Purification and Characterization of Acetylcholinesterase from Clarias Batrachus and Oreochromis Mossambica Brain Tissues
This study reports on the purification and characterization of a soluble AChE (EC 3.1.1.7) from Clarias batrachus and Oreochromis mossambica brain tissues. The purification protocol involved homogenization, centrifugation, ultrafiltration, application of customsynthesized affinity chromatography...
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English English |
| Published: |
2006
|
| Online Access: | http://psasir.upm.edu.my/id/eprint/4838/ http://psasir.upm.edu.my/id/eprint/4838/1/FBSB_2006_34.pdf |
| Summary: | This study reports on the purification and characterization of a soluble AChE (EC
3.1.1.7) from Clarias batrachus and Oreochromis mossambica brain tissues. The
purification protocol involved homogenization, centrifugation, ultrafiltration,
application of customsynthesized
affinity chromatography gel (Edrophonium–
Sephacryl S400)
and the use of high performance liquid chromatography system
(HPLC). The affinity matrix was synthesized by coupling an AChEspecific
inhibitor, edrophonium chloride to epoxyactivated
Sephacryl S400
matrix.
Soluble AChE from C. batrachus and O. mossambica were purified 26.4 and
27.9 fold with a specific activity of 59.7 × 10 3 and 73.1 × 10 3 U/mg proteins,
respectively. The molecular weight of AChE for C. batrachus estimated on
Superose TM gel filtration column under nondenaturing conditions is 311 kDa.
Native polyacrylamide gel electrophoresis (NativePAGE)
under nondenaturing
conditions showed only one major molecular form of protein for C. batrachus
with a molecular weight of about 309 kDa, while AChE from O. mossambica could not be purified. Polyacrylamide gel electrophoresis in the presence of
sodium dodecyl sulphate and betamercaptoethanol
(SDSPAGE)
gave only one
band for C. batrachus with an estimated molecular weight of 74 kDa. Based on
the molecular weights obtained for C. batrachus from both SDSPAGE
and
NativePAGE,
the purified AChE can be postulated as being a tetramer form
linked with disulfide bonds. Acetylcholinesterases purified from brain tissues
samples of C. batrachus and partially purified from O. mossambica have been
analyzed further on substrate and sensitivity to inhibitors to distinguish from
butrylcholinesterase (BuChE). The AChE from C. batrachus and O. mossambica
were most active against acetylthiocholine (ATC) and shows less activity against
propionylthiocholine (PTC) and butyrylthiocholine (BTC). From a kinetic point
of view, the purified AChE from C. batrachus exhibit the Michaelis constants
Km, for ATC, PTC and BTC in the range of 97, 138 and 238 μM and the
maximum velocities Vmax were 347, 64 and 25 μmol/min/mg protein,
respectively. Meanwhile, partially purified AChE from O. mossambica exhibit
Km(app) for ATC, PTC and BTC in the range of 125, 260 and 600 μM and Vmax(app)
were 276, 59 and 36 μmol/min/mg protein, respectively. The turnover number
(kcat) for purified AChE from C. batrachus with ATC as a substrate was 0.19 ×
10 5 min 1
. The inhibition constant (ki) values of eserine, propidium and
carbofuran were 0.34, 81 and 0.51 μM 1
min 1
for C. batrachus and 0.24, 65 and
0.41 μM 1
min 1
for O. mossambica, respectively. This enzyme is apparently an
AChE since it hydrolyzes ATC at a higher rate than other substrates, such as BTC
and PTC, at pH 7.0 and 25ºC, and is inhibited by eserine but not by isoOMPA |
|---|