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

Abstract

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.

• Authors: Dipanjan Basu, Nenggang Zhang, Anil K Panigrahi, Terzah M Horton, Debananda Pati
• Journal: Analytical biochemistry (Anal Biochem) Vol. 392 Issue 2 Pg. 133-8 (Sep 15 2009) ISSN: 1096-0309 [Electronic] United States
• PMID: 19497291 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't, Validation Study)

Chemical References

• Nuclear Proteins
• Oligopeptides
• Peptides
• Phosphoproteins
• acetyl-aspartyl-arginyl-glutamyl-isoleucyl-norleucyl-arginyl-7-amido-4-methylcoumarin
• Endopeptidases

Topics

• Anaphase
• Animals
• Cell Line
• Endopeptidases (analysis, metabolism)
• Enzyme Activation
• Enzyme Stability
• Female
• Humans
• Kinetics
• Leukemia (enzymology)
• Metaphase
• Nuclear Proteins (chemistry, metabolism)
• Oligopeptides (analysis, chemistry)
• Peptides (chemistry, metabolism)
• Phosphoproteins (chemistry, metabolism)
• Spectrometry, Fluorescence (methods)
• Xenopus laevis