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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 59
Nucleotides and Cyclic Nucleotides 60
Column Chromatography of Standard Reaction Mixture Page
Ordered Additions Reactions 61
Radioisotope Assays 62
Nuclear-Chicago Planchet Counter 62
Packard Tri-Carb Liquid Scintillation Counter 62
Cyclic AMP Radioimmunoassay 63
Materials 64
Results 66
Introduction 66
Effects of Reagent Additions on Light Scatter and Packed Volume 67
Phosphate Accumulation Studies with Ortho^ phosphate 32Pi, (y-32P)GTP and (y-32P)ATP 80
Effect of Isolation Medium on Phosphate Accumulation 85
Ordered Addition Reactions Phosphate Accumulation 96
Column Chromatography of the Standard Reaction Mixture: MTP + PA + (y-32P)GTP 100
3H-Protamine Accumulation on Microtubules 103
Microtubules from Newborn Rats and Dogs 104
Inhibition of Phosphate Accumulation with Drugs 116
Structure Activity Relationships 116
Drugs Investigated: Dose Response Data 120
Fine Structure Studies 127
Staining Mechanisms 127
xi Page
Isolated Microtubules 131
Microtubules from the Standard Reaction 132
Ferritin-Labeling Studies 134
Native Ferritin 134
Ferritin-Protamine, Peak I 134
Ferritin-Protamine, Peak II 135
Fine Structure Studies Following Drug Additions 136
Fine Structure Studies Following Additions with Ions and/or EGTA 138
Calcium ions 138
Magnesium ions 138
Calcium and Magnesium ions 138 .
EDTA 138
EGTA and Magnesium ions 139
Fine Structure of Microtubules Following Cyclic Nucleotide: Additions 139
cAMP 139
cGMP 139
D 2O Incubations - Fine Structure 139
DISCUSSION 141
SUMMARY 184
APPENDIX A 187
APPENDIX B 190
BIBLIOGRAPHY 233 xii LIST OF TABLES
Packed Volume and Light Scatter of Rat Brain Microtubules Treated with Guanine Nucleotides, in Vitro
Packed Volume and Light Scatter of Rat Brain Microtubules Treated with Cyclic Nucleotides, in Vitro
Packed Volume and Light Scatter of Rat Brain Microtubules Treated with Calcium and Magnesium Ions, in Vitro
Packed Volume and Light Scatter of Rat Brain Microtubules Treated with Chelating Agents, in Vitro
Packed Volume and Light Scatter of Rat Brain Microtubules Treated with Vinca Alkaloids, in Vitro
Packed Volume and Light Scatter of Rat Brain Microtubules Treated with Colchicine and SCHA-306, in Vitro
Phosphate Accumulation with S2p^ an Time Course of Adult Rat Brai,n Microtubule (y32-p)GTP Accumulation Microtubule Peecipitation with Vinblastine Phosphate Accumulation by Rat Brain Microtubules: Kinetics of Secondary Added Compounds Phosphate Accumulation by Rat Brain Microtubules: Addition of Rat Brain Mitochondria Phosphate Accumulation by Rat Brain Microtubules: Ordered Additions Studies 3 H-Protamine Accumulation on Adult Rat Brain Microtubules Standard Reaction of Phosphate Accumulation by 0-Day Newborn Rat Brain Microtubules 72-Hour Newborn Rat Brain Microtubule Phosphate Accumulation 6-Day Newborn Rat Brain Microtubule Phosphate Accumulation cAMP Radioimmunoassay of Super- nates of 72-Hour and 6-Day Newborn Rat Brain Microtubule Phosphate Accumulation Inhibition of Rat Brain Microtubule Phosphate Accumulation Following Drug Additions - I Inhibition of Rat Brain Microtubule Phosphate Accumulation Following Drug Additions- II Physical Characterization of Microtubules and Neurofilaments xiv LIST OF ILLUSTRATIONS Figure Page 1 Preparation of Microtubules from Adult Rat Brain 26 7. J 2 Standard Treatment for (y- P)GTP Accumulation into Rat Brain Micro - tubule Protein 29 3 Diagram of Microfuge Tube and Epoxy Insert for Preparation of Electron Microscopy Pellet of Microtubules 34 4 Packed Volume Tube in Place in Sorvall SS-34 Rotor 39 5 Mechanism of Salicylaldehyde Addition to Protamine and Reduction by NaB^H. (sodium borotritiide) 44 3 6 Elution of H-Protamine by Column Chromatography 46 7 Ferritin-Protamine Coupling Mechanism 49 8 Spectrophotometric Tracing of Ferritin-Protamine Reaction by Elution from Bio-Rad P-150 Column 51 9 Summary of Fluorometer Deflections by Addition of Compounds to Rat Brain Microtubules 70 10 pH vs. Orthophosphate-32 binding to Washed, Soaked Microtubules . 8 4 11 Microtubule Phosphate Accumulation with (y,32p)GTP and Protamine 102 12 Rat Brain Microtubule Protein Phosphate Accumulation with Added Protamine 107 xv Figure Page 13 Scatch&rd Plot of Rat Brain Micro- tubule Phosphate Accumulation with Increasing Additions of Exogenous Protamine 109 14 Structure Relationships of Drugs in this Study: Propanediols and Carbamates 119 15 Structure Relationships of Drugs in this Study: Chlorproamzine, Diazoxide and Mephenesin 122 16 Structure Relationship of Drugs in this Study: Oxanamide and Phenaglycodol 124 17 Log Dose-Response Inhibition of Phosphate Accumulation on Micro- tubules by Drugs 126 18 Possible Mechanisms for Phosphate Accumulation by Rat Brain Micro - tubules 168 19 Diagrammatic Representation of Microtubule Structure: PTA Stain 175 20 Schema of "Lollipop" Formations: (Side Arm Projections) 177 21 Possible Nucleotide (GTP) Locali zation in Microtubule: Uranyl Acetate Stain 179 Plate I Low Power Magnification of Hexylene Glycol Isolated Microtubules: PTA Stain (1%) -192 xvi Plate Page II High. Power Magnification of Hexylene Glycol Isolated Microtubules: PTA Stain Cl%) 194 III High Power Magnification of Hexylene Glycol Isolated Microtubules: UA Stain CO.5%) 196 IV High Power Magnification of Microtubules Following Reaction with GTP and Protamine; Formation of Lollipop Figures on Microtubule Surface: PTA Stain (1%) 198 V High Power Magnification of Microtubule Following Reaction with ATP and Protamine: PTA Stain (1%) 2 00 VI High Power Magnification of Micro- tubule Following Reaction with Native Ferritin: PTA Stain (1%) 202 VII High Power Magnification of Microtubule Following Reaction with Ferritin- Protamine Conjugate; Peak I: PTA Stain (1%) 204 VIII High Power Magnification of Microtubule Following Reaction with Ferritin- Protamine Conjugate; Peak I: PTA Stain (1%) 206 IX High Power Magnification of Micro tubules Following Reaction with Ferritin-Protamine Conjugate; Peak II: PTA Stain (1%) 208 X High Power Magnification of Micro- tubule Following Reaction with Ferritin-Protamine Conjugate; Peak II: PTA Stain (1%) 210 XI High Power Magnification of Micro tubule Following Reaction with Meprobamate (10 nM): PTA Stain (1%) 212 xvii Plate Page XII High Power Magnification of Microtubule Following Reaction with Hexylene Glycol CIO nM): PTA Stain CIS) 214 XIII High Power Magnification of Microtubule Following Reaction with Chlorpro- mazine, C0.1 mM): PTA Stain CIS) 216 XIV High Power Magnification of Microtubule Following Reaction with Chlorpor- mazine CIO nM): PTA Stain CIS) 218 XV High Power Magnification of Microtubule Following Reaction with Chlor- promazine CIO nM): PTA Stain CIS). 220 XVI High Power Magnification of Microtubule Following Reaction with Magnesium Ion Cl mM): PTA.Stain CIS) 222 XVII High Power Magnification of Microtubule Following Reaction with Calcium Cl mM) and Magnesium Cl mM) Ions: PTA Stain CIS) 224 XVIII High Power Magnification of Microtubule Following Reaction with EGTA Cl mM) and Magnesium Cl mM) Ions: PTA Stain CIS) 226 XIX High Power Magnification of Microtuhule Following Reaction with Cyclic AMP C0.1 mM): PTA Stain CIS) 228 XX High Power Magnification of Microtubule Following Reaction with Cyclic GMP .(0.1 mM): PTA Stain .(1%) 230 XXI High Power Magnification of Micro tubule Following Reaction with 40% Deuterium Water: PTA Stain CIS) 232 xviii Definitions and Abbreviations ATP, ADP, AMP adenosine-5'-triphosphate, adenosine-5'-diphosphate, adenosine-5'-monophosphate GTP, GDP, GMP guanosine-5'-triphosphate, guanosine-51-diphosphate, guanosine-5 r-monophosphate cAMP, cGMP cyclic-3‘:5'-adenosine monophosphate, cyclic 3 1 : 5* - guanosine monophosphate MTP microtubule protein radioactive inorganic phosphate-32 S sedimentation coefficient Svedberg units; the velocity for unit centrifugal field of force with dimensions of time: 10-13sec. PCA, TCA perchloric acid (HCIO^), trichloroacetic acid (C15C-C00H) HGK HEPES, potassium chloride, guanosine-5 *-triphosphate buffer HH hexylene glycol, HEPES Kv kilovolts PA- protamine (sulfate) HEPES N-2-hydroxyethyl-piperizine- N-2-ethane sulfonic acid GMP-PCP B-y-methylene guanosine triphosphate VBL vinblastine sulfate _ ++ ++ w + + Ca , Mg , Mn calcium ions, magnesium ions, manganese ions xix EGTA ethyleneglycol-bis- (3-aminoethyl ether) - N-N1-tetraacetic acid CPZ chlorpromazine PV packed volume mM, uM, nM millimolar (1 x 10“ micromolar (1 x 10"%) nanomolar (1 x 10"%) EDTA ethylenediamine tetraacetic acid p micron (1 x 10"^ meters) This symbol is also used for ionic strength of a solution. XX INTRODUCTION Microtubules as Structural Elements Historically, the description of microtubules was first made by Fawcett and Porter in 1954 when these authors noted their appearance in the shafts of cilia. Palay, in 1956, provided the first report of these structures in neurons. The identification of microtubules as important structural elements in nervous tissue and secretory tissue was dependent upon three major areas of investigation: improved fixation techniques and electron microscopy, axoplasmic flow investigations and neuropathologic degenerations. There are significant differences between these elements and such similar structures as neurofilaments, although they are frequently confused with one another. Before the description of microtubules using electron microscopy, these structures had been observed and described in the literature by early histologists using light microscopy. Such early investigators included Schultze (1871) and Ramon y Cajal (1923) who 1 published more than any light microscopist on these particular structures. In all of these early writings, because of their comparative dimensions, the structures visualized and reported probably represented aggregations of both microtubules and neurofilaments. The structures may be observed unstained in the light microscope but observation is more easily accomplished through the use of either silver or silver and gold stains. Improved Fixation Techniques Increased interest in microtubules developed when glutaraldehyde was introduced as a fixative in electron microscopy techniques. With additional improvements in the resolving power of electron microscopes of the early 1960’s, and the use of this fixative with others (e.g., formalin solutions) many investigators described and documented these structures in a variety of cells. After 1965-1966, the presence of microtubules within cells became nearly universally accepted. In longitudinal section, microtubules in nerve are observed as tubular straw-like elements which pass through the cytosol of a neuron. They faithfully follow all of the contours of the cell membrane, and maintain a discrete distance from other neighboring microtubules. In transverse section, they are seen to be composed of approximately 13 globular elements, so far taken to represent cross sections of the microtubular elements. Such structures are not observed in material which has been thin-sectioned longitudinally in the usual manner of electron microscopy tissue preparation. Evidence for the existence of these elements may be found however in the visualization of mechanically disrupted microtubules, especially when tissues are prepared in a medium which stabilizes microtubular fine structure. In the newborn or immature animal, it is of some significance that neurons contain an abundance of microtubules, but very few neurofilaments when compared to the adult; however, neurofilament structures are in excess in large axons in the adult, and the ratio of neurofilaments to microtubules decreases in small axons. With branching of an axon, neurofilaments are rarely observed. Additionally, it has also been noted that the appearance of neurofilaments in the newborn does not occur in a species like the rat until the fifth post natal day, whereas microtubules are present at birth. 4 Axon Flow The directed, non-random movement of both the cytosol and vesicle-like units within the cytosol has been termed axonal flow or transport. In recent years, the flow direction of cytosol within a cell has been extended to include the cell body (Pomerat et al ., 1967) and dendrites (Globus et a l ., 1968) as well as the axon (Schmitt and Samson, 1968) . Hypotheses have been proposed concerning the exact mechanisms of axonal flow. There appears to be at least three types of axonal flow; a variety of experiments have been designed to involve microtubules in at least one of these types: axonal fast flow. Axonal fast flow is the rapid (up to 400 mm/day) flow of material centrifugally within a neuron. Currently, the mechanisms describing axonal fast flow incorporate the role of microtubules in some manner, whether it be a "sliding vesicle" or sliding filament types of arrangement (Schmitt and Samson, 1968) or the local breakdown and reaggregation of microtubules (Peters and Vaughn, 1967) . The "sliding filament" hypothesis is basically explained by suggesting that only the transported material is moved from the soma to the axon, and not the elements of the transport mechanism. The hypothesis states that a 5 protein "transport" filament is produced in the cell body, enters the axon, and in analogy with muscle slides along microtubules by crosslinks which are ATP activated. The sliding vesicle theory has the same mechanism except that it involves vesicle migration. The sliding filament transport hypothesis implying microtubule involvement is the final step for the actual transport of material centrifugally down the axon. Ochs (1972) has investigated the phenomenon of fast axonal transport following injection of C-leucine into the L-7 dorsal root ganglion. He has demonstrated in this manner a transport mechanism which is operative along the length of a nerve fiber. In these studies, as in some others, the fast transport rate was clearly dependent upon oxidative phosphorylation and a steady state concentration of ATP. At present there is no direct proof of any hypothesis offered since the transport filament has not been isolated. In vivo blockade of axonal fast flow has been accomplished using agents which can be demonstrated to bind to microtubular protein. Colchicine, a drug which binds to tubulin, a protein unique to tubules, has been shown by several investigators to be a drug which specifically blocks flow and/or binds to microtubules. Other drugs, notably the vinca alkaloids, have also been shown to bind MTP, but do not show the same specificity. Vinca alkaloids will precipitate MTP both in vitro and in vivo, but also have an affinity for + + a Ca binding site in other proteins (Wilson et al., 1970). A study by Olmsted however showed that the vinca alkaloids will selectively precipitate easily and rapidly a protein which demonstrates colchicine binding (1970) . Actin- or myosin-like filaments in neurons have been suggested to be present in tissues by several investigators. Gawadi (1971) has recorded tension in mitotic spindles and has concluded that either micro tubules have myosin-like character or these structures are associated with actin or myosin which is lost during isolation. Even more convincingly, Fine and Bray (1971) in an elegant set of experiments have observed a neuronal protein in cell culture which contains many characteristics of one of the microtubule subunits (tubulin II) which also acts like actin. Finally, the observation of vesicle association within microtubules has been visualized by Jarlfors and Smith (1969) who postulate that microtubules may perform a "charging" phenomenon for vesicles near the presynaptic membrane. Neurofibrillary Pathology By light microscopy, neuropathologists have observed at least three structures associated with neurofibrillar degeneration: microtubules, neurofilaments and aggregates of these. Use of the electron microscope has allowed the definition of ultrastructural organelles to be expanded to include five types: the normal neurofilament, averaging 100 X diameter; a morphological equivalent 100 X filament occurring in pathological situations; a normal microtubule, with a diameter of 250 X and a lumen of 150 X; abnormal "twisted" tubules, with a diameter of 200 X, narrowing to 100 X diameter every 800 X length; and abnormal aggregates of filaments GlQQ X). which lie closely associated to sheets of membrane and which resemble beaded structures. The 100 X "twisted" tubules are quite unique to neurofibrillary pathology occurring in lesions of Alzheimer’s disease, forms of senility, Parkinson’s disease and Nieman-Pick's disease. The isolation of these unusual structures has never been reported and currently it is not known whether they represent specific abnormal, malformed tubule protein, or are of a more non-specific nature. Neurofibrillary tangles of 100 X filaments are usually found associated with characteristic biochemical deficiency. In addition to the major fine structure abnormality of the tangles of neurotubules and neuro filaments, there are neuronal varicosities present. Clinical disorders such as neuronal infantile dystrophy, vincristine neuropathy and motor neuron disease are found. In addition, a variety of experimental.disorders can be produced in the laboratory resembling the patho logical fine structure changes of the clinical disease, e.g., encephalomyelopathy produced by aluminum powder, and by mitotic spindle inhibitors. Axons which are impeded in their flow in any manner seem to be the basis for models of these types of degenerative changes. It also is possible that the proliferation of neuro filaments under pathological conditions may possibly represent a non-specific type of change which serves as a marker for neuronal injury (Wisniewski et al., 1970). Physical and Biochemical Characteristics of Microtubules In 1962, Kane observed that chromatid mitotic figures could be isolated under carefully controlled conditions of temperature, pH and appropriate solvents. Using general principles derived from this source, many investigators described fractionation methods for the isolation of both microtubules and neurofilaments Initial studies have defined the physical character of the subunit, tubulin, from various sources such as mitotic spindles, cilia and flagella (Renaud et al., 1968; Stephens, 1967, 1968a; Shelanski and Taylor, 1968) with respect to size, enzymic activity, amino acid composition and precipitability with various agents. Interest has increased concerning the biochemical characterization of these structures in providing a mechanochemical basis for the understanding of the processes of axoplasmic fast flow. Tubulin isolated by any of the various methods seems to resemble actin CRenaud et al., 1968; Stephens, 1968a; Shelanski and Taylor, 1968), erythrocyte spectrin and other structural proteins which are generally classed as tektains (Mazia and Ruby, 1968). Many studies depend upon colchicine binding for both the isolation and chemical characterization of the MTP. Because intact structures are not isolated by these methods and because of the fragile nature of MTP, they allow only the isolation of a 6S protein from tissues rich in microtubules, notably brain (Borisy and Taylor, 1967a,b). Biochemically these subunits bind 2 moles of GTP per mole MTP, and have a molecular weight equal to 120,000. By electrophoretic analysis, the major band of purified tubulin matches the colchicine binding protein and the 6S protein is IQ now felt to be the 120,000 molecular weight subunit. The binding of vinca alkaloids to tubulin occurs at a site other than the colchicine binding locus. Vinblastine causes the formation of microtubular precipi tates both ill vitro and iii vivo which are well charac terized. These precipitates have been used in structural studies of tubulin, but as previously alluded to, this binding is not entirely specific for tubulin. As if matters were not sufficiently complex, several reports now suggest that colchicine binding protein is a component of other structures and not only of microtubules CJames et al,, 1969; Feit and Barondes, 1971). Vinca alkaloid binding to tubulin has been shown to cause the partial release of GTP (Olmsted, 1970), and several laboratories have commented on the close association between this nucleotide and tubulin. Olmsted (1970), in describing the vinblastine precipitation of microtubules found that the presence of GTP and/or M g C ^ would stabilize colchicine binding, to tubules precipitated with vinblastine. Stabilization by GTP of microtubules isolated from brain was also reported by Weisenberg et al. (1968) who found that this nucleotide prevented an appreciable loss of colchicine precipitatable MTP. From ultracentrifuge boundary studies of the 6S dimer, GTP appears to reduce aggregation 11 of the MTP. The relative affinity of nucleotide binding was much greater (although precise measurements have not been made) for GTP rather than GDP. Further, ATP additions were unsuccessful in the stabilization of MTP and did not appear to bind to the subunit. The simplist explanation for the GTP exchange reactions, which demonstrate one mole releasable and one mole bound GTP/mole MTP is a head to tail configuration of two types of tubulin. Other interpretations of course are possible including configurational changes and the possibility that the monomers were non-identical. That this latter suggestion proved to be true is now documented (Feit et al., 1971; Bryon and Wilson, 1971). Two forms of tubulin were independently established with molecular weights of 55,000 and 53,000. The dissimilarity of the monomers is confirmed by differences in amino acid structure and electrophoretic mobility. In vitro biochemical activity of isolated tubulin has also been associated with the nucleotides cAMP and ATP. Goodman et al (1970) have reported the phosphory lation of serine residues in microtubules following cAMP addition and suggests that microtubules serve as a substrate for a protein kinase associated with this subunit. Using purified porcine MTP, Murray and Frascio (1971) also demonstrated cAMP dependent phosphorylation 12 of MTP which behaved quite differently to purification than non-phosphorylated subunits. Soifer et al. (1971) demonstrated in rat brain MTP that stabilized MTP subunits would not undergo phosphorylation by themselves, but required addition of a separate acceptor such as protamine, histone or alpha-casein as a transfer mechanism. From the preceding, it would seem that associated with brain microtubules is a protein kinase activity. In studies with a specific protein kinase isolated from brain by Greengard, (Miyamoto et al., 1969), protamine appears to act as a substrate or phosphate acceptor. This is quite similar to the phosphokinase activity reported in microtubules. Since the exact nature of this interaction has not been defined, it was of interest to know if protamine participated by binding to the microtubule in a non-specific way. + + Interesting events occur with Mg addition to microtubule protein. Direct addition of Mg++ in excess causes a milky-white amorphous precipitate to occur with no apparent structure. If the addition is made slowly, e*g-» by dialysis, fibers may be observed by phase contrast microscopy. Solvation of the MTP plays an integral role in the effects of Mg++. Moderate concentrations of other ions, e.g., 50 mM NaCl or traces of (NH^SO^ will completely inhibit Mg++ induced MTP 13 precipitation. The effects are enhanced by a decreased pH and by cold temperatures. The fibrous aggregations formed by Mg++ are reversible by either warming or cooling. Finally, the active colchicine binding form of + + tubulin is much more easily precipitated by Mg than is a denatured or previously aggregated form. These results, using Mg++, can be duplicated qualitatively by Ca++ (Weisenberg and Timasheff, 1970). Schlepfer (1971) found that alterations in both microtubules and neurofilaments could be observed in whole desheathed nerve following treatment with CaCl2 (1 mM) or AgCl (10 mM)• Equivalent concentrations of EDTA seemed to be protective against the disruptive effects of these two ions. Interestingly, mixtures of MgCl2, PbCl2 or A1C13 did not alter the fine structure of microtubules to concentrations of 20 mM. The Ca induced alterations resembled Wallerian degeneration patterns and the author suggested that + + excess Ca entry into the axon could play a role in this phenomenon. A circular dichroism spectroscopy study of micro- tubules isolated by colchicine precipitation has been reported by Shelanski (Ventilla et al., 1972). These authors found that the native protein at 4°C and pH 6.5 contained alpha-helix, beta-structure and random coil in a ratio 22:30:48. Following colchicine binding the 14 protein undergoes a conformational change with the loss of alpha-helix character, a change which increased the lability of the protein to undergo further irreversible denaturation with aggregation. If GTP, GDP, GMP, GMP-PCP, colchicine or VBL were present, the protein was protected from denaturation. Wilson et al. (1970a) "I* described the precipitation of proteins by VBL and Ca The results suggested that VBL, acting as a cation precipitates proteins including tubulin by combining + + with them at a Ca binding site. Reconstitution Experiments The reconstitution of intact microtubules from the 6S dimer subunit has been attempted by various investi gators. Stephens (1968) first demonstrated partial success with reconstitution of these particles following the dissociation of sea urchin spermatids with a detergent into detergent-subunit complexes. These subunits were later recombined following dilution of the detergent. The reassembly mechanism was complex however, requiring low temperatures, and was not inhibited by colchicine. Weisenberg (1972) was able to repolymerize rat brain MTP in solutions of ATP or GTP plus Mg++. •f + All reconstitutions required the chelation of Ca for trace amounts of this metal would cause the reassociation to fail. In addition, Boisy and Olmsted (1972) have observed nucleated discs in MTP isolations which appear to be required for intact microtubule assembly. These authors suggested that disc-'like structures were particulate, could be demonstrated by electron microscopy, were not observed following repolymerization procedures at 37°C, but reappeared if the reaggregated polymers were broken down with either colchicine, Ca or reduced temperature. These nucleation structures are quite prominent in preparations of microtubules visualized by electron microscopy. Determination of the precise role these elements perform in relationship to phosphate binding and in fine structure changes induced by drugs, remains to be established. Further, the possibility of an equilibrium between the nucleation centers and intact microtubules would be quite exciting if the disc nucleus is required for microtubule assembly. Demonstration of these elements has not been performed vivo, a feature which would be of far reaching importance in dividing cells. Functions of Microtubules Five major functions have generally been accepted as most probable roles for microtubules. Microtubules are found in abundance in mitotic spindles, cilia and flagell.ar elements. The functions to be described have been discussed by Porter (1965) in other non-nervous systems. Mechanical Support Because of sheer numbers of microtubules in both neurons and glia, the assumption has been made that mechanical support and cellular form is an important role for these structures. If microtubules are disrupted, cells become varicosed and neurofilaments become more abundant. Addition of colchicine to nerve cell cultures has been reported to halt elongation of the nerve axon without adversely affecting the growth curve. These types of experiments suggest the cytoskeleton as proposed by Porter (1965). Axoplasmic Flow The work of Schmitt and Samson (1968), Ochs: (1972) and others have established that the microtubule is involved quite intimately in fast flow. A two-component actomyosin system has been proposed (Jahn and Bovee, 1969) such systems called neurostenin have been isolated from brain by Berl and Pusykin (1970). Tubulin can evidently substitute for stenin although it is not chemically 17 identical. Dynein, a protein studied extensively by Gibbons (1966, 1972) has been isolated from sea urchin sperm flagella microtubules. This protein possesses ATPase actively and possibly represents the side arm projections or crosslinks of microtubules. Presumably it could also represent a myosin-like protein. Colchicine has provided most of the indirect evidence for microtubules participating in axoplasmic flow. Following administration of this drug, nerve preparations generally decrease both fast and slow axoplasmic transport; a mechanism has not yet been postulated because it is felt that colchicine treatment involves more than a simple depolymerization of microtubules. Axoplasmic flow represents a special case of cytoplasmic transport or flow. Whether channels exist within the cytoplasm for more rapid transport of express specific materials has not been defined, however a reasonable hypothesis for transport would include the presence of energy coupled contractile proteins. The implication that axoplasmic transport involves MTP is important in view of new concepts regarding neuronal trophic factors and the trans-synaptic passage of proteins. 18 Sensory Transduction Because of the abundance of microtubules in such structures as retinal rods, cones (Marchesi et al., 1969), olfactory structures (Reese, 1965; Frisch, 1967), and hair cells (Wersall et al., 1965) it has been suggested that microtubules are associated in some manner with sensory transduction. Moran and Varela (1970, 1971) demonstrated the failure of transduction following colchicine or VBL treatment of sensory receptor cells in the cockroach. It has not been demonstrated whether microtubules support the plasmalemma, i.e., a cytoskeleton, allowing deforming to occur or whether the sensory mechanism depends upon a current generation which is built into the cilia, i.e., a piezoelectric crystal effect. Ionic Concentration In studies on hydra, Slutterback (1963) suggested that maintenance of ionic concentration was a role of microtubules, principally as the result of observations involving hydration-dehydration. No further work has been reported. The active ionic distribution present in a cell -- particularly neurons -- could be a product of microtubules or neurofilament proteins acting separately 19 or in concert to influence membrane potentials. Loss of excitability is associated with cold, colchicine treat ment, disaggregation of intact microtubules (Rodriguez Echandia et al., 1968; Hinkley and Green, 1971) and with a proliferation of microtubules following halothane anesthesia (Allison and Nunn, 1968). Transmitter Release Lacy et al. (1968), Poisner (1971) and Malaisse-Lagae (1971) have reported the effects of colchicine, vinca alkaloids and D2O on secretion mechanisms of non-neuronal and neuronal systems. All work published to date indicates tubulin is intimately required fox these processes, Elongation and Movement As previously discussed, microtubules seem to be the major element for neuronal elongation in cell cultures (Seeds, 1970; Yamanda et al., 1970, 1971; Wessells et al., 1971). By elegant studies using the drug cytocholasin B which causes the loss of neurofilaments, Schroeder (1970), Yamanda (1970, 1971), Wessells (1971) and others have blocked the proliferation of a growth cone in nerve cell culture. Neurofilaments are best studied on non-neuronal 20 tissues and evidence now available indicates they are responsible for cellular movement in cytokinesis. Summary From the early studies of microtubules as electron microscopy artifact curiosities, investigations have progressed toward transport and release of all varieties of material required by nerve. To relate metabolism and excitability to transport is to relate physiology and morphology. To investigate physiology and morphology with selective drugs is to postulate some apparent order in morphologic, physiologic and biochemical changes observed. From early observations with isolated microtubules on the physical parameters of light scatter and packed volume changes following biochemical stimulation, I postulate a view of microtubules which will undergo morphologic changes in response to varying biochemical stimulations. Because of the ubiquitous nature of phosphate interaction with biologic systems as a supply and use of energy, and because observations on axonal flow incorporated microtubules as a mechanism, I decided to investigate the biochemical character and associated morphologic features of isolated microtubules following exposure to stimulations known to affect microtubule fine structural features. In addition, I will monitor biochemical reactivity as changes in phosphate accumulation on microtubules. Markers, should they be located, will be used to observe changes in either biochemical reactivity or fine structure morphology. Drug additions will be made as a method to separate the various components of reactions which may occur; fine structural detail will be monitored after drug additions 22 MATERIALS AND METHODS Microtubule Preparations Isolation of Microtubules Intact microtubules were prepared from adult rat brain using a modified procedure of Kirkpatrick (1969a). Wistar rats without regard to sex were used. After decapitation with a guillotine, the whole brain was repidly removed including the cerebellum but not the olfactory bulbs. The brain was homogenized in 7.0 ml ice cold 0.01 M HEPES buffer (N-2-hydroxyethyl-piperazine- N-2-ethane sulfonic acid), pH 6.6 containing 1M hexylene glycol (2-methylpentane-l,4-diol) (HH buffer) using 10 up and down strokes with a Potter Elvehjem teflon glass homogenizer. Nuclei and fragments were separated by initial centrifugation at 600 x g/10 minutes in 50 ml polycarbonate centrifuge tubes using an RC2-B Sorvall centrifuge equipped with an SS-34 rotor at 4°C« The supernate was carefully removed with a Pasteur pipet and recentrifuged at 20,000 x g for 20 minutes to isolate a "microtubule" pellet. Following the isolation, the pellet was resuspended in 3.0 ml of the HEPES buffer. Electron 23 microscopy performed on this preparation demonstrated intact, stabilized microtubular structures similar in quantity and fine structural detail to those described by Kirkpatrick (1969a, 1969b, 1970). Following the technique of Weisenberg (1971) , three washfng were performed on the isolated microtubules using a buffer mixture as follows: 0.15 M KC1, 0.2 mM GTP, 0.01 M HEPES, pH 6.6. Protein assays were done using a slight modifi cation of the biuret method of Gornall et al (1949) . This modification was the use of the detergent deoxycholate. Washing-Soaking Protocol for Isolated Microtubules The influence of glycol on microtubules has been described as that of "stabilizing" by several investi gators. It serves effectively, at a concentration of 1 M, to maintain the intactness of microtubule fine structure (Kane, 1962, 1965). The biochemical reactivity as measured by phosphate accumulation of such isolated microtubules was nearly non-existent until the glycol had been removed. The reversibility of the glycol effect has previously been reported in other investigations involving mitotic spindles which also contain the protein tubulin. 24 Standard Reaction Conditions for Phosphate Accumulation by Isolated Microtubules Recentrifugation at 20,000 xg for 20 minutes of the supernate from the initial 600 xg centrifugation provides a microtubule pellet. Figure 1 illustrates the complete preparation of microtubules using a method modified from Kirkpatrick. Due to the presence of hexylene glycol (1 M) in the buffer system, the preparation contains no identi fiable organelle shapes, such as mitochondria. An organic buffer, HEPES, was selected since it offered low change in pH over temperature changes, had negligible binding constants to the metals Ca , Mg and Mn++ and because phosphate buffers could not be used with the phosphate binding studies to be conducted. Upon removing the supernate with a Pasteur pipet, the microtubule pellet was resuspended three times in a buffer system of: 0.15 M KC1, 0.01 M HEPES and 0.2 mM GTP at pH 6.6 (HGK buffer). Volumes of 10 ml each were used in the washing procedure to rid the preparation of the glycol used for the isolation. Following the third washing, the preparation was allowed to soak in the same buffer mixture in the cold (4°C), for 4 to 12 hours after which it was again recentrifuged at 20,000 xg/20 minutes in a Sorvall SS-34 rotor. Final resuspension was to a volume of 3.0 ml KHG buffer/brain. Protein assay was performed by the 25 FIGURE 1 PREPARATION OP MICROTUBULES FROM ADULT RAT BRAIN, 26 FIGURE 1 PREPARATION OF MICROTUBULES FROM ADULT RAT BRAIN* Whole Brain 1 M Hexylene Glycol 0.01 M HEPES, pH 6.6 0.15 M KC1 7.0 ml/brain Homogenize 600 xg/10 min Nuclei and Supernate Fragments 20,000 xg/ 20 min Microtubule (Supernate) Pellet Resuspend in 0.1 M KC1, 0.01 M HEPES 0.2 mM GTP pH 6.6 3X Washing, 4-12 hours, 4°C Soaking 20,000 xg/20 min r Resuspend to 1 Pellet 3.0 ml (brain) Supernate (discard) *Modified from Kirkpatrick (1969). 27 biuret procedure described on page , . All handling of the preparation was done at 4°C. Standard Reaction Treatments for Isolated Washed Brain Microtubules The standard method of assay of all binding studies performed in this investigation is shown in Fig. 2. MTP is the isolated, washed brain microtubule preparation resuspended according to the direction of preparation. All reactions were carried out in individual 10x75 mm soft glass test tubes at 37°C or 4°C. To 0.5 ml of the HGK buffer at pH 6.6, additions were made of the various compounds incorporated into the specific assay. For each assay set the usual number of samples was five. Specifically, the reactions usually included the addition of protamine (2 mg). Radionuclides were added using a Hamilton microliter syringe. The timed reactions were begun following the addition of 0.05 ml (50 pi) of the MTP preparation, after the total incubation mixture had been allowed to equilibrate to the temperature required. Pilot experiments establishing time, temperature and pH were performed in groups of five samples each, and all results were normalized following counting of the various fractions FIGURE 2 STANDARD TREATMENT FOR (y ;-32?)GTP PHOSPHATE ACCUMULATION INTO RAT BRAIN MICROTUBULE PROTEIN. 29 I FIGURE 2 | ; STANDARD TREATMENT FOR (y-32P)GTP ACCUMULATION INTO | • RAT BRAIN MICROTUBULE PROTEIN (MTP) MTP + Protamine + GTP + (y-32P)GTP 5 min, 37UC (VBL, 1 x 10'3M, 30 min/37°C) Spin 3600 rmp 15 sec, I 1 Pellet Supernate (count) Heparin (200 U, 30 min/4°C) 5100 xg/5 min I------1 Precipitate Supernate (count) (count) 30 (microtubule pellet, heparin-protamine complex pellet and supernate) to values of cpm/mg protein. Newborn Preparations Microtubules from newborn rats, 0 to 6 days old, were prepared as pooled samples of at least three animals. The animals were decapitated using scissors, and the brains were treated with hexylene glycol-HEPES buffer, pH 6.4 in the same manner as the adult rat preparations, including the washing-soaking procedure following the isolation of the microtubule pellet. Protein assays were done as previously described on page Newborn mongrel dogs, 9 days old, were treated in the same manner as adult rats for the preparation of brain microtubules,. Electron Microscopy Techniques Parlodian-Coated, Carbon Stabilized Copper Grids Copper grids were washed following the procedure of Pease (1964). Grids (300 mesh) were first soaked and rinsed in acetone and washed in hot soapy water. They were then rinsed in tap water three times, rinsed in distilled water three times, and finally rinsed in 95% 31 ethyl alcohol and air dried. A 2% parlodian solution was prepared in redistilled (62°C, < 1 atm) amyl acetate. One drop of parlodian solution was placed on a dish of water and allowed to spread to form a thin film. When a satisfactory film was prepared, i.e., free of wrinkles and interference fringe lines, the clean copper grids were carefully dropped onto its surface. After approxi mately 25 grids were in place, they were picked up with a xvet filter paper together with the parlodian film and allowed to air dry in a closed petrie dish. After drying, the grids were coated lightly with carbon. Preparation of Samples: Parlodian Carbon Grids In most instances, the material to be examined by electron microscopy was placed on parlodian carbon grids according to the method of Kirkpatrick C1969b) . The microtubule fractions were fixed with an equal volume of 6% glutaraldehyde in 0.15 M Na2HPO^--KH2PO^ buffer at pH 7.3 (^prepared fresh weekly). After 60 minutes in the cold, one drop of the fixed preparation was added to the parlodian-carbon coated grids and held for one minute. Excess fluid was gently blotted with filter paper and a drop of 0.1% bovine serum albumin (6 x recrystalized) was added to the grid as a wetting agent. After 10 seconds, the grids were blotted again. Phosphotungstic acid (1%, pH 6.4), prepared daily and used as a negative stain in these studies was added to the grid using a Pasteur pipet for one minute. The grid was again blotted dry and placed in a grid box. Alternatively, when uranyl acetate (0.51, pH 5.0) was employed for negative staining, grids were placed on individual droplets of the stain and kept in the dark. After 20 minutes, they were blotted dry and placed in a grid box. Preparation of Samples: Thin Sections If specimens were to be thin sectioned, microtubular preparations were treated with glutaraldehyde according to a method used by Stoner (1969) for heart mitochondria as follows. After fixation with an equal volume of 3% buffered glutaraldehyde (charcoal treated) for one hour at 4°C, the specimens were stained with 0.1 ml osmium tetroxide freshly prepared in phosphate buffer for one hour at 4°C according to the method of Millonig as described by Pease (1964) . After staining, the prepara tions were centrifuged against an epoxy mold in poly ethylene tubes in a Beckman microfuge, using a Variac to adjust the speed. Several volumes of 0.5 to 1.0 ml each were needed to pack a pellet against the epoxy insert. Dehydration and embedding in SEM (Spurr Low Viscosity Embedding Medium were carried out in the FIGURE 3 DIAGRAM OF MICROFUGE TUBE AND EPOXY INSERT FOR PREPARATION. OF ELECTRON MICROSCOPY PELLET OF MICROTUBULES. DIAGRAM OF EPOXY INSERT PLACED IN MICROFUGE TUBE FOR MAKING MICROTUBULE PELLET * ROTOR BAR-HOLDER / PELLET EPOXY INSERT(CUT FLAT) * AFTER STONER, (1969) 35 polyethylene microfuge tube (Fig. 3, see Appendix A for details). Final embedding was done with SEM using a firm ratio of polymer to hardener in either flat molds or blocks (Appendix' A) Each mold contained an identifi cation number on a paper placed with the specimen before the final embedding. The blocks were hardened for 8 hours at 70°C, removed from the molds, and were thin sectioned using a glass knife on either a Porter-Blum . MT-1 or MT-2 ultramicrotome. Section thicknesses were approximately 60 to 150 p as judged by a characteristic silver colored interference fringe on the thin section. After sectioning, the individual thin sections were carefully picked up using a cleaned eyelash hair and gently heated in a small petrie dish. Five to ten sections were then mounted on the cleaned copper grids and allowed to dry in the grid box. Counterstaining was performed using a saturated solution of uranyl acetate in the dark (20 minutes) followed by lead hydroxide (20 minutes). Electron Microscopy A Philips Model EM-300 electron microscope using a 60 Kv beam was employed for all studies described. Normally, freshly isolated microtubules in hexylene glycol could be viewed as fields. Fine structural detail was routinely visualized with a 90 to 250,000 X plate magnification. Exposures were made on Kodak Electron Microscope Sheet Film (Kodak #4489) which was developed in either Kodak D-19 (1:3) or Kodak High Resolution Plate (HRP) Developer (1: 4.) ,. washed in tap water, and fixed in Kodak F-5 Fixative for 10 minutes. Light Scattering Material for light scattering studies was used soon after preparation in hexylene glycol-potassium phosphate buffer, pH 6.3 (0.05 y) . Initial studies were begun on a Phoenix-Brice light scattering instrument with a differential refractometer. This instrument proved to be too sensitive for even extremely small additions effecting changes in microtubular fine structures. A Farrand ratio recording fluorometer was used for measuring scatter at 90° to the incident light. Each sample contained 0.9 ml of buffered hexylene glycol and 0.1 ml of the microtubule preparation in hexylene glycol. Following stabilization, an arbitrary baseline setting of 50% of the full scale was established. Addition of reagents were made to test their effect on light scatter. After two minutes the reactions were stopped with 1.0 ml of 3% glutaraldehyde. All the light scatter studies were done with N = 5. Positive or 37 negative deflections were measured in millimeters at the point of greatest deflection within a two minute period; control values usually fell (A = .5-7) in this same period. Samples were measured in ordinary 10 x 75 mm glass test tubes. Recordings were made with a Farrand modified Heathkit chart recorder at a chart speed of 2 inches/minute with a sensitivity setting 10 on the microammeter using quinine standards prepared according to Lowry (1972) . Packed Volume Studies In attempts at relating scatter changes to some physical parameters associated with the dimension of microtubules, packed volume measurements were made. Packed volume (PV) tubes were constructed using 50 ml polycarbonate centrifuge tubes modified for these studies. Construction of these tubes was made by embedding a 0.065 inch piano wire coated with silicone grease in a 1:7:28:35 mixture by weight of 2,4,6 Tris (dimethyl aminomethyl) phenol (DMP-30): Dow Epoxy Resin 732 (DER-732): Dow Epoxy Resin 332 (DER-332) dodecenyl succinic anhydride (DDSA). The tube is schematically diagrammed as prepared and placed in an SS-34 rotor, Fig. 4 (Stoner, 1969). FIGURE 4 PACKED VOLUME TUBE IN PLACE IN SORVALL SS-34 ROTOR. 39 EPOXY k RESIN 40 For calibration, each PV tube slot was filled with 5 pi increments of water and carefully checked for air bubbles. A caliper was used to measure volume changes as a linear function and a graph was constructed which provided a calibration curve for each of the. PV tubes . . Equations to describe these curves were progammed into a Wang 700C computer and the data were corrected to microliters packed volume for each sample, ./. The microtubule preparations were poured into the reservoir at the top of the prepared packed volume tubes and separation by centrifugation for 10 minutes at 12,000 xg in a RC2-B Sorvall centrifuge equipped with an SS-34 rotor. The tubes were immediately removed and the packed volume of the reaction was estimated with a pair of dividers calibrated with a millimeter ruler. All tubes were numbered and careful records were kept to designate the tube used for each measurement. Protamine Additions to the Microtubule Standard Reaction Unlabeled Protamine Protamine sulfate from salmon was routinely added to all phosphate studies. The protamine, kept at a concentration of 2 mg/10 X in HGK buffer frozen at -20°C, was thawed and thoroughly mixed before each use. Protamine Labeling The specific labeling of protamine to monitor the reactivity of this compound in the standard reaction system was carried out in two ways. First protamine was tritium labeled in order to study the participation of this molecule in the phenomenon of phosphate binding to MTP. Later the compounds were labeled with ferritin in order to permit the visualization of the same reaction in the electron microscope. It was hoped that there would be correlation between the two methods in order to allow conclusions to be drawn relating the biochemical changes to fine structural detail. 3 H-Protamine Labeling Protamine was reacted with salicylaldehyde at various pH, and the Schiff's Base reaction between these two molecules monitored at 400 my in a spectrophotometer Controls of each of the reactants separately and together were included. After the preliminary reactions had gone to completion (e.g., no further O.D. change with time) the reaction was made irreversible by the addition of sodium borohydride (NaBH^) to the reaction, 42 and the O.D. values monitored until the solution was again clear (Fig. S).'1' Protamine was labeled with tritium by reacting 1 mg protamine sulfate with 20 equivalents of salicyl- aldehyde at pH = 10, in water. A bright yellow color (Amax. = 400 my) was instantly observed which deepened as the reaction proceeded for five minutes at room temperature. To reduce the Schiff’s Base reaction thus formed between o-hydroxybenzaldehyde and the reactive amino groups on protamine, 2 mCi NaB was added. The reactive amino groups on protamine were assumed to be 3 epsilon amino lysine. Upon the addition of the NaB H^, reduction occurred and the reaction mixture slowly lost its yellow color and turned colorless. To assure complete reduction of the Schiff’s Base, 10 mg cold NaBH^ was added after the initial addition of the borotritiide. After five minutes, a 1.0 ml aliquot of acetone was added to the reaction mixture as a scavenger. Separation of the reaction products was made on a 1 x 10 cm Bio-Rad P-6 column and eluted with water; 1.0 ml fractions were collected and counted in a liquid scintillation spectrometer in Bray’s solution. The labeled protamine was eluded in fraction #7 (Fig. 6). ■'’Personal communication from Dr. Perry Frey, Dept, of Chemistry, The Ohio State University. 43 FIGURE 5 MECHANISM OF SALICYLALDEHYDE ADDITION TO PROTAMINE AND REDUCTION BY NaB3H 4 ,' (SODIUM BOROTRITIIDE) . 44 MECHANISM OF SALICYLALDEHYDE ADDITION TO PROTAMINE AND REDUCTION BY N a B 3 H4 cH ) + HoN-PROTAMINE 6® 6© OH i = N - PROTAMINE + Na B3H 6© 0" 6© ^ M M C 400m /j .) 3H - N —PROTAMINE 0" FIGURE 6 ELUTION OF 3H-PROTAMINE BY COLUMN CHROMATOGRAPHY. lO CPM/5X ALIQUOT XIO" - 0 4 2 260-1 220 200 140- 100 160- 180- 120 - 0 4 0 2 - 0 6 - 0 8 - - - - - 5 UE RCIN NUMBER FRACTION TUBE 10 OUN CHROMATOGRAPHY COLUMN H- T NE, IRD P-6 BIORAD , E IN M 0TA R -P 3H 525 15 20 l.0 m l/M IN ( ( WATER) IN l/M m l.0 I XIO cm XIO I o\ 47 The specific activity of the H-protamine was 2.46 mCi/ Vimole. Protamine-Ferritin Coupling Ferritin was coupled to protamine according to the method of Singer (1964) using protamine sulfate and toluene 2-4 diisocyanate which had been previously coupled to ferritin. Following the two step coupling reaction (Fig. 7), dialysis was performed first against (NH^^COg buffer (10 mM, pH 7.4) and then against HEPES buffer (3.3 mM, pH 7.4) overnight at 4°C with stirring to remove unreacted components. This method of purification gave unsatisfactory results due to denaturation when the product was used with microtubules. Contamination with unreacted ferritin also caused non-specific types of binding. Fractionation of the product was then performed using a Bio-Rad P-150 column, 2 x 32 cm, eluting with water and collecting 5 ml fractions. Two peaks were separately eluted from the column; the first using a gradient of 1 M KC1 and a second using a gradient of 1 M N aOH. These two peaks were exhaustively dialysed for 30 hours against distilled water and finally HEPES buffer, 0.01 M, pH = 6.5. The spectrophotometric data of the two peaks when compared to native ferritin indicates that the second peak (eluded with NaOH) was FIGURE 7 FERRITIN-PROTAMINE COUPLING. MECHANISM. FERRITIN-PROTAMINE COUPLING 3 J H 3 p N = C = 0 0°C, 2 hrs. r Y N==c=o a N=C=0 HN-C-N -FER II I 0 H (rc) N-PROTAMINE STAGE 2 tc + H2N-PROTAMINE --C * — H FIGURE 8 SPECTROPHOTOMETRIC TRACING OF FERRITIN-PROTAMINE REACTION BY ELUTION FROM BIO-RAD P-150 COLUMN: Column was 2.5 x 30 cm, 1.0 ml/min flow, eluted with water, KC1 (1M) and NaOH (1M). SPECTROPHOTOMETRIC TRACING OF FERRITIN - PROTAMINE REACTION BY ELUTION FROM BIO-RAD P -150 COLUMN 1.6 1.0 0.8 0.6 0.4 0.2 0.1 200 300 500 600 tn 52 the ferritin-protamine coupled product, and that the first peak (eluded with KC1) was probably ferritin (Fig. 8). Studies with Orthophosphate, Preparation and Methods Phosphate accumulation by isolated adult rat brain microtubules was measured as orthophosphate, either ■^P^ or ^ P ^ as phosphoric acid in ^ 0,02 N HC1. All reactions were run with N = 5 at room temperature in 20 mM Tris at pH 7.58 containing 0.9 ml of 1 M hexylene glycol. To this mixture was added 0.1 ml of the microtubular suspension and the reaction allowed to occur at room temperature for 5 minutes. After addition of 1.0 ml 16 mM PCA, the reaction was mixed and placed in ice for 5 minutes. Effect of Time on Phosphate Accumulation Incubation was performed using times of 0,2,5 and 10 minutes and temperatures of 0° and 37°C. Protamine (2 mg) was added to a separate set of assays; all assays 32 were performed with N = 5. To eliminate pyrophosphate, 0.05 ml of 6 N HC1 was added to 0.3 ml of the isotope (orthophosphate-32 or sodium phosphate-32) and was 53 heated in a water bath at 60°C/30 minutes in a loosely- capped vial. Phosphate Accumulation with (y-32P)GTP and (v -52P)ATP Using * 20,000 cpm of (y-32P)ATP and (y-32P)GTP (6 yCi/ymole) duplicate assays were run on "soaked" microtubules in HGK buffer. Protamine, 2 mg in 10 yl was routinely added to each assay tube. Duplicate assays were performed at 37°C with controls held at 0°C. The reactions of both groups were stopped with 1.0 ml additions-of 16 mM PCA. Samples were plated on ringed planchets and counted in a Nuclear-Chicago Counter. Microtubules were alternatively precipitated with vinblastine (1 mM final concentration). After 30 minutes at 37°C a Serofuge was used to pack the vinblastine preparation. The supernate was removed and heparin, in stoichiometric amounts + 10% excess, was added to the supernate following removal of the micro- tubular precipitate. After incubating for 30 minutes at 4°C, centrifugation at 5100 xg for 10 minutes resulted in a white protamine-heparin complex pellet which was separated from the supernate. Both fractions were counted in the planchet counter. Further additions of heparin to the supernate did not cause additional 54 precipitation. Supplemental assays were later performed, employing vinblastine (1 mM) to precipitate the microtubules in the standard reaction mixture. Centrifugation at 3600 rpm for 15 seconds in a Serofuge to precipitate microtubules 3 2 also provided a rapid method of assaying accumulation by microtubules. Subsequently, after removal of the supernate from the microtubule pellet, the precipitate was plated in ring planchets. All samples were counted in the Nuclear-Chicago planchet counter with 2 4% efficiency using a ^C-standard. Specific Additions of Compounds to the MTP Standard Reaction System Specific Preparations for Drug Studies Drug studies involved the addition of drugs dissolved in 0.01 , HEPES, 0.15 M KC1 at pH 6.6. Further dilutions were done in the same buffer system. Carisoprodol The drug was made to a stock concentration of 5 x 10”^M in HEPES-KC1 buffer, pH 6.6. This compound is reported to have 6 to 8 times the effectiveness for skeletal relaxation as meprobamate. 55 Chlorpromazine The molecular weight of this compound is 355.3. This drug was routinely used at a final concentration of 1 x 10'^M in investigations with microtubules. Preparr ation was in HEPES-KC1 buffer at pH 6.6. This drug, a major tranquilizer, exhibits a multifaceted pharmacologic response upon administration. It is effective in reducing motor activity in psychoneurotic and psychotic patients. Diazoxide The drug was made up to a stock concentration of 5 x 10 ^M. This drug is an antihypertensive but has also been reported to block insulin secretion, known to be a function of microtubules. Mephenesin The drug was made up to a stock concentration of - 4 5 x 10 M. Structurally it is a monoether of glycerol exhibiting skeletal muscle relaxation. This action is central and appears to depress sites in the mid-brain and brain stem at the level of the thalamus and below. In the spinal cord the depression is thought to be at the internuncial neurons. 56 Meprobamate - 4 The drug was made up to a concentration of 5 x 10 M in HEPES-KC1 buffer at pH 6.6. At this concentration, the drug is completely in solution. Routinely, 10 yl of drug was used in a 0.5 ml assay for a final concen tration of 10”5M/assay. Oxanamide The drug was made up to a concentration of 5 x 10”^M in HEPES-KC1 buffer, pH 6.6. This drug resembles meprobamate in pharmacologic activity, experimentally producing skeletal muscle relaxation by partial blockade of internuncial neurons. Phenaglycodol The drug was made up to a stock concentration of 5 x 1 0 " in HEPES-KC1 buffer, pH 6.6. This drug is chemically related to mephenesin and meprobamate, however it is a butane diol. Pharmacologically it demonstrates similar actions to meprobamate by evoking a selective depressant action of polysynaptic pathways at the spinal and supra-spinal level. 57 2-Methyl-l,3-Propylpropane Diol The drug was made up to a stock concentration of 5 x 10" ^M. This drug is the immediate precursor in the synthesis of meprobamate. Vinblastine and Vincristine Vinblastine and vincristine are known to specifically react with tubulin, a protein present in microtubules. At a final concentration of 1 mM, these drugs were used to selectively precipitate microtubules from the reaction mixture. Drug additions were made following a five minute standard incubation time at 37°C. Prepar ations of the vinca alkaloids were diluted in HEPES- KC1 buffer, pH 6.6, in such a manner that 20 yl provided the desired concentration (400 yl/10 mg vial). Incubations were routinely held at 37°C for a period of 30 minutes. Separation of reactants was the protocol discussed under phosphate accumulation methods, page The drugs were prepared fresh for each assay. 58 Preparation of Other Reagents »2o Deuterium water O^O) was diluted with HGK buffer, pH 6.6, directly in the assay tube to a final percentage ratio Cv/v) , in a final volume of 0.5 ml. The usual concentration employed was 30 to 701. EGTA and EDTA EGTA (ethyleneglycol-bis(beta-aminoethylether)N-N1 vtetracetic acid) and EDTA (ethylenedianine tetracetic acid) were useful in studies of microtubule reaggregation from subunit proteins. These compounds were prepared in HEPES-KC1 buffer at pH 6.6 at a concentration of 1 M and stored frozen at -20°C until used. Ferritin This electron dense iron globin compound was used both in native form, and coupled with protamine as a marker for the possible visualization of protamine attachment to microtubules by electron microscopy. Native ferritin, when employed in reaction with micro tubules, was used at a final concentration of 182 pg/ standard assay of 0.5 m l . 59 Heparin Heparin was used to follow the course of binding between microtubules and protamine. In the assay, 200 U of heparin was found to stoichiometrically precipitate 2 mg protamine; each reaction supernate received 11 yl or 220 U heparin. This was the standard amount of protamine added to each tube of the assay prior to the addition of microtubules. The heparin, supplied in vials of 20,000 U/ml in 1 ml quantities, was thawed and refrozen for each experiment. Hexylene Glycol For studies of hexylene glycol as a reagent to study phosphate accumulation, a solution was routinely made to a concentration of 0.5 mM in HEPES-KC1 buffer. Ten microliter additions to a 0.5 ml sample assay gave a final concentration of 10 yM. Ion Studies Calcium as CaC^, magnesium, as MgSO^ and manganese, as MnCl2 were prepared as stock solutions containing 1 M/liter in HEPES-KC1 buffer at pH 6.6. Additions were made with calibrated microliter pipets to final concen trations of 0.001--10-0 mM per 0.5 ml microtubule assay. 60 Nucleotides and Cyclic Nucleotides ATP, ADP, 5'AMP, cAMP, GTP, GDP, 5'GMP and cGMP were used in this study. All of these compounds were diluted to concentrations of 1 to 10 mM in HEPES-KC1 buffer at pH 6.6. Additions were made to the individual assay tubes by microliter pipets, to provide a concen tration of 1 to 100 pM/assay of 0.5 ml volume. Column Chromatography of Standard Reaction Mixture The standard reaction (MTP + protamine + (y-^P)GTP) was incubated for five minutes at 37°C. Vinblastine _3 was added to a final concentration of 1 x 10 M and allowed to react for 30 minutes at 37°C. To the micro tubule precipitate, 1.0 ml of double distilled, deionized water was added and the pellet mixed thoroughly on a buzzer. The entire 1.0 ml was carefully layered on the top of a 0.9 x 17 cm G-25 Sephadex column, previously equilibrated with deionized double distilled water. A small Buchler pump was used to maintain the flow rate of the solvent at 1,3 ml/minute. One minute fractions were collected using a ISCO Model #272 fraction collector for 106 fractions. Because some radioactivity still remained on the column, further elution was performed 61 using a salt gradient of 1,0 M KC1, Identification of the various peaks was performed using this same column, 32 Protamine (10 mg) and (y- P)GTP were layered separately on the column which was re-equilibrated and eluted in both instances with double distilled deionized water at a flow rate of 1.3 ml/minute. Ordered Additions Reactions Using all or some of the following components: 50 pi prepared microtubules 10 pi protamine sulfate (2 mg) 11 pi heparin (20,000 U/ml) 10 to 20 pi (y-32P)GTP 20 pi vinblastine (1 x 10~3M final concentration) sequential manipulations of addition to 0.5 ml of the HGK buffer (pH 6,6) were performed. All reactions were allowed to incubate for five minutes; however, many initial additions were pre-incubated if microtubules were not present at that time. 62 Radioisotope Assays Nuclear-^Chicago Planchet Counter This instrument was used for the assay of the radio- 32 33 nuclides and P^. Individual samples were pipetted onto aluminum planchets and were evaporated to dryness in a 50°C drying oven. Calibration of the instrument, using a ^ C standard and 7 pounds flow of Q-gas (1.3% butane, 98.7% helium) was determined for each assay group. The efficiency was calculated to be near 24.5%, Samples were counted for one to five minutes each. Data reduction and normalization was done on a Wang 700C calculator using a program written by myself for this purpose. Packard Tri-Carb Liquid Scintillation Counter 14 3 For the assay of the radionuclides C and H, as 32 well as some special studies using P, the method of liquid scintillation was chosen. Samples were counted in Bray’s liquid scintillation solution with the following ingredients: naphthalene, 60 gm, reagent grade methanol, 100 ml, reagent grade ethylene glycol, 20 ml, reagent grade 63 PPO C2,5-diphenyloxazole), 4 gm, scintillation grade POPOP (1,4-bis-C2-(5-phenyloxazolyl))), 200 mg, scintillation grade p-dioxane to make 100 ml, reagent grade Possible complex pellet quenching of the microtubule pellet or heparin-protamine was handled by using an external standardization curve which was determined by the addition of small amounts of nitromethane to the scintillation solution, or by the channels ratio method of determining the amount of this quenching. The data collected were calculated and normalized on a Wang 700C computer using programs written by myself for this purpose. cAMP Radioimmunoassay To determine the presence of adenyl cyclase, cAMP was measured by a commercially available radioimmunoassay kit. ATP was used as substrate with the microtubule preparation serving as a possible source of "cyclase" activity. Following the removal of the supernate from the microtubule pellet, three 1.0 ml extractions were made with diethyl ether. They were pooled and evaporated to dryness under a stream of dry nitrogen. The dried extract liras resuspended in acetate buffer, pH = 6.2, following the 64 protocol provided. Volumes of 1, 10 and 100 yl of the extract were assayed for cAMP content in triplicate. The samples were incubated with the primary antibody over night, and a second antibody was added for 48 hours incubation at 4°C. The individual assay tubes were diluted with 2 ml of water. A pellet was obtained after centrifugation at 2600 rpm for 30 minutes. Fractions- were counted and calculated from the standard curve obtained with various amounts of standards in the range of 0.01 to 10 pmoles. All values were normalized to pinoles of cAMP/mg microtubule protein added. Materials Chemicals and Reagents: ATP, ADP, cAMP, GTP, GDP, cGMP, EGTA, protamine sulfate were obtained from the Sigma Chemical Co, of St. Louis, Missouri. HEPES was purchased from Calbiochem of La Jolla, California. Hexylene glycol was obtained from Polysciences, Inc. of Warrington, Pennsylvania. PTA was obtained from Mallinkrodt Chemical Works, St. Louis, Missouri. Salicylaldehyde was purchased from the Aldrich Chemical Co.,.Inc., Milwaukee, Wisconsin. 65 Amyl acetate, NaBH^, was purchased from the Fisher Scientific Co., Pittsburgh, Pennsylvania. Ferritin and toluene-2,4-isocyanate were purchased from Miles Laboratories, Inc. of Kankakee, Illinois. Radioisotopes, D20, NaB H^, (y- P)GTP, P^ and P^,were purchased from New England Nuclear of Boston, Massachusetts. Planchets and sephadex were purchased from the Sigma Chemical Co., St. Louis, Missouri. Bio-Rad P-6 and P-150 columns were purchased from Bio-Rad Laboratories, Richmond, California. The electron microscopy supplies, DER, SEM, grids, parlodian, uranyl acetate, were purchased from Polysciences, Inc., Warrington, Pennsylvania. Chlorpromazine was donated by Dr. Sarah Tjioe. Carisoprodol, meprobamate, 2-methyl-1,4-propyl- propanediol and mephenesin are products of Wallace Pharmaceuticals. Diazoxide is a product of Schering Corporation. Heparin was obtained from the Upjohn Co. Oxanimide is a product of the William S. Merrell Co. Vinblastine, vincristine and phenaglycodol are products of E. Lilly. 66 RESULTS Introduction Since 19G8, when Weisenberg et al. published a sucrose method for the isolation of microtubules and demonstrated colchicine binding, ill vitro, investigations on the nature of these particles has been abundant. The association of microtubules with fast axoplasmic trans port, with various diseases associated with aging and during fetal and neonatal development have been the subject of many investigations relying in the greater part in the biochemical characterization of the dimer form of tubulin. Most of these studies relied heavily on the colchicine binding property of tubulin for quantitation. Kirkpatrick (1969a) described a method to isolate microtubules from rat brain using a glycol to actively "stabilize" the intact microtubular fine structure. Use was made of the ability to visualize these particles in the electron microscope, and it was decided to further investigate the physical and biochemical character of microtubules. Markers, should they be located, would 67 play an integral part in determining the subtle effects of both chemical and physical treatment and the role of drug treatment on the behavior of these stabilized particles. Effects of Reagent Additions on Light Scatter and Packed Volume Initial studies, begun using buffered hexylene glycol at pH 6.4, were concerned with the effect of nucleotide additions on the physical properties of light scatter and packed volume. The Phoenix-Brice light scattering machine and the differential refractometer were selected for use because of the precision which they were capable of measuring either the scatter of light or the refractive index of a specific solution. Calibration of the instruments was achieved using the homogenizing buffer, nucleotides to be added and solutions of millimolar concentrations of EDTA, Ca , Mg and/or Mn . Addition of 5 pi of the microtubule preparation in a 50 ml quartz cuvette to 50 ml of the hexylene glycol buffer was made and a null balance was found in the ratio recorder of the light scattering machine. Subsequent 1 to 5 microliter additions of various reagents (GTP, ATP, cAMP, cGMP, Ca++, Mg , Mn and EDTA) proved to have a large effect even 68 when the sensitivity of the instrument was at the lowest 2 possible tap. Due to the poor yield of microtubules in available procedures, it was decided to attempt further light scatter studies on the initial preparation, but to measure the scatter on an instrument much less sensitive to such subtle changes. For this work, a Farrand Ratio Recording Fluorometer Model A-4 was chosen. Light scatter was measured at an angle of 90° at 340 my for both excitation and emission, and recorded on a Farrand- Modified Heathkit chart recorder. Individual 10 x 75 my soft glass test tubes were used as cuvettes, containing 0.9 ml of buffered hexylene glycol and 0.1 ml buffer- glycol containing brain microtubules. At a sensitivity range of 10 on the ratio fluorometer, the null baseline was set at 50% on the chart recorder. The addition of various reagents was made directly to the test tube using calibrated microliter pipettes, briefly mixed and replaced in the sample chamber. Figure 9 shows the type of tracings which were recorded from the fluorometer by the modified Heathkit recorder. Packed volume was measured as described under METHODS. 2 Dr. Sylvan Frank, College of Pharmacy, the Ohio State University, suggested that no further investigation be attempted until microtubules in a more purified state were prepared. FIGURE 9 SUMMARY OF FLUOROMETER DEFLECTIONS BY ADDITION OF COMPOUNDS TO RAT. BRAIN MICROTUBULES. ' SUMMARY OF FLUOROMETER DEFLECTIONS BY ADDITION OF COMPOUNDS TO RAT BRAIN MICROTUBULES A. DISAGGREGATION 0 -A B. AGGREGATION - RELAXATION + A 0 C. CONTROL 0 0 1.0 2.0 TIME (MINUTES) 71 In Table 1 are the corrected results for both packed volume and light scatter measurements showing the effect of added guanine nucleotides, GTP and GDP. Increasing the concentration of GTP caused a decrease in packed volume. Light scatter goes through a maximum at 1 x 10 Si GTP, apparent from the large positive _ 8 value of +106 mm deflection which is reversed at 10 M and 10~^M GTP. GDP addition to the microtubule suspension did not significantly change the packed volume, but did cause a gradual increase in the negative deflection of the light scatter. Cyclic nucleotide additions (Table 2) indicated that the cAMP response parallels GTP effects on both packed volume and light scatter measurements. Packed volume changes with increasing concentrations of nucleotide were more regular with cGMP than with cAMP. The light scattering measurements with cGMP additions were all positive (upward) deflections and were all of considerably greater magnitude than those of other additions. This series of measurements indicated that the light scattering property was quite real and of sufficient importance to warrant further investigation using various ions, chelating agents and certain drugs which were known to act selectively on microtubules. + + The addition of Ca (as CaCl2) to the microtubule preparation showed a progressive increase in a positive TABLE 1 PACKED VOLUME AND LIGHT SCATTER OF RAT BRAIN MICROTUBULES TREATED WITH GUANINE NUCLEOTIDES, IN VITRO3, GTP GDP Concentration Packed Volume*3 Light Scatter0 Packed Volume*3 Light Scatter0 (moles/liter) (microliters) (mm) (microliters) (mm) 10’3 -49 -137 1 O 18.97 ± 0.24 -77 24.88 ± 0.65 - 32 10_S 20.20 ± 0.46 - 6 24.71 ± 0.57 - 48 10-6 20.75 ± 0.29 +106 25.18 ± 0,28 - 30 I 00 O - 5 25,10 ± 0,39 - 18 Control 23.24 ± 1.24 - 5 24.46 ± 0.42 - 5 aAll reactions contained 0.1 ml microtubule preparation in 0.9 ml hexylene glycol-phosphate buffer, pH 6.4. ^Following fixation with 1.0 ml 3% glutaraldehyde, pH 6.4. °Light scatter at 340 my was measured at maximum deflection within 2 minutes of microtubule addition at room temperature. 10 TABLE 2 PACKED VOLUME AND LIGHT SCATTER OF RAT BRAIN MICROTUBULES TREATED WITH CYCLIC NUCLEOTIDES, IN VITROa cAMP cGMP Concentration Packed Volume^ Light Scatter0 Packed Volume^ Light Scatter0 (moles/liter) (microliters) (mm) (microliters) (mm) 10-3 -101 10-4 20.76 ± 0.38 - 72 20.95 ± 0.59 +253 10"5 23.10 + 0.41 -68 21.34 ± 0.42 +627 10‘6 21.10 ± 0.46 +123 22.93 ± 0.51 +333 10‘7 21.55 ± 0.31 + 23 22^05 ± 0.42 +108 Control 24.75 ± 0.33 - 5 24.75 ± 0,29 - 5 Q All reactions contained 0.1 ml microtubule preparation in 0.9 ml hexylene glycol-phosphate buffer, pH 6.4. Following fixation with 1.0 ml 31 glutaraldehyde, pH 6.4. °Light scatter at 340 mp was measured at maximum deflection within 2 minutes of microtubule addition ^ at room temperature. ■ w 74 (A-deflection) light scatter property with increasing final concentrations, similar to the cGMP additions in nature and magnitude (Table 3). The packed volume data indicated that the microtubules or some endogenous material *i*' *4* *t" which was responsive to exogenously added Ca and Mg for the dose response curve falls both below and above the control values for each of these determinations. Light scattering data after Mg++ addition, although not as + + great in magnitude as that of Ca addition, was again correlated with an increased positive scatter with increased concentration, similar to cGMP, and in contrast to GDP which showed a negative correlation, i.e., increased negative scatter with increased nucleotide concentration. Chelating agents (Table 4), used so effectively to influence metabolic changes involving calcium and magnesium, were employed next to determine if a clue might be observed concerning the role of ions endogenously present in the initial preparation of microtubules. ? -8 Citrate, as sodium citrate (io to 10 M), caused a reversal of the packed volume shrinkage (Table 4). Citrate also caused light scatter of a negative deflection at concentrations > 10 ~^M, similar to the pattern shown by GDP addition. EGTA also caused a reversal of the packed volume trend, similar to the citrate additions. Finally, four separate drug additions were employed with microtubular preparations and changes in these two TABLE 3 PACKED VOLUME AND LIGHT SCATTER OF RAT BRAIN MICROTUBULES TREATED WITH CALCIUM OR MAGNESIUM IONS, IN VITRO3, Ca„ + + wMg + + Concentration Packed Volume*3 Light Scatter0 Packed Volume*3 Light Scatter0 (moles/liter) (microliters) (mm) (microliters) (mm) 1 to o 21.28 ± 1.15 +340 23.99 ± 1.04 +77 -3 10 23.32 ± 0.97 +183 25.70 ± 0.53 +60 *3* 1 rH o 28.39 ± 0.68 +137 27.19 ± 0.76 -20 Control 25.66 ± 1.15 - 5 25.66 ± 0.45 - 5 aAll reactions contained 0.1 ml microtubule preparation in 0.9 ml hexylene glycol-phosphate buffer, pH 6.4. ^Following fixation with 1.0 ml 3% glutaraldehyde, pH 6.4. °Light scatter at 340 my was measured at maximum deflection within 2 minutes of microtubule addition at room temperature. TABLE 4 PACKED VOLUME AND LIGHT SCATTER OF RAT BRAIN MICROTUBULES TREATED WITH CHELATING AGENTS, IN VITROa Citrate EGTA Concentration Packed Volume^ Light Scatter0 Packed Volume^ Light Scatter0 (moles/liter) (microliters) (mm) (microliters) (mm) 10'2 19.85 + 1.12 -205 23.09 ± 1.00 10-3 21.48 + 1.03 -173 23.31 ± 0.34 -150 10'4 21.76 + 0.40 -122 23.81 ± 0.21 l 10-6 24,03 + 0.72 - 36 10"7 21.27 + 0.51 1 00 + H-» o 21.37 0.32 Control 19.63 + 0.25 - 5 22,29 ± 0,72 - 5 ^Conditions are the same as Table 1. 1- Following fixation with 1.0 ml 3% glutaraldehyde, pH 6.4, ^ 77 physical measurements were noted (Tables 5 and 6). Treatment of the microtubules iifith the vinca alkaloids vinblastine (VBL) or vincristine (VCR) results in increasing volume with concentrations to 10 ^M. Above this concentration of alkaloid a significant decrease in volume occurs, possibly indicative of some action by the drug in the microtubule, possibly a phase change, e.g., crystal formation. That this reasoning may be valid is supported by the light scattering data obtained with vinblastine, which represents a decrease of 50% at _ 2 10 M compared with the large negative deflection for _3 packed volume studies already seen at 10 M. Vincristine results support this view but at total concentrations of drug an order of magnitude lower. Investigation of the effects of colchicine and SCHA-306, a colchicine-like drug, support the trend seen with the vinca alkaloids of increased packed volume measurements combined with a dose-related negative deflection in light scattering. Colchicine and SCHA-306 were equipotent in their microtubular responses when results were expressed as percentage changes to eliminate differences in absolute values from day to day. TABLE 5 PACKED VOLUME AND LIGHT SCATTER OF RAT BRAIN MICROTUBULES TREATED WITH VINCA ALKALOIDS, IN VITRQa Vinblastine Vincristine Concentration Packed Volume Light Scatter0 Packed Volume Light Scatter0 (moles/liter) (microliters) (mm) (microliters) (mm) 10"3 18.93 ± 0.12 -137 10 “4 21.27 ± 0.38 -275 16.03 ± 0.75 10-6 20.28 ± 0.16 -214 23.16 ± 0.67 -227 M f 8 20.00 ± 0.19 -170 22.10 ± 0.30 -150 1 M O M O 19.95 ± 0.22 10-12 - 70 Control 19.24 ± 0.34 - 5 21.24 ± 0.35 - 5 3 Conditions are the same as Table 1, Following fixation with 1.0 ml 3% glutaraldehyde, pH 6.4. TABLE 6 PACKED VOLUME AND LIGHT SCATTER OF RAT BRAIN MICROTUBULES TREATED WITH COLCHICINE AND SCHA-306, IN VITROa Colchicine SCHA-306 v _ v Concentration Packed Volume Light Scatter Packed Volume Light Scatter (moles/liter) (microliters) (mm) (microliters) (mm) 10-3 -730 10"4 18.97 + 0.56 -180 22.88 ± 0.80 -168 10"6 20.17 + 0.55 -114 23.05 ± 0.55 -100 10"8 20.20 + 0.37 - 81 24.14 ± 0.40 - 35 10"10 20.61 + 0.35 10-12 - 40 Control 19.24 + 0.42 - 5 21,24 ± 0.36 - 5 a Conditions are the same as Table 1. ' . ------ h Following fixation with 1.0 ml 31 glutaraldehyde, pH 6.4. VO 80 Phosphate Accumulation Studies with Orthophosphate 32P^, (y-32P)GTP and (y-32P)GTP The physical changes reflected in packed volume and light scatter suggested a possible mechanochemical property similar to that seen in muscle actomyosin systems. Chemical changes involving phosphate would be anticipated and for this reason it was decided to attempt experiments with 32P^ and 3 ^P^ due to the high probability that the microtubules or the subunit tubulin would undergo a phosphate-related reaction. These reactions might reflect enzyme catalysed specific or non-specific binding of the radionuclide under the appropriate conditions. The results of experiments using the radionuclide 32Pi (either as H332P04 or Na232P04) following the experimental approach as outlined in METHODS, are shown in Table 7). The experimental protocol was designed according to the method of Rodbell et al. (1971), which called for precipitating the microtubules with 16 mM PCA. Phosphate accumulation in the microtubule or its subunit would help to determine whether or not phosphory lation had occurred. Hexylene glycol isolated microtubules did not accumulate phosphate in either a time- or temperature-dependent manner. The pH of the reactions over the range of 5.4 -> 7.4 did not significantly influence this binding (Fig. 10). Phosphate adsorbable TABLE 7 32-PHOSPHATE ACCUMULATION WITH INORGANIC PHOSPHATE AND PROTAMINE ON ADULT RAT BRAIN MICROTUBULES, IN VITRO Groupa Time Temperature Cpm/PCA^ %52?i in PCA (minutes) (centigrade) Precipitate Precipitate I Hexylene Glycol** 1 2.0 0 91 < 0.1 37 103 < 0.1 2 5.0 0 56 < 0.1 37 77 < 0.1 3 10.0 0 73 < 0.1 37 70 < 0.1 II HGK Bufferc o 1 2.0 0 00 1.9f 37 2459 10.3 2 5.0 0 808 3.1 37 4076 20.3 00 TABLE 7 Continued Group Time Temperature Cpm/PCA in PCA (minutes) (centigrade) Precipitate Precipitate 3 10.0 0 319 1.5 37 4194 23.2 ^ = 5 for all groups. ^Buffered with 0.01 M HEPES, pH 6 .6; 0.9 ml buffered glycol + 0.1 ml microtubule preparation. hashed three times, soaked 12 hours in 0.01 M HEPES, 0.2 mM GTP, pH 6.6; 0.9 ml HGK buffer, 0.1 ml microtubule preparation (0.6 mg/0.05 ml). ^0.5 ml 16 mM PCA added/assay. eAll counts in Part II of this table refer to microtubule precipitate following VBL addition (1 x 10'3M, final concentration). f I Pi in VBL precipitate (using 32-orthophosphate) 00 FIGURE 10 pH vs. ORTHOPHOSPHATE-32 BINDING TO WASHED, SOAKED MICROTUBULES. % PHOSPHATE ACCUMULATION ON MICROTUBULE PELLET/10M lN/37“C « w w o x g m h o x - o ® X h»" ■o nIs x x ° 5 5 m o I X o roOJ CDS 2— C z r 2 t o ' m z w o *8 85 from the neutralized supernate with activated charcoal was not significant, nor were experiments in which a phosphomolybdate complex was made of the supernate. The latter two measurements were designed to measure organic ^2p formed or ^2p liberated. Incubation of wfth other agents, previously described in packed volume and light scattering measure ments ( e.g., protamine, cAMP and GTP) , did not provide phosphate accumulation data different from the initial ■^P^ accumulation experiments in any fraction examined. Effect of Isolation Medium on Phosphate Accumulation The microtubule preparation was subsequently treated with a washing-soaking procedure using the organic buffer HEPES and then re-examined in an identical assay system as described above except that the microtubule pellet was obtained by centrifugation and not with PCA precipitation. Under these conditions, the microtubule pellet with added protamine showed a phosphate accumulation which was both time- and temperature-dependent with orthophosphate ^ P f (Table 7, part II). The pH curve of the reaction was quite broad, ranging from pH 6.0 to 7.2 (Fig. 10). The radioactive charcoal extractable phosphate did not show any change from the very low values obtained with 86 controls. This same low level of charcoal extractable phosphate was also found in reactions with microtubules which contained either ATP or GTP. The time course of phosphate accumulation (at 37°C., pH 6.6) 70 __ using (y- P)GTP is seen in Table 8 for both hexylene glycol isolated microtubules and a microtubule preparation exposed to the washing- soaking procedure to decrease the glycol concentration.^ The time points 0.1 and 0.3 minutes represent times after initial additions but are not true initial times in this protocol, since the procedure of centrifugation and removal of the supernate required an additional 30 seconds. Since protamine had been used as a secondary acceptor of phosphate in studies of phosphoprotein kinase isolated, from brain by Greengard (Miyamoto et al., 1969) and since microtubules had been reported to contain a phosphokinase, the measurement of this component of reaction in the system was of importance. If protamine was not present in the reaction mixture, the microtubular phosphate binding was substantially decreased to near background levels. Further, reactions which were done outside the pH range 6.3 to 6.9 showed a decreased binding falling to background levels at pH 6.0 and 7.1. If PCA was employed to stop the reaction, the microtubule pellet again showed only background activity. There was a slight decrease in phosphate accumulation in the ^The term phosphate accumulation used henceforth may represent true binding or loose association to the microtubule. Unless indicated this refers to (y- 2P)GTP addition. 87 microtubule pellet when the individual reactions were stopped by the addition of the alkaloid vinblastine. The variance from 25 to 40% binding was \\rithin the limits of the daily preparation of the microtubule pellet following the washing-soaking procedure. Table 9 illustrates the dose response relationship using vinblastine to precipitate the microtubule in a reaction containing protamine and unlabeled GTP and measuring the phosphate accumulation by the pellet. Heparin, commercially prepared from either mucosa or lung, is known to precipitate protamine and it was used to isolate protamine from the supernatant of the reaction mixture. When the heparin-protamine complex isolate was counted, phosphate binding could be demonstrated, and this binding amounted to approximately 8 to 15% of the total added isotope (Table 8 ). Without protamine added, the microtubular pellet with vinblastine- precipitation did not significantly bind labeled phosphate (Table 9). Table 10 shows the dose-dependent binding of phosphate by microtubules and protamine when incubated with GTP. Other known phosphate acceptors such as alpha-casein, type IIA-histone and type III lysine-rich histone were tested for their ability to act as secondary phosphate acceptors. All preparations demonstrated the ability to cause phosphate accumulation on the microtubule pellets analysed. The amount of phosphate accumulated by these various acceptors was not measured, 88 During the course of these studies, observations of the microtubule preparations by electron microscopy failed to reveal any intact organelles such as mito chondria or microsomes. Because of the ’'lollipop" decorations seen following GTP and PA treatment however (see Electron Microscopy, Results section), it was decided to test the crude microtubule preparation with adult rat brain mitochondria, isolated according to the method of Tjioe (1972). Following isolation, an aliquot of the mitochondria were treated with hexylene glycol buffered with HEPES at pH 6.4. This preparation was then submitted to the washing^soaking procedure described for the preparation of microtubules. All of the samples were incubated for 5 minutes at 37°C in HGK medium at pH 6 .6 . The results of the experiments using the two preparations of brain mitochondria and microtubules are seen in Table 11. The total binding of phosphate for controls in these particular assays was decreased somewhat from other experiments, however, the addition of freshly isolate isolated mitochondria or HG-treated mitochondria did not enhance the phosphate accumulation by the VBL. precipitated microtubules. Phosphate accumulation by added protamine was only increased with the addition of untreated mito chondria to the microtubules, likely an additive effect. When the same reaction sequence was made without the addi tion of protamine to the assays, there was no substantial TABLE 8 TIME COURSE OF ADULT RAT BRAIN MICROTUBULE GUANOSINE-5'-TRIPHOSPHATE ACCUMULATION8, Time^ Buffered Hexylene Glycolc HGK Buffer Soaked^ (minutes) ^ e pAf MTPe PAf (corrected %)® (corrected %)® (corrected %)^ (corrected I)® 0.1 4,95 9,21 8,45 6,15 0.3 5.65 16.93 8,52 11.83 1.0 7.50 13.82 11.49 10.68 3.0 7.26 12.20 32.58 8.87 10.0 5.14 11.37 46.45 8.58 30.0 3.10 9.63 41.09 7.42 a2 mg protamine added/assay (10 yl), 10 - 15 myCi (y-^2P)GTP added/assay. = 5 for all time groups. cBuffered with 0.01 M HEPES, pH 6.6; 0.5 ml buffered glycol + 0,05 ml micro tubule preparation (0.65 mg/0.05 ml). Table 8 -- Continued ^0.5 ml HGK buffer, pH 6.6 + 0.05 ml washed, soaked microtubule preparation (0.65 mg/0.05 ml). -3 eMTP (microtubule precipitate); 10 yl vinblastine sulfate added, 1 x 10 M final concentration. fpA (protamine precipitate); 220 U heparin (mucosal) added to supernate, 30 minute incubation at 4 C, centrifuge at 5100 x g/5 minutes to obtain pellet. §Based on: counts recovered x ^oo ' corrected to' (cpm'- blank) „ volume . total counts added efficiency mg 91 TABLE 9 MICROTUBULE PRECIPITATION WITH VINBLASTINE3 T*\ p Group Treatment Microtubule Precipitate Protamine^ No Protamine A p (corrected %) (corrected %) 1 2 x 10“3f 22.9 4.5 2 1 x 10'3 23.9 7.1 3 1 x 10'4 25.4 4.1 4 1 x 10~5 24.5 4.3 5 1 x 10'6 19.7 3.6 6 PCA (10%)g 3.9 3.8 a 32 .. Phosphate accumulation monitored by (y- P)GTP; 5 minute.reaction at 37°C. ^N = 5 for all groups. c0.5 ml HGK buffer, pH 6.6 and 0.05 ml washed soaked microtubule preparation (0.71 mg/0.05 ml); 30 minute incubation at 37°C. ^2 mg protamine/assay (10 pL). eCorrected % the same as Table 8. f Vinblastine sulfate concentration, moles/liter. g0.5 ml 101 PCA. TABLE 10 PHOSPHATE ACCUMULATION BY RAT BRAIN MICROTUBULESa Kinetics of Secondary Added Compounds 1- 0 Compound Concentration Microtubule Precipitate^ Protamine Precipitate £ £ (mg) (corrected %) (corrected %) Protamine 0 4.8 — 0.25 14.9 1.9 0.50 17.0 3.3 1.00 24.3 7.7 2,00 34.4 7.9 4.00 38.6 7.2 Alpha-Casein 2.00 16.1 — Histone (type IIA) 2.00 26.5 — Lysine-Rich Histone 2,00 30.2 Ctype III) VO tsi TABLE 10 -- Continued aPhosphate accumulation monitored by (y-^P)GTP, 5 min/37°C reaction; 0.5 ml HGK buffer pH 6 .6, 0.05 ml microtubule preparation CO.63 mg/0.05 ml). \ T = 5 for all groups. Compounds prepared for 2 mg/10 yl addition. ^VBL at 1 x 10_4M, 30 min/37°C. . e220 U heparin, 30 min/4°C added to supernate. £ Corrected % the same as Table 8, to TABLE 11 ADULT RAT BRAIN MICROTUBULE PROTEIN WITH ADULT RAT BRAIN MITOCHONDRIA3, Group Reaction*3 Phosphate Accumulation Microtubule Precipitate MTPC PAd (no PA) (corrected l)e (corrected %)e (corrected %)e 1 MTP 21.4 8.9 3.7 2 MITOS-HG 16,5 7.7 6.2 3 MITOS 13,7 7.7 5.9 4 MTP+HITOS-HG 21.6 8.1 7.3 5 MTP+MITOS 17.7 12.2 8.9 Conditions are the same as Table 10. •L 0.05 ml mitochondria preparation added; (0.71 mg protein/ml). CMTP (microtubule precipitate). dPA (protamine precipitate). TABLE 11 Continued the same as Table 8. 96 binding in any group tested. This suggests that the mitochondrial preparation may have contained microtubules as a contaminant. In any event, the mitochondrial factors reported by others do not seem to play an essential role in the phenomena of phosphate accumulation. Ordered Addition Reactions Phosphate Accumulation Since sequential ordered addition of reactants has been of value in elucidating biochemical mechanisms, and because I had a method of separating the components of the system, it was decided to perform experiments of this design. The results of addition reactions are described below. In Table 12, group #1 showed the standard ordered reaction system with average values of phosphate accumu lation measured in the microtubule pellet and the heparin- protamine complex pellet. In group #2, when protamine is deleted from the reaction, the microtubular phosphate accumulation is reduced to near background values. Heparin addition does not cause a precipitation of the microtubule alone, suggesting the absence of protamine-like proteins. Only a minimal phosphate accumulation is found when the microtubules are not removed before addition Cof heparin) is made. The reaction of group #4 TABLE 12 ORDERED ADDITIONS FOR ADULT RAT BRAIN MICROTUBULE PROTEIN (Y-32P) -GUANOSINE-5* -TRIPHOSPHATE ACCUMULATION* Group Reaction and Accumulation 1 MTP + GTP + PA -*• VBL -*• Heparin (34.3%) (2.85%) 2 MTP + GTP -► VBL -> Heparin (2.44%) (-0-) 3 MTP + PA + GTP ->■ Heparin (4.22%) 4 GTP + PA + MTP -V Heparin VBL (4.88%) C-o-) 5 MTP + GTP ■+ Heparin -*• VBL (-0-) (2.35%) 6 Protamine + GTP VBL Heparin (-0-) (1.75%) 7 Protamine + GTP Heparin (5.88%) VO TABLE 12 — Continued Group Reaction and Accumulation 8 Protamine + GTP VBL MTP Heparin C-o-) (17.0%) (3.28%) 9 GTP + Protamine -* Heparin MTP T*- VBL (7.0%) (-0-) 10 GTP + PA Heparin + MTP VBL (7.26%) (2.47%) 11 MTP + VBL -*■ GTP + Protamine ->- Heparin (3.16%) (3.28%) 12 GTP + VBL -*■ MTP Heparin (2.98%) (-0-) 13 GTP + MTP -*• VBL Protamine -y Heparin (7.30%) (2.30%) 14 GTP + MTP + Heparin -*• Protamine -»■ VBL (4.30%) (8.61%) ^Conditions are the same as Tables 9 and 10, 0.77 mg microtubule protein/0.05 ml. to 00 99 is the same as group #3, but VBL is added to the supernate of the heparin pellet. Vinblastine appears to be hindered from precipitating a MT which has accumu lated phosphate; however, the protamine-heparin may be blocking the phosphate binding site on the microtubule. Reaction #5 (the deletion of protamine) shows that heparin addition before VBL addition does not affect the MTP phosphate binding. The results obtained with groups #6 through #10, show that neither VBL nor heparin will appreciably bind phosphate using (y-^P)GTP. of particular interest is the reaction of group #8 . These results imply that pretreatment or competitive treatment of MTP with VBL will block or inhibit about one-half of the phosphate label binding. 7,7 If after reacting with (y- P)GTP, protamine is removed from the reaction mixture and MTP are then added, upon VBL addition there is no label associated with the MTP pellet (group #9). Similarly, the simultaneous addition of (heparin + MTP) to (PA + GTP*) provides the identical level of radioactivity in the PA-HEP complex, while VBL addition to this supernate produces a MTP pellet with very minimal uptake. In the final series of reactions (groups #11 to #14) the alkaloid VBL is tested for a possible binding site which would be competitive with phosphate or protamine- phosphate binding sites. If (in group #11) MTP are 100 reacted first with VBL and then (GTP + PA) is added following this pre-incubation, the MTP pellet shows minimal binding. MTP added to (GTP + VBL) does not bind phosphate due to the lack of PA (group #12) . The addition of VBL to (GTP + MTP) shows approximately 20% MTP phosphate of the total binding of phosphate in the standard complete reaction system. Finally, pre-incubation of the reactants (GTP + MTP + HEP) shows that a phosphokinase reaction may be possible since the addition of PA showed slight increase of phosphate binding and since VBL addition to the supernate again showed the 20 to 25% binding seen with the (GTP + MTP) pre-incubation free of PA. The value of 6 to 9% seems more real than the value of approximately 2% in the reactions of group #2 .. Column Chromatography of the Standard Reaction Mixture: MTP + PA + (y-^P)GTP The separation of reactants in biological in Vitro systems is accomplished by several established procedures. Simple column chromatography elution techniques provide a method to assay compatible systems. This was selected in an attempt to provide insights into the character and extent of phosphate-32 accumulation in (MTP + PA) system. The eluate was counted for radio isotope activity presented in Fig. 11. The series of 101 FIGURE 11 MICRQTUJSULE PHOSPHATE ACCUMULATION WITH (y-32P) GTP. AND PROTAMINE: Sephadex G-25 column, eluted with water, pH = 7.0; Flow = 1.0 ml/min, 1 ml fractions. CPM IN 5X SAMPLE i 0 0 0 3 2000 2500- 1000 1500 0 0 4 200 600 800-) 0 0 30 20 10 VJ 0 0 0 0 0 0 0 10 9 20 210 200 190 110 100 90 80 70 60 50 40 WITH G T P -8-32 +PROTAMINE -8-32 P T G WITH TB FATO NUMBER FRACTION TUBE . MTP -PHOSPHORYLATION 102 103 four peaks (fractions 6 to 22) are associated with the MTP-VBL-PA-GTP complexes. Fraction 45-48 was established corresponded to protamine based on results obtained from separate addition-elution to the column; fraction 62-67 was found to be GTP, The initial peak was observed in the void volume, which in a G-25 column would indicate a MW > 50,000. The only candidate for the species would be the MTP (MW = 120,000 dimer) or either monomer forms I or II (MW I = 55,000 and II = 53,000). The abundance of peaks of radioactivity in the early phase of the elution would be an indication of the impurity of the preparation or the complexity of the possible subunits in the standard reaction mixture. In order to establish this, it seemed that further purification of the initial MTP preparation would be required and the method re-evaluated. 3 H-Protamine Accumulation on Microtubules 3 Table 13 demonstrates the binding of H-protamine to microtubules in the standard reaction mixture. The reaction mixture without microtubules is also shown for comparison. In this study, the correlation from 0,25 mg to 4 mg total protamine added is all accounted for by totaling the microtubule and heparin precipitates. The reaction follows an isotope dilution curve which is 104 shown in Fig. 12. The data when replotted according to 'Scatchard is shown in Fig. 13. The curve is biphasic and demonstrates the existence of both low and high affinity sites of binding. Microtubules from Newborn Rats' and Dogs Preparations of MTP from newborn animals were regarded as essential to the development of ideas concen concerning the phosphate binding/accumulation by MTP. Ontologically, the development of microtubules as a mechanism of rapid transit of required nutrients within the axon is quite plausible. The peak of this development is attained during the early neonatal period, at a time when the brain of Mnon-herdM species of animals are passing through biochemically "critical situations. The unit density of MTP in neonates is documented to be greater by more than an order of magnitude from the adult. A second anatomical consideration is the fact that den drite spine processes contain by far the bulk of the total microtubules within any specific neuron., fetus, neonate or adult. It seemed reasonable then to assume these particles would express their biochemical character in a manner likely to be quantitatively different from the adult.. In Table 14 are seen the results of our initial experiments on phosphate accumulation by MTP in our TABLE 13 TRITIUM PROTAMINE ACCUMULATION ON ADULT RAT BRAIN MICROTUBULE PROTEINa Group PA . Reaction Accumulation (mg) Microtubule Protamine Precipitate , Precipitate , (corrected %) (corrected %) 1 2.00 GTP + PA — 96.9 2(a) 0.25 MTPb + GTP + PAC 84.6 10.7 Cb) 0.50 53.9 42.6 (c) 1.00 30.8 61.9 (d) 2.00 17,2 77.0 (e) 4.00 11.1 82.2 Conditions are the same as Tables 9 and 10. ^MTP = 0.68 mg/0.05 ml. cSp. act. ^H-protamine = 2.46 mCi/ymole; ^ 120,000 cpm added. Corrected I the same as Table 8. FIGURE 12 RAT BRAIN MICROTUBULE PROTEIN (y-32P)GTP PHOSPHATE ACCUMULATION WITH ADDED EXOGENOUS PROTAMINE. % PHOSPHATE ACCUMULATION 40- 50i 20 30* RAT BRAIN MICROTUBULE PROTEIN PHOSPHATE PROTEIN MICROTUBULE BRAINRAT 10 - - CUUAIN IH ADDEDPROTAMINE WITH ACCUMULATION LOG PRO TAM INE INE TAM PRO LOG 1.0 (mg) 108 FIGURE 13 SCATCHARD PLOT. OF RAT BRAIN MICROTUBULE 3H-PROTAMINE ACCUMULATION WITH INCREASING ADDITIONS OF EXOGENOUS PROTAMINE. SCATCHARD PLOT OF 3H-PR0TAMINE BINDING TO MICROTUBULES 5 10 20 3 0 nmoles/mg 110 TABLE 14 STANDARD REACTION OF PHOSPHATE ACCUMULATION BY 0-DAY NEWBORN RAT BRAIN MICROTUBULESa MTPb PAC (corrected %)^ (corrected %)^ Protamine 18.27 15.68 No Protamine 11.56 --- Conditions are the same as Tables 9 and 10, (y-^2P)GTP, 10 - 15 myCi. ^MTP (microtubule precipitate); 0.35 mg microtubule protein/0.05 ml. CPA (protamine precipitate). Corrected % is the same as Table 8. Ill standard reaction sequence. In the newborn (< 6 hours old), the total binding of phosphate is approximately 40 to 501 of the values found for the adult preparation CTable 15). The phosphate accumulation by added protamine was increased by as much as five times over the adult preparation; if the PA were deleted from our standard reaction the phosphate binding fell about 40% from the level found with its inclusion. By 72 hours post natal (Table 15) the phosphate binding had increased by 50% in both the MTP and PA pellets. The addition of the cyclic nucleotides cAMP or cGMP did not substantially change this value for phosphate binding in the MTP pellet, but did raise the PA pellet accumulation by nearly 30%. The addition of cGMP was 12 pM (final) while over 16 times that concentration was required to effect the same change for cAMP. If ATP (2 mM) was added to the standard reaction system, the phosphate binding was decreased to the approximate levels of the 0-day newborn. Table 16 provides a summary of the phosphate binding studied in the 6-day newborn and in a comparison with adult preparations. The reactants of all groups are mixed containing 0.2 mM GTP in the standard assay system. By 6-days, the addition of cGMP to the microtubule preparation doubles the total phosphate binding compared with the 0-day newborn and is at adult levels. The peak of the PA binding seems to occur at 72 hours because the 112 TABLE IS 72-HOUR NEWBORN RAT BRAIN MICROTUBULE PHOSPHATE ACCUMULATION3 Group Reaction Accumulation MTPb PAC (corrected %)^ (corrected %)^ 1 GTP (0.2 mM) 27.4 18.2 2 cGMP (12 pM) 27.7 23.0 3 cAMP (200 pM) 25.6 24.3 cAMP (10 pM) 26.4 19.6 4 ATP (2 irM) 17.3 10.7 Conditions are the same as Tables 9 and 10. ^MTP (microtubule precipitate); 0.46 mg microtubule protein/0.05 ml. CPA (protamine precipitate). ^Corrected % is the same as Table 8. 113 TABLE 16 6 DAY NEWBORN RAT BRAIN MICROTUBULE PHOSPHATE ACCUMULATION3- Group Reaction Accumulation MTPb PAC (corrected %)^ (corrected %)^ 1 + cGMP + PA (12 pM) 38.7 23.6 2 + cAMP + PA (200 pM) 18.9 20.7 3 + ATP + PA (2 mM) 24.9 24.6 4 GTP + PA 28.9 14.8 5 + cGMP (12 pM) 15.1 --- 6 + cAMP (200 pM) 18.8 --- 7 + ATP (2 mM) 9.7 --- 8 GTP 14.2 --- 9 (Adult) PA + ATP (2 nM) 19.3 2.4 . 10 (Adult) ATP (2 mM) 2.2 4.2 11 (Adult) GTP + PA 37.6 9.7 Conditions are the same as Tables 9 and 10. ^MTP (microtubule precipitate). CPA (protamine precipitate). TABLE 16 — Continued ^Corrected % the same as Table 8. 115 value at 6 days remained the same as the 7 2 hour preparation. The deletion of PA from the assays reduces the phosphate accumulation with cGMP to a value 60% below the control. Addition of cAMP without PA does not seem at first to have fallen, however when the value is compared to PA addition alone (but still including the GTP in the buffer system) it has decreased the phosphate binding to the MT pellet by 35%. ATP addition, with or without PA also inhibits the phosphate binding which would be present with only the GTP from the buffer. The adult preparation included in the final section of Table 16, indicates that ATP drastically dilutes or inhibits not only the phosphate accumulation on micro- tubules and that it markedly decreases this value even in the presence of PA, but also inhibits the action of the phosphokinase associated with microtubules, as measured by the phosphate binding present on protamine. A final observation from this set of data is the comparisons of standard reactions between the 6-day newborn and the adult. Phosphate accumulation is still not up to adult levels (down 24%), but there remains a 2.2-fold increased binding by the protamine in the 6-day newborn preparation (when compared to the adult). Phosphate accumulation by microtubules, inhibited by the addition of ATP to a standard assay system in 72-hour, 6-day and adult preparations could be the result of an 116 endogenous cyclase reaction acting to produce cAMP, or an inhibition of a cyclase type of reaction by ATP, which would be required for cGMP formation preceding the final phosphate accumulation by MTP. Accordingly, the 72-hour, 6-day and adult preparations were examined for cAMP activity in each of these separate preparations of MT using ATP and PA additions. The comparison of results between adults and newborns, reveals first that the adenyl cyclase activity of the newborn is enhanced by the addition of PA CTable 17), Secondly, it is noted that the value of this reaction group (ATP + PA) is increased more than 2-fold in the 6-day when compared to the 72-hour newborn; ATP addition without PA is not significantly changed during this same time period. The PA addition alone also provides a 2-fold increase in this time period without the addition of ATP as a reactant. The adult ,MTP preparation with PA results in a value substantially lower than either newborn preparation. Inhibition of Phosphate Accumulation with Drugs Structure-Activity Relationships Figure 14 compares the structures of the--isola-t-i»g- hexylene glycol with meprobamate, a carbamate derivative which has been demonstrated to have effects at multiple 117 TABLE 17 CYCLIC-3': 5 ’ -ADENOSINE MONOPHOSPHATE RADIOIMMUNOASSAY IN SUPERNATES OF 3 AND 6 DAY NEWBORN RAT BRAIN MICROTUBULE PROTEIN ACCUMULATION3 Group Reaction cAMP (pmoles) 72-hour 1 ATP + PA 52.4 ± 11.9b 2 ATP 41.6 ± 11.1 3 PA 26.3 ± 9.4 6 day 4 ATP + PA 137.0 ± 13.0 5 ATP 50.0 ± 3.1 6 PA 51.8 ± 13.4 Adult 7 PA 14.5 ± 3.5 aAssayed from supemate of reactions of Tables 15 and 16. t . ■* X ± SEM, corrected. 118 FIGURE 14 STRUCTURE RELATIONSHIPS OF DRUGS IN THIS STUDY: PROPANE DIOLS AND CARBAMATES. 0 CH, II HZN - C - 0-CH£-C-CH£-0 -NH* H 0 -CHt-C-CH,-0 H I I c h 2 CH. I CH2 CHt I I CH, CH, MEPROBAMATE 2-METHYL-2-N-PROPYLPROPANEDIOL c h . c h ,-c h HN NHe I I 0 = C CH, C = 0 CH, I I I I Q-CH,-C-CH,-0 H|Q-C-CH«-CH-OlH i I I c h 2 C H , CH, I c h 2 HEXYLENE GLYCOL I CH, CARISOPRODOL 120 synaptic sites in the central nervous system,' The immediate synthetic precursor to meprobamate is 2-methyl-2-n-propylpropanediol. Mephenesin and carisoprodol were also incorporated for testing. The structures are given in Figs, 14 and 15, Chlorpromazine and diazoxide were also tested for the ability to block microtubule phosphate accumulation CFig. 15) as well as phenaglycodol and oxanamide CFig. 16). Drugs Investigated: Dose Response Data Drug inhibition of phosphate binding to microtubules is found in results shown in Tables 18 and 19, All drugs containing a glycol within its structure were effective inhibitors of phosphate accumulation in microtubules at -12 concentrations to 10 M. Additionally, these compounds were able to effectively block the phosphate accumulation with added protamine. When compared with other drugs tested, the dose response curves were shifted to the left for all glycols (Fig. 17). Drugs with non-glycol structures were somewhat less effective in reducing phosphate accumulation in microtubules. Ethanol, for example, required a concentration of 10% to reduce the phosphate uptake of 25% of control. Chlorpromazine followed a dose response curve which was 4 to 5 orders of magnitude less effective in the blockade of phosphate FIGURE 15 STRUCTURE RELATIONSHIPS. OF DRUGS IN THIS STUDY CHLORPROMAZINE, DIAZOXIDE AND MEPHENESIN. 122 I ch2 I CH jj I CH* N(CHs)2 CHLORPROMAZINE C,Y*VSNH ^ ^ N ^ C H S DIAZOXIDE r * - a^ ^ O C H .- C H - CH.-OH 2 I OH MEPHENESIN 123 FIGURE 16 STRUCTURE RELATIONSHIP OF DRUGS IN THIS STUDY: OXANAMIDE AND PHENAGLYCODOL. CH3-(CH2)-CH —c - c-n h 2 II u o OXANAMIDE HO OH ci^ C M -? -cH3 HjC CH3 PHENAGLYCODOL 125 FIGURE 17 LOG DOSE-? RESPONSE INHIBITION OF PHOSPHATE. ACCUMULATION ON MICROTUBULES BY DRUGS; The system is described under METHODS. Abscissa units are moles/liter. % % INHIBITION OF PHOSPHATE ACCUMULATION ON WASHED, SOAKED MICROTUBULES 100 80 90 0 4 70 60 50 20 30 □ CHLORPROMAZINE ■ OXANAMIDE ■ CHLORPROMAZINE □ • HEXYLENE GLYCOL O MEPROBAMATE O DIAZOXIOE A PROPANEDIOL 3-N-PROPYL YL-l, ETH 2-M GLYCOL A HEXYLENE • NIIIN F PHOSPHATE OF INHIBITION ACCUMULATION ON MICROTUBULES BY DRUGS DOSE 126 127 accumulation than the glycol-carbamate structures. Heavy water (D2O increased the phosphate accumulation above control (Table 18). Table 19 shows the inhibitory effects of the drugs oxanamide and diazoxide. Fine Structure Studies Staining Mechanisms In these studies, two types of stains were incorporated to visualize the fine structure of the microtubules: phosphotungstic acid (PTA) and uranyl acetate (UA) . In both instances the stains were employed as negative stains, i.e., treatment of the organelles in such a manner that they are viewed as relatively light objects objects against a dark background. This technique has proved to be a quite valuable method for the examination of fine structural surface detail in small particulate specimens. The term "negative stain" is completely misleading however, but it is firmly ingrained in the literature. Practically, the embedding, choice of thin film material and choicewof^ stain all..work, together to achieve optimal visualization of any given specimen. Some consideration must be given to all of these factors if results are to be achieved which will reveal the necessary detail of the fine structure to be visualized. 128 TABLE 18 INHIBITION OF RAT BRAIN MICROTUBULE PHOSPHATE ACCUMULATION FOLLOWING DRUG ADDITIONS - Ia Treatment Concentration MTP PA (molar) (corrected %) (corrected %) -6 Meprobamate 5 x 10 5.7 2.9 -8 5 x 10 6.3 3.4 -10 5 x 10 10.6 1.6 -12 5 x 10 28.7 10.9 2-methyl-1,3-N- propyl 5 x 10 5.6 3.6 propanediol -8 5 x 10 4.6 3.5 5 x 10-10 6.9 4.1 -12 5. x 10 17.0 4.2 Hexylene 5 x 10-6 4.6 3.0 Glycol -8 5 x 10 6.0 1.9 -10 5 x 10 17.1 4.8 -12 5 x 10 33.0 8.5 Chlorpro- 1 x 10‘ 6.2 3.5 mazine -5 1 x 10 10.9 5.8 1 x 10 23.1 7.9 -7 1 x 10 32.0 6.4 -8 1 x 10 34.6 7.8 129 TABLE 18 — Continued Treatment Concentration MTPb PAC (molar) (corrected %) (corrected %) Ethanol 1% 8.6 2.2 d 2o 401 38.2 8.7 Control 31.7 7.7 Conditions are the same as Table 10. ^NTTP (microtubule precipitate) CPA (protamine precipitate) 130 TABLE 19 INHIBITION OF RAT BRAIN MICROTUBULE PHOSPHATE ACCUMULATION FOLLOWING DRUG ADDITIONS - i t Treatment Concentration MT^3 PA° (molar) (corrected %) (corrected %) Gxanamide 1 x 10"4 2.2 4.1 1 x 10"6 17.4 5.8 1 x 10"8 29.2 7.9 Diazoxide 1 x 10'6 2.8 2.0 1 x 10"8 8.6 5.1 1 x 10“10 22.0 8.3 1 x 10"12 29.2 8.1 Control 30,7 8.4 3. Conditions are the same as Table 10. ^MTP (microtubule precipitate). £ PA (protamine precipitate). 131 Sodium phosphotungstate, prepared with phospho- tungstic acid and NaOH is by far the most useful of negative stains. Uranyl acetate has been employed by several authors for investigating the fine structure of microtubules (Borisy and Olmsted, 1972; Weisenberg, 1972). The differences in fine structure noted with these two stains are the results of differences in interactions with various chemical groupings on the microtubular surface. Acidic PTA is documented to stain polyanionic and polyhydroxyl containing residues. Uranyl acetate will stain nucleoproteins and most proteins not directly in a membrane. Uranyl acetate is usually thought of as a stain superior to PTA but inferior to lead stains (Pease, 1964). Isolated Microtubules Freshly isolated microtubules buffered in hexylene glycol, when treated according to methods described can be visualized as whole fields using electron microscopy. Plate I demonstrates the type of fields seen under low magnification. In Plate II at a magnification of 210,O00X, the typical structure of the isolated microtubule is seen following PTA staining. The diameter of this particular tubule is 300 ft, somewhat larger than the average. This may be due somewhat to a flattening process 132 of the microtubule on the parlodian thin film. Plate III shows microtubules from the same preparation stained with UA as described. The major differences between these two stains is that the PTA stain allows the identification of six to seven filaments in cross sections of the microtubule, while the UA stain does not reveal this fine structure. In contrast, UA-stained material shows the appearance of two parallel bands, densely stained which could be nucleoprotein associated with the intact microtubular structure. Microtubules from the Standard Reaction Following reaction with GTP and PA the microtubular fine structure is somewhat changed, with approximately 701 of the microtubules, stained with PTA, having the structure shown in Plate IV. The appearance of these "lollipop" structures have not previously been reported. "Decorated microtubules" have been described, but their fine structural detail is considerably different from those observed in this study (Heisenberg, 1972). The remainder of the tubules observed were usually divided in appearance, with 10% demonstrating structure resembling the original isolated microtubule fine structure of Plate II and the remainder (20%) having structures which lack both the lollipop configurations and the filamentous 133 striations or beaded appearance. If reactions are performed using microtubules isolated with GTP, which was dialysed away and replaced with ATP at the same concentration (0.2 mM) the fine structure is not signifi cantly changed from those structures which were freshly isolated (Plate' V) . In all of these procedures the numbers of microtubules observable per grid square was considerably reduced. Immediately following the isolation procedures, approxi mately 40 to 50 intact microtubular structures can be visualized/grid square (300 mesh grid). After soaking in the HGK buffer for 10 to 12 hours, the number of intact structures is reduced to 5 to 15 structures/grid square. Photographs of whole fields are nearly impossible under these circumstances since no detail can be observed at magnifications less than 10,000X-fold. When microtubules are observed following reaction with GTP the fine structure is similar in appearance to those microtubules which lose their filamentous striations, but demonstrate no "lollipop" structures coating the surface of the intact structure. In appearance they resemble the 10% of seemingly "semi-reacted" microtubules seen in the reaction: MTP + PA + GTP as described above. 134 Ferritin Labeling Studies Reaction of microtubules with ferritin-tagged protamine, prepared as previously described, was carried out at 37°C. These reaction demonstrated the binding of protamine to microtubules. The fine structure results are in agreement with spectrophotometric analysis of the two peaks eluded from the Bio-Rad column as described below. Native Ferritin Native ferritin, at a concentration of 0.96 mg/ml (final) was incubated with microtubules and protamine at 37°C for 5 minutes. The microtubule preparation was subsequently visualized by electron microscopy as seen in Plate VI. There was no specific binding of the native ferritin to the intact microtubule structures and no lollipop structures were visualized. Intact tubular structures were difficult to locate in these preparations. Ferritin-Protamine, Peak I Elution of the coupled product ferritin-protamine (see page for synthesis) from the Bio-Rad column with 1 M KC1 provided a peak which had an absorption spectra similar to native ferritin (Fig. 8). Plates VII and VIII show the fine structure of intact microtubules following 135 the reaction: MTP + GTP + ferritin-PA(PI) ■+■ . The appearance of lollipop structures on the surface indicates that there is some ferritin coupled protamine in peak I. The microtubules in general retained their striated appearance while binding the coupled protamine. In the photograph one can also see ferritin particles, recognizable by their dense, electron opaque center cores. Ferritin-Protamine, Peak II Elution with 1 M NaOH cleared the Bio-Rad column of visually observable ferritin. The peak eluded by this procedure had an absorption spectra which was different from that of either ferritin or elution peak I (Fig, 8). The fine structure of intact microtubules incubated with this elution peak and GTP is shown in Plates IX and X. In these photographs the appearance of the lollipop structures are quite prominent on the surface of the microtubules. Apparently the density of surface binding is maximal, for close inspection does not reveal open spaces or areas of non-attachment of these particles to the microtubule. Almost every "lollipop" structure observed on the tubule surface contains a small dot 136 (dense core) indicative of ferritin (shown more clearly in the higher magnification of the inset of Plate X) . The individual ferritin molecules are also observable on the parlodian film as background. Fine Structure Studies Follonring Drug Additions In Plate XI, the fine structure of the isolated washed microtubule is observed following treatment itfith _7 5 x 10 M meprobamate in the reaction: MTP + PA + GTP + meprobamate -> . In these studies neither the fibrils of the microtubule are demonstrable nor was there presence of the "lollipop" decoration observed with other treatments. The average diameter of the microtubule was swollen following meproba mate treatment (300 to 350 &) as seen in Plate XI. Hexylene _7 glycol, when incubated with microtubules at 10 M in the reaction: MTP + GTP + PA + HG ->■ also causes the loss of the fibril appearance. In addition, no "lollipop" figures are seen with this treat ment (Plate XII), and swelling (increased diameter) of the microtubule does not occur. 137 Chlorpromazine treatment of microtubules in the reaction: MTP + PA + GTP + CPZ , -4 at 10 M causes the loss of "lollipop" structures, however a few isolated structures retaining this feature are occasionally visible (Plate XIII) . The striated fibril appearance is also lost, but the edge of the microtubule is more clearly delineated with this treatment than with any other. The appearance is that of a small cylinder sealed on both ends. Some swelling is also detected with this high concentration, i.e., diameters average == 350 S. In contrast to results obtained at high drug concentra- _o tion, at a lower concentration, 1 x 10 M, the "lollipop" structures reappear (Plates XIV and XV). In some instances, the fibrillar structure can be faintly deter mined. At this lower concentration, knobs appear at the ends and within the length of the microtubule structure. These varicosities appear in approximately 75 to 80% of all intact structures observed. 138 Fine Structure Studies Following Addition with Ions and/or EGTA Calcium Ions Following incubation with Ca++ (5 mM), there was no visible intact structures observable on any of three grids stained with both PTA and UA. Magnesium Ions Incubation with Mg (1 mM) in the standard reaction system provided microtubular structures which showed the "lollipop" figures (Plate XVI). The microtubules were not swollen but did contain curious knob-like formations at the ends of the microtubule. Calcium and Magnesium Ions When Ca++ (5 mM) and Mg++ (1 mM) were added to the microtubule reaction mixture, microtubules were observed which were swollen (to > 350 X) and demonstrated fibrillar structure (Plate XVII). EDTA No microtubules were observed following incubation with EDTA, 1 mM. 139 EGTA and Magnesium Ions When incubated with EGTA (1 mM) and magnesium (1 mM), microtubular structures were observed (Plate XVIII). These tubules contained the "lollipop" configurations. Fine Structure of Microtubules Following Cyclic Nucleotide Additions cAMP cAMP additions (0.1 mM) caused the microtubular structures to swell (> 300 ft) and to lose their fibrillar appearance. None of these structures contained "lollipop" figures on the sides of the microtubule (Plate XIX). cGMP cGMP addition (0.1 mM) caused a slight swelling of the microtubule (to near 280 ft) . The appearance of the "lollipop" figures on the sides of the tubule were prominent on all structures examined (Plate XX). D2O Incubations -- Fine Structure By incubation in D2O (40%), microtubules acquired the ability to retain a very different staining pattern with 140 PTA OPlate XXI). The tubules were of normal size and contained ’'lollipop" structures. DISCUSSION Microtubules are one of several organelles which seem to be responsible for maintaining the viability of a neuron. Differences between microtubules and neuro filaments are provided in Table 20. The nearly universal finding of microtubules within cells provides a basis for controlling flow and cytosol movement within axons and secretory cells and for providing shape and movement in cilia and flagella of eukaryotic cells. Evidence that microtubules are present as mitotic spindles in eukaryotic cells has now been accepted; additional investigation is now centered on the relationship of tubulin to actin. The present investigations are related to the events which occur to isolated, soaked microtubules by relatively short treatments which may lead to binding of GTP or protamine and subsequent fine structural changes. These studies are based upon the knowledge that several drugs can cause definite structural changes in microtubules and that by careful ordered treatments it may be possible to describe more correctly the biochemical character of these subunits. By directed treatment, morphology and fine structure analysis merely reflect the presence of the more obvious I 142 TABLE 20 PHYSICAL CHARACTERIZATION OF MICROTUBULES AND NEUROFILAMENTS . MICROTUBULES 1. Long, slender, unbranched, indefinite length. 2. 250 ft diameter, core 150 ft diameter. 3. Dimer, MW approximately 120,000 4. 2 moles GTP/mole MTP. 5. Tubulin. NEUROFILAMENTS 1. Similar to microtubules, 2. 100 ft diameter, core 30 ft diameter. 3. 85,000 MW, acidic. 4. Filarin. 143 fine structural changes of .intact microtubular structures not completely degraded to 6S subunits. Because of the time consuming nature of embedding and sectioning' procedures for electron microscopy, and because the visualization of particulate structures can frequently be of greater resolution with negative staining, the majority of all structures examined in this investigation were of the negative staining technique. Differences in staining between PTA and UA no doubt reflect valuable information which could be utilized in experimental design. For ease of analysis, maximum output and standards of comparison, the utility of sodium phospho- tungstate was the stain of choice. Fine structure investigations are made possible by a series of events. (1) The initial isolation of the microtubules, made in buffered hexylene glycol is responsible for the stabilization of these intact structures and their final high quantity yield; (2) that this initial preparation contains contaminants is most certainly a factor to be considered? however, in these preliminary investigations this probably worked to the advantage of the investigator. From the freshly isolated microtubule structure of adult rat brain, a number of features can be observed with sodium phosphotungstate staining (Plates I and II). 144 1. The diameter of the structures are very consistent, averaging ^ 300 ± 20 X in preparations reported in this study, due to flattening on the grid. 2. This stain is unique for the demonstration of microtubule fibrils. One can observe 6-7 fibrils in all freshly prepared specimens. The ends of the tubule structure are assumed to be open and the entire structure resembles a short soda straw. Negative stain enters the lumen of the tubule, blocks the visualization of the entire three dimensional unit, and allows only 6’ of the 13 fibrils to be examined. 3. The length of the isolated microtubules is 0.27 ± 0.12 y in these preparations and varies only slightly from one preparation to another; occasional tubules will be much longer or shorter. 4. Additional information can be observed in the background from these preparations (Plate II). Nucleated discs, recently described, are observable with a diameter of ^ 270 X and a center core of 150 X (Borisy and Olmsted, 1972) . These structures are evidently required for the aggregation of microtubules from 6S subunits in reconsti tution studies with appropriately controlled conditions. Also, one can observe, from a general appearance of back ground structures, some degree of purification of the microtubule preparation, and can estimate the extent of 145 cytolysis of extraneous material from any cellular shapes when apparent. Examination of microtubules following a washing- soaking procedure discloses that autolysis has occurred consistent with the results of other investigators. The decrease in recognizable intact tubular structures is approximately 60-75% following soaking for 12 hours. Of these structures remaining, approximately 80% demon strate the general appearance of the freshly isolated microtubule; the remainder are of varied form, usually without fibrillar structure and swollen somewhat in diameter (to 340 ± 40 R). When the washed, soaked microtubules are incubated in the presence of protamine in HGK buffer, the curious formation of lollipop structures becomes apparent in ^ 70% of the structures visualized (Plate IV). This effect is not observable if either the protamine is eliminated or if another nucleotide (ATP) is substituted for GTP in the reaction. The lollipop structures are also observed following some drug treatments at low concentrations. For example, CPZ at 10 will block phosphate accumulation on microtubules (Table 18). At this concentration, the fine structure (Plate XIII) exhibits neither decorated tubules nor striated fibrils. The tubules are somewhat swollen 146 but in general appear unremarkable. However, when the _ O CPZ concentration is reduced to 10 M, phosphate accumulation on microtubules is again detected as well as on added protamine. In this treatment, the microtubules have regained the lollipop structures along the outside (Plates XIV and XV). Certain other treatments have also been observed to allow the formation of lollipop configu rations on microtubules: cAMP, cGMP and (EGTA + Mg++) .. When these structures are formed, they completely cover the microtubule surface, for there was no specific instance in which the tubule was only partially coated with these structures. In physical measurement, the lollipop structures resemble the F^-factor described Racker et al. in heart mitochondria (1969). The binding in these studies however is thought to be protamine, from results obtained from fine structure studies performed in this investigation (Plates X and XI). By cAMP radioimmunoassay, ATP treatment does not cause the adenyl cyclase of the tubule to become, responsive. When GTP was used as the substrate in serial dilutions of the standard reaction, it was found that as much as 65% of the GTP was converted to GDP in concentrations to 10 mM by fluorometric measurements. cGMP was not measured in these procedures since no method was operable at this time. From these studies, the formation of the lollipop figures on the surface of the microtubule are related to the physical binding of protamine to the tubule. At the same time, phosphate accumulation has occurred on the exogenously added protamine and probably the microtubule structure as well. Drugs or other agents which block the phosphate accumulation on the micro tubule structures also block this accumulation on exogenously added protamine. Fine structure morphology reflects this decreased binding and lollipop figures can no longer be observed; with this treatment however, increased numbers of microtubules are observable per grid square. Conversely, if enhanced binding of phosphate is observed biochemically, the fine structure of the (remaining) tubules is changed to show the lollipop figures, indicating both protamine and phosphate binding. Fewer intact tubule structures are observed per grid square under the latter condition. There are several conclusions which can be reached from this information. If microtubules are in a mainly 6S dimer form, there will be fewer intact structures observed. It is this situation which reveals most effectively the phosphate accumulation on the microtubule. Quite possibly pmospha£e ■••binding requires the 6S dimer form of tubulin for activity. 148 However, if an increased number of structures are observed following stabilization either by drugs or added reagents, the stabilization or partial fixation of the tubules will not allow phosphate accumulation, and the phosphate binding activity associated.i'iflbth tubules iff trMs- configuration seems to be suppressed. The effect of additions of various reagents to a standard preparation of microtubules examined by light scatter and packed volume changes was investigated in the opinion that changes in physical measurement would be a reflection of previous alteration of biochemical interactions, leading to changes in physical conformation. Since microtubules were known to contain bound GTP, different concentrations of this nucleotide and GDP were tested for light scatter changes by reaction with the microtubule preparation in hexylene glycol. The data of Table 1 shows that 90° light scatter was some index of biochemical-physical conformation reaction since reversal of scatter deflection was concentration-dependent on GTP addition, but were not indicative of any change with GDP addition. Conformational changes of the microtubule were of a dose-response character. All additions of GTP caused a reduction of packed volume, presumably due to aggregation of the subunits and a decrease in numbers of total particles. In contrast, 149 GDP additions demonstrated only minor changes in packed volume. This was not entirely unexpected since previous authors had commented upon the relative refractoriness of microtubules to nucleotides with the exception of GTP. If instead of 5'-nucleotides, 31-5’-cyclic nucleotides were tested in this same system (Table 2), cAMP was found to provide data for light scatter and packed volume similar to GTP addition. cGMP addition in contrast caused profound positive scatter deflections at all concentrations and decreased the packed volume of the incubated microtubules. The data obtained from addition of the ions Ca++ and Mg++ provide somewhat of a paradox. In both studies (Table 3), the packed volume reflecting the state of aggregation of the microtubules appears to be related to the concentration of ion added. At either low or high concentrations there is a significant difference from the control group. Calcium has since been reported to cause disaggregation of intact microtubule structures at concentrations of 5 mM (Weisenberg, 1972) . Magnesium on the other hand seems to be required for tubulin to aggregate and form intact tubular structures . Concentra tions of Mg++ to 20mM can be used in the reaggregation studies (Weisenberg, 1970) although profound aggregation is observable as low as 1 to 2 mM. 150 When the two chelating agents, citrate and EGTA were investigated in this system, it was interesting that the packed volume reflected either no change (citrate) or was significantly increased in volume (EGTA) when compared to control which did not receive treatment. Although light scatter produced negative deflections in direct contrast to the addition of metal ions, this information is not completely unexpected. EGTA has been reported useful in lowering the Ca++ concentration for reaggre gation of the tubulin 6S subunit. Light scatter data of negative deflection from a null would indicate a decrease in numbers of particles, but must also indicate a configu rational change which would at the same time increase the packed volume. Treatment of microtubule preparations with drugs which are known to have a dispersing effect on intact structures caused either little or no significant change in packed volume (Tables 5 and 6). The light scatter associated with these same reagents were always of a negative deflection. Because of the disaggregating effects of these drugs and because of the negative light scatter deflection, the downward motion would seem to represent an increase in numbers of particles. This may be modified to include the possibility that these agents may be binding subunits during the two minute 151 incubation, causing a decreased scatter. The light scatter data then probably is a measure of the reduction in number of particles from the optical path due to interaction with the reagent added. There are many possibilities for interpreting phosphate binding in these studies. One possibility is that binding represents phosphorylation of the micro tubule protein. This possibility is somewhat unlikely because the microtubule pellet or precipitate is acid 32 32 labile following reactions with or (y- P) nucleotides (Table 7), which eliminates serine phosphate but does not exclude imidazole phosphate which is also very labile. Furthermore, phosphate accumulation did not respond to either temperature, time or pH. conditions which would all be indicative of energy-dependent phosphorylation. Another explanation would be that phosphate binding was inhibited in parallel with these changes. This possibility is more real, since as seen in Table 7, following a soaking procedure to remove the glycol used in isolation, the microtubules did undergo phosphate accumulation with orthophosphate-32. The particles also responded in temperature- and time- dependent reaction studies. These particles still do not contain true covalently bound phosphate however, because they continued to demonstrate acid lability. 152 The impurity of the preparation evidently allows other types of phosphate binding (e.g., electrostatic or hydrogen bonding) to the microtubule. The ability to form acid stable phosphate linkages may be an additional reaction following these reactions. By observing the 32 results obtained with phosphate as (y- P)GTP it was found that this reactant served somewhat better as an agent for phosphorylation of microtubule subunit (Table 8). Acid lability was still persistent in these preparations however. Phosphate accumulation studies were performed using vinca alkaloids (VBL) or by centrifugation of a microtubule pellet. These methods provided more gentle separation of the microtubule pellet from the reactants and allowed the monitoring of a quite delicate binding. The concentrations of VBL used (Table 9) were equivalent to concentrations previously published by other investigators (Olmsted, 1970) . Soifer, describing phosphorylation and a phospho- kinase associated with microtubules, noted that the microtubule subunits did not undergo phosphorylation or phosphate binding by themselves (1971) . In order to measure phosphate accumulation the addition of either protamine, histone or alpha-casein was necessary. He found no evidence of phosphate uptake on the microtubule itself, but did observe phosphate binding to the added 153 secondary phosphate acceptor. The phosphate kinase demonstrated time, temperature and pH related charac teristics and was rather consistent from preparation to preparation. A third interpretation which also agrees with the data presented is that this microtubule preparation contains a protein kinase which is quite dependent upon added histone, protamine or alpha-casein as a secondary acceptor. This added substrate (protamine) which is concentration, time and temperature dependent will react with the washed, soaked tubule in such a manner as to bind phosphate (Table 10). The other compounds, alpha-casein, lysine-rich histone and histone also react with this phosphokinase in a similar manner. The interest in this reaction is two-fold. That the binding occurs to the microtubule in a concentration dependent manner is explainable by one of several possibilities. One possible explanation is that the secondary acceptor binds the phosphate and in turn transfers this phosphate to the microtubule. An alternative explanation is the possibility that the phosphate-acceptor binds to the microtubule as a unit. The third possibility exists that the acceptor binds to the microtubule which then separately binds phosphate to either the attached acceptor or the modified configuration of the microtubule. 154 Tentatively then there are four arguments which the data will support: 1. The data support an argument based upon a steady state concentration of phosphate binding to the acceptor (Table 10). 2. The data also support the view that the phosphate, phosphate-acceptor can be inhibited from binding, and that, 3. Significantly increased binding will occur if time, temperature and pH are held carefully to controlled limits. 4. The final argument these data will support is that significantly increased phosphate accumulation will occur if the phosphate is linked to a nucleotide for the reactant rather than being orthophosphate-32. At this time, it was of interest to know if the phosphate binding mechanism was supportable from ordered additions studies, or whether contaminants in the microtubule preparation might be providing significant amount of non-specific phosphate binding. A preparation of brain mitochondria, treated in a manner similar to the homogenate for isolation of microtubules consistently bound less phosphate than did microtubules alone or when protamine was present (Table 11). Mitochondria when untreated did in fact interfere with the binding 155 of phosphate by microtubules. From these indications contaminants obviously could bind phosphate in the standard reaction. If this did occur, the measured binding in MTP would either be decreased or would be unaffected. The possibility still existed that ordered addition reactions would provide information suitable for interpretating the validity of previous arguments. Specifically the data obtained in these studies indicate the following (Table 12): 1. That protamine is necessary in order to achieve significant phosphate binding on microtubules, 2. That heparin does not cause the precipitation of microtubules but blocks precipitation of microtubules subsequently to vinblastine addition. 3. That microtubule preparations contain components w h i c h slightly decreases the reactivity between protamine and GTP. These reactions can be observed by heparin precipitation of the protamine, but this blocks subsequent vinblastine precipitatable MTP. 4. That when microtubules are added following heparin addition a blockade of GTP binding occurs. 5. That pre-incubation of MTP with VBL blocks approximately 50% of the phosphate binding sites. Previous investigations have established that precipitation of microtubules with vinca alkaloids causes 156 the release of up to 151 of the GTP associated with the microtubule. Presumably this is either a configurational change involving the partial release of GTP from the MTP or a competitive displacement reaction of a GTP binding position by VBL. The high percentage of substitutions would indicate that with pre-incubation, VBL substituted for or blocked nearly all of the easily releasable GTP (possibly terminal), and that the remaining nucleotide must be more firmly bound to the structure of the tubulin chains. The initial protamine addition in the standard cannot be considered indicative of knowledge gained from ordered additions studies. The blockade of added protamine by heparin or the separate addition of heparin to the reaction is of some interest. Specifically, this could mean that the phosphokinase reaction has as an intermediate function the transferring of phosphate 3 2 from (y- P)GTP to the secondary acceptor. If the protamine is blocked or masked by the addition of heparin before the phosphate or protamine-phosphate is transferred to the microtubule, the decrease would become apparent. This seems to be an adequate description of the ordered addition reactions. However, an additional problem exists which is: Does the microtubule bind protamine? According to results obtained from the ordered additions reactions, the possibility that protamine is bound to microtubular protein is quite good. For instance 1. When protamine is added to the standard reaction, phosphate is bound to the microtubule, and 2. If the protamine is first removed by reacting with heparin or deleted from the standard reaction mixture, phosphate binding is decreased or eliminated on the microtubule. In order to check more thoroughly on this reaction sequence, the labeling of protamine with both tritium or ferritin was accomplished to obtain additional biochemical and morphological data. Table 13 provides evidence that protamine binds to microtubules using 3 3 H-labeled protamine. This study shows that the H- protamine is bound to microtubules in a concentration dependent manner over a range of 2-800 nmoles/0.6 mg microtubule protein. The data when replotted according to the method of Scatchard (1949) is shown in Fig. 13. The curve is biphasic, indicating the existence of high and low order affinity sites for protamine on microtubules. The plot yields a value of n, for maximal numbers of binding sites per microtubule subunit of 26 nmoles protamine bound/mg microtubule protein for the 3 low affinity site. Since most of the H-protamine is retained after washing the pellet with HGK buffer, the conclusion may be drawn that some protamine must be 158 bound to high affinity sites . By extrapolation of the linear portion of the biphasic curve, a value of n = 10.8 nmoles protamine/mg microtubule protein is obtained. Affinity constants cannot be calculated from these data since the curves do not intersect the ordinate. The loss of H-protamine binding occurred in the cold or with addition of 1 x 10"^M glycol. Confirmatory evidence of protamine binding was provided from electron microscopy studies following incubation with ferritin tagged protamine. There is no obvious binding of native ferritin to the microtubule (Plate VI). A protamine-ferritin complex, purified on a Bio-Rad P-150 column by 1 M KC1 provided a peak which matched the spectrophotometric absorption pattern of native ferritin. This complex, when incubated with a standard reaction mixture, provided microtubules which had the appearance seen in Plates VII and VIII. A second fraction of a protamine-ferritin conjugate was obtained by elution of the column with 1 M NaOH. Following incu bation of microtubules with this fraction, microtubules with the appearance of Plates IX and X were obtained. On extensive enlargement of the photographs containing the microtubules shown in Plate X, it was clear that peak I (KC1 eluate) contains some protamine bound to ferritin 159 since some dense core centers of ferritin can be visualized on the microtubule surface and in the background. It is far more apparent that peak II (NaOH eluate) contained the majority of the reactive protamine-ferritin conjugate. The dense core centers of ferritin are readily apparent on the microtubule. The density of packing of the protamine-ferritin conjugate along the microtubular surface seems to be maximal. From these data, we now have additional support for the argument originally presented concerning the role of protamine in phosphate binding. It is apparent from both biochemical and electron microscopic evidence that protamine binds to microtubules. What is not readily apparent is whether phosphate 32 32 either as orthophosphate or (y- P) nucleotide phos phate will bind to protamine or histone, in order to effect the binding of phosphate to the microtubule. Wiche.tt and Isenberg have discussed the interaction of histone IV, whose structure is known, with nucleotide triphosphates and with various ions, including phosphate (1972, Li et al., al., 1971). Protamine is very similar chemically to histones. They report that conformational changes, monitored by circular dichroism measurements, are induced by phosphate. Upon addition of phosphate, a fast conformational change occurs (time constant = seconds) followed by a slow process (time constant = minutes to hours). The conformational change involved with the fast reaction is primarily one of alpha-helix formation together with dimerization of the histone. They also found that purine nucleotide triphosphates are much less effective than pyrimidine nucleotide triphosphates in inducing conformational changes in histone IV. The binding of ATP or GTP was either cooperative or sequential in nature because binding of > 1 ATP or GTP/ molecule of histone IV produced an even greater conforma tional change than did the initial binding. The binding studies demonstrated both phosphate and nucleotide specificity: e.g., triphosphate > diphosphate > mono phosphate and ATP GTP, Cyclic nucleotides did not produce these changes in realistic concentrations, i.e., * 10"3M. The evidence presented supports the notion that protamine binding and phosphate binding (either ortho phosphate or nucleotide triphosphate) does occur. Binding can be affected by changing either temperature or pH, and the binding rate is time dependent. The phospho- kinase reaction reported to be associated with microtubule preparations (Soifer, 1972) can be viewed as a mechanism to insure the transfer of phosphate in a form required for a transport mechanism to be operative on 161 the surface of the microtubule. Alternatively, phosphate binding may be required to insure that the 6S subunit tubulin becomes aggregated to form the required tubular structure. The investigation of phosphate accumulation by microtubule preparations from newborns can be regarded as critical to the understanding of the role of micro tubules viewed as a purely transport function. Characteristic of studies concerning development of the nervous system is that the maximal formation of synapses occurs at a time following a neuronal "spurt” of growth. Bloom and Agajanian found the plateau of a growth curve, which began at 12 days to be 26 days by counting synaptic junctions in thin sections (Woodward et al., 1971). Phosphate accumulation studies in neonatal brain microtubules (Tables 14 , 15 and 16 ) provide evidence / that added protamine caused increased phosphate accumu lation compared to adult microtubules. However, if protamine were deleted from the reaction the microtubules from the neonate seemed to bind phosphate much more than adult preparations. Additionally, phosphate accumulation with protamine was increased to twelve times that of adult controls. Phosphate binding by neonatal preparations is then a microtubular feature quite different from the adult. The inhibition of phosphate 162 (approximately 5%) could also indicate that the drugs do not block that small fraction of binding to MTP which occurred without the addition of protamine to the stan dard reaction. Ethanol was far less effective in blocking phosphate accumulation, requiring a 1.0% concentration (0.172 M) . The effectiveness of D20 (deuterium water) to increase the phosphate accumulation on MTP in the standard reaction was most likely due to an isotope effect of this. reagent. This substance has been reported to actively reaggregate microtubule subunits following disaggregation with VBL (Malaisse-Lagae, 1971), although in Vitro reports of the activity of D20 have varied. The overall effectivness of the glycol-carbamate derivatives possibly lies in their role as "stabilizers" of intact microtubule structures. Hexylene glycol has been reported to perform this function reasonably well, combining the features of rapid cytolysis, high rates of penetration and lipid character into one compound. It is possibly for the latter two reasons together with the need for critical pH control (6.0--6.4), heavily emphasized by Kane (1962) which is responsible for the decreased phosphate uptake of the MTP in the standard reaction. Several mechanisms are postulated to help understand the results observed in this investigation. The 163 binding by added ATP or cAMP was curious, since these agents provided a slight stimulant effect on phosphate binding in adult preparations. One conclusion from this would be that the early neonate cannot synthesize cyclic nucleotides (Weiss, 1971), and has no mechanism » to allow for their expression. Results obtained with the 6-day old preparation was the only evidence which indicated a possible stimulant role following cGMP addition (Table 16). A second possibility would be that the neonate microtubule preparation contained a kinase activity which was nucleotide dependent and already fully active. Upon addition of nucleotides other than GTP, a binding occurred which displaced the native nucleotide from the kinase. This would cause a decrease in kinase activity and a decreased phosphate accumulation on the MTP, Conformation of the added protamine must play an impor tant role in the binding of phosphate to the microtubule since only cGMP addition stimulated phosphate accumulation on microtubules above the standard reaction system (GTP + PA) stimulation. It is interesting to note that microtubule preparations which demonstrated increased phosphate accumulation where difficult to visualize by electron microscopy. By contrast, preparations which were 164 inhibited in some nianner from phosphate accumulation demonstrated increased numbers of microtubules per grid square following the reaction sequence and fixation procedure required. An explanation of this phenomenon would be the possible loss of expression of phosphokinase activity when microtubules were present as intact structures, since the enzyme would be active only in a disaggregated 6S dimer state of the tubule. Inhibition of phosphate accumulation on microtubules by drugs is reported in Tables 18 and 19. Glycol derivatives blocked phosphate accumulation to concen trations of 5 x 10"12m (Fig. 17), especially when there was no degradation of the molecule required; (see Table 18, hexylene glycol vs meprobamate). These agents also blocked phosphate accumulation on exogenously added protamine. The non-glycol drugs examined in this study were much less effective in reducing phosphate accumu lation on MTP than were the glycol-type structures (Fig. 17). It can also be noted that increased substi tution of the diol on these compounds decreased the blocking effectiveness of the compound. The appearance of curves, which never reached complete inhibition, also indicates the possible presence of a small fraction of "trapped" isotope within the VBL precipitated MTP. This small fraction (approximately 5%) could also indicate that the drugs do not block that small fraction of binding to MTP which occurred without the addition of protamine to the standard reaction. Ethanol was far less effective in blocking phosphate accumulation, requiring a 1.0% concen tration (0.172 M) . The effectiveness of I^O (deuterium water) to increase the phosphate accumulation on MTP in the standard reaction was most likely due to an isotope effect of this reagent. This substance has been reported to actively reaggregate microtubule subunits following disaggregation with VBL (Malaisse-Lagae, et al, 1971) although in vitro reports of the activity of D2O with microtubules have varied. The overall effectiveness of the glycol-carbamate derivatives possibly lies in their role as "stabilizers" of intact microtubule structures. Hexylene glycol has been reported to perform this function reasonably well combining the features of rapid cytolysis, high rates of penetration and lipid character into one compound. It is possibly for the latter two reasons together with the need for critical pH control (6.0 - 6.4), heavily emphasized by Kane (1962) which is responsible for the decreased phosphate uptake of the MTP in the standard reactions until they are washed. Several mechanisms are postulated'to help understand the results observed in this investigation. The stabilizing or partial fixation effects on hexylene glycol on mitotic spindles noted by Kane (1962) is the first mechanism to be considered (Fig. 18). If the tubule or 6S particle is held in a state of partial fixation following drug treatment, the binding or exchange of GTP cannot occur. GTP must be present however for aggregation to occur from 6S particles. Alternatively, in such a system, the presence of a phosphatase activity may be of importance, so that if blocked, GTP hydrolysis does not occur and P^ will not be placed on the micro tubule structure. This analogy is supported from actin polymerization investigations where a nucleoside tri phosphate (ATP) is bound to the subunit and one phos-* phate group is hydrolysed per subunit incorporated into the polymer. The subunits involved in microtubule reaggregatiqn must follow a similar mechanism. Investi gations concerned with microtubule reassembly have demonstrated that only microtubule protein is incorporated into the reassembled microtubule. It has also been demonstrated by Cook and Koshland that many oligomeric enzymes retain the ability to correctly reassemble in the presence of other proteins (1969). Preparations of microtubules contain phosphoprotein kinase activity when isolated in sucrose. Low levels of phosphate accumulate on microtubules which cannot 167 FIGURE 18 POSSIBLE MECHANISMS FOR PHOSPHATE ACCUMULATION BY RAT BRAIN MICROTUBULES. POSSIBLE MECHANISMS FOR PHOSPHATE ACCUMULATION BY RAT BRAIN MICROTUBULES I. STABILIZER GTP ------ (A) ▲ + TUBULE OR ENZYME GLYCOL GTP3 2 ------GTP 32 r- A ■ R ------ (B) GLYCOL GTP 32 ----- 3 + A PROTAMINE PHOSPHATASE PHOSPHORYLASE CGMP ------ 2. INHIBITOR OF INITIAL PHOSPHORYLATION A + GLYCOL GTp32 L I PHOSPHORYLASE ■ (KINASE) 3. INHIBITOR OF PROTAMINE KINASE GLYCOL I, ,1 + o ^ - i, I + CHi I, ? + O-Q A ■ A A CHi 4. CYCLASE INHIBITION (A) A - O GTP CGMP (B) i . n +0+0 ------O-B + i , i q be blocked with drugs. This occurrs without exogenously added protamine. This mechanism allows for the hydrolysis of GTP. In support of this, it is known that GTP binds to the6 S particle and is required for reaggregation of these particles. If this (GTP) is to phosphorylate the subunit which then assumes a correct configuration for reassembly, GTP would likely perform a function of stabilization on the 6S particle nrhile the phosphate is placed on the particle. The stabilizing property of GTP could be due to non-additive effects of a second nucleotide on the subunit structure, since the 6S unit is known to have one tightly bound nucleotide. Only small amounts of phosphate bind to microtubule protein, however this may be a matter of controlling the purity of reagents used and their additive order. An important mechanism, supported by this investi-* gation is the inhibition of a phosphokinase associated with the microtubule preparation. The difference between this mechanism and the inhibition of initial phosphory lation is that exogenously added substrate is phos- phorylated and not the 6S particle. By some mechanism protamine is bound to the intact microtubule after binding phosphate. This reaction sequence demonstrates the importance of time, temperature and pH changes which are sensitive to drugs which stabilize the tubule or 170 6S particle. The hydrolysis of GTP is incorporated into this mechanism since increased phosphate accumulation occurs when nucleotide triphosphate is present rather than orthophosphate-32. A variation of this mechanism is possible because phosphate may be bound to protamine after the protamine is associated with the microtubule. Evidence has been presented in this study that micro tubules do bind protamine, giving rise to structures on the microtubule surface physically resembling the F^-factor of Racker. These structures are not visible unless exogenous protamine is present, A final mechanism for consideration is that of guanyl cyclase inhibition. If cGMP were required for phosphory^ lation of either the protamine or the microtubule through a phosphokinase, the presence of endogenous cyclic nucleotide would be required. In these reactions it might have to be added due to a loss from the isolation procedure. In this connection, addition of cGMP caused changes in packed volume, light scatter and phosphate accumulation of the isolated microtubules. If stabilization, partial fixation, blockade of the guanyl cyclase synthesis or insufficient cGMP were formed or remained following synthesis, subsequent reaction steps would be reflected as lowered phosphate accumulation. The measurement of cGMP was not performed, however 171 fluorometric evidence was obtained for the breakdown of exogenous GTP. It is well known for example that in the chromosomal apparatus, tubulin is present. Also present are nuclear histones which are basic polypeptides. In this investi gation, the basic polypeptide protamine could be considered histones* counterpart. It is held by Bonner C1964) and others that the interaction between nucleic acids and nuclear histones somehow represses the expression of genetic information contained in the chromatid, and that removal of histone brings on depression. How the release of histone occurs from the nucleus of the cell, like the release of mRNA remains to be established. Phosphate accumulation by intact microtubules and 6S tubulin particles can be discussed from the relationship these structures bear to axonal transport mechanisms. From the studies of Wolfe and Mcllwain C1961), it was observed that electrical stimulation of brain tissue resulted in excitation, with marked increases in ion flux, respiration and intermediary metabolism. Electrical stimulation also caused activation of a protein kinase and phosphorylation of a labile protein. The addition or inclusion of protamine or other small basic proteins resulted in a loss of excitability and ion movements, decreased intermediary metabolism and presumably the 172 the phosphorylation process. These inhibitory effects were antagonized by the addition of gangliosides or other acidic compounds. The authors were at a loss to explain these phenomena, but suggested a role for basic proteins and acidic gangliosides in the excitation process. The effect of drugs on microtubular phosphate accumulation is viewed as evidence that agents such as meprobamate do have an effect on organelles within the CNS. Stabilization, partial fixation or reduced solubility of the intact microtubule or 6S particle is the result of drug addition. Lehrer has demonstrated a symbiotic relationship between neurons and glia grown in cell culture (Hyddn and Lange, 1970). From these studies, it was felt that neurons in cell culture were more viable in growth media which contained glia. Lehrer suggested that glia along neuronal axons are involved in the feeding and nutrition of neurons. This relationship could be expanded to include non-nutrients which presumably could originate from glia and influence function on or within the neuronal axons. Hyden and Lange C1970) and others have presented evidence that S-100 protein is unique to glia but appears to be transferred from glia to neurons and accumulates in the neuronal nucleus. 173 Figure 19 illustrates diagramatically a representation of the fine structure of the microtubule following isolation with buffered hexylene glycol. As illustrated, the tubule contains 13 filament structures as demonstrated by PTA staining techniques, shown in Plates II and V. The total diameter of the tubule is ^ 250 ft with a central lumen of 150 X. Figure 20 is a schematic diagram of the '’lollipop” structures observed in the microtubule surface following several types of treatment. These projections, on the iri vivo microtubule, may be representative of "side arm projections" which have been reported. In my study, they likely represent some form of GTP-protamine complex as demonstrated from Plates IV, IX and X. Measurements taken from particularly clear examples of these structures indicate an ellipsoid ball of ^ 75 ft diameter on a stem-like structure of ^ 60 ft length. Finally, in Fig. 21 I have pictured a conceptuali zation of the possible localization of GTP nucleotides on the microtubule following isolation in buffered hexylene glycol and staining with uranyl acetate. Several investigators have proposed a length of 800 ft for tubulin subunits within the intact tubular structure. From Plate III and other micrographs of tubular structures following UA staining, one can see 2 to 3 intensely staining dots/800 ft length. The tubulin FIGURE 19 DIAGRAMMATIC REPRESENTATION OF MICROTUBULE STRUCTURE PTA STAIN. DIAGRAMMATIC REPRESENTATION OF MICROTUBULE STRUCTURE (PTA STAIN) 175 176 FIGURE 20 SCHEMA. QF "LOLLIPOP" FORMATIONS; (SIDE ARM PROJECTIONS). SCHEMA OF "LOLLYPOP" FORMATIONS (SIDE ARM PROJECTIONS) 177 FIGURE 21 POSSIBLE NUCLEOTIDE CGTP) LOCALIZATION IN MICROTUBULE URANYL ACETATE STAIN. POSSIBLE NUCLEOTIDE (GTP) LOCALIZATION IN MICROTUBULE (URANYL ACETATE STAIN) CONTRACTED iv A' RELAXED 180 filaments have also been reported to have a slight helical twist associated with the longitudinal axis which is illustrated. I propose a "contracted" and "relaxed" state of the tubular structures as shown and have provided a coiled spring to further emphasize this overall state of "tone" on the intact microtubule. Future plans for experiments related to understanding the role of phosphate accumulation and phosphate binding to microtubular protein should consider the following topics : 1. The nature of the phosphate binding must be \ determined. In this study, phosphate binding was determined from either orthophosphate-32 or (y-^^P)GTP additions to the assays. Experiments designed to allow a more precise definition of this binding would include the following: a. Thin Layer Chromatography of the supernate from the standard reaction mixtures would aid in determining the relationships of the various breakdown products of added GTP. b. Radioimmunoassay of the supernate should be performed to determine the presence of cGMP formation following incubation with GTP. c. Fluorometry assays using non-specific phosphorylating enzymes can be used to assay for GTP, 181 GDP and GMP. d. Assuming that a phosphokinase or intermediate factor required for stable phosphate binding on the microtubule is lost in the preparation of the micro tubules from hexylene glycol, preparations of micro- tubules can be made according to Boisy and Olmstead (1972) as well as in hexylene glycol. If this initial assumption is correct, addition of the supernate from the non’-glycol isolation might contain the fraction necessary for stable phosphate binding to MTP. e. To isolate serine phosphate formed on the microtubule surface, the use of pronase, tryptic and chymotryptiq digests should be performed and analysed. 2. To study the mechanism of meprobamate inhibition of phosphate accumulation on microtubules, and to examine the possibility of significance of this feature of the drug, I propose that injections of -^C-leucine be made to L-7 spinal roots. A cuff of meprobamate should be applied to the sciatic nerve distal to the injection to determine whether a radioactive crest of protein is stopped or dammed at the site of the cuff. 3. By electropharmacology, I propose that experiments be designed to determine whether meprobamate and other similar compounds shown in this study to block phosphate 182 accumulation on microtubules will block MEPP's of nerve. 4. To further investigate the structure activity relationships proposed briefly in this investigation, experiments must include a glycol series varying both carbon chain length and increased interatomic distance between hydroxyl groups: e.g. 1-2, 1-3, 1-4 diols and 1-2 diols with chain length varying between 2 - 10 carbon atoms. 5. In order to investigate further the actions of various drugs on the blocking of microtubule phosphate accumulation, I propose that additional experiments be made using the following drugs and metabolites: a, diazoxide b. chlorpromazine sulfoxide c, 7-hydrbxychlorpromazine d. 7,8-dihydroxychlropromazine. 6. To further understand the possible relationships of washing and soaking the microtubules, I propose a re-investigation of the physical measurements of light scatter and packed volume using microtubules which have undergone the washing-soaking protocol outlined in this study. Electron microscopy should be performed to visualize the nature of the structural changes correlated with the physical measurements. 183 7. Additional data points must be established on the dose response curves for the drugs used in this study, shown in Fig. 17. 8. In vivo experiments should be planned to develop the concepts of phosphate binding as a necessary link to microtubular function in both the adult and newborn of at least two species. These studies should include both neuronal and secretory tissues since microtubules are integral features of both. 184 SUMMARY The following reflects conclusions which have been drawn from results in my study. These represent initial observations in some instances, and cannot be considered complete; however, these statements may prove useful in the development of a broad view of the character of microtubules both biochemically and morphologically. 1. Stabilization of microtubules is associated with reduced solubility of the tubulin protein 6S subunit. This configuration of the tubule is unresponsive to biochemical stimulation. 2. Reactivation of biochemical activity can be achieved by a washing-soaking procedure which decreases the concentration of the stabilizing agent to levels below that required to induce this stabilization. 3.. The response of isolated, soaked microtubules in packed volume studies is consistent with a view of particles which aggregate and disaggregate in response to exogenously added compounds; i.e. increased or decreased packed volume. 4. Configurational changes are postulated to occur based on light scatter measurements. This reflects the 185 number of particles in the optical path, indicating aggregation and disaggregation of subunits. 5. Increased phosphate accumulation on microtubules, responsive to pH, temperature and time, can be observed with addition of GTP. 6. Protamine and other small basic polypeptides serve as secondary acceptors and increase phosphate accumulation. Lollipop configurations are then observable on the surface of the microtubules, presumably protamine. Other reagents which preserve these structures are: Mg++ cGMP and (EGTA + Mg++) . 7. Drugs which contain a 3-carbon aliphatic diol nucleus block phosphate accumulation by interfering with the interaction of microtubules and exogenous protamine. These compounds seem to stabilize microtubule structure and/or may inhibit an intrinsic protein kinase associated with microtubules. 8. Fluorometric analysis demonstrates that isolated microtubules contain enzyme activity which converts GTP to GDP. 9. Microtubule preparations from newborn rat pups 0 - 6 days of age exhibit quantitatively different phosphate binding activity following treatment identical with that of adult preparations. 186 10. Nuclear histones, postulated to be released from the nucleus of the cell, and in this study designated by protamine, react with microtubules to cause conformational structure changes in microtubule protein. These interactions may be the side-arm projections visualized on microtubular structures. 11. A symbiotic relationship between neurons and glia is suggested to provide contacts for transmembrane trophic factors which may modulate local microtubule function. 187 APPENDIX A TECHNIQUES IN ELECTRON MICROSCOPY Embedding Medium All embedding of pellets was perfomred in SEM (Spurr Low-Viscosity Embedding Medium), using a firm ratio of polymer to hardener as follows: Vinylcyclohexene dioxide (VCD) 10.0 gms Diglycidyl ether of polypropylene glycol (DER) 6.0 gms Nonenylsuccinic anhydride (NSA) 26.0 gms Dimethylaminoethanol (DME) 0.4 gms This mixture must be thoroughly stirred for at least two hours before using. Tissue Preparation Before the pellets were made in the polyethylene inicrofuge tube, they were fixed in 3% buffered glutar- aldehyde and stained with 1% osmium tetraoxide according to Pease (1964) and Stoner (1969). When a sufficiently thick pellet was obtained (4-6 packings of 0.3 ml each), the inicrofuge tube was used to dehydrate and embed the pellet as follows: 50% Ethanol 30 minutes (2 x 15 minutes) 70% Ethanol 30 minutes 188 95% Ethanol 30 minutes 100% Ethanol 30 minutes (2 x 15 minutes) Propylene Oxide 60 minutes 1 SEM : 1 Propylene Oxide 60. minutes SEM 60 minutes When the pellets were ready to be embedded in the mold, the microfuge tube was cut: (1) just above the black, osmium stained pellet and, (2) again just above the end of the tube. By careful use of a wooden applicator stick, the pellet and epoxy mold insert could be gently removed from the lower remaining portion of the tube. If the pellet was packed sufficiently dense, one can now gently separate the pellet from the epoxy mold insert by using a scapel. The pellet could easily be cut, pie-shaped, to wedges or partial wedges for final embedding in the flat or standard molds. The plastic embedding medium was hardened by incubation for 8 hours at 70°C. PTA Staining Phosphotungstic acid, prepared fresh daily, was made by weighing 1.0 gm of PTA and dilution to 100 ml with water. Approximately 5 drops of 10 M NaOH changed the pH to near 6.4, required for staining. 189 Uranyl Acetate Staining Uranyl acetate staining was performed in the dark by placing grids (coated surface down) on droplets of 0.5% uranyl acetate, pH 5.0. Petri dishes containing wax easily held droplets for this purpose. Parlodion Coating of Grids Parlodion was prepared by weighing out 2.0 gm parlodion and dissolving it for 2 days in redistilled amyl acetate. Amyl acetate is distilled at ~62°C., using the vacuum line to lower normal pressure. This redistilled acetate will keep for approximately one month before redistilling is necessary. One drop of the parlodion solution is placed on a film of water, and if a sufficiently thin film is obtained, the washed, dried grids are placed (shiny side down) on this surface. After 10 - 25 grids are positioned, a wet filter paper is laid over the film and grids and both grids and film are removed from the water surface simultaneously. The filter paper containing the film and grids is placed under a lamp, beneath a Petri dish to dry; the grids are stored in a closed Petri dish at 4°C. Following use, they are best preserved in a desiccator at room temperature in the grid box holders. APPENDIX B PLATES I - XXI PLATE I LOW POWER MAGNIFICATION OF HEXYLENE GLYCOL ISOLATED MICROTUBULES: PTA STAIN (1%). 192 f Xi-r ; PLATE II HIGH POWER MAGNIFICATION OF HEXYLENE GLYCOL ISOLATED MICROTUBULES: PTA STAIN (1%), F = filament structures N = nucleation center. p e m h k 195 PLATE III HIGH POWER MAGNIFICATION OF HEXYLENE GLYCOL ISOLATED MICROTUBULES: URANYL ACETATE STAIN (0.5%). Dense staining dots are possibly GTP associated with microtubule surface. m M f l l N r i l ■ M M PLATE IV HIGH POWER MAGNIFICATION OF MICROTUBULES FOLLOWING REACTION WITH GTP AND PROTAMINE; FORMATION OF LOLLIPOP FIGURES ON MICROTUBULE SURFACE: PTA STAIN (1%), L = lollipop structures. PLATE V HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH ATP AND PROTAMINE: PTA STAIN (1%), F = filament structures. sl-iW PLATE VI HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH NATIVE FERRITIN: PTA STAIN (1%). 202 PLATE VII HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH FERRITIN-PROTAMINE CONJUGATE: PEAK I: PTA STAIN (1%), FE = ferritin, K = knob-like terminal, L = lollipop structures. 204 ,0iiJllffi ■ w y n 'rx .. v V d u l *V“. iiifS 205 PLATE VIII HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH FERRITIN-PROTAMINE CONJUGATE; PEAK I: PTA STAIN (1%), F = filament structures, FE = ferritin, S = staining artifact. 206 m B ^ ^ r ? £ ^tiSfiPr'vC \ ^ ^ iA»;jyr t * M b ' V*V - 3# ' ?« ♦ ?r>-& i *_■; v 'V'SE.-J ‘-J ' * ' “ , , ' V’ i ~A « r ‘ PLATE IX HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH FERRITIN-PROTAMINE CONJUGATE; PEAK II PTA STAIN (1%), FE = ferritin, FE-L = ferritin- lollipop structures, N = nucleation center, K = varicose knob-like terminal swelling. 208 PLATE X HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH FERRITIN-*PROTAMINE CONJUGATE: PEAK II PTA STAIN (1%), FE = ferritin, FE-L = ferritin- lollipop structures, S = staining artifact, K = knob-like varicose terminal. 5 0 0 A PLATE XI HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH MEPROBAMATE (10 nM): PTA STAIN (1%). 212 213 PLATE XII HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH HEXYLENE GLYCOL (10 nM): PTA STAIN (1 ©\« 500 A 215 PLATE XIII HGIH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH CHLORPROMAZINE, (0.1 mM): PTA STAIN (1 PLATE XIV HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH CHLORPROMAZINE, (10 riM): PTA STAIN C1 %3, K = varicose knob-like terminal, L *= lollipop structures. 218 0.1 ti PLATE XV HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH CHLORPROMAZINE, (10 nM): PTA STAIN (1%), Note varicosities in both terminal and body of tubule. Oil PLATE XVI HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH MAGNESIUM IONS, (1 mM) : PTA STAIN (1%), K - varicose knob^-like terminal of tubule, L = lollipop structures. 222 , PLATE XVII HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH CALCIUM (5 mM) AND MAGNESIUM (1 mM) IONS: PTA STAIN (1%), F - filament structures. 224 «t , . - j • ' f teAC-wv l^£iv:6‘ PLATE XVIII HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH EGTA (1 mM) AND MAGNESIUM (1 mM) IONS PTA STAIN (II), L = lollipop structures. 2 26 , YJi '.'-A '•r’V\>.» PLATE XIX HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH CYCLIC AMP, (0.1 mM): PTA STAIN (1%). * "V PLATE XX HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH CYCLIC GMP, (0.1 mM) : PTA STAIN (1%), L = lollipop structures, ST ® stem of lollipop structure. 230 PLATE XXI HIGH POWER MAGNIFICATION OF MICROTUBULE FOLLOWING REACTION WITH 40% DEUTERIUM WATER: PTA STAIN (1%), L = lollipop structures. 232 233 BIBLIOGRAPHY Allison, A.C. and Nunn, J.F.: Effects of General Anaesthe tics on Microtubules; a Possible Mechanism of Anaesthesia. 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