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1.
Recently, we demonstrated that angiotensin-(1–7) (Ang-(1–7)) stimulates the Na +-ATPase activity through a losartan-sensitive angiotensin receptor, whereas bradykinin inhibits the enzyme activity through the B 2 receptor [Regul. Pept. 91 (2000) 45; Pharmacol. Rev. 32 (1980) 1]. In the present paper, the effect of bradykinin (BK) on Ang-(1–7)-stimulated Na +-ATPase activity was evaluated. Preincubation of Na +-ATPase with 10 −9 M Ang-(1–7) increases enzyme activity from 7.9±0.9 to 14.1±1.5 nmol Pi mg −1 min −1, corresponding to an increase of 79% ( p<0.05). This effect is reverted by bradykinin in a dose-dependent manner (10 −14–10 −8 M), reaching maximal inhibitory effect at 10 −9 M. Des-Arg 9 bradykinin (DABK), an agonist of B 1 receptor, at the concentrations of 10 −9–10 −7 M, does not mimic the BK inhibitory effect, and des-Arg 9-[Leu 8]-BK (DALBK), a B 1 receptor antagonist, at the concentrations of 10 −10–10 −7 M, does not prevent the inhibitory effect of BK on Ang-(1–7)-stimulated enzyme. On the other hand, HOE 140, an antagonist of B 2 receptor, abolishes the inhibitory effect of BK on the Ang-(1–7)-stimulated enzyme in a dose-dependent manner, reaching maximal effect at 10 −7 M. Taken together, these data indicate that stimulation of B 2 receptors by BK can counteract the stimulatory effect of Ang-(1–7) on the proximal tubule Na +-ATPase activity. 相似文献
2.
We investigated the involvement of prostaglandin E (PGE) receptor subtype EP3 in the regulatory mechanism of duodenal HCO 3− secretion in rats. A proximal duodenal loop or a chambered stomach was perfused with saline, and HCO 3− secretion was measured using a pH-stat method and by adding 2 mM HCl. Mucosal acidification was achieved through 10 min of exposure to 10 mM HCl in the duodenum or 100 mM HCl in the stomach. Various EP agonists or the EP4 antagonist were given i.v., while the EP1 or EP3 antagonist was given s.c. or i.d., respectively. Sulprostone (EP1/EP3 agonists) stimulated duodenal HCO 3− secretion in a dose-dependent manner, and this response was inhibited by AE5-599 (EP3 antagonist) but not AE3-208 (EP4 antagonist). AE1-329 (EP4 agonist) also increased duodenal HCO 3− secretion, and this action was inhibited by AE3-208 but not AE5-599. The response to PGE 2 or acidification in the duodenum was partially attenuated by AE5-599 or AE3-208 alone but completely abolished by the combined administration. Duodenal damage caused by mucosal perfusion with 150 mM HCl for 4 h was worsened by pretreatment with AE5-599 and AE3-208 as well as indomethacin and further aggravated by co-administration of these antagonists. Neither the EP3 nor EP4 antagonist had any effect on the gastric response induced by PGE 2 or acidification. These results clearly demonstrate the involvement of EP3 receptors, in addition to EP4 receptors, in the regulation of duodenal HCO 3− secretion as well as the maintenance of the mucosal integrity of the duodenum against acid injury. 相似文献
3.
Zaltoprofen is a nonsteroidal antiinflammatory drug that has been proposed to inhibit with some selectivity the nociception mediated by the bradykinin (BK) B 2 receptor. In order to test the predictive power of this claim, we applied the drug to vascular smooth muscle assays previously found useful to characterize B 2 receptor antagonists (contractility, human isolated umbilical vein) or B 1 receptor antagonists (contraction, rabbit aorta; relaxation, rabbit mesenteric artery). Zaltoprofen (up to 30 μM) failed to antagonize BK or des-Arg 9-BK-induced contraction in the umbilical vein and aorta, respectively. The drug (1 μM) abated des-Arg 9-BK-induced, prostaglandin-mediated relaxation of the precontracted mesenteric artery, consistent with its known activity as a cyclooxygenase (COX) inhibitor. However, zaltoprofen (10 μM) did not inhibit kinin-stimulated phospholipase A 2 activity in HEK 293 cells expressing recombinant forms of the rabbit B 1 or B 2 receptors. Nonpeptide antagonists of either receptor subtype were active in this respect. The results do not support that zaltoprofen, a COX inhibitor, antagonizes kinin receptors or influences their signaling with selectivity in the tested systems. 相似文献
4.
In the present paper, the modulation of the basolateral membrane (BLM) Na +-ATPase activity of inner cortex from pig kidney by angiotensin II (Ang II) and angiotensin-(1–7) (Ang-(1–7)) was evaluated. Ang II and Ang-(1–7) inhibit the Na +-ATPase activity in a dose-dependent manner (from 10 −11 to 10 −5 M), with maximal effect obtained at 10 −7 M for both peptides. Pharmacological evidences demonstrate that the inhibitory effects of Ang II and Ang-(1–7) are mediated by AT 2 receptor: The effect of both polypeptides is completely reversed by 10 −8 M PD 123319, a selective AT 2 receptor antagonist, but is not affected by either (10 −12–10 −5 M) losartan or (10 −10–10 −7 M) A779, selective antagonists for AT 1 and AT (1–7) receptors, respectively. The following results suggest that a PTX-insensitive, cholera toxin (CTX)-sensitive G protein/adenosine 3′,5′-cyclic monophosphate (cAMP)/PKA pathway is involved in this process: (1) the inhibitory effect of both peptides is completely reversed by 10 −9 M guanosine 5′- O-(2-thiodiphosphate) (GDPβS; an inhibitor of the G protein activity), and mimicked by 10 −10 M guanosine 5′- O-(3-thiotriphosphate) (GTPγS; an activator of the G protein activity); (2) the effects of both peptides are mimicked by CTX but are not affected by PTX; (3) Western blot analysis reveals the presence of the G s protein in the isolated basolateral membrane fraction; (4) (10 −10–10 −6 M) cAMP has a similar and non-additive effect to Ang II and Ang-(1–7); (5) PKA inhibitory peptide abolishes the effects of Ang II and Ang-(1–7); and (6) both angiotensins stimulate PKA activity. 相似文献
5.
Ring segments of rat middle cerebral artery (MCA) were prepared for measurement of isometric force and precontracted with 10 −4 M uridine triphosphate (UTP). Concentration-effect curves (CEC) were constructed for bradykinin (BK, 10 −8–10 −5 M) in segments with functionally intect (E+) or denuded (E−) endothelium. E− segments did not dilate to BK. The BK receptor was characterized by application of specific B 1 or B 2 antagonists [des-Arg 9-Leu 8] BK (10 −5 M) and [
-Arg 0-Hyp 3-Thi 5-
-Tic 7-Oic 8] BK (HOE140,3 × 10 −7 M), respectively, or B 1 agonist [des-Arg 9] BK (10 −8–10 −4 M). Involvement of nitric oxide (NO) was tested with NG-nitro-
-arginine (LNNA, 10 −4 M). BK induced concentration-dependent relaxation with a maximal effect ( Emax) of 40.86 ± 1.50% at 10 −6 M and a pD 2 (−log 10 EC 50) of 6.818 ± 0.044. This relaxation could be prevented with HOE140 or LNNA, but was not influenced by [des-Arg 9-Leu 8] BK. [des-Arg 9] BK did not induce any effect. These results demonstrate that BK induced relaxation via endothelial B 2 receptors and release of NO in isolated rat MCA. 相似文献
6.
Trout bradykinin ([Arg 0,Trp 5,Leu 8]-BK) produces sustained and concentration–dependent contractions of isolated longitudinal smooth muscle from trout stomach, although mammalian BK is without effect. Circular dichroism studies have demonstrated that trout BK, unlike mammalian BK, does not adopt a stable β-turn conformation, even in the presence of sodium dodecyl sulfate (SDS) or trifluoroethanol. The myotropic actions of a series of analogs in which each amino acid in trout BK was replaced by either alanine or the corresponding D-isomer were investigated. The peptides with Ala 4, D-Pro 3, D-Trp 5, D-Ser 6, and D-Pro 7 substitutions were inactive and did not act as antagonists of trout BK. The analog with [Ala 5] was a weak partial agonist. The substitution (Arg 0 → Ala) led to >50-fold decrease in potency but, in contrast to the importance of Phe 8 in both BK and desArg 9-BK in activating the mammalian B 2 and B 1 receptors respectively, substitutions at Leu 8 in trout BK had only a minor effect on potency. Antagonists to the mammalian B 2 receptor generally contain a D-aromatic amino acid at position 7 of BK but the analog [Arg 0,Trp 5,D-Phe 7,Leu 8]-BK was a weak agonist at the trout receptor. Similarly, the potent nonpeptide mammalian B 2 receptor antagonist FR173657 was without effect on the action of trout BK. These data suggest the hypothesis that the receptor binding conformation of trout BK is defined by the central region (residues 3–7) of the peptide but is adopted only upon interaction with the receptor. The bioactive conformation is probably stabilized by an ionic interaction between Arg 0 in the peptide and an acidic residue in the receptor. 相似文献
7.
Intravenous administration of ovokinin(2–7), a cleavage peptide derived from ovalbumin, dose-dependently (0.1–5 mg/kg) lowered the mean arterial pressure (MAP) that was not accompanied by a significant change in the heart rate (HR) of urethane-anesthetized rats. The hypotensive effects of ovokinin(2–7) were five orders of magnitude lower compared to that of bradykinin and were largely prevented by pretreatment with the bradykinin B 2 receptor antagonist HOE140 (81.6±18.4%) and moderately affected by the B 1 receptor antagonist [des-Arg 10]-HOE140 (26.3±15.5%). Intracellular Ca 2+ levels, as measured by Fur 2-AM, were significantly elevated in cultured aorta smooth muscle cells by ovokinin(2–7). The increases were abolished by HOE140 and unaffected by [des-Arg 10]-HOE140. The elevation of intracellular Ca 2+ by ovokinin(2–7) was dependent on Ca 2+ entry from extracellular space as it was reduced in a Ca 2+-free solution. Pretreatment of the cells with the phospholipase C inhibitor U73122 (2 μM) eliminated the Ca 2+ increase by the peptide. PA phosphohydrolase and phospholipase A 2 inhibitors significantly reduced the responses as well. Our results show that ovokinin(2–7) modulates cardiovascular activity by interacting with B 2 bradykinin receptors. 相似文献
8.
Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H 2O 2 induces production of O 2− by activating NADPH oxidase. However, the mechanisms whereby H 2O 2 induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H 2O 2 on O 2− levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H 2O 2 markedly increased intracellular O 2− levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O 2− levels and attenuated cytotoxicity resulting from treatment with H 2O 2. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O 2− levels in PAEC treated with H 2O 2, suggesting that both NOS and NADPH oxidase contribute to H 2O 2-induced O 2− in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O 2− levels in PAEC treated with H 2O 2 to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H 2O 2 produces oxidative stress in endothelial cells by increasing intracellular O 2− levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H 2O 2 and oxidant-generating enzymes that may contribute to endothelial dysfunction. 相似文献
9.
1. Rate constants for reduction of paraquat ion (1,1′-dimethyl-4,4′-bipyridy-lium, PQ 2+) to paraquat radical (PQ +·) by e−aq and CO 2−· have been measured by pulse radiolysis. Reduction by e−aq is diffusion controlled ( k = 8.4·10 10 M −1·s −1) and reduction by CO 2−· is also very fast k = 1.5·10 10 M −1·s −1). 2. The reaction of paraquat radical with oxygen has been analysed to give rate constants of 7.7·108 M−1·s−1 and 6.5·108 M−1·s−1 for the reactions of paraquat radical with O2 and O2−·, respectively. The similarity in these rate constants is in marked contrast to the difference in redox potentials of O2 and O2−· (− 0.59 V and + 1.12 V, respectively). 3. These rate constants, together with that for the self-reaction of O2−·, have been used to calculate the steady-state concentration of O2−· under conditions thought to apply at the site of reduction of paraquat in the plant cell. On the basis of these calculations the decay of O2−· appears to be governed almost entirely by its self-reaction, and the concentration 5 μm away from the thylakoid is still 90% of that at the thylakoid itself. Thus, O2−· persists long enough to diffuse as far as the chloroplast envelope and tonoplast, which are the first structures to be damaged by paraquat treatment. O2−· is therefore sufficiently long-lived to be a candidate for the phytotoxic product formed by paraquat in plants. 相似文献
10.
Resonance Raman measurements have been performed with solutions of iodine-complexed synthetic amyloses ( DP 25–200), malto-oligomers ( DP 3–18, and -cylodextrin. Interest was focused on the minimum chain length for helical complex formation and a possible preferred length for the polyiodine chain. Four fundamental vibrations are observed at 164, 112, 52 and 24 cm −1. The 112 cm −1 Raman line was shown to arise both from free I 3− (enhanced at 363.8 nm excitation) and from bound iodine (relatively most intense at 457.9 nm excitation). The main signal of complexed iodine at 164 cm −1is enhanced at an excitation wavelength close to the long wavelength absorption maximum. This signal is observed firt with malto-octaose and -cyclodextrin. The less intense signals at 52 and 24 −1 are only detected at DP 15 and higher. Raman spectra give no evidence for a preferred length of the polyiodine chain. Significantly identical Raman spectra are obtained when using different molar ratios of I 2/KI solution or I 2 solution initially free of I − ions. The results are discussed in view of previous assignments of the Raman lines to I 2−, I 3−/I 2, and I 5− subunits. Our findings are incompatible with I 3− units as the only bound species. They are compatible with both I 3−/I 2 and I 3− subunits under certain conditions. In the case of I 2 solution used for complexation we favour the polyiodine chain model proposed previously by Cramer 35,36. The I 3− ions formed could function mainly as chain initiators, as has been suggested by Cesàro and Brant 30. 相似文献
11.
We have previously shown that crystals of calcium oxalate (COM) elicit a superoxide (O 2−) response from mitochondria. We have now investigated: (i) if other microparticles can elicit the same response, (ii) if processing of crystals is involved, and (iii) at what level of mitochondrial function oxalate acts. O 2− was measured in digitonin-permeabilized MDCK cells by lucigenin (10 μM) chemiluminescence. [ 14C]-COM dissociation was examined with or without EDTA and employing alternative chelators. Whereas mitochondrial O 2− in COM-treated cells was three- to fourfold enhanced compared to controls, other particulates (uric acid, zymosan, and latex beads) either did not increase O 2− or were much less effective (hydroxyapatite +50%, p < 0.01), with all at 28 μg/cm 2. Free oxalate (750 μM), at the level released from COM with EDTA (1 mM), increased O 2− (+50%, p < 0.01). Omitting EDTA abrogated this signal, which was restored completely by EGTA and partially by ascorbate, but not by desferrioxamine or citrate. Omission of phosphate abrogated O 2−, implicating phosphate-dependent mitochondrial dicarboxylate transport. COM caused a time-related increase in the mitochondrial membrane potential (Δψ m) measured using TMRM fluorescence and confocal microscopy. Application of COM to Fura 2-loaded cells induced rapid, large-amplitude cytosolic Ca 2+ transients, which were inhibited by thapsigargin, indicating that COM induces release of Ca 2+ from internal stores. Thus, COM-induced mitochondrial O 2− requires the release of free oxalate and contributes to a synergistic response. Intracellular dissociation of COM and the mitochondrial dicarboxylate transporter are important in O 2− production, which is probably regulated by Δψ m. 相似文献
12.
A procedure is described for preparing particles from cells of Micrococcus denitrificans which were broken osmotically after treatment with lysozyme. 1. 1. The preparations catalysed ATP synthesis coupled to O2 uptake or NO3− reduction. With NADH or succinate as the electron donors the P:O ratios were about 1.5 and 0.5, respectively; and the P:NO3− ratios were about 0.9 and 0.06, respectively. 2. 2. Addition of ADP or Pi to the reaction mixture increased the rates of NADH-dependent O2 uptake and NO3− reduction. Addition of 1 mM 2,4-dinitrophenol, which inhibited phosphorylation by 50–60%, increased the basal rates of electron transport. 3. 3. Evidence derived from spectrophotometry and from the differential inhibition by antimycin A of O2 and NO3− reduction leads to the conclusion that the nitrate reductase interacted with the respiratory chain in the region of the b-type cytochrome, and that the c-type cytochrome present was not involved in the reduction of NO3− to NO2−.
Abbreviations: TMPD; tetramethyl-p-phenylenediamine 相似文献
13.
The thermoluminescence band observed in chloroplasts after flash excitation at ambient temperatures has recently been identified as being due to recombination of the electron on the semiquinone form of the secondary plastoquinone acceptor, Q B, with positive charges on the oxygen-evolving enzyme, S 2 and S 3 (Rutherford, A.W., Crofts, A.R. and Inoue, Y. (1982) Biochim. Biophys. Acta 682, 457–465). Further investigation of this thermoluminescence confirms this assignment and provides information on the function of PS II. The following data are reported: (1) Washing of chloroplasts with ferricyanide lowers the concentration of Q B− in the dark and predictable changes in the extent of the thermoluminescence band are observed. (2) The thermoluminescence intensity arising from S 2Q B− is approximately one half of that arising from S 3Q B−. (3) Preflash treatment followed by dark adaptation results in changes in the intensity of the thermoluminescence band recorded after a series of flashes. These changes can be explained according to the above assignments for the origin of the thermoluminescence and if Q B− provides an important source of deactivating electrons for the S states. Computer simulations of the preflash data are reported using the above assumptions. Previously unexplained data already in the literature (Läufer, A. and Inoue, Y. (1980) Photobiochem. Photobiophys. 1, 339–346) can be satisfactorily explained and are simulated using the above assumptions. (4) Lowering the pH to pH 5.5 results in a shift of the S 2Q B− thermoluminescence band to higher temperatures while that arising from S 3Q B− does not shift. This effect is interpreted as indicating that Q B− is protonated and the S 2 to S 3 reaction involves deprotonation while the S 1 to S 2 reaction does not. 相似文献
14.
A novel nutrient removal/waste heat utilization process was simulated using semicontinuous cultures of the thermophilic cyanobacterium Fischerella. Dissolved inorganic carbon (DIC)-enriched cultures, maintained with 10 mg l −1 daily productivity, diurnally varying temperature (from 55°C to 26–28°C), a 12:12 light cycle (200 μE sec −1 m −2) and 50% biomass recycling into heated effluent at the beginning of each light period, removed > 95% of NO 3− + NO 2−−N, 71% of NH 3-N, 82% of PO 43− −P, and 70% of total P from effluent water samples containing approximately 400 μg l −1 combined N and 60 μg l −1 P. Nutrient removal was not severely impaired by an altered temperature gradient, doubled light intensity, or DIC limitation. Recycling 75% of the biomass at the end of each light period resulted in unimpaired NO 3− + NO 2− removal, 38–45% P removal and no net NH 3 removal. Diurnally varying P removal, averaging 50–60%, and nearly constant > 80% N removal, are therefore projected for a full-scale process with continuous biomass recycling. 相似文献
15.
Nitrogen dioxide (NO 2•) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO 2• were generated in < 1 μs using pulse radiolysis, and the kinetics of scavenging NO 2• by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO 2• with the thiols (the thiyl radical) was monitored via oxidation of 2,2′-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO 2• with glutathione, cysteine, and urate were estimated as 2 × 10 7, 5 × 10 7, and 2 × 10 7 M −1 s −1, respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO 2• at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of 3 × 10 7 M −1 s −1. The implications of these values are: (i) the lifetime of NO 2• in cytosol is < 10 μs; (ii) thiols are the dominant ‘sink’ for NO 2• in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO 2• is 0.2 μm in the cytoplasm and < 0.8 μm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO 2•; and (v) reactions between NO 2• and thiols/urate severely limit the likelihood of reaction of NO 2• with NO• to form N 2O 3 in the cytoplasm. 相似文献
16.
The perchlorate (ClO 4−)-respiring organism, strain perc1ace, can grow using nitrate (NO 3−) as a terminal electron acceptor. In resting cell suspensions, NO 3− grown cells reduced ClO 4−, and ClO 4− grown cells reduced NO 3−. Activity assays showed that nitrate reductase (NR) activity was 1.31 μmol min −1 (mg protein) −1 in ClO 4− grown cells, and perchlorate reductase (PR) activity was 4.24 μmol min −1 (mg protein) −1 in NO 3− grown cells. PR activity was detected within the periplasmic space, with activities as high as 14 μmol min −1 (mg protein) −1. The NR had a pH optimum of 9.0 while the PR had an optimum of 8.0. This study suggests that separate terminal reductases are present in strain perclace to reduce NO 3− and ClO 4−. 相似文献
17.
Rates of stepwise anation of cis-Cr(ox) 2(H 2O 2) − with SCN −/N 3−, Cr(acac) 2(H 2O) 2+ with SCN − and Cr(atda)(H 2O) 2 with SCN − have been investigated in weakly acidic aqueous solutions. Rate constants, kI and kII for the two steps in each system, are composite as kx = kx0+ kxX[X −] ( x = I, II; X − = SCN −, N 3−). These rate constants have been evaluated also as the corresponding Δ H≠ and Δ S≠ values. The results obtained and the plausible I d mechanism seem to suggest Cr---OOC bond dissociation (hence a strongly negative Δ S≠) generating the transition state in each system with outer-sphere association forming the precursor complex in the X − dependent paths. 相似文献
18.
The preparation and reaction chemistry of 1,3- and 1,2-diene and related complexes derived from metal carbonyl containing anions and allenic electrophiles are addressed. The preparation of some CpFe(CO) 2 η 1-diene complexes and their conversion into CpFe(CO) η 3-diene complexes is presented followed by reactions of CpMo(CO) 3−, CpW(CO) 3− and CpMo(CO) 2PR 3− anions with allenic electrophiles which produce metal complexed cyclobutenones (via CO and alkene insertions from the initially formed product) and 1,2-diene complexes, respectively. Lastly, the reactions of PPh 3(CO) 3Co − anions with allenic electrophiles are outlined which result in several different coordination geometries depending on the reaction conditions used. 相似文献
19.
The reduction of ferricytochrome c by O 2− and CO 2− was studied in the pH range 6.6–9.2 and Arrhenius as well as Eyring parameters were derived from the rate constants and their temperature dependence. Ionic effects on the rate indicate that the redox process proceeds through a multiply-positively charged interaction site on cytochrome c. It is shown that the reaction with O 2− and correspondingly with O 2 of ferrocytochrome c) is by a factor of approx. 10 3 slower than warranted by factors such as redox potential. Evidence is adduced to support the view that this slowness is connected with the role of water in the interaction between O 2−/O 2 and ferri-ferrocytochrome c in the positively charged interaction site on cytochrome c in which water molecules are specifically involved in maintaining the local structure of cytochrome c and participate in the process of electron equivalent transfer. 相似文献
20.
1. Difference spectra, at room and liquid N 2 temperatures, of S 2O 42−-, and NO 2−-reduced intact cells and cell-free preparations of Nitrobacter agilis demonstrated the presence of cytochromes of the c- and a-types. Reduction of cytochromes by succinate, and to a limited extent, by NADPH also occurred, provided KCN (0.1 mM) was also present. 2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O2 was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO2− consumed per mole of O2 consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO2− concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO2 to pH 8.0 at 0.01 M KNO2 with apparent Km's of 0.2 and 1.2 mM NO2−, respectively. Computations of the HNO2 concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO2 which was independent of pH, suggesting the involvement of HNO2 as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN3, NaCN and Na2S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO2− proved to be a competitive inhibitor of the nitrite oxidase system. 3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO3− increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO2 concentration an approximately constant Km of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO3− was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c. 4. The membrane-like composition of the nitrite oxidase system is indicated. 相似文献
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