AN ABSTRACT OF THE THESIS OF EDWARD ELMO ALBIN for the DOCTOR OF PHILOSOPHY (Name of student) (Degree) in Biochemistry presented on September 10, 1973 (Major) (Date) Title:CYCLIC NUCLEOTIDES, CYCLIC NUCLEOTIDE PHOSPHODIESTERASE, PROTEIN KINASE, AND PHOSPHOPROTEIN PHOSPHATASE IN THE CEN- TRAL NERVOUS SYSTEM OF MANDUCA SEXTA Redacted for privacy Abstract approved: R.W. Newburgh Adenosine 3', 5' -cyclic monophosphate (cyclic AMP, cAMP) and guanosine 3', 5' -cyclic monophosphate (cyclic GMP, cGMP) were quantitated in the central nervous system (CNS) of the insect Manduca sexta by competitive protein binding techniques.The M. sexta CNS was found to contain a strikingly high level of cGMP, about 100-fold greater than that of mammalian brain.The level of cAMP, however, was estimated to be about one-sixth that of mammalian brain.The basal ratio of cGMP/cAMP in the insect CNS was approximately ten, whereas this ratio has been reported to be less than one in a variety of vertebrate and invertebrate tissues.Acetylcholine caused a great elevation of cGMP, but not cAMP, in the M. sexta CNS.Short-term incubation with ecdysterone (insect metamorphosis hormone) promoted the accumulation of both cyclic nucleotides. The existence of cyclic nucleotide- stimulable protein kinases (ATP: protein phosphotransferase, EC 2. 7. 1. 37),a system of en- zymes postulated to be instrumental in the biochemical expression of cAMP and cGMP, was demonstrated in the CNS of both larval and adult M. sexta.At low concentrations, cAMP was a much more effective activator of kinase activity than cGMP,Cyclic AMP lowered the Kmof the CNS kinase for ATP, a phenomenon which has been reported to be unique to nervous tissue in mammals. A number of the enzymological properties of the insect kinase were similar to those reported in the literature for thisenzyme in mammalian tis- sues.This insect CNS was also shown to possess a potent enzyme system, viz. phosphoprotein phosphatase, for the dephosphorylation of the phosphorylated products of kinase.Phosphatase activity was gauged using phosphoprotamine as substrate.Both kinase and phos- phatase activities were found to be enriched in particulate fractions of the CNS. An enzyme system for the destruction of cAMP and cGMP, cyclic nucleotide phosphodiesterase (nucleoside 3', 5' -cyclic phosphate nucleoside 5' -phosphate 31 -phosphohydrolase, EC 3.1. 4< c), was examined in the M. sexta larval and adult CNS.Phosphodiesterase (PDE) was found in both soluble and particulate fractions of the CNS, but highest specific activity PDE was noted in a crude mitochondrial preparation, a fraction presumably containing synaptic elements. PDE was greatly enriched in the brain relative to the other CNS ganglia, and was present at higher levels innervous relative to non- nervous tissues.The hydrolysis of both cAMP and cGMP appeared to be the function of a singleenzyme (or similar isozymes) in the larval CNS.Kinetic evidence suggested that PDE in the insect isa cooperative enzyme and is characterized by non-linear, biphasic double-reciprocal plots. PDE could be effectively inhibited bypre- sumed physiological levels of ATP. Cyclic Nucleotides, Cyclic Nucleotide Phosphodiesterase, Protein Kinase, and Phosphoprotein Phosphatase in the Central Nervous System of Manduca sexta by Edward Elmo Albin A THESIS submitted to Oregon State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy June 1974 APPROVED: Redacted for privacy Chairman of Department of Biochemistry and Biophysics and Professor of Biochemistry in charge of major Redacted for privacy Dean of Graduate School Date thesis is presented September 10, 1973 Typed by Opal Grossnicklaus for Edward Elmo Albin TABLE OF CONTENTS INTRODUCTION 1 Purpose of the Studies Described in this Thesis 1 Cyclic AMP 3 Synthesis and Degradation of cAMP 3 The Second Messenger Concept 5 Protein Kinases and Phosphoprotein Phosphatase 6 Reactions Catalyzed by Kinases and Phosphatases 6 Turnover of Phosphoprotein Phosphate 7 Cyclic Nucleotide-Dependent Protein Kinases 8 A Fundamental Hypothesis 9 Protein Substrate Specificity of Kinases 10 The Mechanism of Activation of Cyclic Nucleotide-Dependent Protein Kinases 12 Protein Kinase Modulator 14 The Role of Cyclic AMP and Protein Phosphorylation in the Synaptic Transmission of Nerve Impulses 16 Cyclic GMP 22 GENERAL EXPERIMENTAL MATERIALS AND METHODS 29 Materials 29 Insect Cultures 30 Dissection of Insect Nervous Tissues 31 Protein Determination 34 Polyacrylamide Gel Electrophoresis 34 CYCLIC AMP AND CYCLIC GMP IN THE CENTRAL NERVOUS SYSTEM OF M. SEXTA 41 Materials and Methods 42 Materials 42 Tissue Incubation and Cyclic Nucleotide Extraction Procedure 43 Cyclic AMP Assay 44 Cyclic GMP Assay 46 Results and Discussion 47 PROTEIN KINASE IN THE CENTRAL NERVOUS SYSTEM OF M. SEXTA 60 Materials and Methods 62 Materials 62 Enzyme Source for Kinase Assays 62 Assay of Protein Kinase 63 Choice of Protein Substrate 65 Comments on Experimental Technique 66 Results and Discussion 67 Miscellaneous Observations 67 Endogenous Phosphorylation 68 Sulfhydryl Sensitivity 70 Comparison of the Effectiveness of cAMP and cGMP to Activate Soluble Protein Kinase 72 Time Course of Phosphorylation 78 Effect of Varying Enzyme Concentration 78 Effect of Varying the Amount of Histone 81 Kinase Activity as a Function of ATP Concentration 81 Kinase Activities in the Larval and Adult CNS 85 Effects of Bivalent Cations and EDTA 91 Temperance and pH Dependence 9 5 Effects of Salts 98 Abilities of Various Proteins to Serve as Kinase Substrates 101 Effects of Various Compounds on Kinase Activity 105 Protein Kinase Inhibitor 109 Polyacrylamide Disc Gel Electrophoresis 114 Summary 118 PHOSPHOPROTEIN PHOSPHATASE IN THE CENTRAL NERVOUS SYSTEM OF M. SEXTA 120 Introduction 120 Materials and Methods 121 Materials 121 Enzyme Preparation 121 Assay for Phosphoprotein Phosphatase 121 Choice of Substrate 124 Results and Discussion 125 Miscellaneous Observations 125 Time, Enzyme, and Substrate Dependence of Reaction Velocity 126 Sulfhydryl Sensitivity 128 Effects of Salts and Ionic Strength 130 Effects of Bivalent Metals and EDTA 134 Temperature and pH Dependence 140 Effects of Various Compounds on Phosphatase Activity 143 Distribution of Phosphatase Among Soluble and Particulate Fractions 143 CYCLIC NUCLEOTIDE PHOSPHODIESTERASE IN THE CENTRAL NERVOUS SYSTEM OF M. SEXTA 150 Introduction 150 Materials and Methods 161 Mate rials 16 1 Cyclic AMP Phosphodiesterase Assays 163 Assay I Assay II 167 Cyclic GMP Phosphodiesterase Assays 169 General Comments on Phosphodiesterase Assays 171 Collection of Non-neural Tissues 173 CNS Preparations 174 Preparation of Subcellular Fractions from the Larval CNS 178 Ammonium Sulfate Fractionation 179 Activity Stain for Phosphodiesterase in Polyacrylamide Gels 18 1 Results and Discussion 183 Tissue Distribution of Activity 183 Miscellaneous Properties of Larval CNS Phosphodiesterase 185 Phosphodiesterase Activity as a Function of Incubation Time and Concentration of Protein 19 7 Ammonium Sulfate Precipitation 19 7 Effect of pH and Buffers 20 1 Temperature Dependence of Activity 205 Metal Requirements 207 Inhibition Studies 211 Phosphodiesterase Activities in the Larval and Adult CNS 218 Distribution of Phosphodiesterase Activity Among Larval CNS Ganglia 222 Phosphodiesterase Activities in Larval and Adult Brains 224 Sub cellular Distribution of Phosphodiesterase in the Larval CNS 231 Kinetic Studies 237 Comparison of Phosphodiesterase in the M. sexta With That in Various Tissues 243 Phosphodiesterase Distribution in Polyacrylamide Gels 245 Phosphodiesterase Activators ? 251 Phosphodiesterase Induction 254 General Discussion 259 BIBLIOGRAPHY 267 APPENDIX I 289 APPENDIX II 29 2 APPENDIX III 29 6 ABBREVIATIONS USED IN THE TEXT Ach, acetylcholine Ap( CH2)pp, a, p -methylene -adenosine - 5' -triphosphate ATP, adenosine 5' -triphosphate 32, 32P] ATP - ATP BSA, bovine serum albumin (but)2 cAMP, 6-N-2' -0 - dibutyryl- 3'5'- cyclic-AMP cAMP, adenosine 3', 5' -cyclic-monophosphoric acid cGMP, guanosine 3', 5' -cyclic-monophosphoric acid CNS, central nervous system DTT, dithiothreitol (Cleland' s reagent) EDTA, ethylenediaminetetraacetic acid EGTA, ethyleneglycol-bis-((3-aminoethyl ether)N, N'-tetraacetic acid GTP, guanosine 5'-triphosphate IAA, iodoacetamide Km' Michaelis constant LSC, liquid scintillation counting NEM, N-ethylmaleimide PDE, 3', 5' -cyclic nucleotide phosphodiesterase PHMB, para-hydroxymercuribenzoic acid PMSF, phenylmethylsulfonylfluoride 5, substrate or substrate concentration TCA, trichloroacetic acid Tris, tris-(hydroxymethyl)-arninomethane Vmax,maximum reaction velocity LIST OF FIGURES Figure Page 1, Reactions catalyzed by adenyl cyclase and 4 phosphodiesteras e, 2. Standard curve for the assay of cAMP, 48 3. Standard curve for the assay of cGMP. 49 4. Cyclic nucleotide dependence of soluble histone kinase activity at ATP = 125 p.M. 73 5. Cyclic nucleotide dependence of histone kinase activity at ATP = 12.5 p.M. 74 6. Protein kinase activity as a function of incubation time at various ATP concentrations. 79 7. Dependence of exogenous and endogenous phosphory- lation upon the amount of enzyme. 80 8. Dependence of histone kinase activity upon amount of substrate. 82 9. Kinase activity as a function of ATP concentration. 83 10, Cyclic nucleotide dependence of kinase activity extracted from M. sexta larval CNS membranes by Triton X-100. 89 2+ 11. Kinase activity as a function of Mgconcentration. 93 12. pH dependence of kinase activity in the