title: Measurement of lysine-specific demethylase-1 activity in the nuclear extracts by flow-injection based time-of-flight mass spectrometry.
abstract: Lysine-specific demethylase 1 (LSD1/KDM1A), a histone-modifying enzyme, is upregulated in many cancers, especially in neuroblastoma, breast cancer and hepatoma. We have established a simple method to measure LSD1 activity using a synthetic N-terminal 21-mer peptide of histone H3, which is dimethylated at Lys-4 (H3K4me2). After the enzyme reaction, a substrate of H3K4me2 and two demethylated products, H3K4me1 and H3K4me0, were quantitatively determined by flow injection time-of-flight mass spectrometry (FI-TOF/MS). By using recombinant human LSD1, a nonlinear fitting simulation of the data obtained by FI-TOF/MS produced typical consecutive-reaction kinetics. Apparent K m and k cat values of hLSD1 for the first and second demethylation reactions were found to be in the range of reported values. Tranylcypromine was shown to inhibit LSD1 activity with an IC50 of 6.9 µM for the first demethylation reaction and 5.8 µM for the second demethylation reaction. The FI-TOF/MS assay revealed that the endogenous LSD1 activity was higher in the nuclear extracts of SH-SY5Y cells than in HeLa or PC-3 cells, and this is in accordance with the immunoblotting data using an anti-LSD1 antibody. A simple, straightforward FI-TOF/MS assay is described to efficiently measure LSD1 activity in the nuclear extracts of cultured cells.
authors.affiliation: Molecular and Cellular Biology, Graduate School of Human Health Science, University of Nagasaki, 1-1-1 Manabino, Nagayo, Nishisonogi-gun, Nagasaki 851-2195, Japan.
authors.forename: Chiharu
authors.initials: C
authors.lastname: Sakane
authors.affiliation: University of Nagasaki, 123 Kawashimo, Sasebo, Nagasaki 858-8580, Japan.
authors.forename: Hiromichi
authors.initials: H
authors.lastname: Ohta
authors.affiliation: Molecular and Cellular Biology, Graduate School of Human Health Science, University of Nagasaki, 1-1-1 Manabino, Nagayo, Nishisonogi-gun, Nagasaki 851-2195, Japan.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Shidoji
keywords: consecutive reactions; epigenetics; lysine-specific demethylase 1; time-of-flight mass spectrometry