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 Kinetic mechanism of sheep liver NADPH-dependent aldehyde reductase

1987 ◽  
Vol 242 (1) ◽  
pp. 143-150 ◽  
Author(s):  
K S De Jongh ◽  
P J Schofield ◽  
M R Edwards
Keyword(s):  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Enzyme Mechanism ◽  
Free Enzyme ◽  
Aldehyde Reductase ◽  
Binding Studies ◽  
Sheep Liver ◽  
Low Concentrations ◽  
High Concentrations ◽  
Inhibition Studies

The kinetic mechanism of the major sheep liver aldehyde reductase (ALR1) was studied with three aldehyde substrates: p-nitrobenzaldehyde, pyridine-3-aldehyde and D-glucuronate. In each case the enzyme mechanism was sequential and product-inhibition studies were consistent with an ordered Bi Bi mechanism, with the coenzymes binding to the free enzyme. Binding studies were used to investigate the interactions of substrates, products and inhibitors with the free enzyme. These provided evidence for the binding of D-glucuronate, L-gulonate and valproate, as well as NADP+ and NADPH. The enzyme was inhibited by high concentrations of D-glucuronate in a non-competitive manner, indicating that this substrate was able to bind to the free enzyme and to the E X NADP+ complex at elevated concentrations. Although the enzyme was inhibited by high pyridine-3-aldehyde concentrations, there was no evidence for the binding of this substrate to the free enzyme. Sheep liver ALR1 was inhibited by the ionized forms of alrestatin, sorbinil, valproate, 2-ethylhexanoate and phenobarbitone, indicating the presence of an anion-binding site similar to that described for the pig liver enzyme, which interacts with inhibitors and substrates containing a carboxy group. Sorbinil, valproate and 2-ethylhexanoate inhibited the enzyme uncompetitively at low concentrations and non-competitively at high concentrations, whereas phenobarbitone and alrestatin were non-competitive and uncompetitive inhibitors respectively. The significance of these results with respect to inhibitor and substrate binding is discussed.

scholarly journals Mitochondrial Thioredoxin-Glutathione Reductase from LarvalTaenia crassiceps(Cysticerci)

2010 ◽  
Vol 2010 ◽  
pp. 1-11 ◽  
Author(s):  
Alberto Guevara-Flores ◽  
Irene P. del Arenal ◽  
Guillermo Mendoza-Hernández ◽  
Juan Pablo Pardo ◽  
Oscar Flores-Herrera ◽  
...  
Keyword(s):  
Glutathione Reductase ◽  
Reductase Activity ◽  
Catalytic Efficiency ◽  
Dependent Manner ◽  
Exchange Reactions ◽  
Disulfide Exchange ◽  
Oxidative Inactivation ◽  
Disulfide Reductase ◽  
Thioredoxin Peroxidase ◽  
Thioredoxin Glutathione Reductase

Mitochondrial thioredoxin-glutathione reductase was purified from larvalTaenia crassiceps(cysticerci). The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At25∘Cspecific activities were437  ±  27mU mg-1and840  ±  49mU mg-1with thioredoxin and GSSG, respectively. ApparentKmvalues were0.87  ±  0.04 μM,41  ±  6 μM and19  ±  10 μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

scholarly journals Membrane perturbations induced by the apoptotic Bax protein

2002 ◽  
Vol 367 (3) ◽  
pp. 849-855 ◽  
Author(s):  
Raquel F. EPAND ◽  
Jean-Claude MARTINOU ◽  
Sylvie MONTESSUIT ◽  
Richard M. EPAND
Keyword(s):  
Energy Transfer ◽  
Active Form ◽  
Caspase 8 ◽  
Detergent Concentration ◽  
Bax Protein ◽  
Low Concentrations ◽  
Apoptotic Protein ◽  
Oligomeric Form ◽  
Disulphonic Acid ◽  
High Concentrations

The apoptotic protein Bax, in oligomeric form, is effective in promoting both leakage and lipid mixing in liposomes composed of cardiolipin and phosphatidylethanolamine and/or phosphatidylcholine, upon the addition of calcium. In contrast, monomeric Bax is not active. At low concentrations at which caspase-8-cut Bid (tBid) alone has little effect on leakage, tBid augments the leakage caused by monomeric Bax. When solutions of oligomeric Bax are diluted to lower detergent concentrations than those required for Bax oligomerization, the protein is initially active in inducing liposomal leakage, indicating that the potency of the oligomeric form is not a consequence of being initially added to the liposomes in a high detergent concentration. However, in solutions of low detergent concentration, in the absence of liposomes, the oligomer gradually loses its lytic potency. This is accompanied by a loss of binding of bis-ANS (4,4′-dianilino-1,1′-binaphthyl-5,5′-disulphonic acid), indicating the loss of exposed hydrophobic sites, as well as a loss of the ability of the protein to translocate to membranes. Membrane translocation was measured by an energy-transfer assay. It was demonstrated that membrane binding was greatly enhanced by oligomerization and by the presence of calcium. Thus the membrane-active form of Bax is unstable in the absence of detergent or lipid. In addition, we find that translocation to the membrane is enhanced by oligomerization as well as by the presence of high concentrations of calcium.

scholarly journals Purification and characterization of morphinone reductase from Pseudomonas putida M10

1994 ◽  
Vol 301 (1) ◽  
pp. 97-103 ◽  
Author(s):  
C E French ◽  
N C Bruce
Keyword(s):  
Pseudomonas Putida ◽  
Reductase Activity ◽  
Kinetic Studies ◽  
Thiol Group ◽  
Kinetic Mechanism ◽  
Product Inhibition ◽  
Ph Optimum ◽  
Cell Extracts ◽  
Apparent Homogeneity ◽  
Ping Pong

The NADH-dependent morphinone reductase from Pseudomonas putida M10 catalyses the reduction of morphinone and codeinone to hydromorphone and hydrocodone respectively. Morphinone reductase was purified from crude cell extracts to apparent homogeneity in a single affinity-chromatography step using Mimetic Yellow 2. The purified enzyme was a dimeric flavoprotein with two identical subunits of M(r) 41,100, binding non-covalently one molecule of FMN per subunit. The N-terminal sequence was PDTSFSNPGLFTPLQ. Morphinone reductase was active against morphinone, codeinone, neopinone and 2-cyclohexen-1-one, but not against morphine, codeine or isocodeine. The apparent Km values for codeinone and 2-cyclohexen-1-one were 0.26 mM and 5.5 mM respectively. The steroids progesterone and cortisone were potent competitive inhibitors; the apparent K1 for cortisone was 35 microM. The pH optimum for codeinone reduction was 8.0 in phosphate buffer. No reverse reaction could be detected, and NADPH could not be used as a reducing substrate in place of NADH. Morphinone reductase activity was strongly inhibited by 0.01 mM CuSO4 and p-hydroxymercuribenzoate, suggesting the presence of a vital thiol group. Steady-state kinetic studies suggested a Ping Pong (substituted enzyme) kinetic mechanism; however, product-inhibition patterns were inconsistent with a classical Ping Pong mechanism. Morphinone reductase may, like several other flavoprotein dehydrogenases, operate by a hybrid two-site Ping Pong mechanism.

Physiological responses of Hizikia fusiformis (Phaeophyta) to mercury exposure

2015 ◽  
Vol 58 (2) ◽  
pp. 93-101
Author(s):  
Xifeng Zhu ◽  
Dinghui Zou ◽  
Yanhua Huang ◽  
Junming Cao ◽  
Guangcheng Sheng ◽  
...  
Keyword(s):  
Superoxide Dismutase ◽  
Reductase Activity ◽  
Negative Influence ◽  
Mercury Exposure ◽  
Relative Growth ◽  
Hizikia Fusiformis ◽  
Low Concentrations ◽  
Mercury Stress ◽  
Commercially Important ◽  
High Concentrations

Abstract Marine coastal ecosystems are suffering from metal pollution such as mercury. Hizikia fusiformis is a commercially important brown marine macroalga. Entire thalli of H. fusiformis were cultured in media with different mercury concentrations for 7 days to examine the physiological effects of mercury exposure on this species. The relative growth rate of H. fusiformis was significantly reduced at 0.04 mg l-1 mercury. Chlorophyll a and carotenoid contents were not affected by mercury concentrations ≤0.10 mg l-1, but significantly decreased at 0.20 and 0.40 mg l-1. Optimal quantum yield, maximum net photosynthetic rate, and apparent photosynthetic efficiency were reduced with increasing mercury concentrations. Malondialdehyde contents were significantly increased at 0.04–0.40 mg l-1 mercury. Superoxide dismutase activity increased at 0.02–0.10 mg l-1 mercury. Both superoxide dismutase and catalase activity strongly decreased at 0.20 and 0.40 mg l-1. Nitrate reductase activity declined at mercury concentrations ≥0.04 mg l-1. Consequently, the results suggest that H. fusiformis could tolerate mercury stress at very low concentrations but that mercury at high concentrations has toxic effects on H. fusiformis through its negative influence on photosynthesis and related physiology. The results also illuminate the physiological response mechanisms of algae to mercury exposure.

scholarly journals Primary Human Colonic Myofibroblasts Are Resistant toClostridium difficileToxin A-Induced, but Not Toxin B-Induced, Cell Death

2011 ◽  
Vol 79 (4) ◽  
pp. 1623-1630 ◽  
Author(s):  
N. Mullan ◽  
K. R. Hughes ◽  
Y. R. Mahida
Keyword(s):  
Cell Death ◽  
Smooth Muscle Actin ◽  
Active Form ◽  
Alpha Smooth Muscle Actin ◽  
Toxin A ◽  
Toxin B ◽  
Low Concentrations ◽  
Prolonged Culture ◽  
Mucosal Cells ◽  
High Concentrations

ABSTRACTColonic inflammation inClostridium difficileinfection is mediated by released toxins A and B. We investigated responses toC. difficiletoxins A and B by isolated primary human colonic myofibroblasts, which represent a distinct subpopulation of mucosal cells that are normally located below the intestinal epithelium. Following incubation with either purified toxin A or B, there was a change in myofibroblast morphology to stellate cells with processes that were immunoreactive for alpha-smooth muscle actin. Most of the myofibroblasts remained viable, with persistence of stellate morphology, despite exposure to high concentrations (up to 10 μg/ml) of toxin A for 72 h. In contrast, a majority of the toxin B-exposed myofibroblasts lost their processes prior to cell death over 24 to 72 h. At low concentrations, toxin A provided protection against toxin B-induced cell death. Within 4 h, myofibroblasts exposed to either toxin A or toxin B lost expression of the nonglucosylated form of Rac1, and there was also a loss of the active form of RhoA. Despite preexposure to high concentrations of toxin A for 3 h, colonic myofibroblasts were able to recover their morphology and proliferative capacity during prolonged culture in medium. However, toxin B-preexposed myofibroblasts were not able to recover. In conclusion, primary human colonic mucosal myofibroblasts are resistant to toxin A (but not toxin B)-induced cell death. Responses by colonic myofibroblasts may play an important role in mucosal protection, repair, and regeneration in colitis due toC. difficileinfection.

scholarly journals Adenylate cyclase in bloodstream forms of Trypanosoma (Trypanozoon) brucei sp

1978 ◽  
Vol 175 (1) ◽  
pp. 207-212 ◽  
Author(s):  
B R Martin ◽  
H P Voorheis ◽  
E L Kennedy
Keyword(s):  
Enzyme Activity ◽  
Plasma Membrane ◽  
Adenylate Cyclase ◽  
Trypanosoma Brucei ◽  
Product Inhibition ◽  
Low Concentrations ◽  
Mammalian Enzyme ◽  
High Concentrations ◽  
Opposing Effects ◽  
Substrate Binding Domain

1. The adenylate cyclase in Trypanosoma brucei is located in the plasma membrane. 2. A partial kinetic analysis of the properties of the enzyme revealed a Km for ATP of 1.75 mM and a Km for Mg2+ of 4mM. 3. At low concentrations, Mg2+ activated the enzyme directly in addition to its effect of lowering the concentration of inhibitory free ATP species. 4. At high concentrations, Mg2+ inhibited the enzyme. Furthermore, the enzyme was inhibited at any Mg2+ concentration if the concentration of ATP exceeded that of Mg2+. 5. The opposing effects of Mg2+ at low and high concentrations would be consistent with more than one binding site for Mg2+ on the enzyme. 6. A study of the patterns of product inhibition revealed little or no effect of 3′:5′-cyclic AMP, but a profound inhibition by pyrophosphate, which was competitive with respect to ATP (Ki 0.135 mM). This result suggests that the substrate-binding domain on T. brucei adenylate cyclase interacts mainly with the triphosphate portion of the ATP molecule. 7. The enzyme activity was unaffected by the usual mammalian enzyme effectors glucagon, adrenaline, adenosine, GTP and guanyl-5′-yl imidodiphosphate. 8. The enzyme was not activated by fluoride, instead a powerful inhibition was found. The enzyme was also inhibited by relatively high concentrations of Ca2+ (1 mM).

Neutrophil Elastase Potentiates Cathepsin G-lnduced Platelet Activation

1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak
Keyword(s):  
Neutrophil Elastase ◽  
Cell Activation ◽  
Thromboxane A2 ◽  
Creatine Phosphate ◽  
Dense Granule ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Low Concentrations ◽  
High Concentrations

SummaryWe have previously demonstrated that human neutrophil cathepsin G is a strong platelet agonist that binds to a specific receptor. This work describes the effect of neutrophil elastase on cathepsin G-induced platelet responses. While platelets were not activated by high concentrations of neutrophil elastase by itself, elastase enhanced aggregation, secretion and calcium mobilization induced by low concentrations of cathepsin G. Platelet aggregation and secretion were potentiated in a concentration-dependent manner by neutrophil elastase with maximal responses observable at 200 nM. Enhancement was observed when elastase was preincubated with platelets for time intervals of 10–60 s prior to addition of a low concentration of cathepsin G and required catalytically-active elastase since phenylmethanesulphonyl fluoride-inhibited enzyme failed to potentiate cell activation. Neutrophil elastase potentiation of platelet responses induced by low concentrations of cathepsin G was markedly inhibited by creatine phosphate/creatine phosphokinase and/or indomethacin, indicating that the synergism between elastase and cathepsin G required the participation of ADP and thromboxane A2. On the other hand, platelet responses were not attenuated by the PAF antagonist BN 52021, signifying that PAF-acether did not play a role in elastase potentiation. At higher concentrations porcine pancreatic elastase exhibits similar effects to neutrophil elastase, demonstrating that the effect of elastase was not unique to the neutrophil protease. While neutrophil elastase failed to alter the ability of cathepsin G to hydrolyze a synthetic chromogenic substrate, preincubation of platelets with elastase increased the apparent affinity of cathepsin G binding to platelets. In contrast to their effect on cathepsin G-induced platelet responses, neither neutrophil nor pancreatic elasatse potentiated aggregation or dense granule release initiated by ADP, PAF-acether, arachidonic acid or U46619, a thromboxane A2 mimetic. Moreover, unlike its effect on cathepsin G, neutrophil elastase inhibited thrombin-induced responses. The current observations demonstrate that elastase can potentiate platelet responses mediated by low concentrations of cathepsin G, suggesting that both enzymes may function synergistically to activate platelets under conditions where neutrophil degranulation occurs.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Aggregation of Cat Platelets in Vitro

1970 ◽  
Vol 23 (03) ◽  
pp. 601-620 ◽  
Author(s):  
Th. B Tschopp
Keyword(s):  
Adenosine Monophosphate ◽  
Blood Collection ◽  
Base Line ◽  
Reversible Aggregation ◽  
Low Concentrations ◽  
Irreversible Aggregation ◽  
High Concentrations ◽  
Two Phases ◽  
Improved Technique

SummaryAggregation of cat platelets in the citrated plasma is examined by means of Born’s absorptiometer. A marked tendency of the platelets of this species to spontaneous aggregation necessitated first of all the development of an improved technique of blood collection.A hypothesis according to which 5-HT is released from the platelets, explains the absence of oscillations on the base line of the absorptiometer, the absence of platelet swelling, when ADP is added, and the effect of stirring on the aggregation curves in cat PRP. The average volume of cat platelets amounts to 10.46 μ3 when directly fixed in the blood, when fixed from PRP to 12.17 μ3, when fixed from stirred PRP to 13.51 μ3.In low concentrations (0.3-2 μM) ADP produce reversible aggregation; in narrowly restricted, individually dissimilar mean concentrations irreversible aggregation in two phases and in high concentrations, irreversible aggregation in one phase. Like ADP serotonin produces 2 phase irreversible aggregation in concentrations of 3-10 μM, but unlike ADP, the aggregation velocity decreases again with high 5-HT concentrations (>100 μM). Adrenaline does not produce aggregation and it is likely that adenosine and adenosine monophosphate inhibit the aggregation by serotonin but not by ADP. Species differences in the aggregation of human, rabbit and cat platelets are discussed.

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