scholarly journals The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations

1984 ◽  
Vol 223 (2) ◽  
pp. 337-344 ◽  
Author(s):  
A R Cross ◽  
J F Parkinson ◽  
O T G Jones
Keyword(s):  
Cytochrome B ◽  
Cetyltrimethylammonium Bromide ◽  
Methyl Viologen ◽  
Initial Rate ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
O2 Uptake ◽  
Sharp Maximum ◽  
Low Concentrations ◽  
O2 Production

An NADPH-dependent O2.-−generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.

The Slow Phase of the Electrochromic Shift in Relation to the Q-Cycle in Thylakoids

1987 ◽  
Vol 14 (1) ◽  
pp. 29 ◽  
Author(s):  
AB Hope ◽  
DB Matthews
Keyword(s):  
Electron Transport ◽  
Cytochrome B ◽  
Methyl Viologen ◽  
Slow Phase ◽  
Half Time ◽  
Electrochromic Shift ◽  
Q Cycle ◽  
Reducing Conditions ◽  
Low Concentrations ◽  
Micromolar Range

The electrochromic signal in oxidising conditions with whole-chain electron transport from water to ferricyanide or methyl viologen was compared with that under reducing conditions with electron transport from duroquinol to methyl viologen. Under oxidising conditions, in a train of flashes at 10 Hz, the slow phase in the growth of the electrochromic signal was not apparent after a few flashes whilst under reducing conditions it persisted. The slow phase of the electrochromic signal was also examined particularly under conditions favouring the operation of an apparent Q-cycle, namely oxidising conditions plus the addition of low concentrations of valinomycin and flashes given at 5-10 Hz. The slow phase was retained in the presence of valinomycin at flash rates up to 10 Hz and its decay was accelerated. The half-time for the slow phase was 18-20 ms (whole-chain), or 3-5 ms (reducing conditions, duroquinol/methyl viologen). There was a lag of 3 ms before the rise of the slow phase with ferricyanide. The onset was accelerated by valinomycin under oxidising conditions. The slow phase was sharply inhibited by 2-n-heptyl- and 2-n-nonyl-4-hydroxyquinoline N-oxide (HQNO and NQNO) to at least half its maximum extent by 0.1 �M (HQNO) or 0.05 �M (NQNO); further inhibition took place in the micromolar range. The slow phase is discussed in terms of probable electrogenic events in the cytochrome b/f complexes and their kinetics. We support the hypothesis that its basic cause is the transverse, intramembrane passage of electrons and show that this passage is controlled by the prevailing intramembrane potential difference, an estimated 85-140 mV sufficing for half inhibition of the slow phase.

Effect of cetyltrimethylammonium bromide on the electrochemiluminiscence and electrooxidation of luminol

1983 ◽  
Vol 48 (2) ◽  
pp. 477-483 ◽  
Author(s):  
Jan Lasovský ◽  
František Grambal
Keyword(s):  
Cetyltrimethylammonium Bromide ◽  
Linear Sweep Voltammetry ◽  
Critical Micellar Concentration ◽  
Peak Height ◽  
Anodic Peak ◽  
Alkaline Solutions ◽  
Platinum Electrodes ◽  
Micellar Solutions ◽  
Linear Sweep ◽  
Low Concentrations

The electrooxidation of luminol in alkaline solutions in the presence of cetyltrimethylammonium bromide (I) was studied by linear sweep voltammetry on fixed and vibrating platinum electrodes. The presence of I in low concentrations (below the critical micellar concentration) brings about aggregation of the luminol, which is manifested by an increase in the anodic peak height and its shift towards lower potentials. In micellar solutions the peak height decreases owing to the slower diffusion of the bulkier micelles, the shift to lower potentials being preserved. The light-voltage curves correspond with the voltammetric curves, exhibiting identical shifts of the peak potentials in dependence on the concentration of the surfactant.

scholarly journals NADPH-dependent reduction of 2,6-dichlorophenol-indophenol by the phagocytic vesicles of pig polymorphonuclear leucocytes

1983 ◽  
Vol 210 (2) ◽  
pp. 577-581 ◽  
Author(s):  
H Wakeyama ◽  
K Takeshige ◽  
S Minakami
Keyword(s):  
Oxidation Rate ◽  
Cetyltrimethylammonium Bromide ◽  
Reductase Activity ◽  
Tween 20 ◽  
Polymorphonuclear Leucocytes ◽  
Resting Cells ◽  
Low Concentrations ◽  
Km Value ◽  
Nadph Oxidation

NADPH-dependent 2,6-dichlorophenol-indophenol (DCIP) reductase activity in the homogenate of phagocytosing pig polymorphonuclear leucocytes was twice that of the resting cells and the activity in the phagocytic vesicles corresponded to the activity increment due to phagocytosis. The apparent Km value of the reductase activity in the vesicles for NADPH was 30 microM, which is similar to that of the NADPH-dependent superoxide (O2-) formation. Increasing the DCIP reductase activity by increasing the DCIP concentration caused a decrease in the O2- –forming activity, the NADPH oxidation rate being constant and independent of the dye concentration. p-Chloromercuribenzoate and cetyltrimethylammonium bromide at low concentrations inhibited the O2- –forming activity of the vesicles without inhibiting the DCIP reductase. Quinacrine inhibited both O2- formation and DCIP reduction. The DCIP reductase activity could be extracted with a mixture of deoxycholate and Tween-20, which extracts the O2- –forming activity. The reductase activity in the extract was enhanced 2-fold by the addition of FAD, and its apparent Km was 0.085 microM. These results indicate that the NADPH-dependent DCIP reductase activity of the phagocytic vesicles is catalysed by a flavin-containing component of the O2- –forming system.

scholarly journals Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria

1980 ◽  
Vol 191 (2) ◽  
pp. 421-427 ◽  
Author(s):  
J F Turrens ◽  
A Boveris
Keyword(s):  
Cytochrome B ◽  
Nadh Dehydrogenase ◽  
Submitochondrial Particles ◽  
Bovine Heart ◽  
Biphasic Effect ◽  
Carbonyl Cyanide ◽  
Autocatalytic Process ◽  
Ph Range ◽  
Energy Dependent ◽  
O2 Production

Submitochondrial particles from bovine heart in which NADH dehydrogenase is reduced by either addition of NADH and rotenone or by reversed electron transfer generate 0.9 +/- 0.1 nmol of O2-/min per mg of protein at pH 7.4 and at 30 degrees C. When NADH is used as substrate, rotenone, antimycin and cyanide increase O2- production. In NADH- and antimycin-supplemented submitochondrial particles, rotenone has a biphasic effect: it increases O2- production at the NADH dehydrogenase and it inhibits O2- production at the ubiquinone-cytochrome b site. The generation of O2- by the rotenone, the uncoupler carbonyl cyanide rho-trifluoromethoxyphenylhydrazone and oligomycin at concentrations similar to those required to inhibit energy-dependent succinate-NAD reductase. Cyanide did not affect O2- generation at the NADH dehydrogenase, but inhibited O2- production at the ubiquinone-cytochrome b site. Production of O2- at the NADH dehydrogenase is about 50% of the O2- generation but the ubiquinone-cytochrome b area at pH 7.4. Additivity of the two mitochondrial sites of O2- generation was observed over the pH range from 7.0 to 8.8. AN O2–dependent autocatalytic process that requires NADH, submitochondrial particles and adrenaline is described.

The Physiology of Sea-Urchin Spermatozoa

1950 ◽  
Vol 26 (4) ◽  
pp. 396-409
Author(s):  
LORD ROTHSCHILD
Keyword(s):  
Sea Urchin ◽  
Seminal Plasma ◽  
Human Blood ◽  
Phosphate Buffer ◽  
Dry Weight ◽  
O2 Uptake ◽  
Low Concentrations ◽  
Initial Substrate ◽  
Rough Calculation ◽  
Initial Substrate Concentration

1. Spermatozoa and seminal plasma of Echinus esculentus contain catalase. 2. At 15° C., 4 ml. of a suspension of semen diluted with neutral phosphate buffer in the ratio 1:13 produced in 1 min. 90µl. O2 from an H2O2 solution containing 150 µl. O2. The dry weight of semen in the suspension was 45 mg. and the number of spermatozoa 8.55x109. Under the same conditions, seminal plasma obtained by centrifuging semen produced 50 µl. O2 in 1 min. The dry weight of seminal plasma in the suspension was 12 mg. Human blood, dry weight 229.3 mg./ml., must be diluted with phosphate buffer in the ratio 1:1700 to produce the same amount of O2 in 1 min. as the above suspension of semen. If catalatic activity is defined by the equation Ac = (gt)-1 In {a/(a-x)}, where g = weight in g./ml. of the catalase-containing material, t = 1 min., a = initial substrate concentration (H2O2), and x = amount of H2O2 decomposed in 1 min. at 15° C., Ac = 80-100, 150-200 and 6800 respectively for sea-urchin semen, sea-urchin seminal plasma and human blood. 3. The catalatic activity of semen and seminal plasma is strongly inhibited by hydroxylamine. 4. The O2 uptake and motility of sea-urchin spermatozoa is unaffected by M/5000 H2O2. Higher concentrations of H2O2, M/3000-5000, produce a pronounced ‘shock’ effect, from which the spermatozoa often completely recover. 5. Low concentrations of hydroxylamine, M/3000, reduce O2 uptake and motility. 6. Sea-urchin spermatozoa are almost instantly killed by combinations of hydroxylamine and H2O2, at concentrations which are relatively innocuous when the substances are added separately. 7. A rough calculation indicates that a single spermatozoon contains less than 500 molecules of catalase. 8. A new method of adding H2O2 to catalase-containing material in a manometer is described.

scholarly journals Studies on the electron-transfer mechanism of the human neutrophil NADPH oxidase

1989 ◽  
Vol 262 (2) ◽  
pp. 575-579 ◽  
Author(s):  
J A Ellis ◽  
A R Cross ◽  
O T G Jones
Keyword(s):  
Electron Transfer ◽  
Steady State ◽  
Nadph Oxidase ◽  
Cytochrome B ◽  
Human Neutrophil ◽  
Sodium Deoxycholate ◽  
Human Neutrophils ◽  
Electron Transfer Mechanism ◽  
State Reduction ◽  
O2 Production

A superoxide-generating NADPH oxidase was solubilized from phorbol 12-myristate 13-acetate-activated human neutrophils with a mixture of sodium deoxycholate (0.125%, w/v) and Lubrol-PX (0.125%, v/v). The solubilized preparation contained FAD (577 pmol/mg of protein) and cytochrome b-245 (479 pmol/mg of protein) and produced 11.61 mol of O2-./s per mol of cytochrome b (340 nmol of O2-./min per mg of protein). On addition of NADPH, the cytochrome b-245 was reduced by 7.9% and the FAD by 38% in the aerobic steady state; NADH addition caused little steady-state reduction of cytochrome b and FAD. In this preparation, and several others, the measured rate of O2-. production correlated with the turnover of cytochrome b calculated from the extent of cytochrome b-245 reduction under aerobic conditions. Addition of diphenyleneiodonium abolished the reduction of both the FAD and cytochrome b-245 components and inhibited O2-. production. The haem ligand imidazole inhibited O2-. generation and cytochrome b reduction while permitting FAD reduction. These results support the suggestion that the human neutrophil NADPH oxidase has the electron-transport sequence: NADPH-FAD-cytochrome b-245-O2.

Bioenergetics Studies of the Cyanobacterium Anabaena variabilis

1987 ◽  
Vol 42 (11-12) ◽  
pp. 1280-1284 ◽  
Author(s):  
Siegfried Scherer ◽  
Heike Sadowski ◽  
Peter Böger
Keyword(s):  
Electron Transport ◽  
Photosystem Ii ◽  
Cytochrome B ◽  
Anabaena Variabilis ◽  
Electron Donors ◽  
Free System ◽  
Low Concentrations ◽  
Cyanobacterium Anabaena ◽  
A Cell ◽  
C Complex

A cell-free system exhibiting both photophosphorylation (P/2e= 1) and oxidative phosphoryltion (P/O up to 0.8) is described for the cyanobacterium Anabaena variabilis. NADH ant NADPH were found to be equally effective as electron donors for oxidative phosphorylation. Low concentrations of UHDBT, an inhibitor of the cytochrome b/c complex of mitochondria ant loroplasts, were found to inhibit photosystem-II electron transport reactions, but did not affet the cytochrome b6/f-complex of Anabaena. The inhibition by myxothiazol, antimycin and heptyihydroxyquinoline corroborates the hypothesis that both respiration and photosynthesis share the cytochrome b6/f-complex.

scholarly journals Proton translocation by the mitochondrial cytochrome b-c1 complex is inhibited by NN′-dicyclohexylcarbodi-imide

1982 ◽  
Vol 206 (2) ◽  
pp. 419-421 ◽  
Author(s):  
B D Price ◽  
M D Brand
Keyword(s):  
Electron Transport ◽  
Cytochrome Oxidase ◽  
Cytochrome B ◽  
Rat Liver ◽  
Proton Translocation ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Mitochondrial Cytochrome ◽  
Low Concentrations ◽  
Mitochondrial Cytochrome B

NN'-Dicyclohexylcarbodi-imide at low concentrations decreases the H+/2e ratio for rat liver mitochondria over the span succinate to oxygen from 5.9 +/- 0.3 (mean +/- S.E.M.) to 4.0 +/- 0.1 and for the cytochrome b-c1 complex from 3.8 +/- 0.2 to 1.9 +/- 0.1, but has little effect on the H+/2e ratio of cytochrome oxidase. The decrease in stoicheiometry is due, not to uncoupling or inhibition of electron transport, but to inhibition of proton translocation. NN'-Dicyclohexylcarbodi-imide thus ‘decouples’ proton translocation in the cytochrome b-c1 complex.

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals The effects of acetaldehyde on nitrogenase, hydrogenase and photosynthesis in the cyanobacterium Anabaena cylindrica

1983 ◽  
Vol 212 (3) ◽  
pp. 755-758 ◽  
Author(s):  
B Slatyer ◽  
A Daday ◽  
G D Smith
Keyword(s):  
Methyl Viologen ◽  
Co2 Fixation ◽  
Electron Flow ◽  
Anabaena Cylindrica ◽  
O2 Uptake ◽  
Irreversible Inhibitor ◽  
Photosynthetic Electron Flow ◽  
Separate Electron ◽  
Cyanobacterium Anabaena ◽  
Flow Through

Acetaldehyde was shown to be an irreversible inhibitor of nitrogenase, hydrogenase, CO2 fixation and growth in the cyanobacterium Anabaena cylindrica, but had no effect on photosynthetic electron flow as measured by Methyl Viologen-dependent O2 uptake. The concentration-dependence of the inhibition of nitrogenase and hydrogenase activities was determined, and it was shown that acetaldehyde inhibition poses problems for anaerobic experiments in which the activities of these enzymes are measured in the presence of the frequently used glucose/glucose oxidase/catalase/ethanol O2 trap. It is suggested that acetaldehyde may find use as an inhibitor in experiments designed to separate electron flow through the photosystems from consequent fixation of CO2 and N2.

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