Fluorogenic Assay for Penicillin G Acylase Activity

Ninkovic, Milena; Riester, Daniel; Wirsching, Frank; Dietrich, Rüdiger; Schwienhorst, Andreas

doi:10.1006/abio.2001.5078

Cited by 12 publications

(6 citation statements)

References 32 publications

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“…Homogeneous fluorogenic assays are, however, highly desirable, e.g., assays in which the quenched (nonfluorescent) fluorophore is enzymatically released as a then strongly fluorescent moiety in the scope of the enzymatic reaction. A recent example of this type of fluorogenic assay is the liberation of AMC by penicillin G acylase [41].…”

Section: Hdac Reaction Were Taken At Different Time Points Andmentioning

confidence: 99%

A Fluorogenic Histone Deacetylase Assay Well Suited for High-Throughput Activity Screening

Wegener¹,

Wirsching²,

Riester³

et al. 2003

Chemistry & Biology

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266

238

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Histone deacetylases (HDACs) are important enzymes for the transcriptional regulation of gene expression in eukaryotic cells. Recent findings suggest that HDACs could be key targets for chemotherapeutic intervention in malignant diseases. A convenient and sensitive fluorogenic assay for HDAC activity would therefore expedite studies of HDAC in transcriptional regulation and in vitro screening for drug discovery. In this study, novel fluorogenic substrates of HDACs were synthesized with an epsilon-acetylated lysyl moiety and an adjacent MCA moiety at the C terminus of the peptide chain. Upon deacetylation of the acetylated lysyl moiety, molecules became substrates for trypsin, which released highly fluorescent AMC molecules in a subsequent step of the assay. The fluorescence increased in direct proportion to the amount of deacetylated substrate molecules, i.e., HDAC activity. The nonisotopic, homogeneous assay is well suited for high-throughput HDAC inhibitor screening.

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Section: Hdac Reaction Were Taken At Different Time Points Andmentioning

confidence: 99%

A Fluorogenic Histone Deacetylase Assay Well Suited for High-Throughput Activity Screening

Wegener¹,

Wirsching²,

Riester³

et al. 2003

Chemistry & Biology

Self Cite

266

238

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“…In the design of the traceless linker, the biocatalyzed transformation (i.e., the enzymatic hydrolysis of a carboxamide bond) is thus combined with a domino reaction involving fragmentation of the hemithioaminal 5 and subsequent self-cyclization of the resulting thioalkyl carbonate 6 , yielding to the release of the phenol derivatives (Figure ). To validate the postulated mechanism of this cascade reaction and to check its efficacy under physiological conditions, we have chosen to work with PGA, a commercially available and widely used biocatalyst in the enzymatic synthesis of β-lactam antibiotics, since it allows for the deprotection of phenacetyl-protected amines and the subsequent release of umbelliferone (i.e., 7-hydroxycoumarin), at the blue fluorescent phenolic fluorophore. Thus, it was possible to follow the enzymatic hydrolysis and the following decomposition of the self-immolative spacer by means of a simple and rapid in vitro fluorescence assay.…”

mentioning

confidence: 99%

Development of a New Nonpeptidic Self-Immolative Spacer. Application to the Design of Protease Sensing Fluorogenic Probes

et al. 2008

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The design and synthesis of novel self-immolative spacer systems aiming at the release of phenol-containing compounds are described. The newly designed traceless linkers proved to be conveniently stable under physiological conditions and operate through spontaneous decomposition of an hemithioaminal intermediate under neutral aqueous conditions. Their utility was then illustrated by the preparation of original fluorogenic substrates of penicillin amidase whose strong fluorescence is unveiled through enzyme-initiated domino reactions.

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“…We therefore designed a codon-optimized PGA variant that does not contain the signal sequence. Using a previously described fluorogenic coumarin PGA substrate, we found that PGA produced in human embryonic kidney (HEK) 293T cells was active and not toxic to the cells (Supporting Figure 1).…”

mentioning

confidence: 95%

Cell-specific Profiling of Nascent Proteomes Using Orthogonal Enzyme-mediated Puromycin Incorporation

et al. 2016

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Translation regulation is a fundamental component of gene expression, allowing cells to respond rapidly to a variety of stimuli in the absence of new transcription. The lack of methods for profiling nascent proteomes in distinct cell populations in heterogeneous tissues has precluded an understanding of translational regulation in physiologically relevant contexts. Here, we describe a chemical genetic method that involves orthogonal enzyme-mediated incorporation of a clickable puromycin analogue into nascent polypeptides. Using this method, we show that we can label newly synthesized proteins in a cell-specific manner in cells grown in culture and in heterogeneous tissues. We also show that we can identify the nascent proteome in genetically targeted cell populations using affinity enrichment and tandem mass spectrometry. Our method has the potential to provide unprecedented insights into cell-specific translational regulation in heterogeneous tissues.

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Fluorogenic Assay for Penicillin G Acylase Activity

Cited by 12 publications

References 32 publications

A Fluorogenic Histone Deacetylase Assay Well Suited for High-Throughput Activity Screening

A Fluorogenic Histone Deacetylase Assay Well Suited for High-Throughput Activity Screening

Development of a New Nonpeptidic Self-Immolative Spacer. Application to the Design of Protease Sensing Fluorogenic Probes

Cell-specific Profiling of Nascent Proteomes Using Orthogonal Enzyme-mediated Puromycin Incorporation

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