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Molecular Entomology 2013, Vol.4, No.3, 13-21
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19
highest fraction of amylase inhibitors was dissolved in
ice-cold sodium phosphate buffer (0.02 M and pH 7.0)
and was dialyzed against the same buffer for 20 h.
This dialyzed solution was used as inhibitors in
enzymatic assay tests.
4.5 The effect of inhibitor on α-amylase activity
The effects of the seed extracts on the amylase
activities were determined as described by Mehrabadi
et al. (2010). Enzyme extract was pre-incubated with
Triticale and three cultivars of wheat seed extracts for
30 min at 35 followed by determination of the
℃
enzyme activity as described before using
dinitrosalicilic acid (DNS) procedures. Appropriate
blanks were included in the experiments as well. The
inhibition percentage (%I) was calculated as follow:
%I=100×[(A540 control−A540 Exp)/A540 control]
4.6 The effect of inhibitor on general protease
activity
The effects of the seed extracts on the general protease
activities were determined as described by Saadati et
al. (2011). Enzyme was pre-incubated with triticale
and three cultivars of wheat seed extracts for 30 min at
35
℃
followed by determination of the enzyme
activity as describe before. The inhibition percentage
was calculated as described for amylase.
4.7 In gel assay
Amylolytic activity in the gel was detected using
procedures described by Meharbadi et al. (2011).
Briefly, PAGE was performed in 10% (w/v) gel for
separating gel and 5% for stacking gel with 0.05%
SDS. Electrophoresis was conducted at a voltage of
120V until the blue dye reached the bottom of the gel.
The gel was rinsed with distilled water and washed by
1% (v/v) Triton X-100 buffer for 15 min. Then, the gel
was incubated in Glycine-NaOH buffer (pH 9)
containing 1% starch solution, 2 mM CaCl
2
and 10
mM NaCl for 1.5 h. Finally, the gel treated with a
solution of 1.3% I
2
and 3% KI to stop the reaction and
to stain the un-reacted starch background. Zones of
α-amylase activities appeared at light band against
dark background.
Electrophoretic detection of proteolytic enzymes was
done basically according to the procedures described
by Laemmli (1970) and Walker et al. (1998). PAGE
was performed in 10% (w/v) gel for separating that
co-polymerized with 0.1% gelatin and 4% for stacking
gel with 0.05% SDS. Electrophoresis was conducted
at 4 until the blue dye reached the bottom of the gel.
℃
Then, the gel was rinsed with distilled water and
washed by 2.5% (v/v) Triton X-100 buffer for 60 min
followed by incubation in Glycine-NaOH buffer (pH
10) for about 6 hour. Finally, the gel was stained as
described by Saadati et al. (2011).
4.8 Protein determination
Protein concentration was measured according to the
method of Bradford (1976), using bovine serum
albumin as a standard.
4.9 Statistical analysis
Data was analysed based on a completely randomized
design using SAS software. Mean comparison was
done using Duncan’s test.
Authors’ Contributions
Ehsan is a student and carried out the experiments and drafted the
manuscript. Ali is Ehsan’s supervisor and participated in the design of the
study, helped in statistical analysis and correction of the manuscript. Ali also
paid from his grant for the project expenditure. Both authors read and
approved the final manuscript.
Acknowledgements
This work was funded by a grant from University of Tehran.
References
Baker J.E., 1987, Purification of isoamylases from the rice weevil,
Sitophilus
orizae
L. by HPLC and their interaction with partially
purified amylase inhibitor from wheat, Insect Biochemistry and
Molecular Biology, 17:37-44
http://dx.doi.org/10.1016/0020-1790(87)90141-7
Bandani A.R., Kazzazi M., Mehrabadi M., 2009, Purification and
characterization of midgut α-Amylase of
Eurygaster
integriceps
,
Journal of Entomological Science, 12:25-32
http://dx.doi.org/10.1111/j.1479-8298.2009.00303.x
Behdad E., 2002, Major plant pests in Iran, Yadbood publishing
Bernfeld P., 1955, Amylases, α and β, Method Enzyme, 1:149-158
http://dx.doi.org/10.1016/0076-6879(55)01021-5
Bonavides K.B., Pelegrini P.B., Laumann R.A., Grossi-de-Sa M.F., Bloch
C.Jr., Melo J.A., Quirino B.F., Noronha E.F., Franco O.L., 2007,
Molecular identification of four different alpha-amylase inhibitors from
baru (Dipteryx alata) seeds with activity towards insect enzymes,
Journal of Steroid Biochemistry and Molecular Biology, 40(4):494-500
http://dx.doi.org/10.5483/BMBRep.2007.40.4.494
Bradford, M.M., 1976, A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding, Analytical Biochemistry, 72:248–254
http://dx.doi.org/10.1016/0003-2697(76)90527-3
Durgac C., Ozgen M., Simek O., Kacar Y.A., Kiyga Y., Celebi S., 2008,
Molecular and pomological diversity among pomegranate (
Punica
granatum
L.) cultivars in eastern Mediterranean region of Turkey,
African Journal Biotechnology, 7:1294-1301
Elpidina E.N., Vinokurov K.S., Gromenko V.A., Rudenskaya Y.A.,
Dunaevsky Y.E., Zhuzhikov, Elpidina E.N., Vinokurov K.S.,