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University Microfilms 300 North Zeeb Road Ann Arbor, Michigan 48106 A Xerox Education Company I I 73-11,569 SCHNEIDER, David Russell, 1941- ON THE BIOCHEMICAL AND MORPHOLOGIC CHARACTERIZATION OF MICROTUBULE PHOSPHATE ACCUMULATION. The Ohio State University, Ph.D., 1972 Pharmacology 1 University Microfilms, A XEROX Company, Ann Arbor, Michigan THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED. ON THE BIOCHEMICAL AND MORPHOLOGIC CHARACTERIZATION OF MICROTUBULE PHOSPHATE ACCUMULATION DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by David Russell Schneider, B.A. ****** The Ohio State University 1972 Approved by: Advifser Department of Pharmacology PLEASE NOTE: Some pages may have indistinct print. Filmed as received. University Microfilms, A Xerox Education Company ACKNOWLEDGMENTS A sincere expression of gratitude is first given to Dr. John J. O'Neill, teacher and educator, for his council, guidance and understanding. His acceptance to lead me in both mental and technical training is appreciated. To Dr. Bernard Maries, for providing an environment of critical evaluation and perception for the efforts of his students, an ever increasing appreciation. To Dr. Robert Gardier, for introducing me to the study of pharmacology, for sponsoring and guiding me in my original efforts, my appreciation. To other members of the faculty of the Department of Pharmacology, and A.L. Delaunois, for assistance in my training and impressions which will be retained, I give thanks. To former professors who have guided me in previous training, Drs. Leo Cawley and Gerald Endahl, I owe open acknowledgment. To the memory of Dr. Clare Hannum, whose dedication is more fully understood. To Miss Jane Breitmeier for unraveling the many drafts and for typing this dissertation in final form; to Miss Patricia Marsh for assistance in reference biblio graphy and providing continuous updating service for my research efforts, I express my thanks. Finally, to my wife Betty, how can I ever thank you -- mere words simply cannot be sufficient. Financial support in my training through the National Institutes of Health Trainee Fellowship GM-01417 is acknowledged and appreciated. VITA July 4, 1941 Born, Wichita, Kansas 1963 B.S., University of Wichita, Wichita, Kansas 1963 - 1965 Department of Chemistry, University of Wichita 1965 - 1968 Research Assistant, Dept. of Surgery, The Ohio State University, Columbus, Ohio 1968 - 1972 NIH Trainee, Department of Pharmacology, The Ohio State University College of Medicine, Columbus, Ohio Special Awards and Service 1971 - 1972 Member, Health Center Library Advisory Committee 1972 Chauncey Leake Prize in Pharmacology Publications Cawley, L.P., Eberhardt, L., Goodwin, W.L., Schneider, D. and Harrouch, J.: Electrophoretic and Zymographic Study of Non-hemoglobin Erythrocyte Proteins (NHEP), Trans., 4: 315, 1964. (Abstract) Cawley, L.P., Wiley, J.L., Schneider, D. and Harrouch, J.: Immunoelectrophoretic and Immunochemical Characteri zation of Antibodies Against Bromelain, Trans., 4: 316, 1964. Cawley, L.P., Goodwin, W.L., Schneider, D. and Eberhardt, L.: Electrophoretic and Immunologic Studies of Non-Hemoglobin Proteins (NHEP) , Fed. Proc. 24: 615, 1965. Cawley, L.P., Eberhardt, L. and Schneider, D.: Simplified Gel Electrophoresis: II. Application of Immunoelectrophoresis, J. Lab. Clin. Med. 65: 342, 1965. Cawley, L.P., Schneider, D., Eberhardt, L., Harrouch, J. and Millsap, G.; A Simple Semi-Automated Method of Immunoelectrophoresis, Clin. Chem. Acta 12: 105, 1965. Cawley, L.P., Schneider, D. and Eberhardt, L.: Immunoelectrophoretic and Serologic Study of the Immunologically Active Components of Bromelin with Rabbit Anti-Bromelin, Vox Sang. 11: 81, 1966. Schneider, D.R. and Endahl, G.C.: Production of Antibodies to Gastrin and their Detection, Fed. Proc. 26: 393, 1967. Schneider, D.R., Endahl, G.L., Doud, M.C., Jesseph, J.E., Bigley, N.J. and Zollinger, R.M.: Gastrin Antibodies: Induction, Demonstration and Specificity, Science 156: 391, 1967. v Schneider, D.R. and Gardier, R.JV.: Monoamine Oxidase Inhibition Produced by General Anesthetic Agents, The Pharmacologist 11: 41, 1969 (Abstract) Schneider, D.R. and O'Neill, J.J.: Monoamine Oxidase Activity in Isolated Guinea Pig Hearts: Halothane and Triton X-100, Fed. Proc. 30: 1372, 1971. (Abstract) Schneider, D.R. and O'Neill, J.J.: Neuropharmacologic Observations on Rat Brain Microtubules, The Pharmacologist 13: 507, 1971. (Abstract) Schneider, D.R. and O'Neill, J.J.: Further Observations on Rat Brain Microtubules, Ohio Chap. S.E.B.M., November, 1971. (Abstract) Schneider, D.R. and O'Neill, J.J.: Phosphorylation by Rat Brain Microtubules In Vitro, Abstract, 5th Intl. Cong. Pharmacology, San Francisco, Calif., July,11972. Harris, S.G., Schneider, D.R., Delaunois, A.L. and Gardier, R.W.: Observations on Arrhythmia Production and Monoamine Oxidase (MAO) Activity Following Inhalation Anesthesia, Abstract, 5th Intl. Cong. Pharmacology, San Francisco, Calif., July, 1972. Schneider, D.R., Harris, S.G., Delaunois, A.L. and Gardier, R.W.: Increased Monoamine Oxidase Activity in the Presence of General Inhalation Anesthetics, In Preparation. vi Fields of Study- Major Field; Studies in Anesthesia. Drs. R.W, Gardier and E.B. Truitt, Jr. Studies in Autonomic Pharmacology. Dr, B.H. Marks Studies in Biological Oxidations. Drs. G, Brierly, A.J. Merola and J. Rieske Studies in Cardiovascular Pharmacology. Dr. S. Dutta Studies in Neuroendocrine Pharmacology. Dr. H. Goldman Studies in Electropharmacology. Dr. P.B, Hollander Studies in Electron Microscopy. Drs. N. Baba and J. Lindower Minor Field: Studies in Protein Biochemistry. Drs. J.O. Alberi and R. Matthews Studies in Immunology and Immunological Mechanisms. Drs. R. St. Pierre, M. Dodd and N. Bigley Studies in Endocrinology. Drs. K. Brownell and K. Nishikawara. CONTENTS Page ACKNOWLEDGMENTS ii VITA iv LIST OF TABLES LIST OF ILLUSTRATIONS DEFINITIONS AND ABBREVIATIONS INTRODUCTION 1 Microtubules as Structural Elements 1 Improved Fixation Techniques 2 Axon Flow 4 Neurofibrillary Pathology 7 Physical and Biochemical Characteristics of Microtubules 8 Reconsitution Experiments 14 Functions of Microtubules 15 Mechanical Support 16 Axoplasmic Flow 16 Sensory Transduction 18 Ionic Concentration 18 Transmitter Release 19 Elongation and Movement ' 19 Summary 2 0 viii Page Materials and Methods 2 2 Microtubule Preparation 22 Isolation of Microtubules 22 Washing-Soaking Protocol for Isolated Microtubules 23 Standard Reaction Conditions for Phosphate Accumulation by Isolated Microtubules 24 Standard Reaction Treatments for Isolated, Washed Brain Microtubules 27 Newborn Preparations 30 Electron Microscopy Techniques 30 Parlodian-Coated, Carbon Stabilized Copper Grids 30 Preparation of Samples: Parlodian Carbon Grids 31 Preparation of Samples: Thin Sections 32 Electron Microscopy 35 Light Scattering 36 Packed Volume Studies 37 Protamine Additions to the Microtubule Standard Reaction 40 Unlabeled Protamine 40 Protamine Labeling 41 ^H-Protamine Labeling 41 Protamine-Ferritin Coupling 47 Studies with Orthophosphate, ^Pj 52 Preparation and Methods 52 ix Effect of Time on Phosphate Accumulation 52 Phosphate Accumulation with (y-32p)GTP and (y-^P)ATP 53 Specific Additions of Compounds to the MTP Standard Reaction 54 Specific Preparations for Drug Studies 54 Carisoprodol 54 Chlorpromazine 55 Diazoxide 55 Mephenesin 55 Meprobamate 56 Oxanamide 56 Phenaglycodol 56 2-Methyl-1,3-n-propylpropanediol 57 Vinblastine and Vincristine 57 Preparation of Other Reagents 58 D 20 58 EGTA and EDTA 58 Ferritin 58 Heparin 59 Hexylene glycol 59 Ion Studies