Development and validation of a fluorogenic assay to measure separase enzyme activity

Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpre...

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Published in:Analytical biochemistry 2009-09, Vol.392 (2), p.133-138
Main Authors: Basu, Dipanjan, Zhang, Nenggang, Panigrahi, Anil K., Horton, Terzah M., Pati, Debananda
Format: Article
Language:English
Subjects:
Anaphase
Animals
Cell Line
Endopeptidases - analysis
Endopeptidases - metabolism
Enzyme Activation
Enzyme assay
Enzyme Stability
Female
Humans
Kinetics
Leukemia
Leukemia - enzymology
Metaphase
Nuclear Proteins - chemistry
Nuclear Proteins - metabolism
Oligopeptides - analysis
Oligopeptides - chemistry
Peptides - chemistry
Peptides - metabolism
Phosphoproteins - chemistry
Phosphoproteins - metabolism
Rad21
Securin
Separase
Spectrometry, Fluorescence - methods
Xenopus laevis
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Summary:Separase, an endopeptidase, plays a pivotal role in the separation of sister chromatids at anaphase by cleaving its substrate cohesin Rad21. Recent study suggests that separase is an oncogene. Overexpression of separase induces aneuploidy and mammary tumorigenesis in mice. Separase is also overexpressed and mislocalized in a wide range of human cancers, including breast, prostate, and osteosarcoma. Currently, there is no quantitative assay to measure separase enzymatic activity. To quantify separase enzymatic activity, we have designed a fluorogenic assay in which 7-amido-4-methyl coumaric acid (AMC)-conjugated Rad21 mitotic cleavage site peptide (Ac-Asp-Arg-Glu-Ile-Nle-Arg-MCA) is used as the substrate of separase. We used this assay to quantify separase activity during cell cycle progression and in a panel of human tumor cell lines as well as leukemia patient samples.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2009.05.046