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.

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.

LINGCOD MUSCLE PHOSPHOMONOESTERASES: I. ACID PHOSPHATASES THAT HYDROLYZEp-NITROPHENYL PHOSPHATE

1960 ◽  
Vol 38 (1) ◽  
pp. 605-612 ◽  
Author(s):  
Neil Tomlinson ◽  
R. A. J. Warren
Keyword(s):  
Metal Ions ◽  
The Other ◽  
Acid Phosphatases ◽  
Free Base ◽  
Ph Optimum ◽  
Ophiodon Elongatus ◽  
Nitrophenyl Phosphate ◽  
Diethylaminoethyl Cellulose ◽  
Ph Range ◽  
Acid Phosphomonoesterase

Five fractions (A to E), each possessing acid phosphomonoesterase activity, were separated from an aqueous extract of the muscle of lingcod (Ophiodon elongatus) by stepwise chromatography on diethylaminoethyl cellulose in the free-base form.Fraction A required Zn++or Mn++for activity, was inhibited by heparin, and had its pH optimum at 6.0. Fraction E required Zn++for activity, was not inhibited by heparin, and had its pH optimum at 5.5. Fractions B, C, and D did not require metal ions for activity, and were distinguished from each other by differences in response to pH, cysteine, ethylenediaminetetraacetate, fluoride, and tartrate.The pH range over which fraction A was active was shifted to slightly higher values when Mn++was the activator rather than Zn++. Also, A was inhibited strongly by cysteine when activated by Zn++, but not when activated by Mn++. Data are presented that indicate these differences were due to different properties of the activating ions, rather than to the presence in fraction A of two enzymes, one activated by Zn++and the other by Mn++.

Interaction of human lactoferrin with the rat liver

1985 ◽  
Vol 248 (4) ◽  
pp. G463-G469 ◽  
Author(s):  
M. T. Debanne ◽  
E. Regoeczi ◽  
G. D. Sweeney ◽  
F. Krestynski
Keyword(s):  
Rat Liver ◽  
Plasma Membranes ◽  
Human Lactoferrin ◽  
Dependent Manner ◽  
Ph Optimum ◽  
Concentration Dependent Manner ◽  
Isoelectric Points ◽  
Membrane Bound ◽  
Parenchymal Cells ◽  
Generalized Type

Binding of human lactoferrin (hLf) by purified rat liver plasma membranes was studied to clarify whether the liver possesses specific hLf receptors. The binding was rapid between 4 degrees and 37 degrees C, with a pH optimum close to 5.0. At 22 degrees C and in glycine-NaOH (5 mM, pH 7.4) containing 150 mM NaCl and 0.5% albumin, 1 microgram of membrane bound a maximum of 11.8 ng hLf. The dissociation constant of the interaction was 1.6 X 10(-7) M. Other proteins of high isoelectric points (lactoperoxidase, lysozyme, and particularly salmine sulfate) and a piperazine derivative inhibited hLf binding in a concentration-dependent manner. In contrast, monosaccharides (galactose, N-acetylgalactosamine, mannose, and fucose) were ineffective. By omitting NaCl from the incubation buffer, binding was increased 3.6-fold. Erythrocyte ghosts bound hLf less firmly and alveolar macrophages more firmly than hepatic plasma membranes. Liver cell fractionations performed after the intravenous injection of labeled hLf showed that approximately 88% of the hepatic radioligand was associated with parenchymal cells. When binding was expressed per unit of cell volume, however, more hLf was present in nonparenchymal than in parenchymal cells, implying that the above value was determined by the relative cell masses rather than affinities alone. It is concluded that the binding of hLf by hepatic plasma membranes is electrostatic, i.e., is mediated by the cationic nature of the ligand, and that it is explicable in terms of a "specific nonreceptor interaction" of the generalized type proposed by Cuatrecasas and Hollenberg (Adv. Protein Chem. 30: 251-451, 1976).

scholarly journals Carnosine Decreases PMA-Induced Oxidative Stress and Inflammation in Murine Macrophages

Antioxidants ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 281 ◽  
Author(s):  
Giuseppe Caruso ◽  
Claudia G. Fresta ◽  
Annamaria Fidilio ◽  
Fergal O’Donnell ◽  
Nicolò Musso ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Defense Mechanisms ◽  
Energy State ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Inhibitory Effect ◽  
Nucleoside Triphosphate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Beneficial Effects

Carnosine is an endogenous dipeptide composed of β-alanine and L-histidine. This naturally occurring molecule is present at high concentrations in several mammalian excitable tissues such as muscles and brain, while it can be found at low concentrations in a few invertebrates. Carnosine has been shown to be involved in different cellular defense mechanisms including the inhibition of protein cross-linking, reactive oxygen and nitrogen species detoxification as well as the counteraction of inflammation. As a part of the immune response, macrophages are the primary cell type that is activated. These cells play a crucial role in many diseases associated with oxidative stress and inflammation, including atherosclerosis, diabetes, and neurodegenerative diseases. In the present study, carnosine was first tested for its ability to counteract oxidative stress. In our experimental model, represented by RAW 264.7 macrophages challenged with phorbol 12-myristate 13-acetate (PMA) and superoxide dismutase (SOD) inhibitors, carnosine was able to decrease the intracellular concentration of superoxide anions (O2−•) as well as the expression of Nox1 and Nox2 enzyme genes. This carnosine antioxidant activity was accompanied by the attenuation of the PMA-induced Akt phosphorylation, the down-regulation of TNF-α and IL-6 mRNAs, and the up-regulation of the expression of the anti-inflammatory mediators IL-4, IL-10, and TGF-β1. Additionally, when carnosine was used at the highest dose (20 mM), there was a generalized amelioration of the macrophage energy state, evaluated through the increase both in the total nucleoside triphosphate concentrations and the sum of the pool of intracellular nicotinic coenzymes. Finally, carnosine was able to decrease the oxidized (NADP+)/reduced (NADPH) ratio of nicotinamide adenine dinucleotide phosphate in a concentration dependent manner, indicating a strong inhibitory effect of this molecule towards the main source of reactive oxygen species in macrophages. Our data suggest a multimodal mechanism of action of carnosine underlying its beneficial effects on macrophage cells under oxidative stress and inflammation conditions.

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.

LINGCOD MUSCLE PHOSPHOMONOESTERASES: I. ACID PHOSPHATASES THAT HYDROLYZEp-NITROPHENYL PHOSPHATE

1960 ◽  
Vol 38 (6) ◽  
pp. 605-612 ◽  
Author(s):  
Neil Tomlinson ◽  
R. A. J. Warren
Keyword(s):  
Metal Ions ◽  
The Other ◽  
Acid Phosphatases ◽  
Free Base ◽  
Ph Optimum ◽  
Ophiodon Elongatus ◽  
Nitrophenyl Phosphate ◽  
Diethylaminoethyl Cellulose ◽  
Ph Range ◽  
Acid Phosphomonoesterase

Five fractions (A to E), each possessing acid phosphomonoesterase activity, were separated from an aqueous extract of the muscle of lingcod (Ophiodon elongatus) by stepwise chromatography on diethylaminoethyl cellulose in the free-base form.Fraction A required Zn++or Mn++for activity, was inhibited by heparin, and had its pH optimum at 6.0. Fraction E required Zn++for activity, was not inhibited by heparin, and had its pH optimum at 5.5. Fractions B, C, and D did not require metal ions for activity, and were distinguished from each other by differences in response to pH, cysteine, ethylenediaminetetraacetate, fluoride, and tartrate.The pH range over which fraction A was active was shifted to slightly higher values when Mn++was the activator rather than Zn++. Also, A was inhibited strongly by cysteine when activated by Zn++, but not when activated by Mn++. Data are presented that indicate these differences were due to different properties of the activating ions, rather than to the presence in fraction A of two enzymes, one activated by Zn++and the other by Mn++.

COMPARISON OF ENZYMATIC PROPERTIES OF HUMAN PLASMA FVIIa AND HUMAN RECOMBINANT FVIIa

1987 ◽  
Author(s):  
T Lund-Hansen ◽  
L C Peterson
Keyword(s):  
Human Plasma ◽  
Maximal Activity ◽  
Brain Extract ◽  
Rabbit Brain ◽  
Enhancement Effect ◽  
Dependent Manner ◽  
Chromogenic Substrate ◽  
Ph Optimum ◽  
Concentration Dependent Manner ◽  
Calcium Dependent

Human plasma FVIIa (pFVIIa) and human recombinant FVIIa (rFVIIa) were both purified by immune adsorption chromatography using a calcium dependent monoclonal antibody. The FVII obtained is highly purified and contains only trace contaminants as revealed by SDS-PAGE and reverse phase HPLC chromatography. FVII was fully activated during the purification procedure. A FVIIa activity assay has been developed in microplates using human FX as a substrate and methoxycarbonyl-D-cyclohexal-alanyl-glycyl-arginine-pNA as a chromogenic substrate for the FXa generated. The assay was linear at FVIIa concentrations between 0.5 and 10 nM. The concentration of the chromogenic substrate was 0.5 mM. A pH optimum at about 8 was found. An apparent Km=0.2 μM for FX was found for both pFVIIa and rFVIIa.The results suggest that the kinetics of human FX activation by pFVIIa and rFVIIa are identical. The FVIIa activity was found to be calcium dependent with maximal activity at about 0.25 mM, while the activities at 1 and 2 mM were 20% and 3%, respectively. When rabbit brain extract is used, the well-known dramatic enhancement effect of thromboplastin could be demonstrated with both FVII preparations. Also this reaction is calcium-dependent; however, the profile of the curve is distinctly different. Poly-D-lysine (MW 160,000) was found to enhance the FVIIa activity in a concentration dependent manner. Maximum stimulation (fivefold) was obtained at a concentration of about 10 mg/l.

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.

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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