Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.99.3 (heme oxygenase)
 4,196 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hepatic microsomal heme oxygenase was solubilized, partially purified, and characterized from Co2+-treated rats. The enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis exhibited a minimum molecular weight of greater than or equal to 68,000. The solubilized enzyme was totally devoid of contamination with cytochrome P-450 or b5. The requirement for reduced pyridine nucleotides was absolute, and ascorbate could not support heme oxidative activity. However, both TPNH and DPNH could serve as electron donors, with TPNH being more effective. The presence of an appropriate flavoprotein reductase was essential for heme oxidation. The enzyme had an apparent Km of 40 micrometer, a pH optimum of 7.5, and lost substantial activity upon freezing and thawing. Methemoglobin was 30% as effective a substrate for the enzyme as was heme. Free porphyrins could not serve as substrates for the enzyme. The activity of the enzyme was inhibited by HgCl2, p-chloromercuribenzoate, iodoacetamide, mercaptoethanol, and dithiothrietol indicating that free -SH group(s) is necessary for enzyme activity.
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PMID:Solubilization and partial purification of heme oxygenase from rat liver. 1 77

The hepatic microsomal haem oxygenase activity of rats treated with CoCl2 was studied kinetically by measuring biliverdin, the immediate product of the reaction. Biliverdin was extracted with diethyl ether/ethanol mixture, and was determined by the difference between A690 and A800. The apparent Km value for NADPH (at 50 microM-haematin) was about 0.2 microM when an NADPH-generating system was used, whereas that for NADH was about 630 microM. Essentially the same Vmax. values were obtained for both the NADH- and NADPH-dependent haem oxygenase reactions. No synergism was observed with NADH and NADPH. The NADH-dependent reaction was competitively inhibited by NADP+, with a Ki of about 10 microM. The inhibitoin of the NADH-dependent reaction by the antibody against rat liver microsomal NADPH-cytochrome c reductase was essentially complete, with a pattern similar to that of the NADPH-dependent reaction. The immunochemical experiment and the comparison of the kinetic values with the reported data on isolated NADH-cytochrome b5 reductase and NADPH--cytochrome c reductase indicated the involvement of the latter enzyme in NADH-dependent haem oxygenation by microsomal fraction in situ.
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PMID:Electron-transport pathway of the NADH-dependent haem oxygenase system of rat liver microsomal fraction induced by cobalt chloride. 3 76

1. The microsomal haem oxygenase activity induced by the administration of CoCl2 was found mainly in the smooth-surfaced microsomal fraction, whereas that of the untreated control animals was widely distributed in smooth-surfaced microsomal, rough-surfaced microsomal and Golgi fractions. 2. When microsomal preparation was incubated and the time course of the distribution of biliverdin between the membranes and the medium was followed, most of the biliverdin formed was found first in the medium. This suggests that the active site of haem oxygenase is exposed on the cytoplasmic surface of the membranes. The possible localization of the enzyme at the outer surface of the membranes was also supported by a digestion experiment with trypsin. The haem oxygenase activity was greatly decreased even at low concentration of the proteinase, which did not affected the NADPH-cytochrome c reductase activity. 3. When microsomal preparation was further fractionated by isopycnic centrifugation in the presence of deoxycholate or by partitioning of sonicated microsomal preparation in aqueous-polymer two-phase systems, most of the haem oxygenase activity was found in a fraction different from the main fraction of the NADH- and NADPH-cytochrome c reductase and NADH--ferricyanide reductase activities. This indicates the different distribution of haem oxygenase from the other enzymes mentioned, on the lateral plane of microsomal membranes, and suggests the different localization of the haem oxygenase system from the electron-transport system linked with cytochrome b5 and cytochrome P-450.
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PMID:Topological arrangement in microsomal membranes of hepatic haem oxygenase induced by cobalt chloride. 44 18

Biliverdin reductase is the dual nucleotide-dependent cytosolic enzyme that converts biliverdin to the bile pigment, bilirubin, and displays extensive microheterogeneity in rat organs. The enzyme is unique in having two pH optima. The present study reports on the tissue-dependent pattern of developmental expression of the reductase in rat liver and brain. When analyzed by Western immunoblotting, two closely migrating immunoreactive proteins were detected in the liver cytosol during the first 2-3 weeks after birth; the protein with greater mobility was not detected in the liver of adult aged animals (6 months old) and was present at low levels in rats during the first week of life. The faster migrating protein was not detected in the brain cytosol at any stage of development. Furthermore, in the brain the total amount of enzyme protein increased as the animal matured, whereas in the liver the enzyme protein level decreased with age. When the purified enzyme was analyzed, age-related changes in the variant composition of the enzyme in the liver were noted. Although in both adult and newborn animals (14 days old) the purified enzyme, when subjected to isoelectric focusing, separates into five net charge forms (pl 6.23, 5.91, 5.76, 5.61, and 5.48), the relative abundance of the variants notably differed in the two preparations. In addition, when the purified preparations were subjected to two-dimensional electrophoresis, although both purified preparations separate into three molecular weight forms (Mr 30,400, 30,700, and 31,400) one species (Mr 31,400, pl = 5.77), which was very prominently expressed in the newborn, was essentially absent in the adult. Biliverdin reductase activity of the liver cytosol with both NADPH (pH 8.7) and NADH (pH 6.7) exhibited developmental changes, with the activity increasing after birth, reaching a peak on day 14, and decreasing to low levels in the adult. The existence of a close correlation between development of biliverdin reductase activity in the brain and liver and that of heme oxygenase in these organs is noted. The suggestion is made that the reductase is not a passive component of the heme degradation pathway; rather, its activity could become limiting in the elimination of heme degradation products.
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PMID:Multiple forms of biliverdin reductase: age-related change in pattern of expression in rat liver and brain. 223 89

The present study describes the high degree of sensitivity of the mitochondrial fraction of the striatum to Mn++-mediated perturbations in mixed-function oxidase activity. This study also defines the brain mitochondrial cytochrome P-450 subject to increase by Mn++. In the striatum of Mn++-treated animals (7 days) hydroxylation of benzo(a)pyrene and D-amphetamine was significantly increased in both the mitochondrial and the microsomal fractions. The effects were more pronounced in the mitochondrial fraction where hydroxylation activities were increased by 2- to 3-fold. SKF-525A (2-diethylaminoethyl-2,2-di-phenylvalerate hydrochloride) effectively inhibited NADH-dependent hydroxylation of both substrates by the mitochondrial fraction. In the Mn++-treated animals, significant increases in mitochondrial and microsomal cytochrome P-450 concentration were also noted. In the mitochondria, the hemoprotein concentration was increased by nearly 2.5-fold; in the microsomes the concentration of the cytochrome was increased by about 1.6-fold. Mn++ appeared to selectively increase cytochrome P-450 concentration since that of other cytochromes including the mitochondrial b, c1, c and a, and the microsomal cytochrome b5 was not increased. In addition, the activity of mitochondrial delta-aminolevulinate synthetase was not increased and that of the microsomal heme oxygenase was inhibited by Mn++ treatment. It is suggested that increases in the microsomal and the mitochondrial cytochrome P-450 may reflect intrinsic properties of cytochrome P-450 isozymes in these organelles, including their turnover rate, preferential utilization of heme and/or susceptibility to degradation. The possible relevance of the findings to Mn++ neurotoxicity of dopamine pathways is discussed.
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PMID:Manganese-mediated increase in the rat brain mitochondrial cytochrome P-450 and drug metabolism activity: susceptibility of the striatum. 291 73

The present report describes, for the first time, the identification of two constitutive forms of heme oxygenase, designated as HO-1 and HO-2, in rat liver microsomal fractions. HO-1 was purified to homogeneity and exhibited a specific activity of up to 4000 nmol of bilirubin/mg of protein/h. HO-2 was partially purified to a specific activity of 250 nmol of bilirubin/mg of protein/h. In the native state, the relative activity of HO-2 surpassed that of HO-1 by 2-3-fold. However, a remarkable difference existed in the regulatory mechanism(s) for the production of the two enzyme forms. Whereas the activity of HO-1 was increased up to 100-fold in response to cobalt, cadmium, hematin, phenylhydrazine, and bromobenzene, that of HO-2 was fully refractory to these agents. The two forms differed in their apparent Km, thermolability, ammonium sulfate precipitation, antigenicity, electrophoretic mobility under nondenaturing conditions, and chromatographic behavior. Specifically, for HO-1 the apparent Km value was 0.24 microM, whereas that for HO-2 was 0.67 microM. HO-2 preparation was more susceptible to heat inactivation; nearly 65% activity was retained by HO-1 preparation after exposure to 60 degrees C for 10 min, whereas under the same conditions only about 25% of HO-2 activity was retained. When subjected to ammonium sulfate precipitation the bulk of HO-1 activity precipitated between 0 and 35% saturation, whereas that of HO-2 was precipitated between 35 and 65% saturation. The two forms appeared as immunologically different entities, in so far as a crossreactivity between antibody raised against HO-1 in rabbit and HO-2 could not be detected. Similarities were observed in respect to cofactor requirements for activity, sensitivity to inhibitors, as well as their reactivity towards the substrates used in this study, i.e. hematin, hematoheme, and cytochrome c. Specifically both forms of the enzyme required NADPH-cytochrome c (P-450) reductase, NADPH or NADH, and O2 for activity, and reactions were inhibited by KCN, NaN3, and CO. Both forms cleaved the tetrapyrrole molecule exclusively at the alpha-meso bridge to form biliverdin IX alpha isomer. HO-1 and HO-2 utilized hematin and hematoheme as substrates but not intact cytochrome c.
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PMID:Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. 307 57

We report the identification of an NADH-dependent haem-degrading system in ox heart mitochondria. The activity was localized to the mitochondrial inner membrane, specifically associated with complex I (NADH:ubiquinone oxidoreductase). The mitochondrial NADH-dependent haem-degradation activity was highly effective and displayed a rate nearly 60% higher than that of the microsomal activity. The following observations suggested the enzymic nature of the activity: (i) haem degradation by complex I did not proceed upon exposure to elevated temperature and extremes of pH; (ii) it displayed substrate specificity; (iii) it was inhibited by a substrate analogue; and (iv) it showed a cofactor requirement. Moreover, the activity was distinctly different from the ascorbate-mediated haem-degradation activity. Also, complex I differed from the microsomal NADPH:cytochrome c (P-450) reductase inasmuch as the formation of an effective interaction with the microsomal haem oxygenase could not be detected. Addition of purified haem oxygenase to complex I neither influenced the rate of haem degradation nor resulted in the formation of biliverdin IX alpha. In contrast, addition of haem oxygenase to NADPH:cytochrome c (P-450) reductase enhanced the rate of haem degradation by nearly 8-fold, and more than 60% of the degraded haem could be accounted for as biliverdin IX alpha. The haem-degrading activity of complex I appeared to involve the activity of H2O2, as the reaction was inhibited by nearly 90% by catalase, and propentdyopents were detected as reaction products. Intact haemoproteins such as cytochrome c and myoglobin were not effective substrates. However, the haem undecapeptide of cytochrome c was degraded at a rate equal to that observed for haem. Haematohaem was degraded at a rate 50% lower than that observed for haem. It is suggested that the NADH-dependent haem-degradation system may have a biological role in the regulation of the concentration of respiratory haemoproteins and the disposition of the aberrant forms of the mitochondrial haemoproteins.
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PMID:Characterization of an NADH-dependent haem-degrading system in ox heart mitochondria. 312 Jun 97

Heme oxygenase has been purified to electrophoretic homogeneity from detergent solubilized adult human liver microsomes. Treatment of microsomes with Triton X-100, sodium cholate and subsequent batchwise DEAE-cellulose, 2', 5' ADP-sepharose 4B, Sepharose CLB and hydroxylapatite column resulted in 17% yield of the purified heme oxygenase. The reconsituted system of heme oxygenase, composed of heme oxygenase, NADPH cytochrome c (P450) reductase and biliverdin reductase was equiactive with 1 mM NADPH and 4 nM NADH and showed complete dependence on added heme for catalytic activity. The Km values for NADPH and NADH were .046 and .526 mM, respectively. While NADPH concentration was held constant, the Km value for heme was 1.01 microM with a specific activity of 583 unit/mg protein. The activity of the reconstituted heme oxygenase system was not affected by preincubation with heavy metals despite their inhibitory effect of NADPH cytochrome c (P450) reductase and biliverdin reductase. However, the metalloporphyrins of these heavy metals were found to be strong inhibitors of the reconsituted system with Ki values of 0.015, 0.6, 2.3 and 5 microM for Sn-, Co-, Zn- and Mg- protoporphyrins, respectively. Similarly, the sulfhydryl inactivating reagents, HgCl2, iodoacetamide and p-chloromercurylbenzoate, inhibited the reconstituted heme oxygenase activity. Rabbits were immunized with purified human liver heme oxygenase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Microsomal protein with a molecular weight of 32,000 from rat and human liver as well as HepG2 cells were identified on dot and Western blots by their reaction with the anti-heme oxygenase similar to the purified enzyme protein. Anti-heme oxygenase precipitated quantitatively, the entire heme oxygenase of rat liver microsomes obtained from animals maintained on standard diet. The human bone marrow microsomal heme oxygenase activity was also quantitatively precipitated by this antibody. Antibody inhibition of rat and human heme xoygenase demonstrated a degree of conservation of both enzyme proteins between the species. As judged by Western blotting, the anti-heme oxygenase recognized only a single protein in spleen, liver, kidney, brain, heart, bone marrow, integtine and corneal epithelium. The human heme oxygenase cDNA was isolated by screening a cDNA library in the Okayama-Berg vector with a rat liver cDNA and was subjected to nucleotide sequence analysis. The deducted human heme oxygenase is also composed of 288 amino acids with a molecular mass of 32,800 Da.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Expression of heme oxygenase in hemopoiesis. 314 8

The changes in the cytochrome P-450 enzyme system of rat liver, lung and brain after inhalation of ethylene oxide were studied. When Wistar male rats were exposed to 500 ppm ethylene oxide three times a week for three months, they showed a suppressed gain of body weight. Therefore, the present study was done using pair-fed rats. Haematological examination revealed normocytic and normochromic anemia. Liver and renal functions were normal. The cytochrome P-450 enzyme system in the lung and brain were not affected. However, hepatic cytochrome P-450 and protoheme decreased by 28% and 19%, respectively. Hepatic total microsomal protein, cytochrome b5, NADPH-cytochrome c reductase and NADH-ferricyanide reductase were not affected. The activity of hepatic heme oxygenase showed a 2-fold increase. These results suggest that the heme moiety of hepatic cytochrome P-450 was primarily attacked by exposure of ethylene oxide and the cellular heme balance in liver was altered.
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PMID:[Effects of chronic exposure of ethylene oxide, especially on heme metabolism]. 336 71

The effects of carbon disulfide (CS2) on the liver microsomal drug-metabolizing enzyme system and other enzyme activities were studied 1 hr after the oral administration of 3-300 mg/kg of CS2 in mice. Considerable decreases in drug-metabolizing enzyme activities (such as hydroxylation of aniline, O-dealkylation of p-nitroanisole, 7-ethoxycoumarin and 7-ethoxyresorufin, and N-demethylation of N,N-dimethylaniline), NADPH-cytochrome P-450 reductase (but not NADPH-cytochrome c reductase), and P-450-associated peroxidase activities were already observed at 3 and 30 mg/kg of CS2, dose dependently. At the same dosage levels, the magnitudes of microsomal spectral changes induced by aniline and nicotinamide (type 2 substrates), but not those induced by hexobarbital and SKF-525A (type 1 substrates), were also reduced to a considerable extent. The degrees of these alterations were all greater than that of the measurable loss of P-450 content, i.e. the loss of functional activity of P-450 was much greater than simply expected from the apparent decrease in the hemoprotein content. Cytochrome b5 content and NADH-ferricyanide reductase activity were unchanged at 30 and 300 mg/kg of CS2, although NADH-cytochrome c reductase activity was increased at the latter dose. The following enzyme activities did not change significantly at up to 300 mg/kg of CS2: flavin-containing monooxygenase, UDP-glucuronyl transferase, glucose-6-phosphatase and heme oxygenase in microsomes, and glutathione S-transferases in the soluble fraction. Microsomal conjugated diene levels and liver glutathione content were also unchanged. These observations support the theory that P-450 is a sensitive and selective site for CS2 action, where CS2 itself is bioactivated. It was also shown that the loss of P-450 was reversible after a single, or repeated, administration of CS2.
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PMID:Early, selective and reversible suppression of cytochrome P-450-dependent monooxygenase of liver microsomes following the administration of low doses of carbon disulfide in mice. 377 18


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