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UNIVERSITI PUTRA MALAYSIA
PURIFICATION AND CHARACTERIZATION OF ACETYLCHOLINESTERASE FROM CLARIAS BATRACHUS AND
OREOCHROMIS MOSSAMBICA BRAIN TISSUES
NATARAJAN PERUMAL
FBSB 2006 34
i
PURIFICATION AND CHARACTERIZATION OF ACETYLCHOLINESTERASE FROM CLARIAS BATRACHUS AND
OREOCHROMIS MOSSAMBICA BRAIN TISSUES
By
NATARAJAN PERUMAL
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia in Fulfilment of the Requirement for the Degree of Master of Science
November 2006
ii
“Dedicated to my father, Perumal Sakaravathy and
mother, Thanam Perumal the unconventional scholars,
to my siblings, and to the teachers and lecturers who
have taught me everything…
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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
PURIFICATION AND CHARACTERIZATION OF ACETYLCHOLINESTERASE FROM CLARIAS BATRACHUS AND
OREOCHROMIS MOSSAMBICA BRAIN TISSUES
By
NATARAJAN PERUMAL
November 2006
Chairman : Professor Mohd. Arif Syed, PhD
Faculty : Biotechnology and Biomolecular Sciences
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
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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.
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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
PENULENAN DAN PENCIRIAN ASETILKOLINESTERASE DARIPADA TISU OTAK CLARIAS BATRACHUS DAN OREOCHROMIS
MOSSAMBICA
Oleh
NATARAJAN PERUMAL
November 2006
Pengerusi : Profesor Mohd. Arif Syed. PhD
Fakulti : Bioteknologi dan Sains Biomolekul
Kajian ini melaporkan mengenai penulenan dan pencirian asetilkolinesterase
(AChE) larut yang diekstrak daripada tisu otak Clarias batrachus dan
Oreochromis mossambica. Asetilkolinesterase telah ditulenkan separa
menggunakan cara homogenisasi, pengemparan, penurasan ultra, gel
kromatografi keafinan (Edrofonium–Sephacril S400) yang telah disintesis di
dalam makmal, diikuti dengan kromatografi cecair berprestasi tinggi (HPLC).
Matrik afiniti disintesis dengan menggandingkan perencat spesifik AChE
“edrofonium” klorida kepada matrik Sephacril S400 teraktif yang epoksi.
Asetilkolinesterase terlarut dari C. batrachus dan O. mossambica telah ditulenkan
dengan faktor penulenan masingmasing sebanyak 26.4 dan 27.9 kali ganda dan
aktiviti spesifik masingmasing sebanyak 60 × 10 3 and 73 × 10 3 U/mg protein.
Berat molekul asetilkolinesterase dari C. batrachus dianggarkan seberat 310 kDa
di dalam keadaan tidak ternyahasli dengan menggunakan kolum Superose TM .
Elektroforesis gel poliakrilamida (NativePAGE) di bawah keadaan tidak
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ternyahasli telah menunjukkan hanya satu bentuk utama molekul protein bagi C.
batrachus dengan berat molekul kirakira 310 kDa, manakala AChE daripada O.
mossambica tidak berjaya ditulenkan. Elektroforesis gel poliakrilamida dengan
kehadiran sodium dodesil sulfat dan betamerkaptoetanol (SDSPAGE)
memberikan hanya satu jalur protein untuk C. batrachus dengan anggaran berat
molekul 74 kDa. Berdasarkan berat molekulberat molekul yang diperolehi
daripada C. batrachus bagi keduadua SDSPAGE dan NativePAGE, AChE
yang telah ditulenkan bolehlah dipostulatkan sebagai bentuk tetramer yang
dihubungkan oleh ikatanikatan disulfida. Asetilkolinesterase yang telah
ditulenkan daripada sampel otak C. batrachus dan yang telah ditulenkan separa
daripada O. mossambica telah dianalisis selanjutnya menggunakan substrat dan
kesensitifan kepada perencatperencat bagi membezakannya daripada
butirilkolinesterase (BuChE). Asetilkolinesterase daripada C. batrachus dan O.
mossambica didapati paling aktif terhadap Asetiltiokolin (ATC) dan
menunjukkan aktiviti yang rendah terhadap propioniltiokolin (PTC) dan
butiriltiokolin (BTC). Secara kinetiknya AChE daripada C. batrachus
menunjukkan pekali Michaelis Km bagi ATC, PTC dan BTC masingmasing
dalam julat 97, 138 dan 238 µM dan kelajuan awal maksimum Vmax, masing
masing 347, 64 dan 25 µmol/min/mg protein. Manakala AChE daripada O.
mossambica menunjukkan pekali Michaelis Km(app) bagi ATC, PTC dan BTC
masingmasing sebanyak 125, 260 dan 600 µM dan kelajuan maksimum awal
Vmax(app) masingmasing 276, 59 dan 36 µmol/min/mg protein. Nombor pusingan
(kcat) bagi AChE yang telah ditulenkan dari C. batrachus dengan ATC sebagai
substrat ialah 0.19 × 10 5 min 1 . Nilai pekali perencatan bagi eserine, propidium
dan karbofuran ialah masingmasing 0.34, 81 and 0.51 µM 1 min 1 untuk C.
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batrachus manakala 0.24, 65 and 0.41 µM 1 min 1 bagi O. mossambica. Maka
jelaslah enzim ini adalah AChE kerana ia telah menghidrolisis ATC pada kadar
yang lebih tinggi berbanding dengan lainlain substrat seperti BTC dan PTC
pada pH 7.0 dan suhu 25ºC, dan juga ianya direncatkan oleh eserine tetapi bukan
oleh isoOMPA.
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ACKNOWLEDGEMENTS
First and foremost, I would like to express my deep and sincere gratitude to my
main supervisor, Prof. Dr. Mohd. Arif Syed. I could not have imagined having a
better advisor for my master, and without his endless advice, brilliantness,
perceptiveness and encouragement I would never have finished.
I would like to gratefully acknowledge the enthusiastic supervision of Dr. Mohd
Yunus Abd. Shukor. He is not only a great scientist with deep vision but also and
most importantly a kind person. His trust and scientific excitement inspired me in
the most important moments of making right decisions and I am glad to work
with him. My warmest gratitude also goes to Prof. Dr. Nor Aripin Shamaan for
his detailed and constructive comments which has provided a good basis for the
thesis presentation.
I would also like to express my sincere appreciation to Dr. Johari Ramli who was
my advisor during undergraduate studies and continued to work with me to date.
I want to thank him for valuable advice and friendly help in working under
collaborative project on Acetylcholinesterase enzyme. This research would not
have been completed without the help of En. Ismail Omar and En. Khalid Ithnin
(Department of Biochemistry), where both have given me the permission to use
the equipments and chemicals in their laboratory.
Postgraduates of the Enzymology and Bioremediation Research Group are
thanked for numerous stimulating discussions, help with experimental setup and
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general advice; in particular I would like to acknowledge the help of Ms. Vanitha
Mariappan, Mrs. Azlina, Mr. Shahizal, Mr. Sim Han Koh, Mr. Ariff, Mrs Neni
and Tham Lik Gin. Not forgetting my fellow coursemates, Mr. Syukri, Ms.
Taznim Begam, Ms. Surini, Mrs. Aine and all my friends for their continued
moral support and companionship through thick and thin, thanks.
Finally, I am forever indebted to my parents and to my siblings who gently offer
counsel and unconditional support at each turn of the road.
.
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I certify that an Examination Committee met on 1 st November 2006 to conduct the final examination of Natarajan Perumal on his Master Of Science thesis entitled “Purification and Characterisation of Acetylcholinesterase from Clarias batrachus and Oreochromis mossambica Brain Tissues” in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows;
Suraini Abd. Aziz, PhD Associate Professor Faculty Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)
Johari Ramli, PhD AssociateProfessor Faculty Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)
Muhajir Hamid, PhD Lecturer Faculty Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Internal Examiner)
Hjh Darah Ibrahim, PhD Professor School of Biological Sciences Universiti Science Malaysia (External Examiner)
_______________________________ HASANAH MOHD GHAZALI, PhD Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date: 21 DECEMBER 2006
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This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows.
Mohd Arif Syed, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Chairman)
Mohd. Yunus Abdul Shukor, PhD Lecturer Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)
Nor Aripin Shamaan, PhD Professor Faculty of Biotechnology and Biomolecular Sciences Universiti Putra Malaysia (Member)
____________________________ AINI IDERIS, PhD Professor/Dean School of Graduate Studies Universiti Putra Malaysia
Date: 16 JANUARY 2007
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DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.
_______________________ NATARAJAN PERUMAL
Date:
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TABLE OF CONTENTS
Page
DEDICATION ii ABSTRACT iii ABSTRAK v ACKNOWLEGMENTS viii APPROVAL x DECLARATION xii LIST OF TABLES xvi LIST OF FIGURES xvii LIST OF ABBREVIATIONS xxi
CHAPTER
1 INTRODUCTION 1
2 LITERATURE REVIEW 3 2.1 Acetylcholinesterase 3 2.2 Biochemical and physiological properties of AChE 5
2.2.1 Physiological properties of AChE 5 2.2.2 Structure–function of AChE 7 2.2.3 Relation of AChE to the cholinergic synapse 8 2.2.4 Catalytic mechanism of AChE 11
2.3 The AChE molecule 14 2.3.1 Molecular weight 14 2.3.2 Isoenzymes 17 2.3.3 AChE substrate specificity 18 2.3.4 MichaelisMenten constant 18
2.4 Effect of excessive substrate, pH and temperature on AChE 22
2.5 AntiAcetylcholinesterase 23 2.5.1 AntiAcetylcholinesterase mechanism 25 2.5.2 Biomolecular rate constant ki and inhibition
concentration 50% (IC50) 27 2.5.3 Eserine as a potent inhibitor to AChE 28 2.5.4 Organophosphate as an inhibitor to AChE 29 2.5.5 Carbamate as an inhibitor to AChE 30 2.5.6 Effects of Carbamate and Organaphosphate
pesticides 31 2.6 Biosensor 35
2.6.1 Acetylcholinesterase as biosensor 35 2.7 Acetylcholinesterase purification 38
2.7.1 Affinity chromatography 39 2.7.2 Gel filtration Chromatography 45 2.7.3 High performance liquid chromatography 46
(HPLC)
xiv
2.8 The fishing industry in Malaysia 47 2.8.1 Freshwater fish 48
3 MATERIALS AND METHODS 52 3.1 Chemicals and equipments 52 3.2 Test organisms 52 3.3 Enzyme assay 54 3.4 Synthesis of edrophonium–Sephacryl S400
Affinity gel 57 3.4.1 Epoxy (Bisoxirane) activation 57 3.4.2 Preparation of the affinity matrix 57 3.4.3 Coupling of ligand (Edrophonium)
via epoxyactivated–Sephacryl S400 gel 58 3.5 Preparation of brain extract 59 3.6 Purification of AChE by affinity
chromatography 59 3.6.1 Preparation of affinity column 59 3.6.2 Application of brain extract 61
3.7 Purification of AChE by Superose TM Gel Filtration using HPLC 62
3.8 Acetylcholinesterase molecular weight determination 64 3.8.1 Superose TM gel filtration 64 3.8.2 Sodium Dodecyl Sulphate polyacrylamide
gel electrophoresis (SDSPAGE) 65 3.8.3 Nondenaturing polyacrylmide gel
electrophoresis (NativePAGE) 67 3.8.4 Gel staining 68
3.9 Protein determination 70 3.10 Characterisation of Acetylcholinesterase 70
3.10.1 Determination of optimum pH for AChE assay 71
3.10.2 Determination of AChE pH stability 72 3.10.3 Determination of AChE assay optimum
temperature 72 3.10.4 Determination of AChE storage temperature
stability 73 3.10.5 Effect of AChE substrates 73 3.10.6 MichaelisMenten constant 74 3.10.7 Determination of IC50 74 3.10.8 Biomolecular rate constants (ki) 75 3.10.9 Effect of heavy metals and pesticides
on AChE activity 78
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4 RESULTS AND DISCUSSION 4.1 Purification of soluble AChE 80
4.1.1 Affinity chromatography (Edrophonium–Sephacryl S400 gel) 80
4.1.2 Purification of soluble AChE from C. batrachus and O. mossambica 80
4.1.3 Purification parameter 91 4.1.4 Purification efficiency 93
4.2 Molecular weight determination of AChE 94 4.2.1 Superose TM gel filtration 94 4.2.2 Nondenaturing polyacrylamide
electrophoresis gel (NativePAGE) 97 4.2.3 Sodium Dodecyl Sulphate polyacrylamide
gel electrophoresis (SDSPAGE) 97 4.2.4 Comparisons between globular and subunit
forms of purified AChE 101 4.3 Acetylcholinesterase substrate inhibition constant
(Ksi) 104 4.4 Acetylcholinesterase Km, Vmax and Kcat 108 4.5 Effect of inhibitors 118
4.5.1 Inhibition concentration 50% (IC50) 118 4.5.2 Biomolecular rate constants (ki) 127
4.6 Effect of pH 144 4.6.1 Optimum pH 144 4.6.2 Stability pH 147
4.7 Effect of temperature 150 4.7.1 Optimum and stability temperature 150
4.8 Acetylcholinesterase bioassay system as potential indicators of ecotoxicity 155 4.8.1 Pesticides screening 156 4.8.2 Heavy metal screening 159 4.8.3 Effect of Carbofuran 162
5 CONCLUSIONS 164
REFERENCES 167 APPENDICES 175 BIODATA OF THE AUTHOR 181
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LIST OF TABLES
Table Page
1 Enzymes diffusioncontrolled rate (kcat/Km) association with substrate. 4
2 Comparison of purification method and characteristic of cholinesterase 15
3 Acetylcholinesterase substrate specificity. 21
4 Effects of anticholinesterase compound. 34
5 Biosensors for the detection of organophosphates and carbamates. 37
6 Purification scheme of soluble AChE enzyme from C. batrachus brain tissues. 81
7 Partial purification scheme of soluble AChE enzyme from O. mossambica brain tissues. 82
8 Summary of estimated molecular weights of purified AChE from C. batrachus .
102
9 Kinetics parameters of purified soluble AChE from C. batrachus hydrolyzing various substrates [acetylthiocholine (ATC), propionylthiocholine (PTC) and butyrylthiocholine (BTC)] at pH 8 and 25 ºC. 113
10 Kinetics parameters of partially purified soluble AChE from O. mossambica hydrolyzing various substrates [acetylthiocholine (ATC), propionylthiocholine (PTC) and butyrylthiocholine (BTC)] at pH 8 and 25 ºC. 114
11 Biomolecular rate constants (ki) and IC50 values for in vitro inhibition by eserine, IsoOMPA, propidium and carbofuran from purified AChE (C. batrachus). 119
12 Biomolecular rate constants (ki) and IC50 values for in vitro inhibition by eserine, IsoOMPA, propidium and carbofuran from partially purified AChE (O. mossambica). 120
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LIST OF FIGURES
Figure Page
1 Quaternary structures of the major oligomeric forms of AChE in vertebrates. 6
2 Structural features of AChE enzyme. 9
3 Cholinergic synapse mechanism. 10
4 AChE catalytic mechanism scheme. 13
5
6
General inhibition scheme of AChE by propidium.
Inhibition scheme of AChE by organophosphate pesticides
27
30
7
8
General inhibition scheme of AChE by carbamate compounds
Mechanism of synthesis of Edrophonium–Sephacryl S400 affinity gel.
31
41
9 Binding mechanism of Edrophonium ligand and AChE in affinity chromatography gel (Edrophoniumepoxyactivated Sephacryl S 400 gel). 43
10 Fish as used in experiment, A) C. batrachus and B) O. mossambica. 53
11 Mechanism of action of AChE on ATC. 55
12 Diagram of the brain system from C. batrachus. 60
13 Protein profile of C. batrachus brain tissues in a 10% SDSPAGE. 83
14 Protein profile of O. mossambica brain tissues in a 10% SDS PAGE.
84
15 Elution profile of partially purified AChE from C. batrachus brain tissues on custom synthesized Edrophonium–Sephacryl S400 chromatography column 86
16 The elution profile of pooled fractions (C. batrachus) from affinity chromatography on Superose TM gel filtration column before (A) and after (B) purification and rechromatography. 87
17 Elution profile of partially purified AChE from O. mossambica on custom synthesized Edrophonium Sephacryl S400 chromatography column. 89
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18 The elution profile of pooled fractions (O. mossambica) from affinity chromatography on Superose TM gel filtration column before (A) and after (B) purification and rechromatography 90
19 Relationship between the molecular weight of proteins and retention time on Superose TM gel filtration column. 95
20 Molecular weight determination of purified AChE from C. batrachus on Superose TM gel filtration column. 96
21 Nondenaturing polyacrylamide gel electrophoresis (NativePAGE) of purified AChE from C. batrachus in a 5% polyacrylamide gel 98
22 Nondenaturing polyacrylamide gel electrophoresis (NativePAGE) of partially purified AChE from O. mossambica in a 5 % polyacrylamide gel. 99
23 Denaturing polyacrylamide gel electrophoresis (SDSPAGE) of purified AChE from C. batrachus in a 10% polyacrylamide gel under denaturing condition 100
24 Substrates specificity and effect of substrates concentration on the hydrolysis of ATC, PTC and BTC by purified AChE from C. batrachus at 25°C and pH 7.5 105
25 Substrates specificity and effect of substrates concentration on the hydrolysis of ATC, PTC and BTC by partially purified AChE from O. mossambica at 25°C, and pH 7.5. 106
26 MichaelisMenten curve of ATC, PTC and BTC hydrolyzed by purified AChE from C. batrachus generated by the GraphPad Prism TM software. 109
27 MichaelisMenten curve of ATC, PTC and BTC hydrolyzed by partially purified AChE from O. mossambica. 110
28 LineweaverBurke plot of ATC, PTC and BTC hydrolyzed by purified AChE from C. batrachus. 111
29 LineweaverBurke plot of ATC, PTC and BTC hydrolyzed by partially purified AChE from O. mossambica. 112
30 Inhibition concentration 50% (IC50) curve of purified AChE in the presence of eserine from C. batrachus 121
31 Inhibition concentration 50% (IC50) curve of purified AChE in the presence of propidium from C. batrachus. 122
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32 Inhibition concentration 50% (IC50) curve of purified AChE in the presence of carbofuran from C. batrachus. 123
33 Inhibition concentration 50% (IC50) curve of partially purified AChE in the presence of eserine from O. mossambica. 124
34 Inhibition concentration 50% (IC50) curve of partially purified AChE in the presence of propidium from O. mossambica. 125
35 Inhibition concentration 50% (IC50) curve of partially purified AChE in the presence of carbofuran from O. mossambica. 126
36 Biomolecular reaction constant (ki) curve of purified AChE in the presence and absence of eserine from C. batrachus. 128
37 Biomolecular reaction constant (ki) curve of partially purified AChE in the presence and absence of eserine from O. mossambica. 129
38 Biomolecular reaction constant (ki) curve of purified AChE in the presence and absence of propidium from C. batrachus. 130
39 Biomolecular reaction constant (ki) curve of partially purified AChE in the presence and absence of propidium from O. mossambica 131
40 Biomolecular reaction constant (ki) curve of purified AChE in the presence and absence of carbofuran from C. batrachus. 132
41 Biomolecular reaction constant (ki) curve of partially purified AChE in the presence and absence of carbofuran from O. mossambica. 133
42 LineweaverBurk plots of purified AChE from C. batrachus in the presence and absence of eserine 134
43 LineweaverBurk plots of partially purified AChE from O. mossambica in the presence and absence of eserine 135
44 LineweaverBurk plots of purified AChE from C. batrachus in the presence and absence of propidium 136
45 LineweaverBurk plots of partially purified AChE from O. mossambica in the presence and absence of propidium 137
46 LineweaverBurk plots of purified AChE from C. batrachus in the presence and absence of carbofuran 138
47 LineweaverBurk plots of partially purified AChE from O.
xx
mossambica in the presence and absence of carbofuran 139
48 The effect of pH on purified AChE from C. batrachus at a range of pH from pH 3.5 to 10. 145
49 Effect of pH on partially purified AChE from O. mossambica at a range of pH from pH 3.5 to 10. 146
50 pH stability studies of AChE enzyme from C. batrachus 148
51 pH stability studies of AChE enzyme from O. mossambica 149
52 Effects of assay temperatures on AChE activity with ATC as the substrate (C. batrachus). 151
53 Effects of assay temperatures on AChE activity with ATC as the substrate (O. mossambica). 152
54 Effects of different pre incubation temperatures on AChE stability (C. batrachus). 153
55 Effects of different pre incubation temperatures on AChE stability (O. mossambica). 154
56 Effect of various pesticides on the enzymatic inhibition of partially purified AChE from C. batrachus. 157
57 Effect of various pesticides on the enzymatic inhibition of partially purified AChE from O. mossambica 158
58 Effect of various heavy metals on the enzymatic inhibition of partially purified AChE from C. batrachus. 160
59 Effect of various heavy metals on the enzymatic inhibition of partially purified AChE from O. mossambica 161
xxi
LIST OF ABBREVIATIONS
Å Angstrom
APS Ammonium persulphate
≤ Lesser then or equal
≥ Greater then or equal
ACh Acetylcholine
AChE Acetylcholinesterase
AChR Acetylcholine receptor
ATC Acetylthiocholine iodine
BTC Butyrylthiocholine
BuCh Butyrycholine
BuChE Butyrylcholinesterase
ChAT Cholineacetyltransferase
DTNB 5,5’dithiobis(2nitrobenzoic acid)
g gravity (Relative centrifugal force)
HPLC High performance liquid chromatography
IC50 50% inhibition concentration
IsoOMPA Tetramonoisopropylpyrophosphotetramide
IU International unit
kcat Turnover number
kDa kiloDalton
Ki Inhibition constant
Km Michaelis constant
Ksi Substrate inhibition constant
xxii
L Liter
M Muscarinic receptors
M Molar
m Meter
mAU milliabsorbance unit
mol Mole
NaOH Sodium hydroxide
PAGE Polyacrylamide gel electrophoresis
PAS Peripheral anionic site
ppb Parts per billion
ppm Parts per million
psi Pounds per square inch
PTC Propionylcholine
rpm Revolutions per minute
S.D. Standard deviation
SDS Sodium dodecyl sulphate
TEMED N,N,N’,N’tetramethylethylenediamine
UV Ultraviolet
V Volt
vAChT VesicularACh transporter
Vmax Maximum initial velocity
CHAPTER I
INTRODUCTION
“ACETYLCHOLINESTERASE NEVER CEASED TO AMAZED, EXCITE OR
CHARM US, with its wide ramifications, unexpected roles, strange forms and
complex inhibition,” according to Brzin et al. (1984). These words described
perfectly how important the charm and complexity of Acetylcholinesterase is
becoming more powerful as we learn more about it.
Acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7; AChE) is a
serine hydrolase that serves principally to terminate signal transmission at
cholinergic synapses by rapid hydrolysis of the excitatory neurotransmitter
acetylcholine in the synaptic gap. In accordance with its biological role, AChE is
a very rapidacting enzyme, operating at nearly diffusionlimited rates.
Acetylcholinesterase exhibit genetic and molecular polymorphism and their
distributions and physiological roles differ among species (Forget and Bocquene,
1999). As a consequence, the characteristic associated with biochemical and
physiological properties is also highly variable.
Investigation of AChE in fish was initiated in 1943, when it was demonstrated
that common carp Cyprinus carpio brain tissues contains AChE and further
investigations have found similar results for other teleost (Silver, 1974). Previous
studies also show that the AChE kinetic studies and sensitivity to inhibitor varied
among different fish species (Chuiko, 2000). Although there are numerous
studies of the properties of fish AChE, they have been mainly conducted for a
2
limited number of fish species and have been mostly concerned with non local
source. Currently, characteristic differences of AChE among fresh water fish
species from local source are not well studied.
Most of the studies on AChE have been carried out with relatively crude
preparations which contain other esterases with possible overlapping substrate
specificities. The use of purified AChE has obvious advantages over crude
homogenates in kinetic studies of substrate and inhibitor interactions, especially
when other esterases are incapable of hydrolyzing compounds under
investigation. Although many different methods have been used for the
purification of AChE, affinity chromatography has been demonstrated to be the
most effective technique for purification. It usually provides a high yield with an
adequate purity of AChE which is particularly desirable in many characteristic
and inhibitory studies.
Most studies of AChE enzyme use nonlocal source. In this work the main aim is
to provide fundamental knowledge on AChE from local fish species of Clarias
batrachus and Oreochromis mossambica. This research has been carried out
using C. batrachus and O. mossambica because of their availability, commercial
importance and can be locally produced. The objectives of this study are;
1. To purify and characterize AChE from the brain tissues of C.
batrachus and O. mossambica.
2. To evaluate the effectiveness of AChE as an in vitro inhibition assay
system for pesticides.