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Enzymatic Regulation in the Liver of the Freeze- Tolerant Wood Frog in Metabolically Depressed States

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Enzymatic Regulation in the Liver of the Freeze- Tolerant Wood Frog in Metabolically Depressed States Enzymatic Regulation in the Liver of the Freeze- Tolerant Wood Frog in Metabolically Depressed States by Ranim Saleem A thesis Submitted to the Faculty of Graduate and Postdoctoral Affairs in partial fulfillment of the requirements for the degree of Master of Science Biology, Specialization in Biochemistry Carleton University Ottawa, Ontario © 2020 Ranim Saleem The undersigned hereby recommend to the Faculty of Graduate Studies and Research acceptance of this thesis Enzymatic Regulation in the Liver of the Freeze- Tolerant Wood Frog in Metabolically Depressed States Submitted by Ranim Saleem In partial fulfillment of the requirements for the degree of Master of Science ______________________________ Chair, Department of Biology ______________________________ Thesis Supervisor Carleton University II Abstract The wood frog, Rana sylvatica, can tolerate high degrees of freeze-tolerance, a stress that also requires anoxia, dehydration and hyperglycemia tolerance. Frozen wood frogs show no heartbeat, brain activity, muscle movement or breathing, but phenomenally return to normal once thawed. Control of enzymatic activity is crucial for regulating metabolism and it is imperative for wood frog survival. This thesis investigates the properties of two key enzymes metabolic enzymes, glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase in liver of the wood frogs exposed to freezing, dehydration and anoxia. Current data show that changes in activity, substrate affinity and stability of the enzymes play a major role in their regulation to support the survival of the wood frog during stress, and these regulations are partly controlled by post-translational modifications. Therefore, these enzymes undergo regulation at the level of posttranslational modification to contribute to the overall readjustment of energy production in the wood frog. III Preface This Master’s thesis is composed on two main research papers that are currently awaiting submission to the following journals: Molecular and Cellular Biochemistry Saleem, R. and Storey, K.B. (2020). Insight into the regulation of liver glutamate dehydrogenase during dehydration and anoxia exposure Cell Biochemistry and Function Saleem, R. and Storey, K.B. (2020). Purification and regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from liver of freeze-tolerant Rana sylvatica in response to freezing, dehydration and anoxia IV Acknowledgments First, I would like to thank my supervisor Dr. Kenneth Storey. Ken, thank you so much for agreeing to take me on and giving me the opportunity to work in your lab. Thank you for all your guidance, mentorship and for helping me develop my skills in the research world. I have learned a lot throughout these two years science and non-science related like enlightening me with your music suggestions. I would also like to thank Jan Storey for all her hard work and the things she does to help this lab run smoothly. Her dedication contributed to our success at the lab significantly. I would also like to personally thank Jan for her patience in editing my thesis and answering my questions. Thank you to every Storey lab member that has crossed paths with me these past two years. Thank you so much for making my time spent here so memorable. I would like to give a special thank you to Stuart and Christie for all your guidance in teaching me the basics of enzymes and for all the time you invested in training me. Rasha, thank you for answering all my questions, for all the valuable scientific advice, and for being a great friend. Hanane, thank you for being an amazing friend and for all the fun times. Finally, I would like to thank my mom and dad for all their love, guidance and support throughout the years. Leen, Sharif and Noha thank you for always being there for me and for constantly listening to me talk about my project. I am extremely grateful and blessed to know each and every person I mentioned earlier that has not only contributed to my success, but also believed in my abilities. Thank you all for being part of this journey with me. V Table of Content Table of Contents Title Page ............................................................................................................................... I Acceptance Sheet .................................................................................................................. II Abstract ............................................................................................................................... III Preface ................................................................................................................................. IV Acknowledgments ................................................................................................................. V Table of Content ................................................................................................................... VI List of Abbreviations ............................................................................................................. IX List of Tables ........................................................................................................................ XI List of Figures ...................................................................................................................... XII Chapter 1 .............................................................................................................................. 1 1.1 Survival strategies and metabolic rate depression .....................................................................2 1.2 The wood frog, Rana sylvatica, as a model for adaptation to extreme environments .................4 1.3 Post-translation modification ...................................................................................................8 1.4 Metabolic pathway ................................................................................................................ 11 1.5 Objective ............................................................................................................................... 14 1.6 Hypotheses and Prediction ..................................................................................................... 14 References (chapter 1) ................................................................................................................. 16 Chapter 2 ............................................................................................................................ 22 Abstract ....................................................................................................................................... 24 2.1. Introduction .......................................................................................................................... 25 2.2. Methods ............................................................................................................................... 28 2.2.1 Animals and experimental procedures ..................................................................................................... 28 2.2.2 Preparation of Liver Extracts ..................................................................................................................... 29 2.2.3 Enzyme Assay ............................................................................................................................................ 30 2.2.4 Enzyme Purification................................................................................................................................... 30 2.2.5 Gel electrophoresis and staining ............................................................................................................... 31 2.2.6 Kinetic parameters .................................................................................................................................... 32 2.2.7 Western blots analysis of post-translational modifications ...................................................................... 32 2.2.8 Differential scanning fluorometry ............................................................................................................. 34 2.2.9 Data and Statistical Analysis ...................................................................................................................... 34 2.3. Results .................................................................................................................................. 35 2.3.1 Enzyme purification .................................................................................................................................. 35 2.3.2 Enzyme kinetics ......................................................................................................................................... 36 VI 2.3.3 Structural stability of GDH ........................................................................................................................ 39 2.3.4 Western blots for posttranslational modification ..................................................................................... 40 2.4. Discussion ............................................................................................................................. 40 2.5. Conclusion ............................................................................................................................ 47 Figures ........................................................................................................................................
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