Trypsin inhibition assay as related to limited hydrolysis of inhibitors

1989 ◽  
Vol 178 (1) ◽  
pp. 159-165 ◽  
Author(s):  
Keshun Liu ◽  
Pericles Markakis
Keyword(s):  
Inhibition Assay ◽  
Trypsin Inhibition ◽  
Hydrolysis Of

scholarly journals Catalytic activity and stereoselectivity of engineered phosphotriesterases towards structurally different nerve agents in vitro

2021 ◽  
Author(s):  
Anja Köhler ◽  
Benjamin Escher ◽  
Laura Job ◽  
Marianne Koller ◽  
Horst Thiermann ◽  
...  
Keyword(s):  
Plasma Clearance ◽  
Nerve Agents ◽  
Inhibition Assay ◽  
Ache Inhibition ◽  
Catalytic Activities ◽  
Chiral Lc ◽  
Hydrolysis Of ◽  
Medical Countermeasures

AbstractHighly toxic organophosphorus nerve agents, especially the extremely stable and persistent V-type agents such as VX, still pose a threat to the human population and require effective medical countermeasures. Engineered mutants of the Brevundimonas diminuta phosphotriesterase (BdPTE) exhibit enhanced catalytic activities and have demonstrated detoxification in animal models, however, substrate specificity and fast plasma clearance limit their medical applicability. To allow better assessment of their substrate profiles, we have thoroughly investigated the catalytic efficacies of five BdPTE mutants with 17 different nerve agents using an AChE inhibition assay. In addition, we studied one BdPTE version that was fused with structurally disordered PAS polypeptides to enable delayed plasma clearance and one bispecific BdPTE with broadened substrate spectrum composed of two functionally distinct subunits connected by a PAS linker. Measured kcat/KM values were as high as 6.5 and 1.5 × 108 M−1 min−1 with G- and V-agents, respectively. Furthermore, the stereoselective degradation of VX enantiomers by the PASylated BdPTE-4 and the bispecific BdPTE-7 were investigated by chiral LC–MS/MS, resulting in a several fold faster hydrolysis of the more toxic P(−) VX stereoisomer compared to P(+) VX. In conclusion, the newly developed enzymes BdPTE-4 and BdPTE-7 have shown high catalytic efficacy towards structurally different nerve agents and stereoselectivity towards the toxic P(−) VX enantiomer in vitro and offer promise for use as bioscavengers in vivo.

scholarly journals The Quenching Effect of Flavonoids on 4-Methylumbelliferone, a Potential Pitfall in Fluorimetric Neuraminidase Inhibition Assays

2011 ◽  
Vol 16 (7) ◽  
pp. 755-764 ◽  
Author(s):  
Jarinrat Kongkamnerd ◽  
Adelaide Milani ◽  
Giovanni Cattoli ◽  
Calogero Terregino ◽  
Ilaria Capua ◽  
...  
Keyword(s):  
Pearson Correlation ◽  
Experimental Uncertainty ◽  
Inhibition Assay ◽  
Inhibitory Effects ◽  
Quenching Effect ◽  
Acetylneuraminic Acid ◽  
Naturally Occurring ◽  
Neuraminidase Inhibition ◽  
Hydrolysis Of ◽  
Positive Results

Many assays aimed to test the inhibitory effects of synthetic molecules, and naturally occurring products on the neuraminidase activity exploit the hydrolysis of 2′-O-(4-methylumbelliferyl)-N-acetylneuraminic acid (4-MUNANA). The amount of the released product, 4-methylumbelliferone (4-MU), is then measured fluorimetrically. The authors attempted an analysis of the inhibitory properties of 35 naturally occurring flavonoids on neuraminidase N3, where only 29 of them were sufficiently soluble in the assay medium. During the analysis, the authors noticed a strong quenching effect due to the test compounds on the fluorescence of 4-MU. The quenching constants for the flavonoids were determined according to the Stern-Volmer approach. The extent of fluorescence reduction due to quenching and the magnitude of the fluorescence reduction measured in the inhibition assays were comparable: for 11 of 29 compounds, the two values were found to be coincident within the experimental uncertainty. These data were statistically analyzed for correlation by calculating the pertinent Pearson correlation coefficient. Inhibition and quenching were found to be positively correlated (r = 0.71, p(uncorr) = 1.5 × 10−5), and the correlation was maintained for the whole set of tested compounds. Altogether, the collected data imply that all of the tested flavonoids could produce false-positive results in the neuraminidase inhibition assay using 4-MUNANA as a substrate.

scholarly journals STUDY OF ANTI-INFLAMMATORY POTENTIAL OF WHOLE CELL EXRACTS OF CYANOBACTERIA BY MEMBRANE STABILIZATION & TRYPSIN INHIBITION ASSAY

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Shraddha Ratnaparkhe ◽  
Devyani Mali
Keyword(s):  
Tissue Damage ◽  
Inflammatory Activity ◽  
Inhibition Assay ◽  
Loss Of Function ◽  
Membrane Stabilization ◽  
Whole Cell ◽  
Cell Extracts ◽  
Trypsin Inhibition ◽  
Inflammatory Drugs ◽  
Anti Inflammatory

Inflammation is a complex mechanism in response to any infection, injury, or irritation. The prolonged inflammation leads to tissue damage and loss of function result in various disease conditions like osteoarthritis, and autoimmune diseases like lupus, rheumatoid arthritis, asthma and Crohn’s disease. Anti-inflammatory drugs are used to control inflammatory response and prevent tissue damage .Cyanobacteria are rich sources of phytochemicals like phenolics, flavonoids are known to have anti-inflammatory activity. Because of ease of cultivation and faster growth rate than plants, they are preferred candidates over plants. This study focuses on screening of cyanobacterial isolates for their anti-inflammatory activity. The human erythrocytes (HRBC) membrane stabilization assay indicated the potential of whole cell extracts of cyanobacteria to stabilize lysosomal membrane and thereby prevent tissue damage by lysosomal chemokines and enzymes. The trypsin inhibition assay is an indicator for potential to inhibit proteinases and decelerate tissue damage. The whole cell extracts of 10 cyanobacterial isolates of different genus namely Weistellopsis, Pseudophormidium, Oscillatoria, Nostoc, Phormidium, Chlorella, Hapalosiphon under study showed 85%-94% membrane stabilization in hypotonicity induced hemolysis and 92% to 97% membrane stabilization in heat induced hemolysis. The test extracts also showed 48% to 52% inhibition of trypsin. Thus, the isolates under study have application as anti-inflammatory agent.

scholarly journals ELISA-based competitive trypsin inhibition assay

2021 ◽  
Vol 35 (1) ◽  
pp. 1385-1392
Author(s):  
Bengü Ergenoğlu ◽  
Özlem Ertekin ◽  
Şerife Şeyda Pirinçci Göktürk ◽  
Göknur Gizem Dinç ◽  
Esin Akçael ◽  
...  
Keyword(s):  
Inhibition Assay ◽  
Trypsin Inhibition

Fine Structural Localization of Acyltransferases Involved in Lipid Synthesis in Intestinal Mucosa.

1970 ◽  
Vol 28 ◽  
pp. 54-55
Author(s):  
R. J. Barrnett ◽  
J. A. Higgins
Keyword(s):  
Smooth Endoplasmic Reticulum ◽  
Lipid Synthesis ◽  
Mucosal Cell ◽  
Structural Localization ◽  
Morphological Studies ◽  
Mucosal Cells ◽  
Initial Appearance ◽  
Sh Group ◽  
Hydrolysis Of ◽  
Intestinal Mucosal Cells

The main products of intestinal hydrolysis of dietary triglycerides are free fatty acids and monoglycerides. These form micelles from which the lipids are absorbed across the mucosal cell brush border. Biochemical studies have indicated that intestinal mucosal cells possess a triglyceride synthesising system, which uses monoglyceride directly as an acylacceptor as well as the system found in other tissues in which alphaglycerophosphate is the acylacceptor. The former pathway is used preferentially for the resynthesis of triglyceride from absorbed lipid, while the latter is used mainly for phospholipid synthesis. Both lipids are incorporated into chylomicrons. Morphological studies have shown that during fat absorption there is an initial appearance of fat droplets within the cisternae of the smooth endoplasmic reticulum and that these subsequently accumulate in the golgi elements from which they are released at the lateral borders of the cell as chylomicrons.We have recently developed several methods for the fine structural localization of acyltransferases dependent on the precipitation, in an electron dense form, of CoA released during the transfer of the acyl group to an acceptor, and have now applied these methods to a study of the fine structural localization of the enzymes involved in chylomicron lipid biosynthesis. These methods are based on the reduction of ferricyanide ions by the free SH group of CoA.

Lattice imaging and pore structure of β ferric oxyhydroxide

1978 ◽  
Vol 36 (1) ◽  
pp. 264-265
Author(s):  
T. Baird ◽  
J.R. Fryer ◽  
S.T. Galbraith
Keyword(s):  
Electron Microscopy ◽  
Room Temperature ◽  
Bulk Material ◽  
Model Structure ◽  
Bulk Crystals ◽  
Lattice Imaging ◽  
Thin Sections ◽  
Ferric Oxyhydroxide ◽  
Resolution Electron ◽  
Hydrolysis Of

Introduction Previously we had suggested (l) that the striations observed in the pod shaped crystals of β FeOOH were an artefact of imaging in the electron microscope. Contrary to this adsorption measurements on bulk material had indicated the presence of some porosity and Gallagher (2) had proposed a model structure - based on the hollandite structure - showing the hollandite rods forming the sides of 30Å pores running the length of the crystal. Low resolution electron microscopy by Watson (3) on sectioned crystals embedded in methylmethacrylate had tended to support the existence of such pores.We have applied modern high resolution techniques to the bulk crystals and thin sections of them without confirming these earlier postulatesExperimental β FeOOH was prepared by room temperature hydrolysis of 0.01M solutions of FeCl3.6H2O, The precipitate was washed, dried in air, and embedded in Scandiplast resin. The sections were out on an LKB III Ultramicrotome to a thickness of about 500Å.

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