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Role of Enzyme / Inhibitors from Vigna Radiata in Host

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Role of Enzyme / Inhibitors from Vigna Radiata in Host ROLE OF ENZYME / INHIBITORS FROM VIGNA RADIATA IN HOST – PATHOGEN INTERACTIONS Thesis submitted to the University of Pune for the degree of DOCTOR OF PHILOSOPHY (Ph.D.) in BIOTECHNOLOGY AAROHI A. KULKARNI DIVISION OF BIOCHEMICAL SCIENCES NATIONAL CHEMICAL LABORATORY, PUNE 411008 January 2007 Science is a complex stepwise process of wide observation, detailed understanding, logical analysis and precise conclusions. Experiencing this process leads to evolution and maturation. Discovery is where you don’t know everything but know only one thing in certainty. TABLE OF CONTENTS Page No ACKNOWLEDGEMENT CERTIFICATE DECLARATION BY THE CANDIDATE ABBREVIATIONS ABSTRACT LIST OF PUBLICATIONS CHAPTERS IN BOOKS CONFERENCES /POSTERS/ ABSTRACTS Chapter I 1- 19 General Introduction Abstract 2 Introduction 3-19 Chapter II 20-40 Purification and physiological role of aspartic protease from Vigna radiata Abstract 21 Introduction 22 Materials and Methods 24 Results 28 Discussion 37 Chapter III 41-100 Biochemical studies of aspartic protease from Vigna radiata Abstract 42 Introduction 43 Section I Materials and Methods 52 Results 57 Discussion 65 Section II Materials and Methods 68 Results 72 Discussion 79 Section III Materials and Methods 83 Results 86 Discussion 97 Chapter IV 101-140 Purification and biochemical studies of aspartic protease inhibitor from Vigna radiata Abstract 102 Introduction 103 Materials and Methods 108 Results 115 Discussion 137 Chapter V 141-228 Aspartic protease inhibitors as biocontrol agents Abstract 142 Introduction 143 Introduction to pigeon pea 152 Materials and Methods 157 Results 166 Discussion 199 Introduction to mung bean 204 Materials and Methods 211 Results 215 Discussion 227 Chapter VI 229-266 Synthesis of metal nanoparticles using aspartic proteases Abstract 230 Introduction 231 Synthesis of gold nanoparticles 237 Materials and Methods 239 Results of VrAP 242 Discussion of VrAP 247 Results of pepsin 251 Discussion of pepsin 256 Synthesis of silver nanoparticles 258 Materials and Methods 260 Results of VrAP 262 Discussion of VrAP 266 Bibliography ACKNOWLEDGEMENTS I take this opportunity to acknowledge the guidance and encouragement of my guide Dr. Mala Rao. I have been inspired by her meticulousness, her in depth analysis of the problems and their solutions, her attention to detail and her dynamic approach to any problem. I value her concern and support at all times, good and bad. Her continued interest and involvement in my work and concern for my welfare have greatly motivated me. I have been privileged to have worked with her for the course of this thesis. I thank my seniors and friends CV, Jui and Sudeep, whom I always looked up to for advice and help. I am grateful to Atul B, Atul T, Rashmi, AGP, Rahul, Manoj, Vaishali, Kanchan, AGP, Ajay, Ashok etc for their support and help. I would like to make a mention of all the students of the division who have always maintained a congenial and healthy atmosphere for work. I acknowledge Nilesh and Hrushikesh for their unassuming friendship and willingness to help at all times. I enjoyed the company of my friends and labmates – Ajit, Sharmili, Vinod, Anamika, Rachana and all the project students who have come and worked in the lab during my thesis years. They have maintained a very cordial and enjoyable lab environment. A special mention goes to Nitin who has traveled many a mile with me and has always been a right hand in all my endeavors. I was indeed fortunate to have a friend like Anish with me throughout the course of my Ph.D. His help, faith in me, steadfast support and encouragement has enabled me to ease through many difficult situations. I do not have words to mention the impact on me of Dr Aditi Pant who has always appreciated my efforts and has infused a positive attitude in me. I am grateful to Dr Sastry and Dr Sushama Gaikwad for introducing me to the wonderful fields of nanobiotechnology and protein folding respectively. A special mention is for Dr Vasanti Deshpande who has always loved and encouraged me. I thank Ms Indira Mohandasan and Mr Ramakant Lambharte for their ready and unpretentious help whenever required, even in times of need. I am thankful to Shri Kamthe, Karanjkar, Trehan and Jagtap for their technical assistance for instrumentation and other chores. I thank the Director, National Chemical Laboratory for making the facilities available for my work. I acknowledge the financial assistance by CSIR, New Delhi in the form of Junior and Senior Research Fellowship during the tenure of my work. I thank Dr R. B. Deshmukh, Director of Research, Mahatma Phule Krishi Vidyapeeth (MPKV), Ahmednagar, Dr B. M. Jamadagni, Dr Mandhare, Dr Suryawanshi and the staff of Pulse Research Station, Rahuri for making available the field facilities and for conducting all the detailed analysis of the data. Their selfless and timely help has encouraged me to look at people with a healthy attitude. I acknowledge the several friends and well wishers, whom I have not mentioned above and whose best wishes have always boosted me. I am very fortunate to have understanding parents, who have given me the freedom to choose my own career, encouraged me in all my endeavors and have instilled in me a deep rooted determination and steadfastness to survive under all circumstances and come out with positive results. It would have been difficult for me to complete my Ph. D. without the constant encouragement and support from Sumit, Anuradha and little Amogh. Special thanks are due to Arun for being there in times of need. He has always backed me and has been a solid support. I acknowledge the concern and interest shown by all the near and dear ones in my family. I acknowledge with appreciation and thanks Dada, Vinaya, Pappa and Aai for giving me a homely environment and for the appreciation shown towards my work and efforts. Words fail me in expressing gratitude for my husband Atul and my li’l Nannu without whose ardor, fortitude, cooperation and consideration throughout; this thesis wouldn’t have been possible. Their steadfast faith in my capability, unfailing belief in me and tremendous understanding which cannot be described in mere words has always spurred me to go ahead, especially in difficult times. Aarohi Atul Kulkarni CERTIFICATE Certified that the work incorporated in the thesis entitled: "Role of enzymes / inhibitors from Vigna radiata in host-pathogen interactions", submitted by Mrs. Aarohi A. Kulkarni, for the Degree of Doctor of Philosophy, was carried out by the candidate under my supervision at Division of Biochemical Sciences, National Chemical Laboratory, Pune - 411 008, Maharashtra, India. Material obtained from other sources is duly acknowledged in the thesis. Dr. Mala Rao (Research Supervisor) DECLARATION BY RESEARCH SCHOLAR I hereby declare that the thesis entitled "Role of enzymes / inhibitors from Vigna radiata in host-pathogen interactions", submitted for the Degree of Doctor of Philosophy to the University of Pune, has been carried out by me at Division of Biochemical Sciences, National Chemical Laboratory, Pune - 411 008, Maharashtra, India, under the supervision of Dr. Mala Rao. The work is original and has not been submitted in part or full by me for any other degree or diploma to any other University. Mrs. Aarohi A. Kulkarni (Research Scholar) ABBREVIATIONS µl Microliter Å Angstrom ATBI Alkalo-thermophilic Bacillus inhibitor APS Ammonium persulphate CD Circular dichroism CsCl2 Cesium Chloride Da Dalton DNSA Dinitrosalicylic acid DTNB 5, 5’-dithiobis 2-nitrobenzoic acid EDTA Ethylene diamine tetra acetic acid FProt Fungal aspartic protease g Gram Gdn HCl Guanidine Hydrochloride HAuCl4 Chloroauric acid h hours IC50 50% inhibitory concentration KI Potassium iodide KA Association constant KD Dissociation constant Ki Inhibition constant * Ki Total Inhibition constant Km Michaelis Menten Constant L liter LB Lineweaver Burk M Molar MALDI ToF Matrix assisted laser desorption / ionization time of flight μM Micromolar min Minutes ml Milliliter mM Millimolar nM nanomolar nm Nanometers ºC Degree Celsius PAGE Polyacrylamide gel electrophoresis PHMB Para hydroxyl mercury benzoate PMSF Phenylmethane sulfonyl fluoride rpHPLC Reverse phase high performance liquid chromatography SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis TEMED N,N,N’N’- Tetramethyl ethylene diamine Vmax Maximum velocity WRK N-ethyl-5-phenylisoxazolium-3’-sulfonate. (Woodward’s. reagent K) ABSTRACT Every molecule has a predestined course of action. Its journey of life is a chart of tumultuous upheaval, downfalls and peace. The fulfillment of this journey is what each and every object of life strives for attainment. That is life! Enzyme molecules are the essence of every life systems, the work horses responsible for maintaining all the life processes of the cell and consequently of the organisms. Their study is essential in understanding not only the fundamental concepts of working of the cell but also in comprehending the interactions in the flora and fauna which are a result of their operation. Complex systems are all the more complicated in their working with a lot of enzyme regulation being involved in the process. The regulatory aspects are critical and mostly performed at the enzyme level itself without involving the genomic level. An elegant system of converting enzymes from their inactive forms to the active molecules is that of proteases, enzymes which cleave other proteins to make them functional and suitable for the cell machinery. The regulation of these specialized enzymes is all the more critical since their overwork can cause havoc in the cell with eventual lysis or cell death due to malfunction of the metabolic processes. Out of all the classes of proteases, aspartic proteases are all the more significant due to their specialized functions in the maintenance and working of the cell. The significance of these enzymes can be outlined by gaining a peek into the life processes of some organisms like pathogenic fungi, HIV, Plasmodium, human digestive system, human angiotensin system to name a few wherein this class of enzymes is found to be critical for normal functioning and in gaining nutrition for the cell.
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