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…

iii

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

iv

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.

v

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

xviii

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.