Peptide
1 was purchased
from GenScript USA Inc. (NJ, USA); the purity was stated by the manufacturer
to be >95%. Peptides 2 and 3 were synthesized, purified, and characterized
as described elsewhere.17 (link) The peptide sequences
are shown in Table1 . For inhibition studies
on KDM1A, a peroxidase-coupled assay monitoring hydrogen peroxide
production was performed as previously described.18 (link) KDM1A expression and purification was carried out as described
elsewhere;13 (link) non-GST-tagged KDM1A was purchased
from BPS Biosciences (#50097). The time courses of the reaction were
measured under aerobic conditions using a Beckman Instruments DU series
600 spectrophotometer equipped with a thermostat-controlled cell holder
(T = 25 °C). The 100 μL reactions were
initiated by addition of enzyme (100–200 nM) to the reaction
mixture (HEPES buffer (50 mM, pH 7.5), 4-aminoantipyrine (0.1 mM),
3,5-dichloro-2-hydroxybenzenesulfonic acid (1 mM), horseradish peroxidase
(0.76 μM, Worthington Biochemical Corp.), peptide 1 (100 μM),
and H3K4me2 histone peptide substrate (24 μM)). Absorbance changes
were monitored at 515 nm, and an extinction coefficient of 26 000
M–1·cm–1 was used to calculate
product formation. For curve fitting and data analysis, GraphPad Prism
6.0 was used.
1 was purchased
from GenScript USA Inc. (NJ, USA); the purity was stated by the manufacturer
to be >95%. Peptides 2 and 3 were synthesized, purified, and characterized
as described elsewhere.17 (link) The peptide sequences
are shown in Table
on KDM1A, a peroxidase-coupled assay monitoring hydrogen peroxide
production was performed as previously described.18 (link) KDM1A expression and purification was carried out as described
elsewhere;13 (link) non-GST-tagged KDM1A was purchased
from BPS Biosciences (#50097). The time courses of the reaction were
measured under aerobic conditions using a Beckman Instruments DU series
600 spectrophotometer equipped with a thermostat-controlled cell holder
(T = 25 °C). The 100 μL reactions were
initiated by addition of enzyme (100–200 nM) to the reaction
mixture (HEPES buffer (50 mM, pH 7.5), 4-aminoantipyrine (0.1 mM),
3,5-dichloro-2-hydroxybenzenesulfonic acid (1 mM), horseradish peroxidase
(0.76 μM, Worthington Biochemical Corp.), peptide 1 (100 μM),
and H3K4me2 histone peptide substrate (24 μM)). Absorbance changes
were monitored at 515 nm, and an extinction coefficient of 26 000
M–1·cm–1 was used to calculate
product formation. For curve fitting and data analysis, GraphPad Prism
6.0 was used.
