scholarly journals Cathepsin G is a strong platelet agonist released by neutrophils

1988 ◽  
Vol 251 (1) ◽  
pp. 293-299 ◽  
Author(s):  
M A Selak ◽  
M Chignard ◽  
J B Smith
Keyword(s):  
Biological Activity ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymic Activity ◽  
Calcium Mobilization ◽  
Cathepsin G ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Alpha 1 Antitrypsin ◽  
Acid Gel

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.

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.

Characterization of the interaction between chymotrypsin and heparin

1977 ◽  
Vol 55 (2) ◽  
pp. 134-139 ◽  
Author(s):  
Sally S. Twining ◽  
Arthur S. Brecher
Keyword(s):  
Complex Formation ◽  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Enzymic Activity ◽  
Polyacrylamide Gel ◽  
Activity Data

Heparin forms a complex with chymotrypsin which is active towards glutaryl-L-phenylalanine-p-nitroanilide (GPANA) and glutaryl-L-phenylalanine-β-naphthylamide (GPNA) at pH 7.6. The activity of chymotrypsin towards GPANA at pH 7.6 is enhanced in the presence of heparin. Heparin does not bind at the active site of the enzyme since proflavin is not displaced from the active site of chymotrypsin upon complex formation. The heparin–chymotrypsin complex migrates under basic polyacrylamide disc gel electrophoresis conditions to a position intermediate between heparin and free chymotrypsin. The complex is dissociable under acidic polyacrylamide gel electrophoresis conditions. It is estimated that one to three molecules of heparin can bind to each chymotrypsin molecule on the basis of electrophoretic and enzymic activity data.

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 ◽  
...  
Keyword(s):  
Peptide Mapping ◽  
Enzymic Activity ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Progressive Decline ◽  
Conserved Motif ◽  
Woodward’S Reagent K ◽  
Mapping Analysis ◽  
Cytosolic Side ◽  
Enzyme Protection

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.

scholarly journals Identification of Constituents of Human Neutrophil Azurophil Granules That Mediate Fungistasis againstHistoplasma capsulatum

2000 ◽  
Vol 68 (10) ◽  
pp. 5668-5672 ◽  
Author(s):  
Simon L. Newman ◽  
Lisa Gootee ◽  
Joelle E. Gabay ◽  
Michael E. Selsted
Keyword(s):  
Human Neutrophil ◽  
Histoplasma Capsulatum ◽  
Cathepsin G ◽  
Human Neutrophils ◽  
Dependent Manner ◽  
Subsequent Growth ◽  
Concentration Dependent Manner ◽  
Fungistatic Activity ◽  
Maximum Inhibition ◽  
Azurophil Granule

ABSTRACT Previously we demonstrated that human neutrophils mediate potent and long-lasting fungistasis against Histoplasma capsulatumyeasts and that all of the fungistatic activity resides in the azurophil granules. In the present study, specific azurophil granule constituents with fungistatic activity were identified by incubation with H. capsulatum yeasts for 24 h and by quantifying the subsequent growth of yeasts via the incorporation of [3H]leucine. Human neutrophil defensins HNP-1, HNP-2, and HNP-3 inhibited the growth of H. capsulatum yeasts in a concentration-dependent manner with maximum inhibition at 8 μg/ml. At a concentration of 4 μg/ml, all possible paired combinations of defensins exhibited additive fungistatic activity against H. capsulatum yeasts. Cathepsin G and bactericidal-permeability-increasing protein (BPI) also mediated fungistasis against H. capsulatum in a concentration-dependent manner. The fungistatic activities of combinations of cathepsin G and BPI were additive, as were those of combinations of cathepsin G or BPI with HNP-1, HNP-2, and HNP-3. Lysozyme and elastase exhibited modest antifungal activity, and azurocidin and proteinase 3 exhibited no significant fungistasis against H. capsulatum yeasts. Thus, defensins, cathepsin G, and BPI are the major anti-H. capsulatum effector molecules in the azurophil granules of human neutrophils.

Coagulation Factor XIIa Activates Platelets and Is the Physiological Agonist of Protease-Activated Receptor 3.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 770-770 ◽  
Author(s):  
Yingying Mao ◽  
Todd M Getz ◽  
Jianguo Jin ◽  
Satya P. Kunapuli
Keyword(s):  
Intracellular Calcium ◽  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Shape Change ◽  
Coagulation Factor ◽  
Calcium Mobilization ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Factor Xiia

Abstract Abstract 770 Protease-activated receptors (PARs) are G-protein coupled receptors that are activated by proteases. Thrombin is the major agonist for PAR1 and PAR4, whereas tryptase and coagulation factor Xa are the agonists for PAR2. In addition to these major agonists, PARs can be activated by other coagulation proteases. The physiological agonist of PAR3 has not been identified to date; as a result, the molecular pharmacology and physiology of PAR3 remain poorly understood. The purpose of this study is to identify a physiological agonist to PAR3. We used PAR4 null murine platelets, which are known to express only PAR3. In this study, we tested the effect of several coagulation proteases and found that only coagulation factor XIIa (FXIIa) activated PAR4-/- murine platelets, in a concentration-dependent manner. FXIIa caused murine platelet shape change, aggregation, secretion and thromboxane A2 generation and this activation was abolished by C1 esterase inhibitor, a FXIIa inhibitor. FXIIa-induced murine platelet activation was completely abolished by BMS200261, a PAR1 antagonist, without affecting the catalytic activity of FXIIa. As murine platelets do not express PAR1, these data indicate that BMS200261 acts as an antagonist of PAR3 and hence inhibits FXIIa-induced platelet activation. FXIIa also caused mobilization of intracellular calcium from murine platelets and this calcium increase is abolished by BMS200261 in the presence or absence of the PAR4. PAR1 and PAR4 couple to Gq to cause intracellular calcium increases. YM-254890, a Gq inhibitor, abrogates PAR1- or PAR4-mediated calcium mobilization. However, YM-254890 did not affect FXIIa –induced platelet calcium mobilization in murine platelets. FXIIa caused activation of Gq-/- mice platelets similar to wild -type platelets, suggesting that FXIIa -induced calcium mobilization in platelets is independent of Gq pathways. Furthermore, FXIIa-induced platelet activation was completely abolished by BAPTA-AM, which indicates that calcium is required for FXIIa-induced platelet activation. Furthermore, FXIIa caused phosphorylation of Erk and Akt in PAR4 null murine platelets and this phosphorylation was abolished by BMS200261, but not by YM-254890. These observations may explain previous reports that demonstrated lack of stable thrombus formation in FXII null mice. We conclude that FXIIa activates platelets through PAR3 independently of Gq pathways leading to calcium mobilization and activation of Erk and Akt. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Peptidoglycan O Acetylation and Autolysin Profile of Enterococcus faecalis in the Viable but Nonculturable State

2006 ◽  
Vol 188 (3) ◽  
pp. 902-908 ◽  
Author(s):  
John M. Pfeffer ◽  
Hendrik Strating ◽  
Joel T. Weadge ◽  
Anthony J. Clarke
Keyword(s):  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Hydroxyl Group ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Viable But Nonculturable ◽  
Egg White Lysozyme

ABSTRACT The O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains of Entercoccus durans, E. faecalis, E. faecium, and E. hirae produce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells of E. faecalis in the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins in E. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNC E. faecalis produced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.

scholarly journals Interaction of calmodulin with troponin I and the troponin-tropomyosin-actin complex. Effect of Ca2+ and Sr2+ ions

1987 ◽  
Vol 241 (3) ◽  
pp. 905-909 ◽  
Author(s):  
K Yamamoto ◽  
H Nakayama ◽  
K Nunoi ◽  
M Fujishima
Keyword(s):  
Muscle Contraction ◽  
Gel Electrophoresis ◽  
Troponin I ◽  
Troponin T ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Dependent Manner ◽  
Bivalent Cation ◽  
Regulation Of Muscle Contraction ◽  
Dependent Interaction

In an effort to elucidate the mechanism of calmodulin regulation of muscle contraction, we investigated the interaction between calmodulin and troponin components in the presence of Ca2+ or Sr2+ by the use of ultracentrifugation methods and polyacrylamide-gel electrophoresis. Skeletal-muscle troponin C bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was absent, calmodulin bound to troponin I and dissociated it from the tropomyosin-actin complex in the presence of Ca2+ or Sr2+. When troponin T was present, calmodulin hardly bound to troponin I even in the presence of bivalent cations. Trifluoperazine, a calmodulin antagonist, inhibited the bivalent-cation-dependent interaction between calmodulin and troponin I. Calmodulin migrated more slowly in the presence of Sr2+ than it did in the presence of EGTA but faster than it did in the presence of Ca2+ on polyacrylamide-gel electrophoresis under non-denaturing conditions. It is concluded that troponin T is not required in the calmodulin regulation of muscle contraction because troponin T inhibits the bivalent-cation-dependent interaction between calmodulin and troponin I and because calmodulin binds to troponin I and dissociates it from the tropomyosin-actin complex in a bivalent-cation-dependent manner. Sr2+-induced exposure of the hydrophobic region enables calmodulin to bind to troponin I, as is the case with Ca2+.

scholarly journals Anti-Inflammatory and Neuroprotective Effects of Constituents Isolated fromRhodiola rosea

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Yeonju Lee ◽  
Jae-Chul Jung ◽  
Soyong Jang ◽  
Jieun Kim ◽  
Zulfiqar Ali ◽  
...  
Keyword(s):  
Biological Activity ◽  
Crude Extract ◽  
Therapeutic Potential ◽  
Neuroprotective Effect ◽  
Rhodiola Rosea ◽  
Dependent Manner ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Bv2 Cells ◽  
Proinflammatory Factors

To determine the biological activity ofRhodiola rosea, the protein expression of iNOS and proinflammatory cytokines was measured after the activation of murine microglial BV2 cells by LPS under the exposure of constituents ofRhodiola rosea: crude extract, rosin, rosarin, and salidroside (each 1–50 μg/mL). The LPS-induced expression of iNOS and cytokines in BV2 cells was suppressed by the constituents ofRhodiola roseain a concentration-dependent manner. Also the expression of the proinflammatory factors iNOS, IL-1β, and TNF-αin the kidney and prefrontal cortex of brain in mice was suppressed by the oral administration ofRhodiola roseacrude extract (500 mg/kg). To determine the neuroprotective effect of constituents ofRhodiola rosea, neuronal cells were activated by L-glutamate, and neurotoxicity was analyzed. The L-glutamate-induced neurotoxicity was suppressed by the treatment with rosin but not by rosarin. The level of phosphorylated MAPK, pJNK, and pp38 was increased by L-glutamate treatment but decreased by the treatment with rosin and salidroside. These results indicate thatRhodiola roseamay have therapeutic potential for the treatment of inflammation and neurodegenerative disease.

scholarly journals Identification of the natural product berberine as an antiviral drug

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jiping Shao ◽  
Debin Zeng ◽  
Shuhong Tian ◽  
Gezhi Liu ◽  
Jian Fu
Keyword(s):  
Conformational Changes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Antiviral Drug ◽  
Host Cells ◽  
Heptad Repeat ◽  
Potential Candidate ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Antiviral Activities ◽  
Hiv 1

Abstract Drugs targeting the fusion process of viral entry into host cells have been approved for clinical use in the treatment of AIDS. There remains a great need to improve the use of existing drugs for HIV therapy. Berberine is traditionally used to treat diarrhea, bacillary dysentery, and gastroenteritis in clinics, here our research shows that berberine is effective in inhibiting HIV-1 entry. Native polyacrylamide gel electrophoresis studies reveal that berberine can directly bind to both N36 and C34 to form a novel N36-berberine-C34 complex and effectively block the six-helix bundle formation between the N-terminal heptad repeat peptide N36 and the C-terminal heptad repeat peptide C34. Circular dichroism experiments show that binding of berberine produces conformational changes that damages the secondary structures of 6-HB. Computer-aided molecular docking studies suggest a hydrogen bond with T-639 and two polar bonds with Q-563 and T-639 are established, involving the oxygen atom and the C=O group of the indole ring. Berberine completely inhibits six HIV-1 clade B isolates and exhibits antiviral activities in a concentration-dependent manner with IC50 values varying from 5.5 to 10.25 µg/ml. This compound-peptide interaction may represent a mechanism of action of antiviral activities of berberine. As a summary, these studies successfully identify compound berberine as a potential candidate drug for HIV-1 treatment. As a summary, antiviral activity of berberine in combination with its use in clinical practice, this medicine can be used as a potential clinically anti-HIV drug.

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