title: 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.affiliation: Department of Pediatric Hematology/Oncology, Texas Children's Cancer Center, Baylor College of Medicine, Houston, TX 77030, USA.
authors.forename: Dipanjan
authors.initials: D
authors.lastname: Basu
authors.forename: Nenggang
authors.initials: N
authors.lastname: Zhang
authors.forename: Anil K
authors.initials: AK
authors.lastname: Panigrahi
authors.forename: Terzah M
authors.initials: TM
authors.lastname: Horton
authors.forename: Debananda
authors.initials: D
authors.lastname: Pati
mesh_terms: D000705:Anaphase; D000818:Animals; D002460:Cell Line; D010450:Endopeptidases / Q000032:analysis* / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D007700:Kinetics; D007938:Leukemia / Q000201:enzymology; D008677:Metaphase; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D009842:Oligopeptides / Q000032:analysis* / Q000737:chemistry; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014982:Xenopus laevis
chemical_list: D009687:Nuclear Proteins; D009842:Oligopeptides; D010455:Peptides; D010750:Phosphoproteins; C544000:acetyl-aspartyl-arginyl-glutamyl-isoleucyl-norleucyl-arginyl-7-amido-4-methylcoumarin; D010450:Endopeptidases