Effects of L-Eopa on Lysine Compartmentation And
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EFFECTS OF L-EOPA ON LYSINE COMPARTMENTATION AND PROTEIN T SYNTHESIS IN EAT BEAIN C i V \ K i n A Thesis submitted by lioO, FAETIN M V T D KING a candidate for the Degree of Doctor of Philosophy in BIOCHEMISTRY Department of Biochemistry Royal Holloway College University of London Egham Hill Egham January 1980 Surrey ProQuest Number: 10097487 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10097487 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 — 11 — ACKNOWLEDGEMENTS I wish to express my gratitude to Dr P. W. Beesley for his unending enthusiasm and encouragement while this work was in progress. I am also indebted to Professor J. B. Pridham for his continued interest and support. In addition I owe much to other staff and postgraduate students in the Biochemistry Department at Royal Holloway College for their helpful discussions. My thanks are also due to Mr G. P. Beaumont for his guidance in connection with the statistical treatment of data and who, in collaboration with Mr J. Anderson, provided the results given in Tables A2 and AIJ. I would like to add a special note of thanks to Dr L. Dim of the Miriam Marks Department of Neurochemistry, The Institute of Neurology, for giving generously of his time and for his invaluable criticisms. I am also grateful to Miss A. Bickel who expertly typed this thesis. Finally, I would like to take this oppor tunity to thank the Postgraduate Awards Committee of Royal Holloway College for providing financial support. - Ill - ABSTRACT The effect of L-dopa on rat brain protein synthesis was examined by studying the effect of the drug on the incorporation of radioactive amino acids into TCA-precipitable material. In the majority of experi ments trace quantities of either L-[3>4(n)-^H ] valine or L-[4>5-^h ] lysine were administered subcutaneously. Incorporation was expressed in terms of l) the relative incorporation defined as the final TCA-insoluble radioactivity divided by the final TCA-soluble radio activity, and 2 ) the apparent incorporation ( ) (Isfined as the final TCA-insoluble radioactivity divided by the final specific activity of the soluble amino acid pool. It is shown that I^^^ is not, in general, a reliable index of rates of protein synthesis. L-dopa (500mg/kg,ip), administered 45 min previously, had no significant effect on either the apparent incorporation of L-[] valine, measured after a 15 min'incorporation period, or that of L-[^h ] lysine, measured after a 74 min incorporation period. The drug did, however, cause a significant 55-59;-^ decrease in the apparent incorporation of L-[^h ] lysine measured after a 15 min labelling period. Computer simulation was used to demonstrate that these results can be quantitatively accounted for if it is assumed that L-dopa has no effect on brain protein synthesis but does affect precursor lysine compartmentation. Temperature con trolled experiments were performed in order to rule out the possibility that the difference between the effect of L-dopa on lysine and valine incorporation arose artifactually through the failure to control ambient temperature. Further, in order to obtain more reliable estimates of rates of brain protein synthesis, in two of these experi ments, rats were given a quantity of L-[U-^^C ] valine sufficient to maintain a constant precursor specific activity throughout the incor poration period. The results confirmed those obtained at room temper ature using trace quantities of labelled amino acids. While the possibility that L-dopa has a small inhibitory effect on brain protein synthesis could not be ruled out, it was concluded that a drug-induced effect on precursor lysine c ompartmentat ion was mainly - IV - responsible for the gross inhibition of lysine incorporation observed in some experiments. Preliminary experiments indicated that a similar effect may occur in rat liver. - V- CONTENTS Page No. 1 INTRODUCTION 1 1.1 The coupling of impulse activity and cellular metabolism in nervous tissue 1 1.1.1 The effect of aromatic amino acids on cerebral protein metabolism 3 1.1.1.1 Phenylalanine 3 1.1.1.2 D-dopa and D-3-hydroxytryptophan 8 1.1.2 The effect of D-amphetamine on cerebral protein synthesis 24 1.1.3 The effect of handling and other stressful stimuli on cerebral protein synthesis 30 1.2 Measurement of rates of protein synthesis 32 1.3 Amino acid compartmentation and protein synthesis 46 2 MATERIALS AND METHODS 56 2.1 Materials ^6 2.2 Animal procedures and tissue incubation techniques 36 2.2.1 In vivo experiments 36 2.2.2 In vitro experiments 60 2.3 Preparation of tissue extracts 63 2.3.1 Preparation of extracts for the measurement of TCA-precipitable and TCA-soluble radio activity 63 2.3.2 Chromatographic separation of tissue acidic and basic materials 63 2.3.3 Purification of amino acids 66 2.4 Assay procedures 66 2.4 .1 Liquid scintillation counting techniques 66 2.4 .1.1 Preparation of samples for liquid scintillation counting 66 2.4 .1 .2 Instrumentation J2 2.4 .2 Amino acid determination 75 2.4 .3 Protein determination J6 2.4 .4 Paper chromatographic determination of the radiochemical purity of labelled amino acids 77 2.3 Mathematical methods 78 — Vi — Page No. 3 EXPERIMENTAL RESULTS 80 3.1 Assessment of methods 80 3.1.1 Mode of precursor administration 80 3.1.2 Assessment of methods used in the determin ation of comparative rates of amino acid incorporation into TCA-precipitable material 81 3.1 .3 A study of the metabolism of radioactive amino acids used in the measurement of comparative rates of protein synthesis 86 3.1 .4 Measurement of low levels of radioactivity in aqueous solution 105 3.1 .5 Altered ion-exchange properties of iso- topically substituted amino acids 115 3.2 The effect of L-dopa on animal behaviour and in vivo protein synthesis 123 3.2.1 Behavioural responses to L-dopa 123 3.2.2 The effect of L-dopa on the uptake of radioactive amino acids into brain and liver and their subsequent incorporation into TCA-precipitable material 124 3.2.2.1 Tie effect of L-dopa on the relative incorporation of L-[3,4(n)-3E]valine 124 3.2.2.2 The effect of L-dopa on the relative incorporation of L-[4»5-^]lysine 135 3.2.2.3 Comparison of the effect of L-dopa on the relative incor poration of L-[ valine and L-[%] lysine 133 3.2.2.4 The effect of L-dopa on the apparent incorporation of L-[ 3»4(n)-^H]valine in brain 142 3.2.2.5 The effect of L-dopa on the apparent incorporation of L-| 4>5-^H]lysine in brain 146 3.2.2.6 Comparison of the effect of L-dopa on the apparent incor poration of L-[3,4(a)-3 S] valine and L-[4 lysine in brain I46 - vil - Page No. 3.2.2.7 The effect of L-dopa on lysine metabolism 153 3.2.2.8 The effect of ambient temper ature on L-dopa-induced behavioural changes 164 3.2.2.9 The effect of L-dopa on the incorporation of radioactive amino acids into protein in rats maintained at different ambient temperatures 168 3.3 The effect of L-dopa on in vitro protein synthesis 182 3.3.1 The incorporation of L-[ 4 »5-^h]lysine into TCA-precipitable material in chopped rat brain 182 3.3.2 The effect of L-dopa on the incorporation of L-[4 lysine into TCA-precipitable material in chopped rat brain 185 A COMPUTER .SIMULATION STUDY OP LYSINE COMPARTMENTATION IN THE BRA.IN 192 4 .1 Simulation of in vivo experiments 193 4 .1 .1 A basic model for the simulation of in vivo tracer experiments 195 4.1.2 Limitations of the in vivo model 196 4.1 .3 Models of lysine compartmentation in the brain 201 4 .1 .4 The effect of L-dopa on the incorporation of radioactive lysine into brain protein 218 4 .1 .4 .1 The effect of an expansion of the brain soluble lysine pool 218 4 .1 .4 .2 The effect of L-dopa on the apparent incorporation of lysine 222 4 .1 .5 A summary of the results of the simulation of in vivo tracer experiments 222 4 .2 Evidence for an intracellular nonexchanging lysine compartment in the brain 224 4 .2.1 Computer simulation of infusion experiments 224 4 .2 .2 Computer simulation of brain slice experi ments 230 4 .2.3 Concluding remarks on the simulation study of brain slice and isotopic lysine infusion experiments 24O 4 .3 An assessment of the methods used for estimating comparative rates of protein synthesis 242 - V l l l - Page No. 5 DISCUSSION 253 5.1 The measurement of rates of cerebral protein synthesis 253 5.2 The effect of L-dopa on the incorporation of radioactive amino acids into brain and liver TCA-insoluble material 257 5.3 Models of lysine compartmentation 27O 5.4 Concluding remarks 278 Appendix A Tables 284 Appendix B The compartmental analysis of tracer kinetic data 306 BQ.