• Bulletin of the Korean Chemical Society
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• v.35 no.12
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• pp.3642-3644
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• 2014

• BMB Reports
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• v.44 no.7
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• pp.458-461
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• 2011

Enteropeptidase is a serine protease secreted by the pancreas and converts inactive trypsinogen to active trypsin. Enteropeptidase cleaves the C-terminal end of the substrate recognition sequence Asp-Asp-Asp-Asp-Lys ($D_4K$). The assay for enteropeptidase has utilized $GD_4K$-conjugated 2-naphthylamine ($GD_4K$-NA) as a fluorogenic probe over the last 30 years. However, no other $D_4K$-conjugated fluorogenic substrates of enteropeptidase have been reported. Furthermore, naphthalene is known as carcinogenic to humans. In this study, we used shift in the emission spectrum of $GD_4K$-conjugated 7-amino-4-methylcoumarin ($GD_4K$-AMC) as a fluorogenic method to measure enteropeptidase activity. The kinetic analysis revealed that enteropeptidase has a $K_M$ of 0.025 mM and a $k_{cat}$ of 65 $sec^{-1}$ for $GD_4K$-AMC, whereas it has a $K_M$ of 0.5 to 0.6 mM and a $k_{cat}$ of 25 $sec^{-1}$ for $GD_4K$-NA. The optimum pH of $GD_4K$-AMC hydrolysis was pH 8.0. Our data indicate that $GD_4K$-AMC is more suitable as a substrate for enteropeptidase than $GD_4K$-NA.

• IMMUNE NETWORK
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• v.22 no.3
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• pp.28.1-28.11
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• 2022

The 26S proteasome irreversibly hydrolyzes polyubiquitylated substrates to maintain protein homeostasis; it also regulates immune responses by generating antigenic peptides. An alternative form of the 26S proteasome is the immunoproteasome, which contains substituted catalytic subunits (β1i/PSMB9, β2i/PSMB10, and β5i/PSMB8) instead of constitutively expressed counterparts (β1/PSMB6, β2/PSMB7, and β5/PSMB5). The immunoproteasome expands the peptide repertoire presented on MHC class I molecules. However, how its activity changes in this context is largely elusive, possibly due to the lack of a standardized methodology to evaluate its specific activity. Here, we describe an assay protocol that measures the immunoproteasome activity of whole-cell lysates using commercially available fluorogenic peptide substrates. Our results showed that the most accurate assessment of immunoproteasome activity could be achieved by combining β5i-targeting substrate Ac-ANW-AMC and immunoproteasome inhibitor ONX-0914. This simple and reliable protocol may contribute to future studies of immunoproteasomes and their pathophysiological roles during viral infection, inflammation, and tumorigenesis.

• Rapid Communication in Photoscience
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• v.4 no.4
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• pp.88-90
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• 2015

The design and development of fluorescent chemosensors have recently been intensively explored for sensitive and specific detection of environmentally and biologically relevant metal ions in aqueous solution and living cells. Herein, we report the photophysical results of rhodamine B based fluorogenic and chromogenic receptor for selective copper detection in the complete organic or mixed aqueous-organic media at neutral pH under ambient condition. The ligand exhibited the remarkable increment in the fluorescence emission and UV-visible absorption signal intensities at 587 and 547 nm, respectively, on induction of copper ion while the ligand solution remain completely silent on addition of varieties of other metal ions.

• Macromolecular Research
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• v.16 no.1
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• pp.73-75
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• 2008

• Proceedings of the PSK Conference
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• 2003.04a
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• pp.125.2-126
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• 2003

Human tumor necrosis factor-alpha (TNFa) is a key pro-inflammatory cytokine produced by activated monocytes and macrophage as a part of the self-defence machinery. TNF-a converting enzyme (TACE) is the metalloproteinase that processes the membrane bound precursor of TNFa to the soluble component. (omitted)

• Biomedical Science Letters
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• v.18 no.4
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• pp.438-444
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• 2012

N-acylethanolamines (NAEs) including endocannabinoids, anadamide, are long chain fatty acid ethanolamines and express ubiquitously in animal and plant tissues. NAEs have several pharmacological effects including anti-inflammatory, analgesic and anorexic effects. The levels of NAEs in tissues are strictly regulated by synthesizing and hydrolyzing enzymes because NAEs are not stored in the cell but rather made on demand. NAEs are hydrolyzed to free fatty acids and ethanolamines by fatty acid amide hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA). Here, we suggest the fluorescence-based assay system for NAAA. We developed N-(4-methy-2-oxo-2H-chromen-7-yl)palmitamide (PAAC) as a fluorogenic substrate for NAAA and we also generated NAAA stably expressing COSM6 cell line. When extracts of cells expressing NAAA were incubated with PAAC, NAAA specifically hydrolyzed PAAC to palmitic acids and fluorogenic dye, coumarin. Release of coumarin was monitored by using fluorometer. NAAA hydrolyzed PAAC with an apparent Km of $20.05{\mu}M$ and Vmax of 32.18 pmol/mg protein/min. This assay system can be used to develop inhibitors or activators of NAAA.

• Korean Journal of Microbiology
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• v.28 no.3
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• pp.229-235
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• 1990

A rapid and sensitive method to detect the extracellular enzymatic activity of bacteria colonies grown on agar plates is described. Selective agar media supplemented with protein, starch, chitin, Tween-80, etc. are conventionally used to detect biochemical properties of bacteria. It has been experimentally demonstrated with bacteria pure cultures that fluorogenic Methylumbelliferyl (MUF)-substrates are excellent substrate analogues for normally occurring polymers. Based on MUF-substrate hydrolysis the new method provides reliable qualitative estimates of extracellular enzymatic properties of bacteria within minutes using pure cultures as well as agar p;ates prepared for colony counts. Extracellular enzyme activities of heterotrophic bacteria from freshwater ecosystems and marine sediment using this method are discussed.

• Bulletin of the Korean Chemical Society
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• v.31 no.3
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• pp.615-619
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• 2010

A new coumarin-thiazolobenzo-crown ether based fluorogenic chemosensor BTC (1) was reported. The ion-selective binding properties of 1 with different alkali, alkaline earth metals and transitional metals were investigated in an ethanol-DMSO system. BTC (1) showed the highest binding constant toward $Hg^{2+}$ over $Ag^+$, $Pb^{2+}$ and $Cu^{2+}$.

• Proceedings of the Korean Society of Dyers and Finishers Conference
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• 2010.03a
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• pp.132-132
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• 2010