i REGULATION OF UREA CYCLE RELATED ENZYMES IN RESPONSE TO FREEZING IN THE WOOD FROG, RANA SYLVATICA By Stuart R. Green B.Sc. (Hons.), 2015 A Thesis Submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science Department of Biology Carleton University Ottawa, Ontario, Canada © copyright 2017 ii The undersigned hereby recommend to the Faculty of Graduate Studies and Research acceptance of this thesis “REGULATION OF UREA CYCLE RELATED ENZYMES IN RESPONSE TO FREEZING IN THE WOOD FROG, RANA SYLVATICA” submitted by Stuart R. Green In partial fulfillment of the requirements for the degree of Master of Science Chair, Department of Biology Thesis Supervisor Carleton University iii ABSTRACT The North American wood frog, Rana sylvatica, is one of the few vertebrate species that is capable of surviving freezing. Prevention of intracellular ice formation and maintenance of osmotic balance is facilitated by the production of large concentrations of urea during freezing in the liver. The regulatory roles of three enzymes critical to production of urea; glutamate dehydrogenase (GDH), carbamoyl phosphate synthetase I (CPS1), and ornithine transcarbamylase (OTC), were investigated in the liver in response to freezing. Freeze-exposed GDH had heightened substrate affinity and higher maximal activity than the control. Freeze-exposed GDH also had decreased acetylation and ADP- ribosylation. CPS1 from frozen frogs was demonstrated to have a higher affinity for ammonium and decreased lysine glutarylation relative to the control. Freeze-exposed OTC demonstrated improved affinity for ornithine and increased serine phosphorylation. Taken together, the results suggest that urea production is sustained in the liver of the wood frog during freezing. iv ACKNOWLEDGEMENTS The number of people who have helped me to make it this far in my studies is too long to list so I apologize in advance to anyone whom I will inevitably forget to mention. First, I must thank my parents and my brothers for all of the love, support and guidance they have given me over the years, and for reminding me to have some balance in life outside of university. I cannot thank Dr. Ken and Jan Storey enough for all of the effort they put into training me, both in lending me invaluable insight in conducting experiments and also teaching me how to properly write scientific literature. Their dedication to all the students in the lab is astounding. Then there are the numerous mentors from the Storey lab both past and present that have sacrificed their time in helping me to learn new techniques or procedures. I am particularly grateful to Neal and Ryan B. for teaching me the basics back in my undergrad and also Christie and Bryan for encouraging me to try new techniques. Over the years at Carleton, I have had many friends who have helped me get through the hectic journey of going through undergrad and grad school. I would like to thank Tom and Trong for the many study sessions in undergrad and for helping to lead our softball team (even if the score didn’t always turn out great). I would like to thank everyone whose desk is near mine for putting up with my questions and the sound of crunching sunflower seeds. Lastly, I must thank all of the professors and teachers who have taught me over the years through their seemingly tireless effort and commitment. v TABLE OF CONTENTS Title Page .......................................................................................................................................... i Acceptance Sheet ............................................................................................................................. ii Abstract ........................................................................................................................................... iii Acknowledgements ......................................................................................................................... iv Table of Contents ............................................................................................................................. v List of Abbreviations ...................................................................................................................... vi List of Tables .................................................................................................................................. ix List of Figures .................................................................................................................................. x Chapter 1: General Introduction ..................................................................................................... 1 Chapter 2: Regulation of glutamate dehydrogenase in the freeze tolerant wood frog ................... 17 Chapter 3: Regulation of ornithine transcarbamylase in the freeze tolerant wood frog ............... 48 Chapter 4: Regulation of carbamoyl phosphate synthetase I in the freeze tolerant wood frog ...... 69 Chapter 5: General Disucssion ....................................................................................................... 90 References .................................................................................................................................... 104 Appendices ................................................................................................................................... 118 vi LIST OF ABBREVIATIONS α-KG α ketoglutaric acid ATP adenosine triphosphate ADP adenosine diphosphate BSA bovine serum albumin CM carboxymethyl sepharose (cation exchange material) CP Carbamoyl phosphate CPS1 carbamoyl phosphate synthetase 1 (ammonia consuming) DEAE diethylaminoethanol Sepharose (anion exchange material) DSF differential scanning fluorimetry DTT dithiothreitol Ea activation energy ECL enhanced chemiluminescence EDTA Ethylene diaminetetraacetic acid EGTA Ethylene glycol tetraacetic acid vii GDH glutamate dehydrogenase GTP guanosine triphosphate I50 half maximal inhibitory concentration INP Ice Nucleating Protein KA half maximal activator concentration KDa kilodalton KM Michaelis-Menten Constant (half maximal substrate concentration) MS/MS tandem mass spectrometry NAD+ oxidized nicotinamide adenine dinucleotide NADH reduced nicotinamide adenine dinucleotide NAG N-acetylglutamate NAGS N-acetylglutamate synthase OTC ornithine transcarbamylase Pi inorganic phosphate viii PMSF Phenyl methyl sulfonyl fluoride PVDF polyvinylidene difluoride SDS sodium dodecyl sulfate SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SEM standard error of the mean TBS Tris buffered saline TBST Tris buffered saline Tween-20 TCA Cycle Tricarboxylic acid cycle Tris Tris[hydroxymethyl]aminomethane Tm melting temperature Vmax maximal enzyme velocity ix LIST OF TABLES Table 2.1.. Representative purification scheme for GDH from freeze-exposed liver of Rana sylvatica ......................................................................................................................................... 34 Table 2.2. Comparison of R. sylvatica GDH kinetic parameters from control and freeze-exposed liver with respect to changing temperatures in the forward and reverse reaction .......................... 35 Table 3.1. Partial purification of OTC from freeze-exposed R. sylvatica liver using a two-step column chromatography method ................................................................................................... 59 Table 3.2. Comparison of partially purified R. sylvatica affinities for substrates and OTC activity of crude liver extracts adjusted for total protein concentration ...................................................... 60 Table 4.1. Purification scheme for CPS1 from control R. sylvatica liver using a three-step chromatography method ................................................................................................................ 80 Table 4.2. Comparison of the inhibitory effects of metabolites on the activity of purified CPS1 from control and frozen R. sylvatica livers .................................................................................... 81 Table A.1.1. Demonstrating the predicted phosphorylation sites determined by NetPhos 3.1 and the relevant kinases associated with specific serine, threonine and tyrosine residues in the peptide sequence of OTC from Rana catesbeiana ................................................................................... 120 x LIST OF FIGURES Fig. 1.1. Pictorial representation of the urea cycle and its relationship to cell compartmentalization. .................................................................................................................... 16 Fig. 2.1. Silver stained SDS-PAGE gel demonstrating 2-step purification of GDH to homogeneity from crude control and frozen wood frog liver ........................................................ 36 Fig. 2.2. Demonstrating the effects of metabolites (glucose, urea, ADP) on the affinity of control and frozen wood frog liver GDH for its substrates in the forward and reverse reaction ............... 37 Fig. 2.3. Western blots demonstrating relative degree of various PTMs in the control and frozen wood frog liver GDH ..................................................................................................................... 38 Fig. 2.4. Results of differential scanning fluorimetry comparing control to frozen wood frog liver GDH in terms of thermal stability with and without the