Biomedical Engineering Reference
In-Depth Information
10.9.1
Pharmacological Blocking of HDAC s
The enzymatic activity of HDACs is inhibited by applying their blocker molecules.
HDAC inhibitors are categorized to fi ve groups: (1) short-chain fatty acids, (2)
hydroxamic acids, (3) cyclic tetrapeptides, (4) cyclic peptides, and (5) benzamides.
In the fi eld of neuroscience and neurobiology,
sodium butyrate
(a short-chain fatty
acid; Fig.
10.6e
; Bredy et al.
2007
;),
valproic acid
(a derivative of a hydroxamic
acid; Fig.
10.6f
; Bredy et al.
2007
; Rinaldi et al.
2007
; Bredy and Barad
2008
), and
trichostatin A
(a hydroxamic acid; Fig.
10.6g
; Korzus et al.
2004
; Levenson et al.
2004
; Bredy et al.
2007
; Chen et al.
2010
) are well used to analyze interactions
between histone acetylation and the consolidation of long-term memory. Sodium
butyrate and trichostatin A are not only effective in mammals but also in inverte-
brates (crabs; Federman et al.
2009
). Valproic acid inhibits GABA transaminase
(Löscher
1993
) and thus, by increasing the concentration of GABA in neurons
(Czuczwar and Patsalos
2001
), may interfere with neurotransmission of GABA. An
analog of valproic acid, valpromide (Fig.
10.6h
), also has anticonvulsant and mood-
stabilizing effects but is not an HDAC inhibitor making it useful as a negative con-
trol for valproic acid (Bredy et al.
2007
).
10.9.2
In Vitro Assays for Measurement of HAT Activity
As mentioned above, HATs add acetyl groups to lysine residues of histones and
nonhistone proteins, and their enzymatic activity can be biochemically measured.
For the broad quantifi cation of HAT activity in a sample solution, the easiest way is
to use HAT activity colorimetric assay kits (available from BioVision, Abcam, and
other companies). By using the kit, HAT activity can be quantifi ed by measuring the
intensity of the yellowish color of the HAT substrate packaged in the kits. Another
standard method for measuring HAT activity involves
3
H-marked acetyl coenzyme
A. Samples are incubated with histones and
3
H-marked acetyl coenzyme A in a buf-
fer containing 10 mM sodium butyrate (for blocking any contaminating HDACs in
the sample) at 30 °C for 30 min. Then, sample solutions are dropped onto strong
cation exchange papers (Grade P81, Whatman). The papers are washed with 0.2 M
sodium carbonate solution buffer, and the intensity of radioactivity was measured
with a scintillation counter.
Using
14
C-marked acetyl coenzyme A, target protein molecules of acetylation
can be identifi ed. After incubation under the same conditions described above, but
substituting
3
H-marked acetyl coenzyme A with
14
C-marked acetyl coenzyme A,
sample solutions were separated by standard SDS polyacrylamide gel electrophore-
sis. Gels were stained with Coomassie Brilliant Blue (CBB) and dried on fi lter
paper, and the radioactivity was transferred onto imaging plates. By comparing the
band patterns of CBB-stained gels and the radioactive images, acetylated proteins
could be identifi ed easily.
