had similar requirements for cofactor and flavoprotein reductase and were inhibited by heme-ligands (CO, KCN, NaN3). HO-2 utilized as substrate, Fe-protoporphyrin, Fe-hematoporphyrin, and Fe-hematoporphyrin acetate; it did not degrade intact purified rat liver cytochromes b5 and P-450 LM2, catalase, cytochrome c, hemoglobin, or myoglobin.