scholarly journals Localization of a carboxylic residue possibly involved in the inhibition of vacuolar H+-pyrophosphatase by N,N′-dicyclohexylcarbodi-imide

1999 ◽  
Vol 342 (3) ◽  
pp. 641-646 ◽  
Author(s):  
Su J. YANG ◽  
Shih S. JIANG ◽  
Soong Y. KUO ◽  
Shu H. HUNG ◽  
Ming F. TAM ◽  
...  

A vacuolar H+-pyrophosphatase (EC 3.6.1.1) that catalyses PPi hydrolysis and the electrogenic translocation of protons from the cytosol to the vacuole lumen, was purified from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.). Group-specific modification was used to identify a carboxylic residue involved in the inhibition of vacuolar H+-pyrophosphatase. Carbodi-imides, such as N,N′-dicyclohexylcarbodi-imide (DCCD) and 1-ethyl-3-(3-dimethylamino-propyl)carbodi-imide, and Woodward's reagent K caused a progressive decline in the enzymic activity of vacuolar H+-pyrophosphatase in a time- and concentration-dependent manner. The stoichiometry of labelling of the vacuolar H+-pyrophosphatase by [14C]DCCD determined that DCCD modifies one carboxylic residue per subunit of the enzyme. Protection studies suggest that the DCCD-reactive carboxylic residue resides at or near the substrate-binding site. Furthermore, peptide mapping analysis reveals that Asp283, located in the putative loop V of a tentative topological model of vacuolar H+-pyrophosphatase on the cytosolic side, was labelled by radioactive [14C]DCCD. Cytosolic loop V contains both DCCD-sensitive Asp283 and a conserved motif sequence, rendering it a candidate for the catalytic site of vacuolar H+-pyrophosphatase. A topological picture of the active domain of vacuolar H+-pyrophosphatase is tentatively proposed.

1994 ◽  
Vol 267 (3) ◽  
pp. H1010-H1016 ◽  
Author(s):  
A. Boraso ◽  
A. J. Williams

The effect of hydrogen peroxide (H2O2) on the sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channel has been investigated under voltage-clamp conditions after incorporation of native membrane vesicles into planar phospholipid bilayers. In the presence of micromolar activating calcium concentrations on the cytosolic side of the membrane, H2O2 (3-5 mM) increased open probability of the channels. H2O2 did not affect the conductance of the channel or the response to activating compounds, such as ATP and caffeine. H2O2 did not alter the inhibitory response to magnesium or the modification of channels by ryanodine. At subactivating calcium concentrations (approximately 45 pM) on the cytosolic side of the membrane, 5 mM H2O2 was still able to open the channel. Analysis of single-channel open and closed lifetimes suggested that H2O2 had a direct effect on the gating mechanism of the channel. Open probability of the SR Ca(2+)-release channel is reduced by millimolar concentrations of dithiothreitol, a sulfhydryl-protecting compound, in a concentration-dependent manner. In conclusion, it is probable that H2O2 activates the SR Ca(2+)-release channel via an oxidation of cysteine thiol groups in the channel protein.


1988 ◽  
Vol 251 (1) ◽  
pp. 293-299 ◽  
Author(s):  
M A Selak ◽  
M Chignard ◽  
J B Smith

The present studies were undertaken to characterize a serine protease released by N-formyl-L-Met-L-Leu-L-Phe (fMet-Leu-Phe)-stimulated neutrophils that rapidly induces platelet calcium mobilization, secretion and aggregation. The biological activity associated with this protease was unaffected by leupeptin, was only weakly diminished by N-p-tosyl-L-Lys-chloromethane, but was strongly inhibited by alpha 1-antitrypsin, soyabean trypsin inhibitor, N-tosyl-L-Phe-chloromethane and benzoyloxycarbonyl-Gly-Leu-Phe-chloromethane (Z-Gly-Leu-PheCH2Cl). These observations indicated that the biological activity of neutrophil supernatants could be attributed to a chymotrypsin-like enzyme such as cathepsin G. Furthermore, platelet aggregation and 5-hydroxytryptamine release induced by cell-free supernatants from fMet-Leu-Phe-stimulated neutrophils were found to be blocked by antiserum to cathepsin G in a concentration-dependent manner but were unaffected by antiserum to elastase. The biological activity present in neutrophil supernatants co-purified with enzymic activity for cathepsin G during sequential Aprotinin-Sepharose affinity chromatography and carboxymethyl-Sephadex chromatography. SDS/polyacrylamide-gel electrophoresis of the reduced, purified protein, demonstrated three polypeptides with apparent Mr values of 31,500, 29,000 and 28,000 and four polypeptides were resolved on acid-gel electrophoresis. Purified cathepsin G from neutrophils cross-reacted with anti-(cathepsin G) serum in a double immunodiffusion assay and elicited platelet calcium mobilization, 5-hydroxytryptamine secretion and aggregation. Calcium mobilization and secretion induced by low concentrations of cathepsin G were partially dependent on arachidonic acid metabolites and ADP, while stimulation by higher enzyme concentrations was independent of amplification pathways, indicating that cathepsin G is a strong platelet agonist. These results suggest that pathological processes which stimulate neutrophils and release cathepsin G can in turn result in the recruitment and activation of platelets.


1996 ◽  
Vol 317 (2) ◽  
pp. 495-501 ◽  
Author(s):  
Faika A. GHAZALEH ◽  
George A. OMBURO ◽  
Robert W. COLMAN

cAMP is a major regulator of platelet function. cGMP-inhibited phosphodiesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cAMP. The pH–rate profile plot for this enzyme yields pKa values of 6.5 and 9.0, consistent with histidine and cysteine residues respectively. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and concentration-dependent manner, and this effect was rapidly reversed by hydroxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as deduced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzymic activity. N-Ethylmaleimide (NEM) and 5,5´-dithiobis-(2-nitrobenzoic acid) (DTNB) inactivate cGI-PDE in a time- and concentration-dependent manner, suggesting the selective modification of a cysteine residue. AMP protects the enzyme against DEP, NEM and DTNB, suggesting the presence of histidine and cysteine residues at the active site of cGI-PDE. [14C]DEP incorporation in the presence of AMP or cGMP indicates the protection of two histidine residues by each nucleotide. These residues are different for each agent, since the combination of AMP and cGMP protects four histidine residues. [3H]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PDE was protected by AMP, but not by cGMP. We conclude that cGI-PDE possesses two essential histidine residues for activity, two additional histidines for cGMP inhibition, and one cysteine residue at the active site.


2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamin’s anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.


1992 ◽  
Vol 68 (05) ◽  
pp. 570-576 ◽  
Author(s):  
Mary A Selak

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.


1993 ◽  
Vol 69 (03) ◽  
pp. 286-292 ◽  
Author(s):  
Che-Ming Teng ◽  
Feng-Nien Ko ◽  
Inn-Ho Tsai ◽  
Man-Ling Hung ◽  
Tur-Fu Huang

SummaryTrimucytin is a potent platelet aggregation inducer isolated from Trimeresurus mucrosquamatus snake venom. Similar to collagen, trimucytin has a run of (Gly-Pro-X) repeats at the N-terminal amino acids sequence. It induced platelet aggregation, ATP release and thromboxane formation in rabbit platelets in a concentration-dependent manner. The aggregation was not due to released ADP since it was not suppressed by creatine phosphate/creatine phosphokinase. It was not either due to thromboxane A2 formation because indomethacin and BW755C did not have any effect on the aggregation even thromboxane B2 formation was completely abolished by indomethacin. Platelet-activating factor (PAF) was not involved in the aggregation since a PAF antagonist, kadsurenone, did not affect. However, RGD-containing peptide triflavin inhibited the aggregation, but not the release of ATP, of platelets induced by trimucytin. Indomethacin, mepacrine, prostaglandin E1 and tetracaine inhibited the thromboxane B2 formation of platelets caused by collagen and trimucytin. Forskolin and sodium nitroprusside inhibited both platelet aggregation and ATP release, but not the shape change induced by trimucytin. In quin-2 loaded platelets, the rise of intracellular calcium concentration caused by trimucytin was decreased by 12-O-tetradecanoyl phorbol-13 acetate, imipramine, TMB-8 and indomethacin. In the absence of extracellular calcium, both collagen and trimucytin caused no thromboxane B2 formation, but still induced ATP release which was completely blocked by R 59022. Inositol phosphate formation in platelets was markedly enhanced by trimucytin and collagen. MAB1988, an antibody against platelet membrane glycoprotein Ia, inhibited trimucytinand collagen-induced platelet aggregation and ATP release. However, trimucytin did not replace the binding of 125I-labeled MAB1988 to platelets. Platelets pre-exposed to trimucytin were resistant to the second challenge with trimucytin itself or collagen. It is concluded that trimucytin may activate collagen receptors on platelet membrane, and cause aggregation and release mainly through phospholipase C-phosphoinositide pathway.


2018 ◽  
Author(s):  
Luke Jordan ◽  
Nathan Wittenberg

This is a comprehensive study of the effects of the four major brain gangliosides (GM1, GD1b, GD1a, and GT1b) on the adsorption and rupture of phospholipid vesicles on SiO2 surfaces for the formation of supported lipid bilayer (SLB) membranes. Using quartz crystal microbalance with dissipation monitoring (QCM-D) we show that gangliosides GD1a and GT1b significantly slow the SLB formation process, whereas GM1 and GD1b have smaller effects. This is likely due to the net ganglioside charge as well as the positions of acidic sugar groups on ganglioside glycan head groups. Data is included that shows calcium can accelerate the formation of ganglioside-rich SLBs. Using fluorescence recovery after photobleaching (FRAP) we also show that the presence of gangliosides significantly reduces lipid diffusion coefficients in SLBs in a concentration-dependent manner. Finally, using QCM-D and GD1a-rich SLB membranes we measure the binding kinetics of an anti-GD1a antibody that has similarities to a monoclonal antibody that is a hallmark of a variant of Guillain-Barre syndrome.


1998 ◽  
Vol 38 (6) ◽  
pp. 147-154 ◽  
Author(s):  
Hideo Utsumi ◽  
Sang-Kuk Han ◽  
Kazuhiro Ichikawa

Generation of hydroxyl radicals, one of the major active species in ozonation of water was directly observed with a spin-trapping/electron spin resonance (ESR) technique using 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) as a spin-trapping reagent. Hydroxyl radical were trapped with DMPO as a stable radical, DMPO-OH. Eighty μM of ozone produced 1.08 X 10-6M of DMPO-OH, indicating that 1.4% of •OH is trapped with DMPO. Generation rate of DMPO-OH was determined by ESR/stopped-flow measurement. Phenol derivatives increased the amount and generation rate of DMPO-OH, indicating that phenol derivatives enhance •OH generation during ozonation of water. Ozonation of 2,3-, 2,5-, 2,6-dichlorophenol gave an ESR spectra of triplet lines whose peak height ratio were 1:2:1. ESR parameters of the triplet lines agreed with those of the corresponding dichloro-psemiquinone radical. Ozonation of 2,4,5- and 2,4,6-trichlorophenol gave the same spectra as those of 2,5- and 2,6-dichlorophenol, respectively, indicating that a chlorine group in p-position is substituted with a hydroxy group during ozonation. Amounts of the radical increased in an ozone-concentration dependent manner and were inhibited by addition of hydroxyl radical scavengers. These results suggest that p-semiquinone radicals are generated from the chlorophenols by hydroxyl radicals during ozonation. The p-semiquinone radicals were at least partly responsible for enhancements of DMPO-OH generation.


2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.


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