A tryptophan residue involved in the inhibition of plant vacuolar H+-ATPase by 2-hydroxy-5-nitrobenzyl bromide

1998 ◽  
Vol 25 (6) ◽  
pp. 679
Author(s):  
Soong Yu Kuo ◽  
May Whei Lin ◽  
Shih Sheng Jiang ◽  
Shu Hsien Hung ◽  
Chi Meng Tzeng ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Conformational Change ◽  
Atp Hydrolysis ◽  
Proton Translocation ◽  
Order Rate Constant ◽  
Tryptophan Residue ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Significant Conformational Change ◽  
State Dissociation

Treatment of the vacuolar H+ -ATPase from mung bean seedlings (Vigna radiata L.) with the tryptophan modifying agent 2-hydroxyl-5-nitrobenzyl bromide (HNBB), caused a progressive decline of the ATP hydrolysis activity and proton translocation in a time- and concentration-dependent manner. Dithiothreitol could not restore the inhibition of H+ -ATPase by HNBB, indicating possible involvement tryptophan, and not cysteine residues. Protection studies suggested that modified sites might not locate in the active domain. Kinetic analysis shows that Vmax but not Km of H+ -ATPase was changed by HNBB. The reaction order of inactivation by HNBB was calculated as 0.98, implying that at least one tryptophan was labelled. The steady-state dissociation constant (Ki) and the pesudo-first-order rate constant of inhibition (k2) were determined as 1.61 mM and 0.22 min-1, respectively. Furthermore, stoichiometry experiments indicated that 8 mol tryptophan/mol ATPase were modified, when the enzyme activity was completely inhibited. However, a Tsou analysis showed that only one out of these modified tryptophans was crucial to the enzymatic activity. In addition, modification of the vacuolar H+ -ATPase by HNBB led to a decrease in intrinsic fluorescence, suggesting a possible conformational change of the enzyme. Taken together, our data indicate that the tryptophan residue is indispensable to vacuolar H+ -ATPase and the modification of this residue may induce a significant conformational change, consequently resulting in the loss of enzymatic activity.

Cytotoxic Effects of Ferula Latisecta on Human Glioma U87 Cells

Drug Research ◽  
2019 ◽  
Vol 69 (12) ◽  
pp. 665-670 ◽  
Author(s):  
Mohammad Jalili-Nik ◽  
Hamed Sabri ◽  
Ehsan Zamiri ◽  
Mohammad Soukhtanloo ◽  
Mostafa Karimi Roshan ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Gelatin Zymography ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Induced Apoptosis ◽  
Natural Herb ◽  
First Time ◽  
U87 Cells

AbstractGlioblastoma multiforme (GBM) is the fatal type of astrocytic tumors with a survival rate of 12 months. The present study, for the first time, evaluated the cytotoxic impacts of Ferula latisecta (F. latisecta) hydroalcoholic extract on U87 GBM cell line. The MTT assay measured the cellular toxicity following 24- and 48 h treatment with various doses of F. latisecta (0–800 μg/mL). Apoptosis was evaluated by an Annexin V/propidium iodide (PI) staining 24 h after treatment by F. latisecta. Moreover, to determine the cellular metastasis of U87 cells, we used a gelatin zymography assay (matrix metalloproteinase [MMP]-2/-9 enzymatic activity). The outcomes showed that F. latisecta mitigated the viability of U87 cells in a concentration- and time-dependent manner with IC50 values of 145.3 and 192.3 μg/mL obtained for 24- and 48 h treatments, respectively. F. latisecta induced apoptosis in a concentration-dependent manner after 24 h. Also, MMP-9 activity was significantly decreased following 24 h after treatment concentration-dependently with no change in MMP-2 enzymatic activity. This study showed that F. latisecta induced cytotoxicity and apoptosis, and mitigated metastasis of U87 GBM cells. Hence, F. latisecta could be beneficial as a promising natural herb against GBM after further studies.

scholarly journals ATP-dependent thermostabilization of human P-glycoprotein (ABCB1) is blocked by modulators

2019 ◽  
Vol 476 (24) ◽  
pp. 3737-3750 ◽  
Author(s):  
Sabrina Lusvarghi ◽  
Suresh V. Ambudkar
Keyword(s):  
Conformational Changes ◽  
Drug Transport ◽  
Atp Hydrolysis ◽  
Atp Binding ◽  
Dependent Manner ◽  
Deficient Mutant ◽  
Concentration Dependent Manner ◽  
Glycoprotein P ◽  
Binding Domains ◽  
P Glycoprotein

P-glycoprotein (P-gp), an ATP-binding cassette transporter associated with multidrug resistance in cancer cells, is capable of effluxing a number of xenobiotics as well as anticancer drugs. The transport of molecules through the transmembrane (TM) region of P-gp involves orchestrated conformational changes between inward-open and inward-closed forms, the details of which are still being worked out. Here, we assessed how the binding of transport substrates or modulators in the TM region and the binding of ATP to the nucleotide-binding domains (NBDs) affect the thermostability of P-gp in a membrane environment. P-gp stability after exposure at high temperatures (37–80°C) was assessed by measuring ATPase activity and loss of monomeric P-gp. Our results show that P-gp is significantly thermostabilized (>22°C higher IT50) by the binding of ATP under non-hydrolyzing conditions (in the absence of Mg2+). By using an ATP-binding-deficient mutant (Y401A) and a hydrolysis-deficient mutant (E556Q/E1201Q), we show that thermostabilization of P-gp requires binding of ATP to both NBDs and their dimerization. Additionally, we found that transport substrates do not affect the thermal stability of P-gp either in the absence or presence of ATP; in contrast, inhibitors of P-gp including tariquidar and zosuquidar prevent ATP-dependent thermostabilization in a concentration-dependent manner, by stabilizing the inward-open conformation. Altogether, our data suggest that modulators, which bind in the TM regions, inhibit ATP hydrolysis and drug transport by preventing the ATP-dependent dimerization of the NBDs of P-gp.

Low concentrations of A23187 increase calcium uptake by cardiac sarcoplasmic reticulum

1985 ◽  
Vol 249 (6) ◽  
pp. H1211-H1215
Author(s):  
J. J. Murray ◽  
A. V. Kuzmin ◽  
P. W. Reed ◽  
D. O. Levitsky
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Uptake ◽  
Atp Hydrolysis ◽  
Ionophore A23187 ◽  
Uptake System ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Calcium Dependent ◽  
Uptake Measurement

The divalent cation ionophore A23187 at a concentration of 1 nM produced an increased rate of oxalate-supported calcium uptake by isolated cardiac sarcoplasmic reticulum as determined by absorbance changes of the calcium-sensitive dye murexide. Addition of a higher concentration of A23187 (0.1 microM) produced a decreased rate of calcium uptake. Measurement of the time during which ATPase was activated by calcium addition also suggested an increased rate of calcium uptake in the presence of 1 nM A23187 and an inhibition of calcium uptake at a higher concentration of the ionophore (0.1 microM). Ca2+-stimulated ATPase activity and incorporation of 32Pi from [gamma-32P]ATP into sarcoplasmic reticular proteins were increased by A23187 at concentrations of 1 nM or greater. An increased coupling of calcium uptake to ATP hydrolysis was observed at 1 nM A23187, while concentrations of the ionophore greater than or equal to 10 nM produced a decreased coupling. Addition of an inhibitor of cyclic AMP-dependent protein kinase decreased the rate of calcium uptake, and this inhibition was reversed in a concentration-dependent manner by 0.01–1 nM A23187. These data suggest that A23187 can activate a mechanism involving the calcium-dependent phosphorylation of protein that may regulate the activity of the calcium uptake system of the sarcoplasmic reticulum. These observations appear to provide an explanation for some of the contractile effects of A23187 in intact cardiac muscle that suggest that treatment with the ionophore results in an increased sequestration of calcium from the cytoplasm.

scholarly journals Inactivation of Recombinant Monocyte cAMP-Specific Phosphodiesterase by cAMP Analog, 8-[(4-Bromo-2,3-Dioxobutyl)thio]Adenosine 3′,5′-Cyclic Monophosphate

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 1019-1026 ◽  
Author(s):  
George A. Omburo ◽  
Theodore J. Torphy ◽  
Gilbert Scott ◽  
Susanne Jacobitz ◽  
Roberta F. Colman ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Enzyme Inhibitor ◽  
Order Rate Constant ◽  
Competitive Inhibitor ◽  
Dependent Manner ◽  
Amino Acid Residues ◽  
Concentration Dependent Manner ◽  
Cyclic Monophosphate ◽  
Inhibitor Complex ◽  
Camp Hydrolysis

Abstract Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl) thio]adenosine 3′,5′-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase (PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5 μmol/L) of cAMP hydrolysis by PDE4a. 8-BDB-TcAMP irreversibly inactivates the enzyme in a time- and concentration-dependent manner with a second order rate constant of 0.022 mmol/L−1min−1. The rate of inactivation of PDE4a is reduced by the presence of the substrate cAMP and specific inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol of the affinity label/mol of enzyme was incorporated. The radiolabeled peptide is composed of 10 amino acid residues (697 to 706) located near the carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has seven nonpolar and aliphatic residues, of which four are proline, giving the peptide a highly structured conformation. This peptide is the first to be identified in the putative catalytic domain involved in substrate recognition.

scholarly journals The 110-kD protein-calmodulin complex of the intestinal microvillus is an actin-activated MgATPase.

1987 ◽  
Vol 105 (1) ◽  
pp. 313-324 ◽  
Author(s):  
K A Conzelman ◽  
M S Mooseker
Keyword(s):  
Enzymatic Activity ◽  
Nucleoside Triphosphate ◽  
Dependent Manner ◽  
Binding Interaction ◽  
Ph Optimum ◽  
Concentration Dependent Manner ◽  
Nitrophenyl Phosphate ◽  
Ph Range ◽  
Mgatpase Activity ◽  
Actin Concentration

The microvillus 110-kD protein-calmodulin complex (designated 110K-CM) shares several properties with all myosins. In addition to its well-defined ATP-dependent binding interaction with F-actin, 110K-CM is an ATPase with diagnostically myosin-like divalent cation sensitivity. It exhibits maximum enzymatic activity in the presence of K+ and EDTA (0.24 mumol P1/mg per min) or in the presence of Ca++ (0.40 mumol P1/mg per min) and significantly less activity in physiological ionic conditions of salt and Mg++ (0.04 mumol P1/mg per min). This MgATPase is activated by F-actin in an actin concentration-dependent manner (up to 2.5-3.5-fold). The specific MgATPase activity of 110K-CM is also enhanced by the addition of 5-10 microM Ca++, but in the isolated complex, there is often also a decrease in the extent of actin activation in this range of free Ca++. Actin activation is maintained, however, in samples with exogenously added calmodulin; under these conditions, there is an approximately sevenfold stimulation of 110K-CM's enzymatic activity in the presence of 5-10 microM Ca++ and actin. 110K-CM is relatively indiscriminant in its nucleoside triphosphate specificity; in addition to ATP, GTP, CTP, UTP, and ITP are all hydrolyzed by the complex in the presence of either Mg++ or Ca++. Neither AMP nor the phosphatase substrate p-nitrophenyl phosphate are substrates for the enzymatic activity. The pH optimum for CaATPase activity is 6.0-7.5; maximum actin activation of MgATPase occurs over a broad pH range of 6.5-8.5. Finally, like myosins, purified 110K-CM crosslinks actin filaments into loosely ordered aggregates in the absence of ATP. Collectively these data support the proposal of Collins and Borysenko (1984, J. Biol. Chem., 259:14128-14135) that the 110K-CM complex is functionally analogous to the mechanoenzyme myosin.

scholarly journals Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39

Marine Drugs ◽  
2021 ◽  
Vol 19 (2) ◽  
pp. 51
Author(s):  
Vittoria Lopez ◽  
Laura Schäkel ◽  
H. J. Maximilian Schuh ◽  
Michael S. Schmidt ◽  
Salahuddin Mirza ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Extracellular Atp ◽  
Sulfated Polysaccharides ◽  
Nucleoside Triphosphate ◽  
Nucleotide Receptors ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Mechanism Of Inhibition ◽  
Immunotherapy Of Cancer ◽  
Dual Inhibitors

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

scholarly journals Calcium-dependent inhibition of adrenal TREK-1 channels by angiotensin II and ionomycin

2011 ◽  
Vol 301 (3) ◽  
pp. C619-C629 ◽  
Author(s):  
John J. Enyeart ◽  
Haiyan Liu ◽  
Judith A. Enyeart
Keyword(s):  
Atp Hydrolysis ◽  
Water Soluble ◽  
Zona Fasciculata ◽  
Dependent Manner ◽  
Ang Ii ◽  
Patch Clamp Recording ◽  
Open Probability ◽  
Concentration Dependent Manner ◽  
Whole Cell ◽  
Dependent Inhibition

Bovine adrenocortical cells express bTREK-1 K+ (bovine KCNK2) channels that are inhibited by ANG II through a Gq-coupled receptor by separate Ca2+ and ATP hydrolysis-dependent signaling pathways. Whole cell and single patch clamp recording from adrenal zona fasciculata (AZF) cells were used to characterize Ca2+-dependent inhibition of bTREK-1. In whole cell recordings with pipette solutions containing 0.5 mM EGTA and no ATP, the Ca2+ ionophore ionomycin (1 μM) produced a transient inhibition of bTREK-1 that reversed spontaneously within minutes. At higher concentrations, ionomycin (5–10 μM) produced a sustained inhibition of bTREK-1 that was reversible upon washing, even in the absence of hydrolyzable [ATP]i. BAPTA was much more effective than EGTA at suppressing bTREK-1 inhibition by ANG II. When intracellular Ca2+ concentration ([Ca2+]i) was buffered to 20 nM with either 11 mM BAPTA or EGTA, ANG II (10 nM) inhibited bTREK-1 by 12.0 ± 4.5% ( n=11) and 59.3 ± 8.4% ( n=4), respectively. Inclusion of the water-soluble phosphatidylinositol 4,5-bisphosphate (PIP2) analog DiC8PI(4,5)P2 in the pipette failed to increase bTREK-1 expression or reduce its inhibition by ANG II. The open probability ( Po) of unitary bTREK-1 channels recorded from inside-out patches was reduced by Ca2+ (10–35 μM) in a concentration-dependent manner. These results are consistent with a model in which ANG II inhibits bTREK-1 K+ channels by a Ca2+-dependent mechanism that does not require the depletion of membrane-associated PIP2. They further indicate that the Ca2+ source is located in close proximity within a “Ca2+ nanodomain” of bTREK-1 channels, where [Ca2+]i may reach concentrations of >10 μM. bTREK-1 is the first two-pore K+ channel shown to be inhibited by Ca2+ through activation of a G protein-coupled receptor.

FIBRIN PROTOFIBRIL AND FIBRINOGEN BINDING TO ADP-STIMULATED PLATELETS:IS THERE A COMMON MECHANISM

1987 ◽  
Author(s):  
R R Hantgan ◽  
J C Lewis
Keyword(s):  
Electron Microscopy ◽  
Order Rate Constant ◽  
Common Mechanism ◽  
Immunogold Electron Microscopy ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Fibrinogen Degradation Product ◽  
Fibrinogen Binding ◽  
Common Receptor ◽  
Platelet Receptor

We have investigated the molecular basis of platelet:fibrin binding by studying interactions between platelets and protofibrils, soluble two-stranded polymers of fibrin which are intermediates on the fibrin assembly pathway. We have taken advantage of the well-characterized anticoagulant properties of the fibrinogen degradation product, fragment D, to prepare solutions of short protofibrils, composed of fewer than twenty fibrin monomer molecules per polymer. Fibrin protofibrils bound to ADP-activated platelets in a time- and concentration-dependent process which was effectively blocked by excess unlabelled fibrinogen, i.e. the binding was specific and appeared to involve a common receptor. ADP-stimulated cells bound approximately 3 micrograms of fibrin protofibrils/108 platelets, compared to 4 micrograms of fibrinogen/108 cells, following a 30 min incubation period at room temperature. The apparent first order rate constant for fibrin protofibril binding was found to be five-fold slower than that measured for fibrinogen. Two monoclonal antibodies directed against the glycroprotein Ilbtllla complex inhibited the binding of fibrin protofibrils and fibrinogen in a similar, concentration-dependent manner, providing strong evidence for a common receptor. Neither fibrin protofibrils nor fibrinogen bound to -Glanzmann’s thrombasthenic platelets, further supporting the hypothesis that fibrinogen and fibrin bind to a common platelet receptor present on the glycoprotein IIb:IIIa complex. The specificity of these interactions was examined with transmission electron microscopy, which clearly showed thin fibers of lengths up to 150 nm attached to the surface of normal, stimulated platelets. Immunogold electron microscopy using rabbit anthihuman fibrinogen as the first stage antibody verified identity of the surface bound molecules and the immunogold distribution paralleled that observed with the fibrin/fibrinogen molecules alone. Contacts between the ends of the fibers and the platelets were frequently observed, but lateral contacts were also evident

The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

2014 ◽  
Vol 84 (1-2) ◽  
pp. 79-91 ◽  
Author(s):  
Amin F. Majdalawieh ◽  
Hyo-Sung Ro
Keyword(s):  
Cholesterol Efflux ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Foam Cell ◽  
Mapk Signaling ◽  
Luciferase Reporter ◽  
Foam Cell Formation ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Macrophage Cholesterol Efflux

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.

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.

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