Development and validation of a simple cell-based fluorescence assay for dipeptidyl peptidase 1 (DPP1) activity.
J Biomol Screen
; 16(1): 36-43, 2011 Jan.
Article
em En
| MEDLINE
| ID: mdl-21088147
ABSTRACT
Dipeptidyl peptidase 1 (DPP1) (EC 3.4.14.1; also known as cathepsin C, cathepsin J, dipeptidyl aminopeptidase, and dipeptidyl aminotransferase) is a lysosomal cysteinyl protease of the papain family involved in the intracellular degradation of proteins. Isolated enzyme assays for DPP1 activity using a variety of synthetic substrates such as dipeptide or peptide linked to amino-methyl-coumarin (AMC) or other fluorophores are well established. There is, however, no report of a simple whole-cell-based assay for measuring lysosomal DPP1 activity other than the use of flow cytometry (fluorescence-activated cell sorting) or the use of invasive activity-based probes or the production of physiological products such as neutrophil elastase. The authors investigated a number of DPP1 fluorogenic substrates that have the potential to access the lysosome and enable the measurement of DPP1 enzyme activity in situ. They describe the development and evaluation of a simple noninvasive fluorescence assay for measuring DPP1 activity in fresh or cryopreserved human THP-1 cells using the substrate H-Gly-Phe-AFC (amino-fluoro-coumarin). This cell-based fluorescence assay can be performed in a 96-well plate format and is ideally suited for determining the cell potency of potential DPP1 enzyme inhibitors.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Bioensaio
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Catepsina C
/
Dipeptídeos
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Aminocumarinas
/
Corantes Fluorescentes
/
Lisossomos
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Biomol Screen
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2011
Tipo de documento:
Article
País de afiliação:
Reino Unido