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PubMed2003

Protective role of heme oxygenase-1 in the intestinal tissue injury in an experimental model of sepsis.

OBJECTIVE: The aim of this study was to examine the role of heme oxygenase-1 induction in the intestinal tissue injury in a rat model of sepsis. DESIGN: Randomized, masked, controlled animal study. SETTING: University-based animal research facility. SUBJECTS: Sprague-Dawley male rats, weighing 220-250 g (n = 126). INTERVENTIONS: Rats were injected with lipopolysaccharide (10 mg/kg) intraperitoneally. Another group of rats was injected with interleukin-6 (10 microg/kg) intravenously. In some rats, tin mesoporphyrin (1 micromol/kg) was administered intravenously 1 hr before lipopolysaccharide treatment. MEASUREMENTS AND MAIN RESULTS: Following lipopolysaccharide treatment, expression of heme oxygenase-1 and nonspecific delta-aminolevulinate synthase (ALAS-N), the rate-limiting enzymes of heme catabolism and biosynthesis, respectively, was examined in various regions of the intestine. Lipopolysaccharide treatment markedly increased heme oxygenase-1 messenger RNA and protein concentrations in the mucosal epithelial cells in the duodenum and the jejunum, whereas its expression in the ileum and the colon was hardly detectable and was not influenced by the treatment. ALAS-N messenger RNA was also more markedly increased in the duodenum, the jejunum, and the ileum than in the colon following lipopolysaccharide treatment. Interleukin-6 administration also induced heme oxygenase-1 and ALAS-N gene expression in a pattern similar to that following lipopolysaccharide treatment. In contrast to the marked heme oxygenase-1 expression in the upper intestine, lipopolysaccharide-induced mucosal injury and inflammation in the upper intestine were far less than observed in the lower intestine as judged both by tumor necrosis factor-alpha gene expression and by histologic analysis. Of note, inhibition of heme oxygenase activity by tin mesoporphyrin produced a significant tissue injury in the upper intestine of the lipopolysaccharide-treated animals. CONCLUSIONS: Intestinal heme oxygenase-1 and ALAS-N gene expression was regulated in a site-specific manner in a rat model of sepsis. Our findings also suggest that heme oxygenase-1 induction may play a fundamental role in protecting mucosal epithelial cells of the intestine from oxidative damages that occur in sepsis.

5-Aminolevulinate Synthetase↗

PubMed1997

2-oxo-1,2-dihydroquinoline 8-monooxygenase: phylogenetic relationship to other multicomponent nonheme iron oxygenases.

2-Oxo-1,2-dihydroquinoline 8-monooxygenase, an enzyme involved in quinoline degradation by Pseudomonas putida 86, had been identified as a class IB two-component nonheme iron oxygenase based on its biochemical and biophysical properties (B. Rosche, B. Tshisuaka, S. Fetzner, and F. Lingens, J. Biol. Chem. 270:17836-17842, 1995). The genes oxoR and oxoO, encoding the reductase and the oxygenase components of the enzyme, were sequenced and analyzed. oxoR was localized approximately 15 kb downstream of oxoO. Expression of both genes was detected in a recombinant Pseudomonas strain. In the deduced amino acid sequence of the NADH:(acceptor) reductase component (OxoR, 342 amino acids), putative binding sites for a chloroplast-type [2Fe-2S] center, for flavin adenine dinucleotide, and for NAD were identified. The arrangement of these cofactor binding sites is conserved in all known class IB reductases. A dendrogram of reductases confirmed the similarity of OxoR to other class IB reductases. The oxygenase component (OxoO, 446 amino acids) harbors the conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and the mononuclear iron. In contrast to known class IB oxygenase components, which are composed of differing subunits, OxoO is a homomultimer, which is typical for class IA oxygenases. Sequence comparison of oxygenases indeed revealed that OxoO is more related to class IA than to class IB oxygenases. Thus, 2-oxo-1,2-dihydroquinoline 8-monooxygenase consists of a class IB-like reductase and a class IA-like oxygenase. These results support the hypothesis that multicomponent enzymes may be composed of modular elements having different phylogenetic origins.

Amino Acid Sequence↗

PubMed2004

Haem oxygenase augments porcine granulosa cell apoptosis in vitro.

Haem oxygenases produce carbon monoxide, which, like nitric oxide, is a gaseous messenger molecule that is one of several important survival factors in ovarian follicles. However, little is known about the expression and possible functions of these enzymes in granulosa cells. The purpose of this study was to investigate the expression and possible role of haem oxygenases in porcine granulosa cells (PGCs). We obtained frozen sections of porcine ovaries and PGCs from ovarian follicles of various sizes by needle aspiration, and examined the expression of haem oxygenase-1 (HO-1; inducible type) and HO-2 (constitutive type) in PGCs by immunohistochemistry, RT-PCR, western blotting and flow cytometry. Both types of haem oxygenase were identified in PGCs throughout follicular development, but HO-1 was expressed primarily in granulosa cells in atretic follicles. We also investigated the effect of haem oxygenases on apoptosis of granulosa cells (flow cytometry to detect subdiploid DNA fluorescence) and on expression of Fas ligand (quantitative analysis of western blotting and flow cytometry). In tightly bound PGCs, the mean proportion of apoptotic cells treated with 1 microM haemin (a haem oxygenase substrate) was approximately 1.7-fold greater than that in untreated controls, and zinc protoporphyrin IX (ZnPP IX; a haem oxygenase inhibitor) completely inhibited the increase in apoptosis induced by haemin in 24-h culture. Conversely, in weakly associated PGCs, the proportion of apoptotic cells was not altered by haemin. The quantity of Fas ligand protein was increased in a dose-dependent manner in tightly bound PGCs treated with haemin compared with controls, and the haemin-induced increase in Fas ligand protein was inhibited by ZnPP IX. Thus we identified inducible HO-1 and constitutive HO-2 in PGCs throughout follicular development, and we conclude that products of reactions catalysed by haem oxygenases are likely to be important autocrine/paracrine factors that regulate apoptosis in PGCs.

Animals↗

PubMed1993

Elevated levels of heme oxygenase-1 activity and mRNA in peripheral blood adherent cells of acquired immunodeficiency syndrome patients.

Patients with the acquired immunodeficiency syndrome (AIDS) commonly develop hematological abnormalities, including anemia, leukopenia, and thrombocytopenia. Heme synthesis and heme degradation are critical to the maintenance of cellular heme homeostasis and to hematopoietic differentiation. We examined heme oxygenase activity and expression of the heme oxygenase gene in adherent cells (monocytes-macrophages) obtained from the peripheral blood of AIDS patients and normal controls. Heme oxygenase activity in normal control cells was 43 +/- 16 pmol bilirubin formed/4 x 10(5) cells/hr as compared to 133 +/- 30 pmol bilirubin formed/4 x 10(5) cells/hr in the AIDS patients. Via blot hybridization analysis with human heme oxygenase cDNA, heme oxygenase mRNA levels in cells of the normal and the AIDS patients were compared. Total RNA from normal cells displayed only weak hybridization with the cDNA probe. In contrast, cells from peripheral blood of the AIDS patients displayed marked increases over normal levels in heme oxygenase mRNA. Heme oxygenase activity could be substantially suppressed by the competitive inhibitor of the enzyme, Sn-mesoporphyrin. Elevated heme oxygenase activity in cells of AIDS patients could produce a decrease in cellular heme needed for transductional signalling for the growth factor network, which regulates the hematopoietic microenvironment, and for other metabolic purposes. Suppression of heme catabolism by inhibitors of this enzyme may thus be useful in potentiating erythropoietic responses in this disorder.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Mitochondrial heme oxygenase of Mastomys coucha.

While studying the fate of heme generated during malaria infection, it was observed that mitochondrial preparations were highly enriched with heme compared to other subcellular particles. With this background, the present study aimed to determine the status of mitochondrial heme oxygenase and compare it with the microsomal enzyme. Mitochondrial and microsomal preparations were obtained from liver, spleen, kidney and brain of normal, inducer (cobalt chloride and hemin)-treated and Plasmodium berghei-infected Mastomys coucha. Heme oxygenase activity was determined by monitoring the formation of bilirubin. Biliverdin reductase activity was assayed by following the decrease in biliverdin content. Heme levels were measured by pyridine haemochromogen formation. Mitochondria from different tissues showed significant activity of heme oxygenase only after inducer (CoCl2 and hemin) treatment. In contrast, cerebral mitochondria did not show any enzyme activity. Hepatic, splenic and renal mitochondria of P. berghei-infected M. coucha showed noticeable heme oxygenase and biliverdin reductase activity. The response of hepatic mitochondrial heme oxygenase towards Triton X-100, trypsin, hydrogen peroxide, temperature, freezing and thawing and hemin was distinguishable from microsomal heme oxygenase. It is concluded that the mitochondria of different tissues from Mastomys display stress (biological and chemical)-induced activity of heme oxygenase. In addition, distinct differences between microsomal and mitochondrial heme oxygenase were observed.

Animals↗

PubMed1995

Induction of heme oxygenase in toxic renal injury: a protective role in cisplatin nephrotoxicity in the rat.

Cellular content of heme is regulated by heme oxygenase, the rate limiting enzyme in the degradation of heme. Induction of heme oxygenase is a protective response in an in vivo model of heme protein mediated renal injury, the glycerol model of acute renal failure. In addition to heme, heme oxygenase is induced by diverse forms of oxidative stress, the functional significance of which is currently unknown. We examined whether heme oxygenase is induced, and the functional significance of such induction, in two in vivo models of oxidant-induced toxic nephropathy, namely, cisplatin and gentamicin nephropathies; nephrotoxicity in these models is not dependent on the delivery of a burden of heme proteins to the kidney as occurs in the glycerol model. We demonstrate induction of heme oxygenase mRNA and protein in the kidney as early as 6 and 12 hours after a single dose of cisplatin (6 mg/kg i.v.). Pretreatment with tin protoporphyrin, a competitive inhibitor of heme oxygenase, led to higher serum creatinine values on days 3 through 5 and lower inulin clearances on day 5; tin protoporphyrin also exacerbated renal injury in this model. Renal hemodynamics studied at day 2 after cisplatin demonstrate reduced renal blood flow rates, increased renal vascular resistance and increased fractional excretion of sodium in rats treated with tin protoporphyrin. Tin protoporphyrin alone had no significant effect on serum creatinine and renal hemodynamics in rats with intact, disease-free kidneys. We confirmed that tin protoporphyrin prevented the increase in heme oxygenase activity induced by cisplatin. Induction of heme oxygenase by cisplatin was associated with increased kidney heme content and ferritin content.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

The role of inorganic metals and metalloporphyrins in the induction of haem oxygenase and heat-shock protein 70 in human hepatoma cells.

The role of inorganic metals and metalloporphyrins in the induction of mRNAs for haem oxygenase and heat-shock protein 70 (hsp70), the two heat-shock proteins, was examined in human HepG2 and Hep3B hepatoma cells. SnCl2, but not Sn-protoporphyrin, was found to be a potent inducer of both haem oxygenase and hsp70 mRNAs. In contrast, CoCl2, ZnCl2 and FeCl2 caused little induction of haem oxygenase and hsp70 mRNAs, whereas the porphyrin complexes of these metals strongly induced haem oxygenase mRNA, without influencing the level of hsp70 mRNA. The induction process was largely transcriptional, as judged by the inhibition of induction by actinomycin D, but not by cycloheximide, and by increased transcription demonstrated by nuclear run-off analysis. Since CoCl2 is a potent inducer of haem oxygenase in vivo in animals, the possibility of the biosynthesis of Co-protoporphyrin was examined in human hepatoma cells by incubating them with CoCl2 and protoporphyrin, or delta-aminolaevulinate (ALA), the precursor of protoporphyrin. Both types of treatment led to a potent induction of haem oxygenase mRNA. Co-protoporphyrin formation was also spectrally demonstrated in cells incubated with the metal and ALA. The results of this study indicate that certain metals, e.g. SnCl2, may directly induce haem oxygenase mRNA, whereas with other elements, incorporation of the metal into the porphyrin macrocycle is necessary for induction. Therefore CoCl2, like haemin, may activate the haem oxygenase gene via a haem-responsive transcription factor, whereas SnCl2 may exert its effect via a metal-responsive transcription factor.

Carcinoma, Hepatocellular↗

PubMed1993

Structure/activity relationships in porphobilinogen oxygenase and horseradish peroxidase. An analysis using synthetic hemins.

The apo-enzymes of porphobilinogen oxygenase and horseradish peroxidase were reconstituted with hemin IX, deuterohemin IX, 2,4-diacetyldeuterohemin IX, 2-vinyl-4-deuterohemin IX and hemin I. The apoproteins did not reconstitute with the dimethyl or diethyl esters of hemin IX. The native enzymes and the synthetic hemoproteins showed similar oxygenase activities toward porphobilinogen in the presence of dithionite and oxygen. They also showed peroxidase activity in the presence of H2O2, which was affected by the side-chain substitution pattern of the hemes. Oxygenase activities, however, were not affected by the heme structure. Iron chelators completely inhibited the oxygenase, but not the peroxidase activities. The EPR spectra of the native and synthetic porphobilinogen oxygenase showed that dithionite reduction produced a rapid disappearance of the high-spin heme-iron signal at g = 6.0. It reappeared 1 min later but the enzyme retained its catalytic activity. The changes in the EPR spectra could be correlated with the biphasic kinetics of the oxygenase reaction which was very fast during the first minute and then decreased to a half-value rate. The oxygenase reaction was inhibited by addition of superoxide dismutase during the fast rate phase, but not during the slower phase. These results could be explained by the formation of a superoxide anion during the first minute of the oxygenase reaction, after which a protein-stabilized radical (g = 2.0) is generated (very likely a tyrosyl radical). The latter then oxidizes the substrate porphobilinogen and facilitates its reaction with O2 to give oxopyrrolenines.

Animals↗

PubMed2006

Spectroscopic characterization of a higher plant heme oxygenase isoform-1 from Glycine max (soybean)--coordination structure of the heme complex and catabolism of heme.

Heme oxygenase converts heme into biliverdin, CO, and free iron. In plants, as well as in cyanobacteria, heme oxygenase plays a particular role in the biosynthesis of photoreceptive pigments, such as phytochromobilins and phycobilins, supplying biliverdin IX(alpha) as a direct synthetic resource. In this study, a higher plant heme oxygenase, GmHO-1, of Glycine max (soybean), was prepared to evaluate the molecular features of its heme complex, the enzymatic activity, and the mechanism of heme conversion. The similarity in the amino acid sequence between GmHO-1 and heme oxygenases from other biological species is low, and GmHO-1 binds heme with 1 : 1 stoichiometry at His30; this position does not correspond to the proximal histidine of other heme oxygenases in their sequence alignments. The heme bound to GmHO-1, in the ferric high-spin state, exhibits an acid-base transition and is converted to biliverdin IX(alpha) in the presence of NADPH/ferredoxin reductase/ferredoxin, or ascorbate. During the heme conversion, an intermediate with an absorption maximum different from that of typical verdoheme-heme oxygenase or CO-verdoheme-heme oxygenase complexes was observed and was extracted as a bis-imidazole complex; it was identified as verdoheme. A myoglobin mutant, H64L, with high CO affinity trapped CO produced during the heme degradation. Thus, the mechanism of heme degradation by GmHO-1 appears to be similar to that of known heme oxygenases, despite the low sequence homology. The heme conversion by GmHO-1 is as fast as that by SynHO-1 in the presence of NADPH/ferredoxin reductase/ferredoxin, thereby suggesting that the latter is the physiologic electron-donating system.

Amino Acid Sequence↗

PubMed1996

Glomerular inflammation induces resistance to tubular injury in the rat. A novel form of acquired, heme oxygenase-dependent resistance to renal injury.

Considerable attention is directed to a surprising biologic phenomenon wherein tissues exposed to one insult acquire resistance to another. We identify a novel example of acquired resistance to acute renal failure and a mechanism that contributes to such resistance. Nephrotoxic serum, administered to rats 24 h before the induction of glycerol-induced acute renal failure, reduces functional and structural injury that occurs in this model. Since heme oxygenase, the rate-limiting enzyme in heme degradation, protects against heme protein-induced renal injury, we questioned whether induction of heme oxygenase underlies the protection afforded by nephrotoxic serum. Kidney heme oxygenase (HO-1) mRNA was induced 6 h after nephrotoxic serum and renal tubules were identified as the site of expression of heme oxygenase protein. Induction of heme oxygenase was accompanied by increased renal content of ferritin but not by induction of other antioxidant enzymes. Inhibition of heme oxygenase prevented the protection afforded by nephrotoxic serum. Nephrotoxic serum did not protect against ischemic acute renal failure, a model in which heme oxygenase is not induced. Thus, nephrotoxic serum protects against glycerol-induced acute renal failure by inducing heme oxygenase in tubules. This study provides the first demonstration of resistance to tubular injury acquired from glomerular inflammation, uncovers a mechanism for such resistance, and exposes the dialogue that occurs between glomeruli and tubules.

Acute Kidney Injury↗

PubMed1982

Purification and characterization of an alpha-ketoisocaproate oxygenase of rat liver.

Rat liver contains a cytosolic alpha-ketoisocaproate oxygenase which oxidatively decarboxylates and hydroxylates alpha-ketoisocaproate to form beta-hydroxyisovalerate. This oxygenase was purified to near homogeneity. The oxygenase is unstable during purification, unless 5% monothioglycerol is added. The purified enzyme is stable in the presence of 5% monothioglycerol for 3 weeks at 4 degrees C and at least 10 weeks at -80 degrees C. The molecular weight of the alpha-ketoisocaproate oxygenase as determined to be 46,000 and 51,000 using denaturing and nondenaturing conditions, respectively, indicating a monomer. The alpha-ketoisocaproate oxygenase requires Fe2+; other metal ions did not replace Fe2+. Ascorbate activates the enzyme at subsaturating levels of Fe2+, by regenerating Fe2+. The activity is markedly affected by the type of buffer used. For example, the oxygenase activity increased 2- to 3-fold when 0.1 M maleate was used. Iron chelators, such as ADP and EDTA, are inhibitory. The ratio of decarboxylation of 1 mM alpha-[1-14C] ketoisocaproate (as measured by 14CO2 release) to decarboxylation of 1 mM alpha-[1-14C]keto-gamma-methiolbutyrate is 1.0 for all purification fractions, indicating that a single enzyme catalyzes the decarboxylation of both substrates. The apparent Km and Vmax values of the alpha-ketoisocaproate oxygenase using optimized assay conditions are 0.32 mM and 130 nmol/min/mg of protein for alpha-ketoisocaproate and 1.9 mM and 247 nmol/min/mg of protein for alpha-keto-gamma-methiolbutyrate. The principal product of the purified alpha-ketoisocaproate oxygenase, using alpha-ketoisocaproate as a substrate, is beta-hydroxyisovalerate, although small amounts of a compound, which has the chromatographic properties of isovalerate, are also produced.

Animals↗

PubMed1994

Regulation of heme oxygenase activity in Cyanidium caldarium by light, glucose, and phycobilin precursors.

Cyanobacteria, red algae, and cryptophytes contain phycobiliproteins which function as photosynthetic light-harvesting pigments. The chromophores of phycobiliproteins are phycobilins, open-chain tetrapyrroles that are synthesized from protoheme. The first step of phycobilin formation is the conversion of protoheme to biliverdin IX alpha in a reaction that is catalyzed by heme oxygenase. In the unicellular red alga, Cyanidium caldarium, light is required for the accumulation of phycobiliproteins. It has been reported previously that the synthesis of the apoprotein components of allophycocyanin and phycocyanin is induced by light in C. caldarium, that the phycobilin precursors, delta-aminolevulinic acid (ALA), protoporphyrin IX, and protoheme can substitute for light, and that the regulation is exerted at the level of mRNA synthesis. We have determined that a key enzyme of phycobilin formation is induced by light in C. caldarium. Extractable heme oxygenase activity is low in dark-grown cells, and it increases approximately 6-fold during the first 24 h after the cells are illuminated. After 24 h, the activity decreases to a level approximately equal to the initial activity. Heme oxygenase is induced in unilluminated cells by administration of ALA. D-Glucose, which is known to inhibit phycocyanin accumulation in C. caldarium, inhibits the induction of heme oxygenase by light or ALA. Induction of heme oxygenase by light or ALA is blocked by cycloheximide, an inhibitor of cytoplasmic protein synthesis, but not by chloramphenicol, an inhibitor of chloroplast protein synthesis. Rifampicin, an inhibitor of algal chloroplast RNA synthesis, and gabaculine, a competitive inhibitor of ALA biosynthesis, block the induction of heme oxygenase by light but not by ALA. These results indicate that heme oxygenase in C. caldarium is induced by phycobilin precursors. The induction by light and the repression of the induction by D-glucose are probably indirect effects mediated by the effects of light and D-glucose on phycobilin precursor formation. The results also indicate that heme oxygenase is encoded by a nuclear gene and is synthesized on cytoplasmic ribosomes.

Aminolevulinic Acid↗

PubMed1999

Cellular localization of cyclo-oxygenase isozymes in Crohn's disease and colorectal cancer.

Deregulation of cyclo-oxygenase isozyme expression has been shown to be a consistent feature of inflammatory bowel diseases and colorectal cancer in humans. This study investigated the cellular localization of aberrant cyclo-oxygenase expression in normal and diseased colon. Biopsies of seven normal colonic tissues, eight tissue samples from patients suffering from Crohn's disease, five polyps from patients with familiar adenomatous polyposis coli, and ten sporadic adenocarcinomas were analyzed using isozyme-selective immunoprecipitation, western blotting, and immunohistochemistry. Cyclo-oxygenase-1 expression was demonstrated in normal human colon, Crohn's disease, and colorectal tumors. In normal colon and also in adenomatous polyps, cyclo-oxygenase-1 specific immunosignals were localized to epithelial cells of the upper part of the crypts and endocrine cells of the lower part. In Crohn's disease cyclo-oxygenase-1 expression was restricted to cells of the inflammatory infiltrate. While barely detectable in normal colon, cyclo-oxygenase-2 protein was strongly increased in epithelial cells located in the uppermost part of the crypts, in surface epithelial cells, and in mononuclear cells of the lamina propria of Crohn's disease. The constitutive overexpression of cyclo-oxygenase-2 protein observed in the majority of the adenomatous polyps and all adenocarcinomas was attributed to both epithelial and interstitial cells in that the latter predominated in adenomas, and epithelial cells were the prevailing cyclo-oxygenase-2 expressing cell type in adenocarcinomas. In conclusion, both autocrine and paracrine effects of aberrant cyclooxygenase-2 expression may contribute to the development of Crohn's disease and colonic tumor development.

Adenocarcinoma↗

PubMed2006

Systematic review: Cyclo-oxygenase-2 in human oesophageal adenocarcinogenesis.

BACKGROUND: Published in vitro and animal in vivo studies have demonstrated that cyclo-oxygenase-2 plays an important role during oesophageal adenocarcinogenesis. However, the extent to which these studies are directly relevant to events in the human lower oesophagus is questionable. AIM: To perform a systematic review of all available human studies that have evaluated levels of cyclo-oxygenase-2 expression during the progression from Barrett's metaplasia to oesophageal adenocarcinoma. METHODS: A literature search was performed to identify all studies which qualitatively or quantitatively assessed cyclo-oxygenase-2 protein or gene expression in either Barrett's, dysplastic or adenocarcinoma tissue in humans. RESULTS: A total of 27 studies met the inclusion criteria. There was general agreement that cyclo-oxygenase-2 was either absent or very weakly expressed in normal oesophageal squamous mucosa, but considerable disagreement regarding the presence of cyclo-oxygenase-2 in Barrett's and low-grade dysplasia. All studies agreed that high-grade dysplasia and adenocarcinoma expressed cyclo-oxygenase-2 to some extent although levels varied considerably between tissue samples. CONCLUSIONS: There is conflicting evidence in the literature for cyclo-oxygenase-2 playing an important role in early oesophageal adenocarcinogenesis. Other non-cyclo-oxygenase-2 targets may account for the epidemiological data supporting the use of non-steroidal anti-inflammatory drugs in the chemoprevention of oesophageal adenocarcinoma.

Adenocarcinoma↗

PubMed1994

The modulation of enzyme reaction rates within multi-enzyme complexes. 2. Information transfer within a chloroplast multi-enzyme complex containing ribulose bisphosphate carboxylase-oxygenase.

Octameric ribulose bisphosphate carboxylase-oxygenase binds in an independent manner its substrate (ribulose bisphosphate) and a substrate analog (6-phosphogluconate). The eight active sites of the free enzyme are thus independent. The kinetic behaviour of the active site becomes different if ribulose bisphosphate carboxylase-oxygenase is inserted in the five-enzyme complex previously isolated from chloroplasts. Ribulose bisphosphate carboxylase-oxygenase then becomes more active than the corresponding free enzyme form. By comparing the behaviour of the same enzyme in the free state and in the associated state it then becomes possible to study how the thermodynamics of protein-protein interactions alters the kinetic behaviour of ribulose bisphosphate carboxylase-oxygenase. This alteration may be expressed in terms of stabilization-destabilization energies exerted upon the various intermediate states of the enzyme reaction, within the multi-protein complex. Heterologous interactions within this complex exert a constant stabilization energy on the enzyme ground states along the reaction co-ordinate of -1.68 kJ/mol and a constant stabilization energy of -3.79 kJ/mol on the enzyme transition states. These stabilization energies express how information propagates within the multi-enzyme complex as to increase the apparent affinity of the substrate for the active sites of ribulose bisphosphate carboxylase-oxygenase, as well as to increase the catalytic rate constant. The binding of the substrate analog 6-phosphogluconate to free ribulose bisphosphate carboxylase-oxygenase is non-co-operative. It becomes positively co-operative if this enzyme is inserted in the multi-protein complex. Under these conditions, only one type of enzyme-inhibitor complex is detected experimentally. Here again heterologous interactions stabilize this enzyme-inhibitor complex relative to that expected if ribulose bisphosphate carboxylase oxygenase is free. The extent of stabilization is -1.03 kJ/mol. Neither free nor associated ribulose bisphosphate carboxylase-oxygenase display any co-operativity relative to substrate binding. However, in the presence of the substrate analog 6-phosphogluconate, this enzyme displays positive co-operativity relative to substrate, although not if it is naked. These results can be explained theoretically and show that the maximum value of the Hill coefficient is < or = 2. As 6-phosphogluconate and other substrate analogs are present in chloroplasts under normal conditions, this co-operativity might be of functional importance in vivo.

Binding Sites↗

PubMed1984

Cyclo-oxygenase inhibitors antagonize indirectly evoked contractions of the guinea-pig isolated ileum by inhibiting acetylcholine release.

The effects of indomethacin, sodium meclofenamate and ketoprofen on the contractile responses of the guinea-pig isolated ileum to directly and indirectly evoked stimuli were investigated. The effects of the cyclo-oxygenase inhibitors on acetylcholine (ACh) release from plexus containing longitudinal muscle strips were also studied. The cyclo-oxygenase inhibitors reduced contractile responses to transmural stimulation (TMS) and nicotine at concentrations which had no effect on ACh-induced contractions. In whole ileum preparations (WIP) indomethacin and ketoprofen (40 micrograms ml-1) reduced TMS responses by 17 +/- 1.8% and 12 +/- 1.8% (n = 6), respectively (30 min incubation). In longitudinal muscle strips (LMS) in which Auerbach's plexus is exposed, indomethacin and ketoprofen (1 microgram ml-1) reduced TMS responses by 28 +/- 2.3% and 34 +/- 2.7% (n = 6), respectively (10 min incubation). Thus the cyclo-oxygenase inhibitors were up to 80 times more effective in LMS than in WIP. The drugs were similarly more effective in blocking nicotine contractions in LMS than in WIP. The cyclo-oxygenase inhibitors reduced basal and stimulated ACh release from LMS. For example, indomethacin (1 microgram ml-1) reduced stimulated ACh release by 35% after 10 min incubation. The percentage inhibition increased to 79% after 40 min incubation (n = 6). Prostaglandin E2 (PGE2) (0.1-2.5 ng ml-1) restored the contractile responses and ACh release depressed by the cyclo-oxygenase inhibitors but not the contractile responses depressed by atropine. PGF2 alpha had no effect on mechanical responses or ACh release depressed by the cyclo-oxygenase inhibitors. 6 It is concluded that the cyclo-oxygenase inhibitors studied reduced responses to transmural stimulation and nicotine by inhibiting ACh release. The site of action is the postganglionic parasympathetic nerve. 7 It is suggested that the reason why previous investigators needed to use high doses of cyclooxygenase inhibitor in the ileum is because the action of the inhibitor is limited by diffusion barriers. There was no evidence to support the view that there is more than one pool of cyclo-oxygenase in guinea-pig gut.

Acetylcholine↗

PubMed2002

Effects of inhibitors of the lipo-oxygenase family of enzymes on the store-operated calcium current I(CRAC) in rat basophilic leukaemia cells.

In non-excitable cells, the major Ca2+ entry pathway is the store-operated pathway in which emptying of intracellular Ca2+ stores activates Ca2+ channels in the plasma membrane. In many cell types, store-operated influx gives rise to a Ca2+-selective current called I(CRAC) (Ca2+ release-activated Ca2+ current). Using both the whole-cell patch clamp technique to measure I(CRAC) directly and fluorescent Ca2+ imaging, we have examined the role of the lipo-oxygenase pathway in the activation of store-operated Ca2+ entry in the RBL-1 rat basophilic leukaemia cell-line. Pretreatment with a variety of structurally distinct lipo-oxygenase inhibitors all reduced the extent of I(CRAC), whereas inhibition of the cyclo-oxygenase enzymes was without effect. The inhibition was still seen in the presence of the broad protein kinase blocker staurosporine, or when Na+ was used as the charge carrier through CRAC channels. The lipo-oxygenase blockers released Ca2+ from intracellular stores but this was not associated with subsequent Ca2+ entry. Lipo-oxygenase blockers also reduced both the amount of Ca2+ that could subsequently be released by the combination of thapsigargin and ionomycin in Ca2+-free solution and the Ca2+ influx component that occurred when external Ca2+ was re-admitted. The inhibitors were much less effective if applied after I(CRAC) had been activated. This inhibition of I(CRAC) could not be rescued by dialysis with 5(S)-hydroxyperoxyeicosa-6E,8Z,11Z,14Z,tetraenoic acid (5-HPETE), the first product of the 5-lipo-oxygenase pathway. Our findings indicate that exposure to pharmacological tools that inhibit the lipo-oxygenase enzymes all decrease the extent of activation of the current. Our results raise the possibility that a lipo-oxygenase might be involved in the activation of I(CRAC). Alternative explanations are also discussed.

Adenosine Triphosphate↗

PubMed1995

Cyclo-oxygenase distribution in human placenta and decidua does not change with labour after term or preterm delivery.

Our objective was to determine, in an immunohistochemical study, the distribution of cyclo-oxygenase in placental and decidual tissues before and after labour at varying gestations. Subjects were pregnant women undergoing singleton delivery after idiopathic preterm labour at less than 34 weeks' gestation (n = 13); spontaneous term labour at 37-42 weeks' gestation (n = 11); preterm caesarean section at less than 34 weeks' gestation for pre-eclampsia or intrauterine growth retardation (n = 8); elective term caesarean section at 37-42 weeks' gestation for cephalopelvic disproportion (n = 7). Within the placental basal plate cyclo-oxygenase was localised in decidual stromal cells, extravillous trophoblast, and the apical border of villous syncytiotrophoblast, both before and after labour, regardless of gestational age. Within the placental membranes, decidual stromal cells showed intense cytoplasmic cyclo-oxygenase labelling and the chorion laeve strong membrane-associated cyclo-oxygenase reactivity before and after labour, regardless of gestational age. The amnion showed a heterogeneous staining pattern. After labour at term there was either positive cytoplasmic (5/10) or apical staining (5/10). Cyclo-oxygenase was demonstrated in an apical distribution in only 50% of the other subject groups. Cyclo-oxygenase is present in both decidua and placental tissues prior to the onset of labour at term. Demonstration of decidual and trophoblastic cyclo-oxygenase in both preterm and term subjects suggests that differential availability of cyclo-oxygenase is unlikely to be important in preterm delivery. Research into the withdrawal of inhibition or increased substrate availability is most likely to shed light on the signal pathways involved in the onset of labour, regardless of gestation.

Cell Membrane↗