Quenched BODIPY Dye-Labeled Casein Substrates for the Assay of Protease Activity by Direct Fluorescence Measurement

Jones, Laurie J.; Upson, Rosalyn H.; Haugland, Rosaria P.; Panchuk-Voloshina, Nataliya; Zhou, Mingjie; Haugland, Richard P.

doi:10.1006/abio.1997.2259

Cited by 171 publications

(172 citation statements)

References 15 publications

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“…BODIPY s FL casein is a conjugate consisting of casein labelled with the fluorophore 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY s FL) (Jones et al, 1997). The casein is heavily labelled with the fluorescent BODIPY s FL dye, resulting in almost total quenching of the conjugate's fluorescence.…”

Section: Cell Extraction Of Mmpsmentioning

confidence: 99%

“…The casein is heavily labelled with the fluorescent BODIPY s FL dye, resulting in almost total quenching of the conjugate's fluorescence. The assay detects protease activity of a wide range of enzymes including serine proteases, sulphydryl proteases, acid proteases and metalloproteinases (Jones et al, 1997). The culture medium, or cell extracts, was added to an equal volume of BODIPY s casein at a concentration of 50 mg ml À1 in a 10 mM Tris-HCl pH 7.8 assay buffer under low light conditions.…”

Section: Cell Extraction Of Mmpsmentioning

confidence: 99%

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TNF-α increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes

et al. 2003

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Inflammatory mediators have been reported to promote malignant cell growth, invasion and metastatic potential. More specifically, we have recently reported that tumour necrosis factor alpha (TNF-a) increases melanoma cell attachment to extracellular matrix (ECM) substrates and invasion through fibronectin. In this study, we extend these investigations asking specifically whether the TNF-a effect on cell invasion and migration involves activation of proteolytic enzymes. We examined the effect of TNF-a on melanoma expression/activation of type IV gelatinases matrix metalloproteinases 2 and 9 (MMPs -2 and -9) and general proteolytic enzymes. Stimulation with TNF-a significantly increased both melanoma cell migration at 24 h ( þ 21%) and invasion through fibronectin ( þ 35%) but did not upregulate/activate the expression of latent MMP-2 constitutively produced by these cells and did not upregulate their general protease activity. However, the increased cell migration and invasion through fibronectin observed following stimulation with TNF-a were inhibited by the general protease inhibitor a 2 macroglobulin. These findings suggest that the promigratory and proinvasive effect of TNF-a on this melanoma cell line may be mediated to some extent by induction of localised cell membrane-bound degradative enzyme activity, which is not readily detected in biochemical assays.

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Section: Cell Extraction Of Mmpsmentioning

confidence: 99%

Section: Cell Extraction Of Mmpsmentioning

confidence: 99%

TNF-α increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes

et al. 2003

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“…25,26 According to the sample standard curve obtained with the EnzChek protease assay, the detection limit of this assay was between c trypsin = 1-5 ng/mL for trypsin in 10 mM Tris-HCl at pH 7.8 with a fluorescence microplate reader using 200 μL reaction mixtures. We set the concentration range for our experiment between c trypsin = 1 ng/mL and c trypsin = 1 μg/mL to include the above-mentioned detection limit.…”

Section: Dynamic Range Assessmentmentioning

confidence: 99%

Increase of Reaction Rate and Sensitivity of Low-Abundance Enzyme Assay Using Micro/Nanofluidic Preconcentration Chip

et al. 2008

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We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a micro/nanofluidic preconcentration chip. The disposable preconcentration device made out of PDMS with a surface-patterned ion-selective membrane increases local enzyme/ substrate concentrations for rapid monitoring of enzyme activity. As a model system, we used trypsin as the enzyme and BODIPY FL casein as the fluorogenic substrate. We demonstrated that the reaction rate of trypsin-BODIPY FL was significantly enhanced by increasing the local concentrations of both trypsin and BODIPY FL casein in the preconcentration chip. The reaction time required to turn over substrates at 1 ng/mL was only ~10 min compared to ~1 h without preconcentration, which demonstrates significantly higher reaction rate through the increase of the concentrations of both the enzyme and substrate. Furthermore, trypsin activity can be measured down to a concentration level of 10 pg/mL, which is ~100 fold enhancement in sensitivity compared to the result without the preconcentration step. This micro/nanofluidic preconcentrator chip could be used as a generic micro reaction platform to study any enzyme-substrate systems, or other biochemical reaction systems in low concentration ranges.

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“…Although non-specific proteolysis of endocytosed proteins such as membrane receptors or extracellular proteins is largely confined to the lysosomal/ vacuolar system 22,55,56,61,66,67 proteolysis also takes place in other cell compartments. The staining procedure was conducted according to Hutter et al 46 with casein substrates labelled with quenched BODIPY ® FL dye, which is a substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative 35,53 . This casein-dye conjugate provides spectrally distinct substrates for detecting metallo, serine, acid and sulfhydryl proteinases.…”

Section: Intracellular Proteinase Activitymentioning

confidence: 99%

“…The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population 19 . BODIPY FL casein and their hydrolysis products show green fluorescence with maximal excitation at about 503 nm and emission maxima near 512 nm 53 .…”

Section: Intracellular Proteinase Activitymentioning

confidence: 99%

The Potential of Confocal Imaging for Measuring Physiological Changes in Brewer's Yeast

Schlee

Miedl

Leiper

et al. 2006

Journal of the Institute of Brewing

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This study focused on the implementation of fluorescence optical methods and laser scanning confocal microscopy for monitoring brewing yeast performance. Physiological parameters and cell compounds in yeast cells (glycogen, neutral lipids, trehalose, bud scars, DNA and intracellular proteinases) have been successfully visualised with the aid of highly specific fluorochromes. The expression and sub cellular localisation of proteinase A during fermentation has been studied employing a Saccharomyces cerevisiae green fluorescent protein clone. This novel approach to monitoring brewing yeast performance provides new insights into physiological events that occur during wort fermentation.

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Quenched BODIPY Dye-Labeled Casein Substrates for the Assay of Protease Activity by Direct Fluorescence Measurement

Cited by 171 publications

References 15 publications

TNF-α increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes

TNF-α increases human melanoma cell invasion and migration in vitro: the role of proteolytic enzymes

Increase of Reaction Rate and Sensitivity of Low-Abundance Enzyme Assay Using Micro/Nanofluidic Preconcentration Chip

The Potential of Confocal Imaging for Measuring Physiological Changes in Brewer's Yeast

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