2
N. Boostan, R. Yazdanparast / Biochemical and Biophysical Research Communications xxx (xxxx) xxx
membrane integrity by the peptide [9].
phosphate, pH ¼ 6.8, 5 mM glutamate, 250
phate, 0.4% mercapthoethanol) was added to 100
homogenated sample. After incubation at 38 C for 60 min, the re-
action was stopped by adding 60 L of 0.25 N HCl and the tubes
were kept at 60 C for 10 min. The GABA content of each sample was
then determined by measuring the NADPH content, using an
m
M pyridoxal phos-
Regarding the structural homology between CLN and Mag II, in
this investigation we were aimed to evaluate the influence of Mag II
on the mouse hypothalamic GABA content via evaluating the in-
fluence on the GABA shunt pathway enzymes namely GAD, GABA-T
and SSADH. Our in vitro and in vivo results confirmed a positive
modulating role on the GAD enzyme, while the other two enzymes
mL of each of the
m
enzymatic cycling method [10]. Briefly, 430
mL of GABA assay re-
(
GABA-T and SSADH) were inhibited by Mag II administration.
agent (0.3 M Tris-HCl, pH ¼ 8.9, 8 mM
a-ketoglutarate, 0.5 mM
NADP , 0.01% W/V mercaptoethanol, and 0.1 mg GABAse) was
appended to the GAD assay solution followed by 30 min incubation
þ
2
. Materials and methods
at 37 C. The reaction was stopped by adding 60
incubating at 60 C for 10 min. Then, 40 L of the GABA assay
mixture was transferred to 500 L of NADPH cycling reagent (0.1 M
Tris-HCl, pH ¼ 8.0, 4 mM -ketoglutarate, 1.5 mM glucose 6- phos-
phate, 0.1 mM ADP, 0.02% bovine serum albumin, 20 mM ammo-
nium chloride, 11 g/mL glucose 6-phosphate dehydrogenase and
25 g/mL glutamate dehydrogenase). The mixture was incubated
mL of 0.25 N NaOH,
2
.1. Chemicals
m
m
0
Adenosine 5 -diphosphate, Aminoethylisothiouronium bromide
a
(
AET), 4-Aminobutyric acid (GABA), GABAse,
D
-
Glucose 6-
- Glucose 6-phosphate dehydrogenase, L-Glutamic
-Nicotinamide adenine dinucleotide, -Nicotin-
amide adenine dinucleotide (reduced form), -Nicotinamide
adenine dinucleotide phosphate, -Nicotinamide adenine dinu-
phosphate,
dehydrogenase,
D
m
a
a
m
a
for half an hour at 38 C and heated at 100 C for 2 min afterward. At
the end, 1 mL of 6-phosphogluconate assay reagent (0.1 M Tris-HCl,
a
þ
cleotide phosphate (reduced form), Oxoglutaric acid, 6-
Phosphogluconic dehydrogenase, Pyridoxal 5 -phosphate, Suc-
pH 8.0, 0.1 mM EDTA and 0.1 mM NADP ) was subjoined to the
0
droplets. After keeping the mixture at room temperature for
cinic semialdehyde, Triton X-100, Tris-HCl and Magainin II (dis-
solved in physiological saline) all were purchased from Sigma
Chemical Company (Paris, France).
30 min, the fluorescence was measured at
l
¼ 455 nm. The control
sample has been subjected to the same procedure. It is important to
note that the degradation probability of the produced GABA is
negligible as the optimum pH for the GABA degrading enzyme is far
from pH ¼ 6.8 of the GAD assay mixture. Finally, the GAD activity
2.2. Laboratory animals
was expressed in
mmol/g hypothalamus/h by using NADPH cali-
̊
Male NIH mice, weighing about 25e30 g, were purchased from
bration graph in the range of 0.001e0.005 mg/mL.
Razi institute (Karaj, Iran) and kept under standard conditions in
the animal house of the institute of Biochemistry and Biophysics,
University of Tehran. Ad libitum access to food and water at a
controlled temperature of 20 ± 2 C under a 10 h light cycle was
provided for the mice. All animal experiments and methods were
conducted according to the guidelines of animal ethics committee
of University of Tehran.
̊
2.7. GABA-T and SSADH assay
A coupled enzyme system was used for assaying the activity of
GABA-T enzyme. In this system, the endogenous SSADH is utilized
to convert the preformed succinic semialdehyde (SSA) to succinate
þ
(SA) in the presence of NAD . The concurrently produced NADH
̊
was used as a measure of GABA-T activity. The incubation mixture
̊
2.3. Experimental design and procedures
was composed of 50 mM Tris-HCl, pH ¼ 8.5, 3.0 mM GABA, 2 mM 2-
þ
oxoglutaric acid, 20 mM 2-mercaptoethanol, 1.1 mM NAD and
Mice were randomly assigned into groups of six. Freshly pre-
pared Magainin II (50 mg/kg body weight) injected intraperitonealy
0.2 mL of Triton-treated homogenate in a total volume of 1.125 as
described by Th. De Boer [11]. After a preincubation period of
30 min at 22 C, 0.1 mL of 33.75 mM GABA was added to the ice-cold
incubation solution and the reaction was started by incubating the
to the treated groups. An equal volume of physiological saline was
used for control group administration. At different time intervals,
the mice decapitation was followed by rapid brain dissection from
the skull and removal of hypothalamus. Finally, the collected tis-
sues were weighed and subjected to homogenization using a Teflon
glass homogenizer.
̊
̊
sample tubes at 22 C. Finally, the formation of NADH was measured
spectrophotometrically at
l
¼ 340 nm based on the NADH calibra-
tion graph. The enzyme activity was expressed in
thalamus/h.
mmol/g hypo-
By an almost similar approach, the activity of SSADH was
determined for each sample. The enzyme (SSADH) in the hypo-
thalamic homogenate would convert SSA to SA in the presence of
2
.4. Homogenate preparation for GAD assay
þ
Each sample was minced and homogenized in 100 mM sodium
NAD . The incubation solution contained 50 mM Tris-HCl, pH 8.5,
þ
phosphate buffer (pH ¼ 7.0), containing 20
m
M pyridoxal phos-
0.25 mM NAD , 0.3 mM SSA, 8.2 mM 2-mercaptoethanol, and
phate, 0.1% Triton X-100 (W/V), and 1 mM AET.
0.2 mL of each homogenate. The mixture was subjected to the same
preincubation condition as mentioned for the assay of GABA-T.
Finally, 0.1 mL of SSA solution (3.375 mM solution) was added to
the reaction mixture and the same steps were followed for
measuring the SSADH enzyme activity, as mentioned for GABA-T
assay.
2.5. Homogenate preparation for GABA-T and SSADH assay
A 10% (W/V) tissue homogenate was obtained using an ice-cold
̊
solution
containing
0.32 M
sucrose
and
4.5 mM
2-
mercaptoethanol. The mixture was ready by adding 3 vol of the
ice-cold Triton medium (%0.67 W/V Triton X-100, 50 mM Tris-HCl,
pH ¼ 8.5 and 4.5 mM 2-mercaptoethanol) to 1 volume of the ho-
mogenate and allowed to stand for an hour before use.
2.8. In vitro enzymatic assays
Aliquotes of each of the homogenated samples were exposed to
different concentrations of Mag II (0e0.03 mmol/mL), and incu-
bated for 2 h on ice. The enzymes’ activities were determined
2.6. GAD assay
(before and after dialysis) by the relevant methods which were
An aliquot (170
mL) of the GAD assay solution (0.1 M sodium
previously described for each enzyme.
̊
̊
Please cite this article as: N. Boostan, R. Yazdanparast, Augmentation of endogenous GABA pool size induced by Magainin II peptide, Biochemical