Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates

Ganesh, Subramaniam; Klingel, Sven; Kahle, H.; Valet, G.

doi:10.1002/cyto.990200409

Cited by 12 publications

(10 citation statements)

References 11 publications

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“…2). These data concur with earlier observations of Valet and his colleagues who measured L-aminopeptidase activity in white blood cells by flow cytometry (8). Populations of monocytes, granulocytes, or lymphocytes were also very heterogeneous, and there was significant overlap between these populations in the rate of hydrolysis of di-(leucyl)-rhodamine 110 (Fig.…”

Section: Measurement Of L-aminopeptidase and Esterase Activities And supporting

confidence: 92%

“…Aminopeptidases are of different categories and are present both on the cell surface as well as within the cell, in different cell compartments. Introduction of fluorogenic substrates utilizing the fluorochrome rhodamine 110 attached to peptides or single amino acids (19) made it possible to measure the activity of these enzymes by flow cytometry (8,30).…”

Section: Measurement Of L-aminopeptidase and Esterase Activities And mentioning

confidence: 99%

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Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry

1998

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This study was designed to explore the utility of a microscope‐based cytofluorometer, the laser scanning cytometer (LSC), for time‐resolved kinetic measurements. This instrument measures fluorescence of individual cells rapidly, with high sensitivity and accuracy. Also recorded in a list mode fashion are the spatial X‐Y coordinates of the cell on the slide as well as the time of individual cell measurement. Repeated measurement of each of a group of cells within a selected area of the slide, thus, yields information on their fluorescence parameters (integrated value, maximal pixel intensity, or fluorescence area) as a function of time. Using the fluorogenic substrate di‐(leucyl)‐rhodamine 110, we measured the kinetic activity of L‐aminopeptidase in HL‐60 cells and in monocytes, granulocytes, and lymphocytes from human blood. Likewise, the rate of fluorescein diacetate (FDA) hydrolysis by esterases was measured in HL‐60 cells. Also studied was the rate of uptake of the lysosomo‐trophic fluorochrome acridine orange (AO) by human leukocytes. Several hundred cells per sample were measured rapidly with a time resolution of 20–60 s. The resolution was inversely proportional to the number of cells within the measured population. The kinetic curves constructed for individual cells had clearly defined slope and plateau regions. During data analysis the kinetic plots were matched with the respective cells; the latter were identified by their position on the slide and classified by their fluorescence or image after staining with Giemsa. Great intercellular variability was noted in enzyme kinetics among cells of the same type, and differences were seen in rate of AO uptake between granulocytes, monocytes, and lymphocytes. Fluorescence fading and recovery was observed especially in the case of FDA hydrolysis and AO uptake. Our results indicate that LSC can be used to rapidly measure the rate of uptake or accumulation of a particular fluorochrome or the kinetics of enzymatic reactions in individual cells of large populations to reveal intercellular variability or the presence of a cell subpopulation with different kinetic properties. Cytometry 33:1–9, 1998. © 1998 Wiley‐Liss, Inc.

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Section: Measurement Of L-aminopeptidase and Esterase Activities And supporting

confidence: 92%

Section: Measurement Of L-aminopeptidase and Esterase Activities And mentioning

confidence: 99%

Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry

1998

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“…For control purposes, also dD 2 R was applied in the assay. We have expected that caspases would not cleave substrates containing D-amino acids as it has been described for aminopeptidases (21) and hence thought that dD 2 R could serve as a negative control. However, the cleavage of dD 2 R, detected in Jurkat but not in CEM cells, shows that caspases or caspase-like enzymes are able to use rhodamine 110 linked to the D-enantiomer of aspartic acid.…”

Section: Resultsmentioning

confidence: 94%

Rhodamine 110-Linked Amino Acids and Peptides as Substrates To Measure Caspase Activity upon Apoptosis Induction in Intact Cells

Hug¹,

Łos²,

Hirt³

et al. 1999

Biochemistry

124

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Caspases (cysteine aspartate-specific proteases) are a structurally related group of cysteine proteases that cleave peptide bonds following specific recognition sequences. They play a central role in activating apoptosis of vertebrate cells. To measure apoptosis induced by various stimuli and at an early apoptotic stage, caspases are an ideal target. This is especially the case when apoptotic cells have to be analyzed ex vivo before phagocytes remove them. A new and sensitive caspase assay is based on a substrate that contains only aspartate residues linked to rhodamine 110. With this and similar substrates, we are able to detect intracellular caspase activation by flow cytometry after apoptosis induction in intact hematopoetic cell lines.

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“…Serine proteases [271] Plasmin [272], Thrombin [272], Subtilisin [37], Tripeptidyl peptidase I [273], Hepatitis C viral protease [274], chymotrypsin [274] Metalloproteases Aminopeptidases [275,276] Cysteine proteases Caspases [269,270], Cathepsin B [274], Cathepsin C [277], Cathepsin K [278], SARS main protease [279], papain [274] Glycosidases β-glucosidase [213] Phosphodiesterase/Phosphatases [225,280] Lipase/esterase [86] Other hydrolases β-N-Acetylglucoaminidase [281],…”

Section: Enzyme Class Specific Enzymementioning

confidence: 99%

Activatable Optical Probes for the Detection of Enzymes

Drake¹,

Miller²,

Jones³

2011

COS

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The early detection of many human diseases is crucial if they are to be treated successfully. Therefore, the development of imaging techniques that can facilitate early detection of disease is of high importance. Changes in the levels of enzyme expression are known to occur in many diseases, making their accurate detection at low concentrations an area of considerable active research. Activatable fluorescent probes show immense promise in this area. If properly designed they should exhibit no signal until they interact with their target enzyme, reducing the level of background fluorescence and potentially endowing them with greater sensitivity. The mechanisms of fluorescence changes in activatable probes vary. This review aims to survey the field of activatable probes, focusing on their mechanisms of action as well as illustrating some of the in vitro and in vivo settings in which they have been employed.

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Flow cytometric determination of aminopeptidase activities in viable cells using fluorogenic rhodamine 110 substrates

Cited by 12 publications

References 11 publications

Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry

Enzyme kinetic reactions and fluorochrome uptake rates measured in individual cells by laser scanning cytometry

Rhodamine 110-Linked Amino Acids and Peptides as Substrates To Measure Caspase Activity upon Apoptosis Induction in Intact Cells

Activatable Optical Probes for the Detection of Enzymes

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