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 Superoxide-forming NADPH oxidase preparation of pig polymorphonuclear leucocyte

1982 ◽  
Vol 205 (3) ◽  
pp. 593-601 ◽  
Author(s):  
H Wakeyama ◽  
K Takeshige ◽  
R Takayanagi ◽  
S Minakami
Keyword(s):  
Nadph Oxidase ◽  
Enzyme System ◽  
Polymorphonuclear Leucocyte ◽  
Tween 20 ◽  
Polymorphonuclear Leucocytes ◽  
Resting Cells ◽  
Superoxide Formation ◽  
Km Value ◽  
Nadph Oxidation ◽  
Relationship Of

A phagocytic vesicle fraction with high NADPH-dependent superoxide-forming activity was obtained in large quantity from pig blood polymorphonuclear leucocytes, phagocytosing oil droplets in the presence of cyanide. The activity of the homogenate of the phagocytosing cells was 40 times that of the resting cells, and 70% of the activity in the homogenate was recovered in the phagocytic vesicle fraction. Essentially all of the superoxide-forming activity was extracted by repeated extraction with a mixture containing deoxycholate and Tween 20. The extract had a superoxide-forming activity of 1 mumol/min per mg of protein with NADPH, and one-fifth of this with NADH, Km values being similar to those of the vesicle fraction (40 microM for NADPH and 400 microM for NADH). A stoichiometric relationship of 1:2 for NADPH oxidation and superoxide formation was obtained, in agreement with the reaction NADPH +2O2 leads to NADP+ + 2O2 -. + H+. The activity of the extract was enhanced 2-fold by the addition of FAD, suggesting that the flavin is a component of the enzyme system. The Km value for FAD was 0.077 microM. The activities in both vesicle fraction and extract were labile even on refrigeration, but could be kept for several months at −70 degrees C.

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.

Inhibition of Escherichia coli Trimethylamine-N-oxide Reductase by Food Preservatives1

1982 ◽  
Vol 45 (3) ◽  
pp. 241-243 ◽  
Author(s):  
M. KRUK ◽  
J. S. LEE
Keyword(s):  
Escherichia Coli ◽  
Benzoic Acid ◽  
Sorbic Acid ◽  
Reductase Activity ◽  
Resting Cells ◽  
E Coli

Trimethylamine-N-oxide (TMA-O) reductase activity of resting cells of Escherichia coli was inhibited by tetrasodium ethylenediaminetetraacetate (Na4EDTA), benzoic acid (BA and methylparaben (MP). The 50% inhibitory concentrations of Na4EDTA, BA and MP were 20.2, 1.2 and 32.4 mM, respectively. BA at pH 6.5 or below most effectively inhibited the TMA-O reductase. Sorbic acid (SA), up to 0.70 mM, had no effect on TMA-O reductase activity, but SA inhibited the growth and subsequent TMA production in E. coli at or above 0.3S mM.

scholarly journals Insight into the Mechanistic Basis of the Hysteretic-Like Kinetic Behavior of Thioredoxin-Glutathione Reductase (TGR)

2018 ◽  
Vol 2018 ◽  
pp. 1-17 ◽  
Author(s):  
Juan L. Rendón ◽  
Mauricio Miranda-Leyva ◽  
Alberto Guevara-Flores ◽  
José de Jesús Martínez-González ◽  
Irene Patricia del Arenal ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Active Form ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Hysteretic Behavior ◽  
Low Concentrations ◽  
Mechanistic Basis ◽  
High Concentrations ◽  
Thioredoxin Glutathione Reductase

A kinetic study of thioredoxin-glutathione reductase (TGR) from Taenia crassiceps metacestode (cysticerci) was carried out. The results obtained from both initial velocity and product inhibition experiments suggest the enzyme follows a two-site ping-pong bi bi kinetic mechanism, in which both substrates and products are bound in rapid equilibrium fashion. The substrate GSSG exerts inhibition at moderate or high concentrations, which is concomitant with the observation of hysteretic-like progress curves. The effect of NADPH on the apparent hysteretic behavior of TGR was also studied. At low concentrations of NADPH in the presence of moderate concentrations of GSSG, atypical time progress curves were observed, consisting of an initial burst-like stage, followed by a lag whose amplitude and duration depended on the concentration of both NADPH and GSSG. Based on all the kinetic and structural evidence available on TGR, a mechanism-based model was developed. The model assumes a noncompetitive mode of inhibition by GSSG in which the disulfide behaves as an affinity label-like reagent through its binding and reduction at an alternative site, leading the enzyme into an inactive state. The critical points of the model are the persistence of residual GSSG reductase activity in the inhibited GSSG-enzyme complexes and the regeneration of the active form of the enzyme by GSH. Hence, the hysteretic-like progress curves of GSSG reduction by TGR are the result of a continuous competition between GSH and GSSG for driving the enzyme into active or inactive states, respectively. By using an arbitrary but consistent set of rate constants, the experimental full progress curves were successfully reproduced in silico.

scholarly journals Coproporphyrinogenase in tobacco (Nicotiana tabacum L.)

1970 ◽  
Vol 117 (2) ◽  
pp. 215-220 ◽  
Author(s):  
W. P. Hsu ◽  
G. W. Miller
Keyword(s):  
Enzyme Activity ◽  
Nicotiana Tabacum ◽  
Differential Centrifugation ◽  
Enzyme Protein ◽  
Sodium Amalgam ◽  
Low Concentrations ◽  
Nicotiana Tabacum L ◽  
Km Value ◽  
Ammonium Sulphate Fractionation ◽  
Final Purification

1. Coproporphyrinogenase was extracted and purified from tobacco (Nicotiana tabacum L.). Enzyme activity was mainly located in mitochondria rather than in chloroplasts. The enzyme was purified by differential centrifugation, ammonium sulphate fractionation, calcium phosphate gel adsorption and dialysis. A 69-fold final purification was obtained. 2. An apparent Km value of 3.6×10−5m was found, the value being largely dependent on the amount of coproporphyrin III recovered after reduction with sodium amalgam to coproporphyrinogen III. Protoporphyrin formation was linear up to 3h and decreased with further incubation. The enzyme activity increased with the concentration of enzyme protein up to 30μg/ml of solution. 3. Enzyme activity was greatly enhanced by increasing Fe2+ concentrations up to 0.5mm, beyond which inhibition occurred. Co2+ and Mn2+ were also found to activate at low concentrations (0.1mm) and inhibit at higher concentrations (5mm). Fe3+ and Cu2+, both at 0.1mm, and o-phenanthroline and EDTA, each at 1mm, were found to be inhibitory.

scholarly journals The mechanism of proton translocation driven by the respiratory nitrate reductase complex of Escherichia coli

1980 ◽  
Vol 190 (1) ◽  
pp. 79-94 ◽  
Author(s):  
Robert W. Jones ◽  
Alan Lamont ◽  
Peter B. Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Mutant Strain ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Proton Translocation ◽  
Deficient Mutant ◽  
Benzyl Viologen ◽  
Low Concentrations ◽  
Nitrite Reductase Activity

Low concentrations (1–50μm) of ubiquinol1 were rapidly oxidized by spheroplasts of Escherichia coli derepressed for synthesis of nitrate reductase using either nitrate or oxygen as electron acceptor. Oxidation of ubiquinol1 drove an outward translocation of protons with a corrected →H+/2e− stoichiometry [Scholes & Mitchell (1970) J. Bioenerg.1, 309–323] of 1.49 when nitrate was the acceptor and 2.28 when oxygen was the acceptor. Proton translocation driven by the oxidation of added ubiquinol1 was also observed in spheroplasts from a double quinone-deficient mutant strain AN384 (ubiA−menA−), whereas a haem-deficient mutant, strain A1004a, did not oxidize ubiquinol1. Proton translocation was not observed if either the protonophore carbonyl cyanide m-chlorophenylhydrazone or the respiratory inhibitor 2-n-heptyl-4-hydroxyquinoline N-oxide was present. When spheroplasts oxidized Diquat radical (DQ+) to the oxidized species (DQ++) with nitrate as acceptor, nitrate was reduced to nitrite according to the reaction: [Formula: see text] and nitrite was further reduced in the reaction: [Formula: see text] Nitrite reductase activity (2) was inhibited by CO, leaving nitrate reductase activity (1) unaffected. Benzyl Viologen radical (BV+) is able to cross the cytoplasmic membrane and is oxidized directly by nitrate reductase to the divalent cation, BV++. In the presence of CO, this reaction consumes two protons: [Formula: see text] The consumption of these protons could not be detected by a pH electrode in the extra-cellular bulk phase of a suspension of spheroplasts unless the cytoplasmic membrane was made permeable to protons by the addition of nigericin or tetrachlorosalicylanilide. It is concluded that the protons of eqn. (3) are consumed at the cytoplasmic aspect of the cytoplasmic membrane. Diquat radical, reduced N-methylphenazonium methosulphate and its sulphonated analogue N-methylphenazonium-3-sulphonate (PMSH) and ubiquinol1 are all oxidized by nitrate reductase via a haem-dependent, endogenous quinone-independent, 2-n-heptyl-4-hydroxyquinoline N-oxide-sensitive pathway. Approximate→H+/2e− stoichiometries were zero with Diquat radical, an electron donor, 1.0 with reduced N-methylphenazonium methosulphate or its sulphonated analogue, both hydride donors, and 2.0 with ubiquinol1 (QH2), a hydrogen donor. It is concluded that the protons appearing in the medium are derived from the reductant and the observed→H+/2e− stoichiometries are accounted for by the following reactions occurring at the periplasmic aspect of the cytoplasmic membrane.: [Formula: see text]

scholarly journals Regulation of 5-hydroxyeicosanoid dehydrogenase activity in monocytic cells

2007 ◽  
Vol 403 (1) ◽  
pp. 157-165 ◽  
Author(s):  
Karl-Rudolf Erlemann ◽  
Chantal Cossette ◽  
Gail E. Grant ◽  
Gue-Jae Lee ◽  
Pranav Patel ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reverse Reaction ◽  
Eicosatetraenoic Acid ◽  
Resting Cells ◽  
U937 Cells ◽  
Forward Reaction ◽  
Monocytic Cells ◽  
Low Concentrations ◽  
Ping Pong ◽  
1,25 Dihydroxy Vitamin D3

The 5-lipoxygenase product 5-oxo-ETE (5-oxo-eicosatetraenoic acid) is a highly potent granulocyte chemoattractant that is synthesized from 5-HETE (5-hydroxyeicosatetraenoic acid) by 5-HEDH (5-hydroxyeicosanoid dehydrogenase). In the present study, we found that 5-HEDH activity is induced in U937 monocytic cells by differentiation towards macrophages with PMA and in HL-60 myeloblastic cells by 1,25-dihydroxy-vitamin D3. We used PMA-differentiated U937 cells to investigate further the regulation of 5-HEDH. This enzyme exhibits approx. 10000-fold selectivity for NADP+ over NAD+ as a cofactor for the oxidation of 5-HETE, which is maximal at pH 10.2. In contrast, the reverse reaction (5-oxo-ETE→5-HETE) is NADPH-dependent and is maximal at pH 6. Although the Km for the forward reaction (670 nM) is about twice that for the reverse reaction at neutral pH, the Vmax is approx 8-fold higher. The oxidation of 5-HETE to 5-oxo-ETE is supported by very low concentrations of NADP+ (Km 139 nM), inhibited by NADPH (Ki 224 nM) and is consistent with a ping-pong mechanism. The amount of 5-oxo-ETE synthesized by 5-HEDH depends on the ratio of NADP+ to NADPH. Exposure of U937 cells to oxidative stress (t-butyl hydroperoxide) increased the ratio of NADP+ to NADPH from approx. 0.08 in resting cells to approx. 3, and this was accompanied by a dramatic increase in 5-HETE oxidation to 5-oxo-ETE. We conclude that differentiation of monocytic cells towards macrophages results in enhanced 5-oxo-ETE synthesis and that the ability of cells to synthesize 5-oxo-ETE is tightly regulated by the ratio of intracellular NADP+ to NADPH.

PHOSPHATASE OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES

1949 ◽  
Vol 27e (5) ◽  
pp. 290-307 ◽  
Author(s):  
D. M. Cram ◽  
R. J. Rossiter
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Activity ◽  
Acid Phosphatase ◽  
Bile Salts ◽  
Polymorphonuclear Leucocytes ◽  
Alkyl Sulphate ◽  
Low Concentrations ◽  
Activity Curve ◽  
Surface Active ◽  
Active Substances

Rabbit polymorphonuclear leucocytes contain an active phosphatase that readily hydrolyzes disodium phenyl phosphate. The pH activity curve of the enzyme was found to have two maxima, one in the region of pH 10 and the other in the region of pH 5. The alkaline phosphatase was much more active than the acid phosphatase. The concentration of alkaline phosphatase in rabbit white cells was approximately one thousand times that of the enzyme in the serum. Under the conditions of study, the alkaline phosphatase activity was proportional to the concentration of the enzyme. The effect of substrate concentration on the enzyme activity was studied and the Michaelis constant (Ks) determined. An excess of substrate inhibited the enzyme. The course of the reaction was linear with time for the first 60 min.; after 90 min. the activity fell off faster than would be expected if the reaction were of the first order.Magnesium and glycine, in low concentrations, caused an increase in the enzyme activity, whereas zinc, cyanide, borate, phosphate, bile salts, and glycine, in higher concentrations, were inhibitory. Fluoride had no demonstrable effect. Surface-active substances, such as saponin, bile salts, or alkyl sulphate, liberated the enzyme from the cells. Similar results were obtained when α-glycerophosphate or β-glycerophosphate was used as the substrate.The alkaline phosphatase can be considered to belong to Class AI of Folley and Kay (22) and the acid phosphatase to Class AII. The alkaline phosphatase can also be considered to be a Phosphatase II of Cloetens (9).

Reverse-Phase High Performance Liquid Chromatography of Metal Chelates of 4-(2-Pyridylazo)resorcinol and 4-(2-Thiazolylazo)resorcinol. Simultaneous Determination of Low Concentrations of Co, Ni and Fe

1994 ◽  
Vol 59 (10) ◽  
pp. 2209-2226 ◽  
Author(s):  
Josef Doležal ◽  
Lumír Sommer
Keyword(s):  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Cetyltrimethylammonium Bromide ◽  
Ion Pair ◽  
Metal Chelates ◽  
Reverse Phase ◽  
Rp Hplc ◽  
Low Concentrations ◽  
Methanol Solutions

The behaviour of stable and inert metal chelates of 4-(2-pyridylazo)resorcinol and 4-(2-thiazolylazo)resorcinol in RP HPLC and in its ion-pair modification (IP RP HPLC) on Separon SGX RPS silica gel was studied. Good results were obtained by the ion-pair variant in water methanol solutions at pH 7 in the presence of cetyltrimethylammonium bromide. The technique proved to be convenient for the preconcentration, separation and quantitation of low concentrations of Fe, Co, and Ni in waters.

scholarly journals Effect of Photosensitization on Inactivation of Aspergillus flavus in Maize

Proceedings ◽  
2020 ◽  
Vol 36 (1) ◽  
pp. 116
Author(s):  
Rafael Nguenha ◽  
Maral Seidi Damyeh ◽  
Hung T. Hong ◽  
Yasmina Sultanbawa
Keyword(s):  
Fungal Growth ◽  
Tween 80 ◽  
Green Technology ◽  
Tween 20 ◽  
Maize Kernel ◽  
Low Concentrations ◽  
Food And Feed ◽  
Potential Alternative ◽  
Maize Kernels

Mycotoxins are naturally occurring toxins produced by certain types of fungi thatcontaminate food and feed, posing serious health risks to human and livestock. Photosensitizationis a light-based technique, which has emerged as a novel and promising green technology to controlmicrobial growth in food and feed. This study aimed to evaluate the effect of solvent mediumincluding ethanol (EtOH), 50% (v/v) propylene glycol (PG), 20 % (v/v) tween 20 (TW-20), and 20 %(v/v) tween 80 (TW-80), on curcumin-mediated photosensitization to inactivate Aspergillus flavusspores in vitro and on the surface of yellow and white maize kernel and flour. Results showed areduction in the phototoxic activity of curcumin in TW-20 and TW-80. However, curcumin-basedphotosensitization using EtOH and PG as solvents led to a significant decrease in the colony formingability of A. flavus spores in vitro, up to 2.04 and 3.33 log colony-forming unit (CFU), respectively.Interestingly, fungal growth was delayed in photosensitized maize kernel and flour for 14 and 7days, respectively, which were stored at 25 °C. Consequently, no Aflatoxin B1 (AFB1) was detectedin maize kernels after 20 days of storage at 25 °C, whereas accumulation of the toxin was reducedby 91% in photosensitized flour. Thus, photosensitization showed to be a potential alternative toreduce A. flavus contamination on maize kernel and flour, giving rise to low concentrations of AFB1.This technique has the potential for use in feed applications resulting in the reduction of postharvestlosses in maize.

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