UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Molecular properties of acetylcholinesterase Webb, Geoffrey 1978

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1978_A1 W42.pdf [ 7.49MB ]
Metadata
JSON: 831-1.0059491.json
JSON-LD: 831-1.0059491-ld.json
RDF/XML (Pretty): 831-1.0059491-rdf.xml
RDF/JSON: 831-1.0059491-rdf.json
Turtle: 831-1.0059491-turtle.txt
N-Triples: 831-1.0059491-rdf-ntriples.txt
Original Record: 831-1.0059491-source.json
Full Text
831-1.0059491-fulltext.txt
Citation
831-1.0059491.ris

Full Text

MOLECULAR PROPERTIES OF ACETYLCHOLINESTERASE by GEOFFREY WEBB B . S c , U n i v e r s i t y o f B r i t i s h Co lumbia , 1974. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR*THE DEGREE OF THE FACULTY OF GRADUATE STUDIES We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA June , 1978 (c) Geof f rey Webb, 1978 DOCTOR OF PHILOSOPHY in (Department o f Chemistry ) In presenting th i s thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree l y ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thesis for scho lar l y purposes may be granted by the Head of my Department or by his representat ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my written permission. Department of CsYl £- ifa 1 f> The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT This thesis describes the affinity purification of the enzyme acetylcholinesterase from the electric organ tissue of the electric eel (Electrophorus electricus) and the characterization of the enzyme by selective proteolytic cleavage monitored by sucrose gradient sedi-mentation, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel chromatography. It describes conditions, using N-nmethylacri-dinium-Sepharose 2B, for the purification of the asymmetric forms of the enzyme from high salt extracts of electric tissue and for the purification of the globular form of the enzyme subsequent to treatment with the enzyme trypsin. In addition it describes for the first time the selective purification of either asymmetric or globular acetyl-cholinesterase from mixtures containing both forms of the enzyme. A distinction between autolytic and tryptic degradation of asymmetric acetylcholinesterase is described for the first time and two new forms of the enzyme generated by collagenase proteolysis of the asym-metric 18S and 14S forms are described. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.IS and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The changes in the sodium dodecyl sulphate-polyacrylamide gel electrophoresis migration pattern of acetylcholinesterase fragments following trypsin or col-lagenase proteolysis and the changes in sedimentation coefficient and Stokes radius with collagenase proteolysis are compared to identify a component with a molecular weight of 45,000 daltons on electrophoresis gels, that contributes greatly to the asymmetry but only minimally to i i i the mass of the 18S and 14S forms of acetylcholinesterase. An appendix discusses some efforts at the purification of the individual subunits of the 18S and 14S forms of acetylcholinesterase and describes several observations made on the proteolytic instability of even highly purified asymmetric acetylcholinesterase. iv TABLE OF CONTENTS CHAPTER PAGE 1. GENERAL INTRODUCTION 1. 1-1 Neural Transmission 1 l^I-I the cell membrane. 2 1 —I — 11 the axon or nerve fiber and axonal 4 transmission 1 - 1 - 1 1 1 the synapse and synaptic transmission 7 l-II Electrophorus electricus 8 1- III Acetylcholinesterase 11 1-111-1 history 11 1 — III — II acetylcholinesterase at the synapse 1.1 1—III—III globular acetylcholinesterase 12 1-III-IV asymmetric acetylcholinesterase 13 1-III-V molecular properties of globular 15 acetylcholinesterase 1-III-VI molecular properties of asymmetric 16 acetylcholinesterase 2. APPROACHES TO THE CHARACTERIZATION OF THE ASYMMETRIC 1 8 FORMS OF ACETYLCHOLINESTERASE 2- 1 Purification of Asymmetric Acetylcholinesterase 18 2-11 Identification of Acetylcholinesterase 21 Protein Fragments 2-1II Reassociation of Purified Enzyme With 23 Synaptic Fragments 2-IV Additional Work 26 27 3. BIOCHEMICAL METHODS APPLICABLE TO THE CHARACTERI-ZATION OF ACETYLCHOLINESTERASE FORMS 3-1 ultracentrifugation and Sedimentation Analysis 27 V CHAPTER PAGE 3-1-I sedimentation velocity 28 3-1-11 sedimentation equilibrium 32 3-1-111 density gradient sedimentation 33 3-I-IV density gradient sedimentation 33 equilibrium 3-I-V density gradient sedimentation 34 velocity 3-I-VI isokinetic sucrose gradient 37 sedimentation 3-I-VII reliability of data obtained by 44 sucrose .gradient sedimentation 3-11 Gel Chromatography 45 3-1I-I theory of gel chromatography 46 3-II-II reliabil ity of data obtained by 48 Sepharose 4B gel chromatography 3-III Sodium Dodecyl Sulphate-Polyacrylamide 48 Gel Electrophoresis (SDS-PAGE) 3-11I-I theory of SDS-PAGE 50 3-111-11 reliability of data obtained by 51 SDS-PAGE 3-IV Methods 52 3-IV-T isokinetic sucrose gradient 52 sedimentation 3-1V-11 Sepharose 4B gel chromatography 53 3-IV-III sodium dodecyl sulphate-polyacryl- 55 amide gel electrophoresis 3-IV-IV acetylcholinesterase assay 62 3-V Materials 62 3-V-I biological materials 62 vi CHAPTER PAGE 3-V-II other materials 63 3- V-III further purification of collagenase 64 4. OPTIMIZATION OF THE CONDITIONS FOR PURIFICATION OF 65 THE ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE BY COLUMN CHROMATOGRAPHY ON AN AFFINITY MATRIX CON-TAINING THE N-METHYLACRIDINIUM LIGAND 4-1 Introduction 65 4-11 Methods 65 4- 1I-I high salt extraction of AChE 65 from electric tissue 4-11-II coupling of N-methylacridinium 66 derivative to Sepharose 2B 4-11-111 analytical affinity chromatography 67 4-11-IV preparative affinity chromatography 67 4-II-V characterization of purified AChE 68 4-III Results 68 4-111-1 ligand coupling to Sepharose 2B 68 4-111-11 enzyme extraction from tissue 68 fragments 4-111-111 analytical affinity chromatography 69 4-III-III-a purification from high salt 69 extracts of electric tissue 4-1II-III-b selective elution of globular 72 AChE with retention of asymmetric AChE 4-III-III-c purification of globular AChE 76 from proteolytic digests 4-111-IV preparative affinity chromatography 80 4-1V Discussion 91 vii CHAPTER PAGE 5. CHARACTERIZATION OF. THE ASYMMETRIC FORMS OF ACETYL- 97 CHOLINESTERASE: SEDIMENTATION COMPOSITION, ACTIVITY ASSAYS, AMINO ACID COMPOSITION, SUBUNIT COMPOSITION, ISOTOPIC LABELLING, PROTEOLYTIC DEGRADATION AND GEL CHROMATOGRAPHY 5-1 Introduction 97 5-II Methods 97 5-11-1 acetylcholinesterase purification 97 and assays 5-11-11 active-site labelling of acetyl- 98 cholinesterase with ^H-diisopropyl-phosphofluoridate 5-II-III collagenase proteolytic cleavage of 100 acetylcholinesterase 5-II-IV trypsin proteolytic cleavage of 101 acetylcholinesterase 5-11-V radioactive labelling of acetyl- 102 chol inesterase with '25jocj-jne 5-11-V-a lactoperoxidase iodination 102 5-II-V-b 1 2 5I-TAGIT labelling of acetyl- 102 cholinesterase 5-11-VI amino acid composition of asym- 103 metric acetylcholinesterase 5-11-VII characterization of intact and 103 proteolytically modified forms of acetylcholinesterase 5-1II Results 104 5-111-1 sedimentation composition of 104 affinity purified acetyl-cholinesterase 5-111-II titrimetric assay, comparison 104 with spectrophotometric assay 5-111-111 hydroxyproline content and amino 104 acid composition of affinity . purified acetylcholinesterase v i i i CHAPTER PAGE 5-III-IV subunit composition of affinity 105 purified acetylcholinesterase 5-111-V isotopic labelling of ?acetyl- 108 cholinesterase with iodine 5-111-VI trypsin cleavage of affinity 111 purified acetylcholinesterase 5-111-VII collagenase proteolysis of 115 affinity purified acetyl-cholinesterase 5-III-VIII gel chromatography; Stokes radius 124 and molecular weight of collagenase modified acetylcholinesterase 5-1V Discussion 127 6. 'REASS0CIATI0N' OF PURIFIED ASYMMETRIC ACETYLCHO- 138 LINESTERASE WITH MEMBRANE FRAGMENTS DERIVED FROM ELECTRIC TISSUE 6-1 Introduction 138 6-II Methods 138 6-1I-I purification of acetylcholin- 138 esterase and assays 6-11-11 preparation of membrane 139 fragments 6-11-111 sucrose gradient sedimentation 140 of membrane fragments 6-1I-IV sucrose gradients sedimentation 140 of membrane fragments in the presence of excess added acetyl-cholinesterase 6-II-V sucrose gradient sedimentation 141 of pure acetylcholinesterase in the absence of membrane fragments 6-11-VI labelling of membrane proteins with 141 1 "iodine ix CHAPTER PAGE 6-II-VII SDS-PAGE analysis of membrane 142 fragments 6-III Results 142 6-III-I preparation of membrane fragments 142 6-III-II sucrose gradient separation of 144 membrane fragements 6-111-111 separation of membrane fragments 144 in the presence of excess pure acetylcholi nesterase 6-111-IV centrifugation of pure acetyl- 147 cholinesterase under the conditions used for the separation of membrane fragments 6-III-V radioiodination of membrane frag- 147 ment proteins 6-III-VI SDS-PAGE analysis of membrane 149 fragments 6- IV Discussion 153 7. CONCLUDING REMARKS 155 7- 1 Affinity Purification of Acetylcholinesterase 155 7-II The Tail of Acetylcholinesterase as a Membrane 155 Anchor 7-111 Basement Membranes 157 7-IV Synaptic Complexes 159 7-V Acetylcholinesterase at the Synapse 161 Bibliography 162 Appendix 171 X LIST OF TABLES TABLE PAGE 3-1 Data for Standard Proteins Used in Sucrose 54 Gradient Sedimentation, Gel Filtration and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis 3- II Stock Solutions for SDS-PAGE and Storage 59 Conditions for Solutions 4- 1 Stoichiometrics of Reactants and Coupling 70 Obtained for the Cyanogen Bromide Activated Sepharose 2B Coupling to N-Methyl-9-[Ng-(6-ami noheaxonyl)-6-ami nopropylami no]acri di ni urn dibromide 4-II Preparation of Electric Tissue Fragments and 71 High Salt Extraction of Acetylcholinesterase 4-III Conditions for Affinity Chromatography at 73 Six Ligand Concentrations: Retention, Yield . and Specific Activity 4-IV Recovery of AChE Activity from Affinity Columns 77 by Sequential Elution with Increased Salt, Decamethonium and Guanidine-HCl 4-V Recovery of AChE Activity from Affinity Columns 81 by Sequential Elution with Decamethonium under Low and High Salt Conditions 4- VI Column Load, Retention, AChE Recovery and 90 Specific Activity of Purified AChE for Pre-parative Affinity Chromatography 5- 1 Amino Acid Composition of Affinity Purified 107 Acetylcholinesterase xi LIST OF FIGURES FIGURE PAGE 1-1 The Fluid Mosaic Model of Cell Membranes 3 1-2 Axonal Transmission 6 1-3 Schematic Portrayal of the Neuromuscular 9 Junction of Synapse 1-4 Electroplax Cell of the Electric Eel 10 1- 5 Multiple Forms of Acetylcholinesterase 14 from E. Electricus 2- 1 Binding Sites on AChE and Ligand Molecules 20 Used.During Affinity Purification 2-2 Catalytic Mechanism of Acetylcholinesterase 24 and Active Site Labelling Procedure 2- 3 Isotonic Labelling of Acetylcholinesterase 25 with ^"Iodine 3- 1 Diagram of a Sedimentation Experiment 30 3-2 Detection of Boundary Formation and Movement 31 in the Analytical Ultracentrifuge 3-3 Final State in Density Gradient Sedimentation 35 Equilibrium 3-4 Density Gradient Sedimentation Velocity 36 3-5 Form of Isokinetic Sucrose Gradient and 40 Exponential Approximation 3-6 Pre-forming an Exponential Gradient to 41 Approximate an . Isokinetic Gradient 3-7 Fractionation of Sucrose Gradients 42 3-8 Diagram Illustrating the Distinction Between 47 Gel Chromatography and Gel Filtration 3-9 Correlation of Stokes Radius with Distribution 49 Coefficient xi i FIGURE PAGE 3-10 Diagram of SDS-PAGE Apparatus Showing Location 58 of Electrodes and Sample Layered on Gel Immedi-ately Prior to Electrophoresis 3- 11 Diagram of Polyacrylamide Gel Following Staining 61 of Protein Bands and Sketch of Densitometric Profile Obtained for Gel 4- 1 Optimization of Conditions for the Affinity 74 Chromatography Purification of AChE Using N-Methyl-acridinium-Sepharose 2B 4-2 Release of AChE Activity From 0.22 ymol/ml 78 Affinity Resin by Salt Elution Followed by Decamethonium Elution: Sedimentation Com-position of the Eluted Species. 4-3 Enzyme Composition on Sucrose Gradients Before 83 and After Preparative Affinity Purification Enzyme Composition on Sucrose Gradients Before and After PreparativerAffinity Purification at Low Column Load on Two Ligand Concentrations 4- 5 Elution Profile and AChE Composition of Pro- 88 duct Obtained by Different Elution Pro-cedures Following Loading of Affinity Columns With Crude Extract at Low Ionic Strength. 5- 1 Sedimentation of Profile of Affinity Purified 106 AChE 5-2 SDS-PAGE of Fully Reduced AChE Components 109 5-3 Calibration Curve for SDS-PAGE 110 5-4 SDS-PAGE of Fully Reduced AChE Following 112 Isotopic Labelling With 125i0dine 5-5 SDS-PAGE of Fully Reduced AChE Before and 113 After Trypsin Proteolysis 5-6 SDS-PAGE of Fully Reduced AChE Before and 116 After Collagenase Proteolysis 5-7 Sucrose Gradient Sedimentation Profiles 119 for Affinity Purified AChE Following Exposure to Collagenase xi i i FIGURE - PAGE 5-8 Progress of Collagenase Cleavage 123 5-9 Elution Profiles for AChE Forms When Chromato- 125 graphed on Sepharose 4B 5-10 Calibration of Gel Chromatography; (-log Kp) 1 / 2 126 Versus Stokes Radius (Re) for Standard Proteins. 5- 11 Calibration Curve of the Product of Stokes 128 Radius and Sedimentation Coefficient Versus Molecular Weight 6- 1 Preparation of Membrane Fragments From Electric 143 T i s s u e 6-2 Sucrose Gradient Sedimentation of Membrane 145 Fragments 6-3 Sucrose Gradient Sedimentation of Membrane 146 Fragments in the Presence of Excess Pure AChE 6-4 Centrifugation of Pure AChE Under the Con- 148 ditions Used for Separation of Membrane Fragments 6-5 Distribution of Radioactivity, AChE Activity and 150 Protein After Separation of Radiolabeled Membrane Fragments on Sucrose Gradient 6-6 SDS-PAGE Densitometric Profiles for Membrane 151 Fragment Proteins Al Preparative SDS-PAGE 173 A2 Preparative SDS-PAGE 174 A3-A19 Proteolytic Instability of Purified AChE 178-196 ACKNOWLEDGEMENTS I would like to express my sincere appreciation to Dr. D.G. Clark whose helpful advice and optimistic approach provided excellent supervision of my work. I am also indebted to Dr. G. Weeks of the Department of Microbiology, Dr. B.D. Roufogalis of the Faculty of Pharmaceutical Sciences and to Dr. F.G. Herring of this department for their support and encouragement during the latter part of this work. I am very grateful for the support of the Ogilvie Fellowship from the Chemical Institute of Canada during the periods 1974-75 and 1975-76 and for the support of a Postgraduate Scholarship from the National Research Council of Canada during the periods 1976-77 and 1977-78. The technical assistance of L. F. Nazar during the summer of 1977 is also greatly appreciated. I would also like to thank G. Hewlet, Curator of the Vancouver Public Aquarium and aquarium staff member F. Larsen for the excellent care provided for the electric eels used in this work. Finally I would like to dedicate this thesis to my wife Ronna and two daughters, Dawn (6) and Fern (3) who have tolerated neglect and irascibility throughout the course of this work. - 1 -CHAPTER ONE GENERAL INTRODUCTION The subject of this thesis is the determination of some of the molecular properties of the enzyme1 acetylcholinesterase (AChE) located at the neuromuscular junction and at cholinergic nerve synapses in the central and peripheral nervous systems. This enzyme aids in the termination of synaptic transmission by catalyzing the hydrolysis of acetylcholine to acetate and choline. 0 + AChE + CH3-C-0-CH2CH2-N(CH3)3 > CH3-C00" + H+ + H0-CH2CH2-N(CH3)3 H20 Enzymes with this catalytic function occur at locations other than cholinergic synapses and the neuromuscular junction. These enzymes in some cases are structurally distinct from the forms of AChE that are present at the neuromuscular junction and the work for this thesis was done entirely on enzyme from a location similar to the neuromuscular junction. A brief description of the mechanism of neural transmission will be given followed by an introduction to the electric eel, Electro- phorus electricus, the source of the enzyme used for this thesis and a summary of previous work on AChE. A more detailed introduction to the particular aspects investigated in this work will follow in chapter two. 1-1. Neural Transmission Two mechanisms exist by which an impulse is transmitted - 2 -through the nervous system of animals; axonal conduction, the propaga-tion of a signal along a nerve fiber; and synaptic transmission, the passage of a signal through the extracellular medium between two nerve cells or between a motor nerve and a muscle cell. l-I-I. the cell membrane Nerve cells, in common with all cells, are separated from their external environment by a cell membrane composed of two types of macro-molecules; lipid and protein. The lipid portion of a cell membrane is comprised primarily of phospholipids which are amphipathic molecules having a polar 'head' and an apolar ' t a i l 1 . CH3(CH2) 1J-0-CH2 A Typical Phospholipid tail o CH3(CH2)1()C-0-CH 9" H,C-0-P-0-CHo-CHo-N(CH,)o head C || c L 6 6 0 In a membrane these molecules are oriented in a bilayer in which the polar heads of the phospholipids are exposed to the intracellular or extracellular medium and the apolar tails form a hydrophobic lamella. The proteins contained within the membrane structure may either span the bilayer and communicate with both the internal and external medium or may simply penetrate into the bilayer from one side. This model, depicted in cross-section in Figure 1-1 is variously known as the "Fluid Mosaic Model" or as the "Singer and Nicolson" model after the proponents of this description ( 1 ) . The proteins which penetrate the hydrophobic FIGURE 1-1 - 3 -Fig 1-1. The Fluid Mosaic Model of Cell Membranes (1) The polar head groups of the phospholipids are represented by the f i l led circles while the hydrocarbon chains are indicated by wavy lines. Two protein molecules are shown with their polypeptide backbone folded to present charged groups to the outer surfaces of the membrane. The por-tion of the protein within the bilayer must have a non-polar character. - 4 -region of the l ipid bilayer, and which are tightly associated with the membrane are described as integral membrane proteins; these proteins can be separated from their normal membrane environment by extraction into a detergent solution. A second class of membrane proteins has been designated extrinsic (peripheral) membrane proteins and this class, while firmly associated with cell membranes under conditions similar to those existing in vivo, can be dissociated intact by mild treatment such as an increase in the ionic strength of the medium or by the addi-tion of chelating agents. Extrinsic membrane proteins do not associate hydrophobically with phospholipids and the interactions responsible for their membrane association are mainly electrostatic. l-I-II. the axon or nerve fiber, and axonal transmission The axon consists of a tubular extension of the nerve ce l l ; the walls of the tube are formed by the cell membrane and in higher animals the tube is wrapped in a myelin sheath, the purpose of the sheath is to increase the speed of propagation of the nerve impulse. However, the basic mechanism of transmission is the same for both myeli-nated and non-myelinated nerve fibers and this feature will be omitted in this description. Included among the proteins within the membrane of the axon are two particular types; ion pumps and ion channels. In the resting axon the ion pumps utilize energy from the hydrolysis of adenosine t r i -phosphate to move sodium and potassium ions across the cell membrane maintaining an increased potassium concentration and decreased sodium concentration inside the axon relative to the external medium. The - 5 -concentration differences cause a polarization of the membrane with a potential difference of up to 90 mV interior negative. During axonal conduction a band of sodium permeability travels down the axon.Within this band sodium ions flow into the cell and the membrane repolarizes to approximately 60 mV interior positive. Closely following the band of sodium permeability is a band of potassium permeability within which potassium ions flow out of the axon. The flow of potassium ions reduces the potential difference created by flow of sodium ions.and, as the concentration gradients for both ions decrease, influx and efflux cease. Finally the membrane becomes once more impermeable to these ions and the ion pumps restore the original polarization (2). Figure 1-2 schematically portrays axonal conduction. The mechanism by which the membrane of the axon becomes selectively permeable first to sodium ions and then to potassium ions is being elucidated only slowly. The opening and closing of the ion channels which permit passage of the ions across the otherwise imperme-able cell membrane is dependent on the electric field experienced by the protein of the ion channel. The reversal of the membrane polariza-tion in the region of sodium ion influx causes additional sodium ion channels to open. Thus the band of sodium permeability moves forward. Potassium ion channels respond similarly but slowly, therefore the region of potassium permeability lags behind that of sodium permeability. Both channels close when the membrane potential returns to zero and remain closed when a negative polarization is present. These events are ex-plained more fully in a recent summary (3) which also presents additional references from which the foregoing description was compiled. - 6 -— + 1 0 0 -cn o Fig 1-2. Axonal Transmission (Adapted from 2). The axon is portrayed in cross-section showing how Na+ is moved out and K+ moved in to give the resting potential. The action potential results from selective changes in the permeability of the membrane as described in the text. - 7 -The initiation of the action potential is described in the following section describing synaptic transmission. l-I-III. the synapse and synaptic transmission The synapse is a specific structure connecting two nerve cells, or a nerve cell and a muscle ce l l , which permits transmission of a nerve impulse between the two cells without direct physical contact (4,5). An axon terminates in a bulbous structure the distal face of which forms the presynaptic membrane separated from the post-synaptic membrane on the proximal face of the adjacent cell by a synaptic cleft f i l led with a filamentous material, the ectolemma. The structure and function of the ectolemma is not clearly known but removal of this material by enzymatic digestion results in the separation of the synapse and, as will be discussed later in this thesis, also causes the release of acetylcholinesterase (AChE) located within the synaptic cleft (6). Within the bulbous structure terminating the axon are many small vesicles each containing about 10,000 molecules of the neurotrans-mitter which carries the nerve impulse across the synapse. At cholinergic synapses this neurotransmitter is acetylcholine and reception of an action potential at the presynaptic membrane causes several vesicles to fuse with the membrane and release quanta of acetylcholine molecules into the synaptic cleft. The acetylcholine diffuses across the synaptic cleft, a distance of about 50 nm, and binds to an integral protein in the post-synaptic membrane. This protein, the acetylcholine receptor (AChRj, is associated with an ion.channel and on the binding of acetylcholine the channel opens permitting a rapid influx of sodium ions and causing a - 8 -depolarization of the surrounding membrane. The depolarization initiates the action potential portrayed in Figure 1-2. Synaptic transmission is terminated by the diffusion of acetylcholine away from the region of the receptor and by the hydrolysis of acetylcholine by AChE. Following termination of synaptic transmission the pre- and post-synaptic membranes return to their resting state. Figure 1-3 portrays a neuromuscular junction; the spread of the action potential through the surrounding membrane in the muscle cell triggers contraction of the muscle. 1 — II. Electrophorus electricus The electric eel, Electrophorus electricus, is one example of a unique group of animals; the electric fishes. In common with other members of this group the electric eel has the capacity to emit electric discharges from a specialized electric organ. This ability was re-cognised in the latter part of the 18th century and, when it was firmly established in the 19th century that neural transmission was accompanied by electrical discharges, the electric tissue of E_. electricus was taken as a readily obtainable large scale model for the events which accompany the generation of an action potential in nerve cells (7). The electric organ of the electric eel is phylogenetically derived from muscle tissue and the region of contact between the in-dividual cells, or,electroplax, of this tissue and the 'motor' nerve which transmits the impulse to trigger an electric discharge, is analogous to the neuromuscular junction. Figure 1-^- shows a simplified picture of the cells of the electric organ showing the asymmetry of the cells and the series arrangement which gives rise to the extremely powerful dis-charge emitted by the electric eel. FIGURE 1-3 9 -motor axon tasement membrane ^ neurotransmitter vesicle synaptic cleft and ectolemma AChE AChR presynaptic membrane junctional folds muscle cell Fig 1-3. Schematic Portrayal of the Neuromuscular Junction or Synapse The postsynaptic membrane is deeply convoluted with the acetyl-choline receptor being present toward the top ends of the folds, The ectolemma is continuous with a filamentous sheath known as the basement membrane which surrounds the cells. One neuro-transmitter vesicle is portrayed intact while another has just discharged its contents into the synaptic cleft. Acetylcholin-esterase is shown adjacent to the postsynaptic membrane. FIGURE \-L - 10 -'motor' axons and innervated faces non-innervated face OmV - 9 0 mV h60mV 150mV - 9 0 mV H60mV resting discharge Fig 1-4. Electroplax Cell of the Electric Eel (7). A single complete cell is sketched with portions of the cells above and below also shown; parallel cells are completely omitted. Only the innervated face of the cell has synapses . and AChE is localised almost exclusively in this face. The normal resting potential is shown on the right side of the picture, when the motor axon transmits a signal to the cell only the innervated facing repolarizes leading to a net potential difference of ca_ 150 mV along the ce l l . - 11 -1 — 111 - Acetylcholinesterase 1 - 1 1 1 - 1 . history The enzymatic hydrolysis of acetylcholine in nervous tissue was suggested more than sixty years ago (8) and in 1938 a remarkably high concentration of the enzyme was found in the electric tissue of the electric eel (9). It was not until the late nineteen forties, however, that AChE was purified from this source and not until the mid nineteen sixties that sufficient enzyme was available for elucidation of its molecular properties. Acetylcholinesterase has been extensively reviewed; most recently in 1975 the kinetic properties were presented (10) while in 1976 the molecular properties were discussed (11). An earlier review in 1971 summarized both kinetic and structural properties (8) as did additional presentations in 1970 (9) and 1971 (12). 1—III—II. acetylcholinesterase at the synapse The early investigations on AChE were carried out before the composition and structure of cell membranes was understood. This led to considerable misinterpretation of data and i t was init ia l ly thought that the function of the acetylcholine receptor, AChIR and the acetyl-choline esterase, ACh£ were both present on the same protein unit (7). Despite this misconception a great deal of knowledge was gained about the in vivo properties of AChE particularly with respect to inhibitors of the enzyme. Both reversible and irreversible inhibitors of the enzyme are known, the reversible inhibitors, in general, contain a quaternary nitrogen function analogous to that of choline while the irreversible inhibitors are typified by the organophosphates, or nerve - 12 -gases. Examples from both these classes of compounds are used in mole-cular investigations on AChE as aids in either the purification of the enzyme or the identification of polypeptide fragments derived from the enzyme protein. These will be introduced later in more detail. The location of AChE, within the 'neuromuscular junction' of the electroplax ce l l , which lead to the misconception referred to above also caused considerable problems during the elucidation of the molecular properties of the enzyme. It is now known that AChE can be solubilized by homogenization of the electric tissue in the presence of high con-centrations of salt plus a chelating agent (vide infra) while the enzyme remains insoluble under conditions of low ionic strength (13). The first purification of AChE, however, was done using electric tissue of the electric eel which had been stored under toluene for several years (8). This storage resulted in the spontaneous solubilization of the enzyme, which could then be purified by standard techniques. More importantly this treatment introduced an artefact into the studies on AChE which did not become apparent until late in the nineteen sixties. l-III-III. globular acetylcholinesterase Storage of tissue under toluene was an accepted technique in biochemistry to protect material from bacterial degradation. This procedure however does not prevent degradation of the stored tissue by endogeneous proteolytic enzymes and it was realised that the solubiliza-tion of AChE under these conditions was due to endogeneous proteolysis. In order to more rapidly obtain larger quantities of AChE this pro-teolysis was augmented by the addition of trypsin or other proteolytic - 13 -enzymes. Acetylcholinesterase isolated in this manner is a globular protein with a molecular weight of 260,000 to 300,000 and ' contains approximately 8% carbohydrate by weight (14). It was this form of the enzyme that was reviewed in 1970 and 1971 (12,9,8). In 1969 however, additional forms of AChE were reported with vastly different molecular weights than those previously encountered (15). 1 -111-IV. asymmetric acetylcholinesterase During the period 1969-75 several accounts of the additional forms of AChE appeared in the literature. The new forms were found to behave in the manner of large asymmetric molecules during gel chromato-graphy (vide infra chapter three) and two classes of AChE were described (16). The globular form of the enzyme comprised one class, while the asymmetric forms, for which three distinct species were identified, comprised the second class (15,16,17). The asymmetric forms of AChE were visualised in the electron microscope (18,19) and the forms pictured in Figure 1-5 were identified. Acetylcholinesterase, isolated from the electric tissue of the electric eel by homogenization of this material in the presence of 1-2 M NaCl and the chelating agent, ethylenediamine-tetraacetic acid, exists predominantly in the larger of the four forms shown in Figure 1-5 (13). The various forms of AChE are identified by their sedimentation coefficients in the ultracentrifuge (vide infra chapter three); small variations exist in the reported values and the nominal values used are; 18S for the largest species containing three tetrameric units attached to an elongated ' t a i l ' ; 14S for the species with two tetrameric units and 9S for the species with only one tetramer FIGURE 1-5 FORM SEDIMENTATION MOLECULAR COEFFICIENT 3 WEIGHT » . ' b to ta l ta i l 18.4 S 1,150,000 157,000 U.2S 796,000 134,000 9.1 S ° 410,000 79 ,000 11.8S 331,000 Fig 1-5. Multiple Forms of Acetylcholinesterase from E. Electricus. .The best determined values for both sedimentation coefficient and molecular weight are shown (21). The manner in which the individual tetramers are attached to the elongated ' t a i l ' is not clearly known. The determination of sedimentation coefficients is described in the introduction to Methods. ^Calculated from the total mw minus the sum of the mw of the tetrameric units present. c This form is not readily obtained under the conditions for purification used in this work. - 15 -attached to the t a i l . The individual tetramer, which is the globular form isolated following proteolytic digestion of electric tissue, has become known as the 1.1 S form. The catalytic properties of the different forms of AChE appear to be essentially identical particularly with reference to the action of inhibitors (20). Under some conditions the larger forms display reduced enzymatic activity. This is thought to result from micro-environmental changes in substrate and product concentrations in the vicinity of the cluster of catalytic tetramers in these forms (13). 1-III-V. molecular properties of globular acetylcholinesterase All of the AChE forms shown in Figure 5 share the catalytic tetramer which exists alone in the globular form of the enzyme. This form of the enzyme has been well characterized and is now known to consist of four apparently identical subunits which are covalently joined in pairs by disulphide bonds to form two dimers and non-covalently associated to yield the tetrameric structure. The individual monomers are susceptible to a well defined proteolytic cleavage which converts the 80,000 molecular weight polypeptide chain of the monomer into a fragment, containing the active site of the enzyme, with a molecular weight of ca 55,000 daltons and two additional fragments with molecular weights of 25,000 to 28,000 daltons. This conversion occurs during the proteolytic solubilization of the enzyme described earlier but does not become apparent until inter- and intra-monomer disulphide bonds stabilizing the structure of the enzyme are broken. The precise nature of this conversion was not described, and the actual monomer molecular - 16 -weight was not determined, until 1974 (22,23,24). It was also demon-strated at this time that the asymmetric forms of the enzyme were pro-teolytically degraded to the globular form, even in 'purified' enzyme preparations, and this degradation preceded the conversion of the in-dividual monomers to smaller fragments (22). The totally converted form of the enzyme has been crystallised and preliminary X-ray structural data reported; the information was meager and the only conclusion given was that the maximum dimensions of this globular form of AChE are 9.3 nm x 8.1 mn x 9.7 nm (25). I T I I I - V I . molecular properties of asymmetric acetylcholinesterase Very l i t t l e information about these forms of AChE was avail-able prior to 1975. The appearance of the ' t a i l ' in electronmicrographs suggested that i t was a rodlike structure (19) and this observation coupled with the release of AChE from its synaptic location by ex-posure of the tissue to collagenase suggested that the tail had some resemblance to collagen (13,6,26). The asymmetric forms of the enzyme have the unique property of forming very large aggregates in the presence of less than 0.4 M NaCl and the removal of this feature by reaction of these forms with maleic anhydride was taken to indicate that the e-ami no function of the amino acid lysine was involved in the aggregation (27). The ionic nature of the interactions responsible for the in vivo locali-zation of the enzyme, suggested by the high salt solubilization, was further indicated by the observation that calcium ion stabilized this association (27) and by the observation that the inclusion of the calcium chelater ethylenediamine-tetraacetic acid in the homogenization medium - 17 -greatly increased the degree of solubilization (13). During the course of the work described in this thesis a con-siderable amount of additional information on the properties of the asymmetric forms of AChE was reported. These accounts will be intro-duced during discussion of the findings of this thesis. The following chapter introduces the intent of this thesis and explains the approach taken to realise this intent. - 18 -Chapter Two APPROACHES TO THE CHARACTERIZATION OF THE ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE The aims of this thesis were threefold; f irst ly, a procedure for the purification of large quantities of the asymmetric forms of the enzyme was to be optimized; secondly, the composition of the tail component of the asymmetric forms was to be investigated; and finally, the nature of the association between the enzyme and the synapse was to be studied using purified enzyme and. cell fragments derived from the region of the synapse. 2-1. Purification of Asymmetric Acetylcholinesterase In 1970 the technique of affinity chromatography was intro-duced to the purification of AChE (28). This method of enzyme purifica-tion depends on the specific interaction between the enzyme and an organic ligand which can be covalently coupled and immobilized on an insoluble matrix. When a solution of proteins, containing the enzyme to be purified, is percolated through the matrix this specific inter-action with the immobilized ligand leads to a preferential retardation of the enzyme protein. It is often possible to wash the non-enzyme proteins clear of the matrix before freeing, or eluting, the retarded enzyme by introducing a soluble ligand which competes with the immobilized ligand for the enzyme. The chemical proecedures by which the ligand molecule may be immobilized have been extensively reviewed (29); the most frequently chosen supporting matrix is the agar based Sepharose 2B gel chromatography - 19 -material marketed by the Pharmacia company. This material can be 'activated' by reaction with cyanogen bromide and molecules containing a primary amine may be readily coupled to the activated gel. 'activated' gel r-OH agarose matrix CNBr —0 OH (activation) \ C=NH H2N-R (coupling) R = -spacer-1igand hOH 0-C-NH-R I  NH affinity matrix In general i t has been found necessary to interpose a spacer molecule between the agarose matrix and the ligand, and this requirement is often met simultaneously with the requirement for a primary amine in the derivatization of the ligand molecule prior to coupling as illustrated in Figure 2-1. Acetylcholinesterase is uniquely suited to purification by affinity chromatography due to the range of small organic inhibitor molecules which bind to specific sites on the enzyme. Each catalytic subunit of the enzyme contains an active site at which the ester function of the substrate binds and is hydrolysed, an anionic substrate binding site at which the quaternary nitrogen of the choline binds, and a peripheral anionic binding site which also binds quaternary nitrogen functions (8-11 and refs therein). These sites are shown schematically in Figure 2-1 along with three organic molecules which are inhibitors - 20 -FIGURE 2-1 acetylcholine P CH3-C-0-CH2-CH2-N(CH3)3 Phenyltrimethylammoni urn (PTA)  N-methylacridinium (MAC) N(CH3)3 decamethonium (CH 2) 1 Q N(CH3)3 PTA = H2N-(CH2)5-C'Y^-'N(CH3)3 0 /C H3 MAC = H 2 N - ( C H 2 ) 5 - C - N H - ^ N Fig 2-1. Binding Sites on AChE and Ligand Molecules Used During Affinity Purification. A schematic representation of the catalytically significant sites on the enzyme subunit. The distance between the sites can be deduced from the length of the molecules which span two sites but the angular orientation is unknown. The site of binding for the substrate and three inhibitor molecules is indicated. Decamethonium is used as a common eluting molecule for both PTA and MAC based affinity chromatography. Both PTA and MAC are shown in the deri-vatized form for coupling to the agarose matrix. - 21 -of the enzyme and which are used in two techniques for the affinity purification of AChE. The binding affinity for ligands at the anionic sites on the enzyme varies greatly with the ionic strength of the medium, and with the phenyltrimethylammonium affinity matrix effective retardation of AChE is obtained only in the presence of 0.1 M NaCl (30). When this molecule is used as the immobilized ligand the purification of the asymmetric forms of AChE, which aggregate under these ionic conditions, is not feasible because the aggregated forms are only poorly retarded and, furthermore, considerable proteolytic degradation occurs during dialysis to reduce the high salt concentration used for the initial solubilization of these forms of the enzyme. The N-methylacridinium derivative shown in Figure 2-1 was introduced in 1972 (31).for the purification of the asymmetric forms of AChE in the presence of high concentrations of salt. This ligand was l i t t le used during the period 1972 to 1976 due to theintractabi1ity of the original synthesis but was obtained by modifications of the published synthetic procedure by two groups in 1976 (32,33). This ligand was used routinely for the.purification of the asymmetric forms of AChE for the work described in this thesis and was also characterized with respect to the purification of the globular forms of the enzyme and the properties of the immobilized ligand in the presence of low and high salt concentrations. 2-II. Identification of Acetylcholinesterase Protein Fragments The investigation of the tail protein components of AChE is - 22 -hindered by three important considerations. Firstly, the tail protein comprises less than 20% of the total protein (see Fig. 1-5), and, in the separation and identification of the tail protein components, even a relatively small level of catalytic protein contaminant will introduce serious errors. Secondly, the tail protein cannot be identified by any specific property such as the enzymatic activity of the catalytic protein components. Finally, the tail protein cannot be specifically purified by an affinity adsorption technique as has been described for the catalytically active enzyme. The selective identification and characterization of tail pro-tein components was approached by the specific isotopic labelling of catalytic protein, to identify fragments deriving from this source, combined with the isotopic labelling of both catalytic protein and tail protein by two additional, semi-specific techniques, to permit the identification of fragments deriving only from tail protein. The specific labelling of the catalytic site of AChE is possible due to the two step mechanism by which the enzyme catalyzes the hydro-lysis of acetylcholine and the discovery of specific organophosphate compounds which irreversibly inhibit the enzyme by covalent attachment at the active site. The active site of the enzyme contains the amino acid serine which bears a primary alcohol function rendered highly nucleophilic by its juxtaposition to charged groups also present at the active site. This nucleophile attacks the carbonyl carbon of the ester function of acetylcholine, displacing choline and resulting in an acetyl-enzyme intermediate which is then hydrolysed by water. The organophosphate inhibitors of the enzyme are mixed acid anhydrides with -23-R 0 the general formula which react with the displacement of R X HX to produce a phosphoryl-enzyme product. The catalytic sequence of the enzyme and the particular organophosphate compound used to introduce 3 the H isotope to the active site are shown in Figure 2-2. a radioactive isotope of iodine and two labelling techniques previously applied to the globular form of the enzyme (34). One procedure speci-fically labels the phenol moiety of the amino acid tyrosine (35) while the second labels the amino function of lysine (36) as shown in Figure 2-3. Following radioactive labelling AChE was subjected to degradation with the proteolytic enzymes trypsin and collagenase and changes in the molecular properties of the enzyme were detected by gel chromatography, sodium dodecyl sulphate polyacrylamide-gel electrophoresis and iso-kinetic sucrose gradient centrifugation (vide infra chapter three). 2-1II. Reassociation of Purified Enzyme With Synaptic Fragments The cellular structure of the electric tissue of the electric eel, diagrammed in Figure 1-4, confers on this tissue a unique pro-perty. Under controlled conditions of homogenization in sucrose solu-tion i t is possible to cause disruption of the cells such that the innervated face and non-innervated face are obtained on separate frag-ments which can be separated. The separate fragments 'vesiculate' to form small sacs which retain many of the functions of that particular region of the cell membrane. These vesicles have been extensively characterized with respect to the functions of the proteins associated with the two faces of the electroplax cell (37-40). Similar studies have also been carried out on cell fragments obtained from the electric The semi-specific isotopic labelling of AChE protein utilized - 24 -F I G U R E 2-2 CH3-C-0-Choline OH B CH, V c^° 6 . . . . +HO-Choline OH .HB/ +CH3C00H i O H . acetyl-enzyme +HF phosphoryl-enzyme Fig 2-2. Catalytic Mechanism of Acetylcholinesterase and Active Site Labelling Procedure. A The catalytic mechanism produces the short lived acetyl-enzyme intermediate which is hydrolysed to regenerate the active enzyme. B_ The organophosphate compound, diisopropylphosphofluoridate (DipF), reacts with the serine hydroxyl at the active site and is re-sistant to attack by hydroxide. The n-tritiated compound was used to introduce a radioactive label to active site protein fragments. - 25-FIGURE 2-3 A 1 2 5 j A C h E - ^ - O H + 1 2 5 I " L a c t g P ^ r o x i d a s e > AChE (^—m 125, C H 2 - C H 2 - C — r N-hydroxy-succinimide-3-(4-hydroxyphenyl)propionate (TAGIT) 125, Chloramine-T 125 ^ HO I" -pi I25j' 125 I-TAGIT 0 125j AChE Lys-NH2 + I-TAGIT -> AChE Lys-NH-C-CH2-CH2 ^ ^ - O H 125, 'I Fig 2-3. Isotopic Labelling of Acetylcholinesterase with 1 2 5Iodine A The lactoperoxidase mediated iodination of protein bound tyrosine resiudes was introduced in 1972 (35). This procedure is specific for tyrosine residues and greatly facilitates the detection of small quantities of protein. B_ The introduction of the reagent TAGIT in 1973 (36) extended the 125 isotopic labelling of proteins with iodine to include the primary amine of the amino acid lysine. -26-tissue of a different electric fish, the electric ray (Torpedo cali- fornicus), (41) and in 1976 an account appeared of the apparent re-association of purified asymmetric AChE with these fragments (42). The lactoperoxidase mediated radioiodination technique (Fig 2-3A) has been applied to the labelling of membrane proteins as a probe to investigate the topology of membrane proteins and to detect changes in exposed portions of the proteins during treatment with proteolytic enzymes (43-46). It was presumed that the same technique could be applied to the identification of those protein components of AChE in-volved in, and shielded by, the association of AChE with membrane fragments from the electroplax cells. The generation of membrane fragments by controlled homogenization of electric tissue was developed and the lactoperoxidase mediated iodination of these fragments was in-vestigated. Little success was attained in this aspect of the work for this thesis and this approach is presented in less depth than other, more successful, approaches. 2-IV. Additional Work During the work for this thesis some attempts were made to isolate and directly identify the tail protein components of AChE. These efforts were unsuccessful and this approach was abandoned. This work is briefly described in an appendix to this thesis. Also included in the appendix are some observations made with respect to the proteo-lytic instability of purified AChE when stored for long periods in the presence of calcium ion. The following chapter introduces the experimental techniques common to the approaches described above. Detailed individual methods are given in the chapters dealing with each of the three aspects of this work. - 27 -Chapter Three BIOCHEMICAL METHODS APPLICABLE TO THE CHARACTERIZATION OF ACETYLCHOLINESTERASE FORMS The work described in this thesis utilized many techniques common to biochemical studies. Three techniques in particular were used very extensively and the data obtained were fundamental to most of the conclusions drawn. These three; Isokinetic Sucrose Gradient Sedimentation, Sepharose 4B Gel Chromatography and Sodium Dodecyl Sul-phate Polyacrylamide Gel Electrophoresis, are explained in greater detail in the following pages in order to provide a more complete under-standing of their uses and limitations for readers unfamiliar with these techniques. This detailed explanation may be omitted without detracting from the completeness of the experimental methods which follow. This chapter also describes the specific enzyme assay which was used to detect the presence of AChE and lists the sources of supply for the materials used in this work. 3-1. Ultracentrifugation and Sedimentation Analysis The measurement of macromolecular weights by the use of sedi-mentation analysis is frequently done in biochemistry. Sucrose density gradient sedimentation is a technique whereby macromolecules of undeter-mined size or shape can be characterized by comparison of their sedi-mentation behaviour with that of previously identified standard proteins. The technique cannot, in general, be used to obtain macromolecular weights directly but in combination with gel chromatography (to be - 28 -described shortly) i t can lead to reliable determinations of macro-molecular weights and can also provide information relating to the shape of the macromolecule. The ultracentrifuge was introduced to biochemistry some 40 years ago (47) and has undergone enormous improvement since that time. Two types of ultracentrifuge are available, the analytical ultracentri-fuge, which is used in the primary determination of macromolecular weights, and the preparative ultracentrifuge. This latter type is used for the preparative separation of macromolecules with distinct sedi-mentation properties, and can also be used for the secondary determina-tion of sedimentation behaviour by comparative density gradient sedi-mentation as mentioned above. Two procedures are available for the primary determination of macromolecular weights in the analytical ultracentrifuge; sedimentation velocity and sedimentation equilibrium. 3-1-1. sedimentation velocity . A macromolecule in solution rotating about an axis is subjected to three forces as shown in Figure 3-1; the centrifugal force F c = w xm, 2 - dx the buoyant force F^ = -u xvp and the frictional force F^ = -f^- where a) is the angular velocity (radians/sec), m is the mass of the macro-molycule, x is the radius of rotation, p is the density of the solution, v is the partial specific volume, f the frictional coefficient and dx the velocity of the macromolecule. The macromolecule will accelerate until the sum of the forces is zero: - 29 -and the sedimentation equation on a.mole basis can be derived (48). - ( A ) - 1 Ai - s ( ID In this form the macromolecular parameters are placed on the left and experimentally measured quantities on the right. M is the macromolecular weight and N is Avogadro's number. dx The velocity (^r) divided by the centrifugal field strength (w x) is called the sedimentation coefficient, s and, as shown in equation (II), i t is proportional to the molecular weight and density of the macromolecule and inversely proportional to the friction coefficient. -13 The units of s are seconds and the quantity 1 x 10 sec is denoted by the symbol S (One Svedberg unit). The velocity of the macromolecules is obtained from the move-ment of the concentration boundary which forms as the macromolecules migrate away from the meniscus of the rotating solution (Fig. 3-2). The right half of equation (II) can be arranged to the form: 2 2^ 303 = X ^ a n c * the sedimentation coefficient obtained by plotting log x^ . versus time t where x t is the radial distance to the boundary (48,49). The boundary blurs with time due to diffusion and this blurring sets a lower limit on the range of molecular sizes that can be deter-mined by this method. It is evident from equation (II) that the sedimentation co-efficient alone does not give sufficient information for the determination - 30 -FIGURE 3-1 AXIS BOTTOM Figure 3-1 Diagram of a sedimentation experiment. A sector shaped cell of the type used in an analytical centri-fuge is shown. - 31 -FIGURE 3-2 A AXIS moving mirror-meniscus m mi o v i n g \ irror %k light source s e c t o r shaped cell in side v iew •^detector t a> o c o _ Q o (/) JD D B t a; o c o -DI i_ O l/) JD a A boundary A x Fig 3-2. meniscus meniscus Detection of boundary formation and movement in the analytical ultracentrifuge. The rotating cell has transparent sides and moving mirrors are used to scan the length of the cell at an appropriate wave length (j_e 280 nm). The absorbance of the cell contents is plotted versus distance. Immediately after starting the centrifuge the macromolecules are evenly distributed. As sedimentation occurs the region near the meniscus becomes depleted in macromolecules and the concentration increases near the bottom (The dotted line depicts the final condition in a sedimentation Equilibrium experiment, (see Text)). - 32 -of molecular weights. Independent measurements are required to deter-mine the partial molal volume and the diffusion coefficient from which the frictional coefficient is derived. 3-1-11. sedimentation equilibrium This procedure for the determination of macromolecular weights in the ultracentrifuge does not require the measurement of diffusion coefficients and can also be used for the determination of molecular weights of a smaller size than is possible using sedimentation velocity. Prolonged centrifugation under the conditions described for Figure 3-2 eventually results in an equilibrium distribution of the macromolecules (Fig 3-2C) in which the concentration varies as a function of the molecular weight, the angular rotation squared, the solution density, the partial molal volume of the molecule and the radial distance from the axis of rotation as shown in equation (IV); r - r /U) 2M(l-v P)(x 2-a 2)v , n n ° ( x ) " C(a)exp( 2RT } ( I V ) where a is the radial distance to the meniscus, C\ is the concentration a at the meniscus, Cv is the concentration at a radial distance x, R is A the gas constant and T is the absolute temperature (48). Equation (IV) can be rearranged to eq. (V) where M is shown to be proportional to 2 the slope of a graph of log C versus x with the only unknown value A in the proportionality being the partial molal volume, when p is assumed to be constant (49). - 33 -M = 4.606 RT d(log C) ( v ) (l-vp)a>2 d(x2) In the case of pure samples for which the partial molal volume is known the measurement of macromolecular weights by sedimentation equilibrium is surpassed in accuracy only by direct techniques based upon the chemical composition of the macromolecule (i.e. knowledge of the precise amino acid composition in the case of proteins)(48). 3-1-111. density gradient sedimentation Both the analytical and preparative ultracentrifuges can be used in density gradient sedimentation analysis of macromolecules. Two procedures can again be used, namely, velocity sedimentation and equilibrium sedimentation, but in the case of density gradient sedi-mentation analysis each of the two techniques yields entirely in-dependent information on macromolecular characteristics. The analytical ultracentrifuge is more suited to the equili-brium procedure while the preparative ultracentrifuge has greater uti l ity in sedimentation velocity determinations. 3-1-IV. density gradient sedimentation equilibrium As described above, prolonged centrifugation of a solution results in an equilibrium distribution of solute where the concentra-tion of the solute and the density of the solution vary with radial distance in a definable manner (eq. IV). This equilibrium state is reached only slowly with small molecules but can be accelerated by pre-forming a linear gradient prior to centrifugation. When a macro-- 34 -molecule is introduced at low concentration into a solution containing a high concentration of small molecules, and a density gradient is generated by centrifugation, the macromolecule will migrate to that point (xQ) in the gradient at which the local density of the solution p(xQ) is equal to ( v ) ~ ^ where (v) is the specific volume of the macro-molecule (Fig 3-3). While this method may be used for the determination of molecular weights its greatest uti l i ty is found in the measurement of density ratios. Clear resolution of nucleic acids differing only in that one contained and the other has been demonstrated (50). When proteins are analysed by density gradient sedimentation i t is necessary to use cesium chloride to obtain suitable density ranges and high concentrations of salt are present in the solution containing the macromolecule. The molecular weights and specific volumes obtained by this method are for the solvated macromolecule which may bear l i t t l e resemblance to the form of the macromolecule under more natural conditions. 3-I-V. density gradient sedimentation velocity The principle of this method is the same as the sedimentation velocity method described earlier, however, the preparative ultra-centrifuge may be used (48,49). The macromolecules are present init ia l ly in a thin band at the meniscus of the rotating column of solution and the migration of this band is measured (Fig. 3-4). The purpose of the density gradient is to stabilize the migrating band; the gradient is pre-formed using a small molecule such as sucrose and the solution of macromolecule is - 35 -FIGURE 3 -3 CD O C a o if) _ Q a / / / / / / / / / / / / / 1 A I I c o o (/) o >> CO c CD meniscus Fig 3-3. Final state in density gradient sedimentation equilibrium. The small molecules have formed a density gradient and the macro-molecules, detected by their absorbance, are distributed in a Gaussian band centered on the point xQ in the gradient with a density p(xQ) equal to ( v ) ~ ^ as described in text. FIGURE 3-4 AXIS B "AXIS 36 -small band of so lut ion containing macromolecules -top of gradient m a c r o m o l e c u l e s have m i g r a t e d Fig 3-4. Density Gradient Sedimentation Velocity. The parallel sided plastic tube used in the preparative ultracentri-fuge is shown. The gradient is pre-formed in the tube in a vertical position and the solution of macromolecules is layered on top. The plastic tube'! .is placed inside a metal carrier which hangs vertically in the centrifuge and swings up to a horizontal position when centri-fugation is started. A Initial position of macromolecules immediately after starting centri-fuge. B_ Position of macromolecules following centrifugation for some time. - 37 -layered on top of the sucrose gradient prior to centrifugation. The solution density of the macromolecular solution is less than the solution density of the top of the sucrose gradient but the density of the macromolecules is greater than the solution density at any point in the gradient. The velocity of migration can be used to deter-mine the molecular weight of the macromolecule but complications arise from the changes in density and viscosity of the bulk solution through which the macromolecule is migrating. In addition, when the preparative centrifuge is used, direct observation of the macromolecule is not possible and migration can only be measured at discrete intervals by stopping the centrifuge. Direct determination of sedimentation co-efficients using this technique is feasible but the least complicated use is the comparative determination of sedimentation coefficients using macromolecules of known partial specific volume and sedimentation co-efficient as reference standards (51). 3-I-VI. isokinetic sucrose gradient sedimentation In the previous discussion the sucrose gradient was a linear increase in percent (w/v) concentration with radial distance. Both the density and viscosity of the solution increase in a non-linear manner with increasing sucrose concentration. Therefore the buoyant force and frictional force affecting a macromolecule increase non-linearly with the distance sedimented. The centrifugal force increases linearly and the sedimentation velocity decreases with increasing distance of migration. When comparative determination of sedimentation coefficients is carried out cumbersome computations are required to - 38 -correct for the non-linear effects unless standard macromolecules are found which migrate closely to the unknown macromolecule. The isokinetic sucrose gradient was developed to solve the problems caused by variations in sedimentation velocity on linear sucrose gradients. The theory for this gradient was derived from a combination of equations describing the sedimentation behaviour of a macromolecule in a combined gradient of density and viscosity with functions relating the density and viscosity to the percent concentra-tion of the solution (52). The combination can be reduced not to an equation yielding the concentration as a function of radius but rather an expression (eq. (VI)) which defines the radius at which solutions of different density and viscosity should rotate to produce identical sedimentation velocity for the same macromolecule in each solution. x = x t (P p - P t ) [ n t ( P p - P m ( x ) ) ] _ 1 nm(x) (VI) radius to the meniscus density of the macromolecule density at the meniscus density at a radius = x viscosity at a radius = x viscosity at the meniscus The radius to the meniscus is determined by the particular ultracentri-fuge that is used and the density of the macromolecule is generally where: x t is the pp is the p t is the pm.(x) is the \(x) i s the n+ is the - 39 -taken as the average density of the standard macromolecules. The sucrose concentration at the meniscus, from which the density and viscosity are obtained by reference to standard tables (53), is ar-bitrarily chosen. With these initial conditions i t is possible to calculate a series of radii for different sucrose concentrations and, using the cross-sectional area of the centrifuge tube, graph the form of the gradient in concentration versus volume as shown in Fig 3-5. The gradient exhibits a convex curvature and can be closely approximated by equation (VII) (solid line, Fig 3-5) which describes the sucrose concentration :CV as an exponential function of the volume V. Cv =C R - (CR - C t) exp " V / V m (VII) Concentration gradients described by equation (VII) are readily pre-formed using the device shown in Fig 3-6 in which an initial volume Vm of solution with concentration C^ is rapidly stirred while a solu-tion with concentration CR is added from a reservoir at the same rate as the stirred solution is removed from the mixing device to the centri-fuge tube. Centrifugation is performed exactly as depicted in Fig 3-4 for linear sucrose gradients and after centrifugation the sucrose gradient in the centrifuge tube is fractionated as shown in Fig 3-7. Each fraction contains an increment of the gradient volume and plotting macromolecule concentration per fraction versus fraction number is equivalent to plotting macromolecule concentration as a function of radial distance. - 40 -FIGURE 3-5 24i VOLUME (ml) Fig 3-5. Form of isokinetic sucrose gradient and exponential approxi-mation. The circles indicate actual values calculated by eqn (VI) using Ct=10%, xt=7.0 cm, and p^=1.343 for a centrifuge tube with a cross-sectional area of 1.60 cm . The line indicates the exponential gradient calculated by eqn (VII) using 0^ =10%, CD=29.3% and Vm=11.7 ml. FIGURE 3 -6 - 41 cent rif uge tube V, m / pumpA^ mixing chamber reservoir Fig 3-6. Pre-forming an exponential gradient to approximate an iso-kinetic gradient. The mixing chamber initially contains a volume Vm of concentration Ct, two pumps are used to add solution and remove solution at the same rate from the mixing chamber. The sucrose gradient is added to the centrifuge tube starting with the lowest concentration which is displaced upwards as the concentration increases. Addition is stopped when the meniscus reaches a position in the tube corresponding to the desired radius x.. 42 -Figure 3-7. Fractionation of sucrose gradient and location of macro-molecules . A The centrifuge tube is removed from the metal carrier tube described in Figure 4 and a thin glass capillary is inserted to the bottom of the tube. The sucrose solution is pumped out through a tube attached to the capillary and is collected in fraction tubes. B_ The original gradient solution is distributed equally among 25 or more fraction tubes and the location of the macromolecule is detected by a specific enzyme assay, or by chemically attached radioactive isotopes or by a specific absorbance in the spectrophotometer. fj The results of the assay or other detection procedure in B_ are plotted versus fraction number. Similar graphs are made for standard macromolecules and the the known sedimentation coefficients are then graphed versus the fraction number corresponding to the center ot the distribution of protein and unknown sedimentation coefficients obtained by interpolation on this graph. - 43 FIGURE 3-7 _ _ X n t o p of g r a d i e n t macro -molecules f ina l p o s i -t i o n o f r a c t i o n tube centrifuge tube 1 2 3 4 5 i CD c o u -+-' CD 2 o C CD O c o o E o o a E X f r a c t i o n number - 44 -3-I-VII. reliability of data obtained by sucrose gradient sedimentation During al l of the preceding explanation and discussion the effect of solution conditions upon macromolecular properties and the effect of macromolecular interactions has not been considered. In the determination of molecular weights by sedimentation velocity or equili-brium in the analytical ultracentrifuge the measurements are taken at different macromolecular concentrations and extrapolated to zero con-centration. In addition the values reported in standard references (53) are corrected to the standard solution condition of water at 20° . When comparative properties are measured using sucrose gradient sedimentation the implicit assumption is that the sedimentation behaviour of both standard and unknown macromolecules is affected equally by changes in sucrose concentration. Variations in partial molal volume between standard and unknown macromolecules are not serious. 3 Most proteins have a partial molal volume between 0.700 cm /g and 3 0.750 cm /g and i t has been shown that using an average value of 3 0.725 cm /g introduces an error not greater than 3% (51). This error is less than the minimum error inherent in determining the location of the different proteins in the sucrose gradient when the gradient is separated into 25 fractions. Collecting a greater number of fractions is not in general feasible as this reduces the individual fraction size and there is then insufficient solution to perform replicate assays to precisely locate the fractions containing particular proteins. In the work described in this thesis the implicit assumption described above was extended to include the composition of the buffer solution which is required to maintain acetylcholinesterase in a - 45 -non-aggregated form. Sucrose gradients were prepared in 1M NaCl and standard proteins were dissolved in a like solution before addition to the acetylcholinesterase sample. The assumption was made that the standard macromolecules maintained their normal conformation in the presence of this salt concentration. The validity of the assumption was supported in some experiments involving a modified form of acetyl-cholinesterase which could be centrifuged in a sucrose gradient pre-pared in 'OilM NaCl without aggregation. The relative positions of the standard proteins and these forms of acetylcholinesterase in the gradient fractions were similar for centrifugation in the presence of either 1M NaCl or 0.1M NaCl, suggesting that alterations, i f any, in sedimentation properties, were similar for all proteins. 3-11. Gel Chromatography This technique, as mentioned in the introduction to centri-fugation techniques, is complementary to sucrose gradient sedimentation and a combination of data obtained by the two methods can yield in-formation on macromolecular properties that is not accessible by each technique separately. In gel chromatography a long column is f i l led with the chromato-graphic material which consists of a porous polymer in the form of small beads, and the sample being chromatographed is passed through the column in a small band occupying less than one percent of the total volume of the column. The most commonly used polymers are polysaccharides such as dextran or agar and the work described in this thesis utilized the agar based material Sepharose 4B obtained from the Pharmacia company. - 46 -3-11-1. theory of gel chromatography The mechanism of gel chromatography is not well defined; a distinction (54) is made between what can be described as gel filtration and gel chromatography as shown in Figure 3-8. The process taking place during chromatography on Sepharose 4B is probably a combination between gel chromatography, which is the differential partitioning of molecules between the moving liquid within the column and the stationary interior of the polymer beads, and gel f i ltration, which is the situation when the molecules migrate at different rates through the porous mesh f i l l ing the column and no freely moving liquid exists (Fig 3-8CJ. Theoretical treatment of results obtained by gel chromatography is limited due to the undefined nature of the interior of thepolymer beads (55) but an empirical distribution coefficient (see Fig 3-8) can be defined to characterize the interaction between the molecular species passing through the column and the column material (56, 57). The interpreta-tion of the distribution coefficient in terms of molecular processes must provide some basis for differential retardation of different sized molecules. One explanation postulates the existence of microregions of inaccessible volume ('pore' volume) such that equilibrium partitioning between the interior of the polymer bead and the freely flowing solu-tion depends on the volume of the region accessible to a particular molecule. This situation strictly applies only during gel chromato-graphy (Fig 3-8A) and a more general interpretation defines an 'effective pore size' which is then used to relate the distribution coefficient to the Stokes radius of the macromolecule (57). The effective pore size is calculated from the distribution coefficients for molecules of - 47 -FIGURF 3-8 A B Fig 3-8. Diagram Illustrating the Distinction Between Gel Chromato-A Gel chromatography; the molecules can partition between the stationary interior of the polymer beads and the freely flowing solution in channels between beads B Partial restriction of channels between beads resulting in combined chromatography and filtration. (Probable condition of "Gel Chromato-graphy" see text) C_ No channels between beads, the molecules are filtered through the porous structure of the polymer beads, (cf. the description of SDS-"PAGE which follows) An empirical distribution coefficient Kn can be defined as: where Vo is the volume external to the polymer beads ('channel1 volume), Vi is the volume internal to the polymer beads ('pore' volume), and Ve is the volume accessible to a particular molecule. The volume Vo is measured as the void volume accessible to a very large molecule which is totally excluded from the beads and Vi is the included volume accessible to a very small molecule minus the volume Vo. graphy and Gel Filtration. = Ve - Vo Vi (D - 48 -known Stokes radius. Several methods exist to graphically portray the relationship between the distribution coefficient obtained during chromatography and the Stokes radius of a macromolecule in a manner which produces a straight line for easy interpolation in comparative determinations (58). The method used in published work on acetylcholinesterase was retained for the work described in this thesis in order to maintain the validity of comparisons (17). 3-II-II. reliability of data obtained by Sepharose 4B Gel chromatography Gel chromatography yields comparative values for the Stokes radius of macromolecules and cannot be used to determine absolute values. The precision of the method depends primarily upon careful manipulation of the equipment used and may be quite high when replicates are averaged and standard deviations obtained. The accuracy of Stokes radius data obtained by gel chromatography is dependent on the accuracy of the in-formation on the molecules used as standards and on the assumption that molecules of different composition and size are behaving in a similar manner during chromatography. This' latter point can be judged from the scatter of the data points in the graph correlating the distribu-tion coefficient and Stokes radius for a group of molecules, Fig 3-9 shows the graph obtained for 5 proteins of widely different Stokes radius; serious deviations from the norm are readily apparent. 3-1II. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis  (SDS-PAGE) This technique, in contrast to the chromatographic and - 49 -F I G U R E 3 - 9 myosin^ • f i b r i n o g e n • / 2 - g a l a c t o s i d a s e • c a t a l a s e bovine se rum albumin U 0 0!1 02 0.3 O'M 0.5 0.6 07 0:8 0.9 ( - l o g K DJ ; 2 Fig 3-9. Correlation of Stokes radius with distribution coefficient. The distribution coefficient, KD calculated as described in the text was correlated with the Stokes radius (Re) by plotting (-log K) 1 / / 2 versus Re for the standard proteins listed in Table 3-1. - 50 -centrifugation procedures described earlier, can be used to determine individual molecular weights for protein subunits. The method is analogous to gel filtration in that the molecules are caused to migrate electrophoretically in the form of a narrow band through the porous structure of the polymer formed with acrylamide and N,N'-methylene bisacrylamide. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) originated with the observation that proteins electrophoresed on a polyacrylamide support in the presence of sodium dodecyl sulphate (SDS) migrated at a rate proportional to the logarithm of the molecular weight of the polypeptide chains (59). This initial observation was confirmed and extended to show that a smooth curve could be drawn con-necting points on a graph of log MW versus migration for polypeptide chains with molecular weights ranging from 11,000 to 200,000 (60). The apparent molecular weight for an unknown polypeptide chain could be obtained by interpolation on the curve, in addition it was possible to identify covalent disulphide bonds between individual polypeptide chains by performing SDS-PAGE with or without prior reduction of these bonds. 3-1II-I. theory of SDS-PAGE In conventional electrophoresis protein molecules migrate in an electric field at a rate predominantly dependent on the ratio of the net charge to the mass of the molecule and separation occurs between molecules with different charge to mass ratios. In the presence of SDS however all proteins bind a constant amount of SDS per unit weight and the resulting complexes have essentially identical charge to mass ratios - 51 -due to the large negative charge component of the bound SDS (61,62). In this condition all proteins would have similar rates of electro-phoretic mobility and separation of different sized molecules during conventional electrophoresis would not occur. The separation that does occur during SDS-PAGE is due to the sieving action of the porous polyacrylamide gel and it is this sieving action that provides the parallel with the mechanism of gel filtration (63). The porosity may be varied by polymerizing gels at different concentrations of the monomer acrylamide so that the rate of migration for a protein-SDS complex can be determined as a function of porosity and extrapolated to zero polyacrylamide concentration to obtain the apparent free electro-phoretic mobility (64). From this a retardation coefficient can be obtained, which in some cases is directly proportional to molecular weight and can be more generally related the effective radius or Stokes radius of the protein-SDS complex (61,62,65,66). The smooth curve observed in the graph of log MW versus migration referred to above occurs at molecular weights greater than 70,000 and indicates that for protein-SDS complexes with molecular weights greater than this size the retardation coefficient is not proportional to molecular weight (65).. The effective radius of the complex is reduced due to the migra-ting complexes orienting in a manner which minimizes their frictional resistance to the gel (67). 3 - I I I r I I . reliability of data obtained by SDS-PAGE The validity of molecular weights obtained by SDS-PAGE is only assured i f both unknown and known proteins have similar rates of - 52 -free electrophoretic mobility (65). Several examples are known of proteins that migrate anomalously during SDS-PAGE and a rigorous deter-mination of molecular weight using this technique requires the measure-ment of both the free electrophoretic mobility and the retardation co-efficient (64 and refs therein). During the work described in this thesis such a rigorous determination was not done, the reason for this being twofold. Firstly, other workers had shown that the subunit mole-cular weight for acetylcholinesterase obtained from SDS-PAGE was closely comparable to the subunit molecular weight obtained by direct techniques (11) and secondly, the use of SDS-PAGE in this thesis was more concerned with the relative sizes of particular polypeptide fragments derived from the subunits of acetylcholinesterase than with the precise mole-cular weight of the fragments. 3-IV. Methods 3-IV-I. isokinetic sucrose gradient sedimentation The sedimentation coefficients for different forms of AChE were determined on isokinetic sucrose gradients in the presence of 1M NaCl, 20mM sodium phosphate pH 7.0. The form of the gradients was calculated by a published procedure (52) for the Beckman SW 41 swinging bucket rotor and had a meniscus concentration of 10% (w/v) and a radial distance to the top of the gradient of 7.3 cm. The gradients were approximated by an exponential gradient generated with a mixing volume of 70.1 ml and a reservoir concentration of 29.3% using a multichannel peristaltic pump to form 6 gradients simultaneously (Fig 3-6). Gradients were cooled to 4° and protein samples were carefully layered on top - 53 -and centrifuged at 4° in a Beckman L3-50 refrigerated ultracentrifuge for 19 h at 40,000 rpm. Following centrifugation the gradients were fractionated (Fig 3-7) using a modified fraction collector and a peri-staltic pump to collect fractions from three separate gradients simul-taneously (68). Samples for centrifugation contained acetylcholinesterase either alone or in combination with one or more of the four standards; 3-galactosidase, catalase, lactoperoxidase and myoglobin. The molecular weights, sedimentation coefficients and Stokes radii for the standard proteins are listed in Table 3-1. Catalase, lactoperoxidase and myo-globin were all located in gradient fractions by their absorbance at 405 nm while 3-galactosidase was detected by a specific enzyme assay (15). Acetylcholinesterase was either detected by its enzymatic activity 3 or by the presence of specifically attached H at the active site of the enzyme. Before fractionation of gradients containing AChE, 0.10 ml of a 1 mg/ml solution of bovine serum albumin in the buffer described above was added to the fraction tubes to prevent loss of AChE enzyme activity during storage of the fractions at 4 ° . 3-IV-II. Sepharose 4B gel chromatography All chromatography operations were carried out in a cold room at 4 ° . A chromatography column 100 cm long and 0.8 cm internal diameter was f i l led with Sepharose 4B suspended in 1M NaCl, 20 mM sodium phos-phate pH 7.0 and packed at a flow of ca_ 8 ml/h until the chromatographic material had settled to a stable level with a useful column length of 96 cm. The top of the gel was protected from disturbance by inserting Table 3-1. Data for Standard Proteins Used in Sucrose Gradient Sedimentation (a), Gel Filtration (b) and Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (c). Protein Sedimentation Coefficient (S) Stokes Radius (nm) V Monomer Molecular Weight (daltons) Multimer Molecular Weight (daltons) Aldolase0 — — — 40,000 — Bovine Serum Albumin'3 4.36 3.5 0.73 65,000 — Catalase a ' b ' c 11.3 5.2 0.730 60,000 247,500 Fibrinogen 7.9 10.7 0.71 — 330,000 B-Galactosidase a'k' c 15.9 8.2 0.76 130,000 540,000 Glyceraldehyde-3-phosphate-dehydrogenasec — — — 36,000 — Lactoperoxidase3 5.37 — — — — Myoglobin3'0 2.04 — 17,200 — Myosin 6.4 21.5 0.74 — 493,000 Phosphorylase a — — — 94,000 — (Catalase)cc — — — 120,000d — (Catalase)3c — — — 180,000d — (Catalase)4c — — — 240,000d — This protein was reacted with dimethylsuberimidate using a published procedure (69) to obtain covalent cross-links between E primary amines of the amino acid lysine in adjacent subunits of the multimeric form. eData from refs 17, 21 and 53. - 55 -a firmly fitting disc of porous polyproplylene 1.5 mm thick into the column and pushing i t down into contact with the gel. Sucrose was added to samples for chromatography to a final concentration of ca_ 5% and the chromatography column was loaded by carefully layering 0.6 ml of sample on the plastic disc with a hypodermic syringe without removing the buffer above the column material. The top of the column was then fitted and elution commenced at a flow of 6 ml/h. Eighty fractions with a volume of 2 ml were collected. The void volume, Vo, (Fig 3-8) was obtained from the position of elution of the dextran polymer Blue Dextran 2000 and the included 3-volume (Fig 3-8) obtained with the small ion Fe(CN)g . The column was calibrated with the standard molecules given in Table 3-J and also with known forms of AChE (21). Blue Dextran 2000 was located in column 3-fractions by its absorbance at 650 nm, Fe(CN)g by the absorbance at 412 nm, catalase at 405 nm and fibrinogen and myosin by the absorbance at 280 nm. ..Bovine serum albumin was located by labelling the protein with 125 iodine (35) and determining radioactivity in the column fractions. 3-galactosidase was located by a specific enzyme assay (15). Acetyl-cholinesterase was located by its enzymatic activity or by the presence 3 of specifically attached H at the active site of the enzyme. When fractions were collected during chromatography of AChE 0.20 ml of a 1 mg/ml solution of bovine serum albumin in the buffer described above was added to the fraction tubes prior chromatography. 3-1V-111. sodium dodecyl sulphate-polyacrylamide gel electrophoresis The procedure used was based upon a published method with - 56 -minor modifications (60). The stock solutions listed in Table 3-11 were prepared and stored as noted. Polymerization of gels was done in glass tubes 12 cm long and 0.4 cm internal diameter inserted v vertically into soft rubber stoppers to seal the bottom end of the tube. The gel mixture consisted of 9.0 ml of solution I, 2.0 ml of solution II, 20.0 ml of solution III and 9.0 ml of h^ O to give a final acrylamide concentration of 5%. This mixture was degassed under partial vacuum with gentle swirling for 5 minutes. Polymerization was initiated by adding 30 yl of N,N,N' ,N-'-tetramethylethylenediamine mixed by gentle swirling and the glass tubes were f i l led to a height of 10 cm using a plastic hypodermic syringe with a long needle. During f i l l ing of the glass tubes the tip of the hypodermic needle was kept immersed below the level of the polymerization mixture to reduce the re-introduction of air to the degassed mixture and immediately after f i l l ing the glass tubes a small amount of water was layered carefully over the solution to prevent formation of a curved meniscus and to exclude oxygen which inhibits polymerization. The f i l led tubes were left undisturbed for a minimum of 6 hours before use to permit complete polymerization to take place. Protein samples for SDS-PAGE were dialysed against 200-1000 volumes of solution IV and in the case of AChE samples were concentrated by ultrafiltration on a f i l ter membrane with a retention of solute greater than 25,000 daltons mw. Alternatively some AChE samples were dialysed against solution IV which had been diluted tenfold and following this dialysis these samples were lyophilized and re-dissolved in one tenth of their original volume in solution IV. The final protein - 57 -concentration obtained during either concentration procedure was ca_ 2 mg/ml. Following dialysis (and concentration for AChE samples) the protein was denatured by adding an equal volume of solution V\(or solution V containing dithbthreitol for reduction of disulphide bonds) and heating to 100° for five minutes. The glass tubes containing the polymerized gels were removed from the rubber stoppers and the top face of the gel rinsed with water and the tubes inserted through rubber seals in the bottom of a plexiglas tank and solution IV added to both tanks to totally immerse the tops and bottoms of the tubes (Fig 3-10). A cathode was immersed in the upper tank and an anode in the lower tank. In some cases a power supply was connected to the electrodes immediately and the electric field was applied with a current flow of 2 mA per tube to electrophoretically remove the ammonium persulfate remaining from the polymerization reaction. No difference was observed in SDS-PAGE results when this step was omitted during the latter part of this work. The denatured (and reduced) protein samples were cooled and 20 - 100 yl was carefully layered on top of the gel beneath solution IV. (Fig 3-10) A power supply was then connected to the electrodes and electrophoresis performed at a current flow of 5 mA per tube. The progress of electrophoresis was monitored by the migration of the tracking dye Pyronin Y and was terminated when the dye had traversed 95% of the gel length. When electrophoresis was complete the glass tube was removed from the tank assembly and the polyacrylamide gel was released from the tube by introducing water between the gel and the wall of the tube FIGURE 3-10 - 58 -upper tank containing solution IV rubber seal^ lower tank containing solutionlV sample layered on polyacryl-amide gel -cathode -glass tube containing polyacrylamide gel -anode Fig 3-10. Diagram of SDS-PAGE Apparatus Showing Location of Electrodes and Sample Layered on Gel Immiedately Prior to Electrophoresis. The equipment used had the capacity for holding twelve glass tubes containing polyacrylamide in the manner shown. The electrodes consisted of loops of platinum wire around the periphery of the tanks. The sample is applied to the top of the polyacrylamide gel after the upper tank has been f i l led with solution IV. - 59 -Table 3-II. Stock Solutions for SDS-PAGE and Storage Conditions for Solutions Solution Composition Storage Conditions I 22.2 g acrylamide 0.6 g N,N'-methylenebisacrylamide H20 to 100 ml Sealed container in dark at 4 ° . II 30 mg ammonium persulfate H20 to 2 ml Made fresh before use III 0.2 M sodium phosphate pH 7.2 0.2% sodium dodecyl sulphate Sealed container at room temperature, used within 10 days IV 1 volume of solution III 1 volume H20 Made fresh before use V 0.01 M sodium phosphate pH 7.2 1% sodium dodecyl sulphate Sealed container at 4 ° , warmed to room (V only 40mM dithiothreitol) 10% sucrose 0.002% pyronin Y temperature before use VI 45% methanol 10% glacial acetic acid 45% H20 0.25% coomassie blue Sealed container at room temperature VII 5% methanol 7.5% glacial acetic acid 87.5% H20 Sealed container at room temperature - 60 -using a fine hypodermic needle. The position of the tracking dye was marked by inserting an ink soaked hypodermic needle into the gel at the leading edge of the dye and the gel was placed in a tube fi l led with solution VI for staining protein bands. Staining was carried out for 24 hours at room temperature or 5 hours at 50° following which the gels were placed in a tube containing solution VII for destaining at room temperature. The destaining solution was replaced daily for several days until the background of the polyacrylamide gels between protein bands was clear of Coomassie blue dye. Densitometric profiles of the stained gels were obtained at 550 nm using the gel scanning attachment for the Gilford 2400 spectro-photometer. The migration of polypeptide fragments was measured from the densitometric profiles and the relative migration calculated with reference to the migration of the tracking dye (Fig 3-11). A standard curve of log MW versus relative migration was constructed using the proteins of known molecular weight given in Table 3-1. The apparent molecular weights for acetylcholinesterase polypeptide fragments were obtained by interpolation on this curve. Gels containing isotopically labelled AChE were cut laterally into 1.5 mm slices, individual slices incubated overnight at ca^  50° in 1 ml of a 9:1 mixture of NCS tissue solubilizer: h^O, 15 ml of Scintiverse liquid scintillation cocktail was added and radioactivity determined in a Nuclear Chicago Mk II liquid scintillation counter. 3 H was counted on the internal settings of the liquid scintillation counter and the counting efficiency was monitored by the external 125 channels ratio method. I was counted on the manual settings of the - 61 -F I G U R E 3 - 1 1 polyacrylamide g e l top bottom d i s t a n c e (x) Fig. 3-11. Diagram of a Polyacrylamide Gel Following Staining of Protein Bands and Sketch of Densitometric Profile Obtained for Gel.. The migration of SDS-protein complexes relative to the tracking dye was calculated from the distance between the top of the gel and the ink trace showing the tracking dye position and the distance between the top of the gel and the top of the absorbance peak in the densitometric profile x_ (Rf = Y~ ' s o m e experiments described in the text the migration of dye different protein fragments was calculated with reference to each other, x not to the tracking dye position; ie_ migration p^  relative to p-, = — v" 3 .) - bZ -liquid scintillation counter optimized for counting this isotope. 3-IV-IV. acetylcholinesterase assay Spectrophotometric assays for AChE activity were done by a published method (70) using the substrate analogue acetylthiocholine. Assays were performed in a 1 cm path cuvette in a Zeiss PM QII spectro-photometer at room temperature in a final volume of 3.00 ml of 0.1 M sodium phosphate pH 8.0 containing 0.6 mM acetylthiocholine, 0.3 mM 5,5'-dithiobis(2-nitrobenzoic acid) and 50 - 100 yl of AChE in 1 M NaCl, 20 mM sodium phosphate pH 7.0 (Buffer H). The rate of hydrolysis was followed by the increase in absorbance at 412 nm due to the thiol ate anion, 5-thio-2-nitrobenzoic acid, produced from the reaction of the enzymatic hydrolysis product, thiocholine, with the disulphide com-pound. Acetylcholinesterase solutions were diluted to obtain a linear rate of hydrolysis for approximately 2 minutes. Enzyme activity is reported as units = khn-in min - 1 or as specific activity = ;ymol n m -1 -1 acetylthiocholine hydrolyzed min mg , where the protein concentra-tion (mg/ml) for the AChE solution was determined using the published 1 °l value e2go = 18.0 (10) and the rate of hydrolysis for acetylthio- . nm choline was calculated from the extinction coefficient, z,-,0 = the '^^ nm T.36 x 10 M - 1 cm"1, for/thiolate anion at pH 8.0 (70). 3^V. Materials 3-V-I. biological materials Electric eels, Electrophorus electricus, were obtained live from World Wide Scientific Animals, Ardsley, N.Y., and maintained at - 63 -the Vancouver Public Aquarium until used. Trypsin and collagenase (CLSPA) were obtained from the worthington Biochemical Corporation, Phosphorylase a_ was from Boehringer Mannheim and rabbit muscle myosin was the gift of I. E. P. Taylor, Department of Botany, University, of British Columbia. All other biochemicals were obtained from the Sigma Chemical Company. 3-V-II. other materials Polyacrylamide gel reagents, Pyronin Y, 5,5'-dithiobis-(2-nitrobenzoic acid), cyanogen bromide and 2,4,6-trinitrobenzensul-phonic acid were obtained from the Eastman Kodak Company, Acetylthio-choline chloride, decamethonium bromide, o-nitrophenyl-B-D-galacto-pyranoside, hydroxyproline, glycine, sodium dodecyl sulphate, dithio-threitol, ouabain, N-chloro-p-toluenesulphonamide sodium salt (Chlora-mine T), adenosine triphosphate, soybean trypsin inhibitor, p-toluene-sulphonyl-L-arginine-methyl ester hydrochloride, p-dimethylaminobenzal-dehyde, ethylenediamine-tetraacetic acid disodium salt, guanidine hydro-chloride, tris(hydroxymethyl)aminomethane and Coomassie Brillant Blue R were obtained from the Sigma Chemical Company. N-hydroxysuccinimide-3-(4-hydroxyphenyl)propionate (TAGIT) was obtained from Calbiochem and 3 fluroescamine from Hoffman-La Roche Limited. H-Diisopropylphospho-125 fluoridate and carrier free sodium iodide were from New England Nuclear and NCS tissue solubilizer was a product of the Amersham Corporation. Sepharose 2B, Sepharose 4B, Sephadex G25 and Blue Dextran 2000 were products of pharmacia Fine Chemicals. Sucrose, dialysis membrane and Scintiverse liquid scintillation cocktail were from the - 64 -Fisher Scientific Company. All inorganic chemicals and all solvents were obtained locally and were the best grade available. Water was filtered through a 0.2 urn Ulttpor f i l ter from the Pall Corporation and deionized on Bion Exchangers from the Pierce Chemical Company. The N-methylacridinium derivative, N-methyl-9-[N6-(6-amino-hexanoyl)-3-aminopropylamino]acridinium dibromide, for coupling to Sepharose 2B was synthesized by D. G. Clark as described elsehwere (33). 3-V-III. further purification of collagenase The collagenase was further purified by a published procedure (71) in the laboratory of P. Bornstein, Department of Biochemistry, University of Washington and was the kind gift of Dr. Bornstein. - 65 -OPTIMIZATION OF THE CONDITIONS FOR PURIFICATION OF THE ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE BY COLUMN CHROMATOGRAPHY ON AN AFFINITY MATRIX CONTAINING THE N-METHYLACRIDINIUM LIGAND. 4-1. Introduction This chapter presents the conditions for optimum purification of the asymmetric forms of AChE and demonstrates that the N-methylacri-dinium based affinity purification procedure can be applied to the purification of the globular form of the enzyme from proteolytic digests. In addition i t shows that under the appropriate conditions the selective purification of either form of the enzyme from mixtures of the globular and asymmetric species is possible. 4-11. Methods 4-11-1, high salt extraction of AChE from electric tissue Electric eels were killed by packing them in crushed ice for 30 min and the electric tissue of the main electric organ was removed by blunt dissection. Electric tissue not used immediately was frozen in liquid nitrogen and stored at -78° until needed. All the following procedures were carred out at 4 ° . Fresh or frozen electric tissue was finely diced and homogenized in ca 4 volumes of 5% sucrose in a Virtis blender for 20 sec at the high setting. The homogenate was centrifuged at 45,000 x g for 5 min in the Sorval RC2B refrigerated centrifuge and the supernatant decanted. The pellet was re-homogenized, in 1/3 of the original volume, in 5% sucrose for 10 sec and centrifuged as before. The pellet from this second centri-fugation was homogenized for 60 sec, in 1/3 of the original volume, in 2 M NaCl, I mM EDTA pH 7.0 and centrifuged at 180,000 x g for 4 h in the Beckman L3-50 refrigerated preparative ultracentrifuge and the high, speed supernatant decanted. The high salt extraction was repeated up to four times on the high speed pellet to obtain maximum yield of soluble enzyme. Generally the first two high speed supernatants were the only ones used; these were pooled and diluted with an equal volume of 20 mM sodium phosphate pH 7.0 and the loading of affinity chromatography columns begun immediately. 4-11 —11. coupling of N-methylacridinium derivative to sepharose 2B The procedures for the cyanogen bromide activation of the Sepharose 2B, the ligand coupling to the activated gel, and the washing of the coupled gel to remove unreacted ligand followed published methods (72). The coupling reaction however was carried out overnight at room tempeature rather than at 4° as suggested in the published method. N-methylacridinium-Sepharose 2B conjugates with different concentrations of covalently attached ligand/ml packed gel were obtained by varying the cyanogen bromide : gel ratio in the activation step and the ligand : activated gel ratio in the coupling step; Table 4-1 lists these ratios and the ligand concentrations obtained. The concentration of unreacted ligand after coupling was determined from the absorbance at 41-2 (e = 9.3 x 10 ) of the 0.2 M sodium bicarbonate solutions (pH 9.5 - 10.0) used to wash the coupled gel and this value was used to calculate the percent of added ligand that reacted with the gel (33). - 67 -4— 11 — 111. analytical affinity chromatography Analytical scale affinity chromatography was performed using small chromatography columns constructed from 5 to 10 ml disposable hypodermic syringes by inserting to the bottom of the syringe a tight fitting disc of porous polypropylene (obtained from the Ami con Corpora-tion). Columns were packed with affinity gel and equilibrated with buffer before use. The particular conditions for a chromatography ex-periment are detailed under Results. Solutions containing AChE were applied to the chromatography columns at a flow of 0.5 column volumes per hour and non-absorbed proteins were washed clear of the affinity gel using buffer at the same flow rate. Elution was done with buffer containing decamethonium and a flow rate of 0.2 column volumes per hour was used. A multichannel peristaltic pump was used to load, wash and elute up to four columns in parallel and fractions were collected simultaneously from all columns on a modified Gilson fraction collector (68). Columns were regenerated by washing with a solution of 5 M guanidine hydrochloride which was permitted to remain on the columns overnight following which the columns were washed with at least 5 volumes of the appropriate buffer for reuse. All operations were done at 5°. 4-11-1V. preparative affinity chromatography Preparative scale affinity chromatography was carried out in Buffer hf using columns constructed from 20 ml hypodermic syringes connected in series depending on the amount of enzyme to be purified. The ligand concentration used for preparative procedures was ca 0.44 *1M NaCl, 20mM sodium phosphate pH 7.0 - 68 -ymol/ml and the specific activity and recovery of purified enzyme were both optimized when columns were loaded with about 9000 units of enzyme activity per ml of affinity resin (see Results). Preparative columns were operated under gravity at the same flow rates as used for analytical columns. Elution was done by applying 0.5 column volumes of 20 mM decamethonium in the above buffer to the column and washing it through with an excess of buffer. Columns were regenerated as described above and all operations were carried out at 5 ° . 4-II-V. characterization of purified AChE The composition of purified enzyme was monitored by isokinetic sucrose gradient sedimentation. 4-111. Results 4-111-1. ligand coupling to Sepharose 2B Six different concentrations of ligand per ml of Sepharose-2B were obtained using two ratios of cyanogen bromide to Sepharose gel in the activation reaction and different ratios of ligand to Sepharose gel in the coupling step. Coupling efficiences were greater than 80% in all cases. The stoichiometrics of reactants, ligand concentration obtained and coupling efficiency for five affinity resins are given in Table 4 -1 . The sixth resin had a ligand concentration in excess of 2 ymol/ml but this particular concentration was not precisely determined. 4-111-11. enzyme extraction from tissue fragments Table 4-II shows the extraction of AChE in soluble form during - 69 -each of the homogenization procedures. Some enzyme was released from the tissue during homogenization in sucrose solution but the relative activity demonstrates that the bulk of the soluble protein in the two sucrose homogenizations was not AChE. The first high salt extract contained the bulk of the soluble enzyme and was ten times more con-centrated in AChE protein relative to other proteins than the first sucrose homogenization. This distribution was typical of all the pre-parations carried out. 4-III-III. analytical affinity chromatography 4-III-III-a. purification from high salt extracts of electric tissue Affinity chromatography on an analytical scale was carried out on high salt extracts to determine the influence of ligand concen-tration on the retention of AChE and to determine the decamethonium concentration required for efficient elution of AChE from the acridinium affinity resin. Buffer H was used and chromatography conditions were identical for all ligand concentrations. The ligand concentration; column load, retention and yield; and the specific activity of the purified AChE are given in Table 4-III, while the elution profiles for selected ligand concentrations are shown in Figure 4-1. Four features are noteworthy in these results. Firstly, appreciable quantities of non-AChE protein were retained on the acridinium affinity matrix even at a ligand concentration too low to retard AChE effectively; this protein was partly released by increasing decamethonium concentration and further release occured with 5M guanidine-HCl (Fig 4-1). Secondly, the specific activity of the eluted AChE was strongly dependent - 70 -Table 4-1. Stoichiometries of Reactants and Coupling Obtained for the Cyanogen Bromide Activated Sepharose 2B Coupling to N-Methyl-9-[Ng-ami nohexanoly)-e-ami nopropyl ami no]acri di ni urn dibromide. CNBR : Gel Ligand : Gel Ligand Coupled % Coupling (g/ml) (mg/ml) (ymol/ml) 0.05:1 0.063:1 0.12 100 0.05:1 0.125 0.22 90 0.05:1 0.25 0.44 90 0.10:1 0.50:1 0.98 100 0.10:1 1.10:1 1.55 80 Table V-II. Preparation of Electric Tissue Fragments and High Salt Extraction of Acetylcholinesterase.9 Homogenization Medium and Volume AChE Activity and Protein Concentration of Supernatant • Activity/ml (AA^min^mr 1 ) Total Activity A280nm Relative activity (AA412min"•ml"1A2g0) 5% Sucrose 500 ml 91 60,060 2.77 33 5% Sucrose 200 ml 125 26,000 1.76 71 2 M NaClb 200 ml 272 175,934 2.06 353 2 M NaCl 200 ml 258 59,355 1.25 206 2 M NaCl 200 ml 114 29,486 0.637 179 2 M NaCl 200 ml 77 18,000 0.528 146 aThis preparation used 130 g of frozen electric tissue bThe composition was 2M NaCl, ImM EDTA, 20mM sodium phosphate pH 7.0. - 72 -on ligand concentration (Table 4-111). Thirdly, the retention of AChE on this affinity matrix and the release upon elution with decamethonium was strongly dependent on ligand concentration; with increasing ligand concentration retention increased while yield diminished. Finally, elution occured at the same point in the decamethonium gradient (about 15 mM) for all ligand concentrations from which activity was eluted (Fig 4-1). 4-III-III-b. selective elution of globular AChE with retention of asymmetric AChE The retention of both globular and asymmetric AChE and the selective elution of globular enzyme was investigated for the four intermediate ligand concentrations shown in Table 4-1II. Acetylcholin-esterase purified by preparative affinity chromatography as described under Methods was subjected to proteolysis with trypsin as described for Chapter Five to obtain a preparation containing both globular and asymmetric forms of the enzyme. This mixture was loaded onto affinity columns at low ionic strength and elution was effected first by an increase in ionic strength followed by elution with decamethonium at high ionic strength. The relative amounts of AChE eluted by increased salt concentration and decamethonium are shown in Table 4-1V and the elution profile and composition of the eluted AChE are shown for a ligand concentration of 0.22 ymol/ml in Figure 4-2. With increasing ligand concentration, the amount of enzyme eluted by increasing ionic strength was reduced and no AChE was re-covered by salt elution from columns with ligand concentrations of 0.98 and 1.55 ymol/ml. Similarly, the recovery of enzyme from these ligand - 73 -Table 4-III- Conditions for Affinity Chromatography at Six Ligand Concentrations: Retention, Yield and Specific Activity. Ligand Column Column Yield in Specific cl b e Concentration Load Retention Decamethonium Activity Eluate (umol/ml gel) ( A ^ j n i r f 1 ) (35) (%) . { y m o l m i n - l m g - l ) 0.12 16900 9 . 0 : 0.22 16900 49 63 4180 0.44 16900 97 88 6040 0.98 13800 100 65 7870 1.55 13800 100 43 4430 2 16900 100 od _ _ _ _ _ "Column retention is that percentage of the load activity that was not accounted for in the effluent from the load and Buffer H wash. ^Yield is that percentage of the retained activity which is obtained in the fractions collected during decamethonium elution. cThe value reported here is an average for all fractions in the peak. dFor this ligand concentration 22% of the load activity was recovered in the guanidine-HCl eluate. No appreciable activity was detected in this eluate for the other five ligand concentrations. - 74 -Figure 4-1. Optimization of Conditions for the Affinity Chromatography Purification of AChE Using N-Methyl-Acridinium-Sepharose 2B. Affinity columns containing 12ml of affinity resin were loaded with 90 - 110 ml of a high salt extract with an AChE activity (AA d 1 ? min"^ ml" ) and protein absorbance (A280nm^ ° f 1 9 0 a n d 0 , 7 4 f o r c o l u I T m s A and B and 130 and 0.47 for columns C and D. Ligand concentrations were; A, 0.12 mol/ml; B, 0.44 mol/ml; C, 0.98 mol/ml and D, 1.55 mol/ml. At the first arrow marked "Buffer", Buffer H was applied to the columns to remove non-specifically adsorbed protein. At the arrow marked "Deca", a 0 - 50 mM linear gradient of decamethonium in Buffer H was applied, eluting AChE at approximately 15 mM decamethonium. At the end of the decamethonium gradient, Buffer H elution was resumed at the position indicated by the second arrow marked "Buffer" which was followed by a 0 - 5M linear gradient of guanidine-HCl in Buffer H initiated at the arrow marked "GuHCl". Following the guanidine-HCl gradient the columns were again washed with Buffer H. Eluate Volume (mis) - 76 -concentrations during elution with 20 mM decamethonium at high ionic strength was lower with increased ligand concentration (Tabl3 4-IV). The material eluted by increased ionic strength at a ligand concentration of 0.22 ymol/ml was 100% globular (US) AChE (Figure 4-2B). The composition of the salt eluate at a ligand concentration of 0.44 ymol/ml was also 100% globular enzyme. The material eluted by decameth-onium from a ligand concentration of 0.22 ymol/ml was a mixture of 1.1 S globular and 18S and 14S asymmetric enzyme (Figure 4-2C). At higher ligand concentrations the decamethonium eluates contained relatively lower amounts of asymmetric forms of the enzyme, and at the highest con-centration (1.55 ymol/ml) the decamethonium eluate was 100% globular enzyme. These results clearly show the preferential retardation of asymmetric enzyme under conditions where the globular enzyme was eluted, thus allowing selective isolation of pure globular enzyme. 4-III-III-c. purification of globular AChE from proteolytic digests Purification of IIS AChE from proteolytic digests containing only this form of the enzyme was investigated for the ligand concentra-tions of the above experiment. Proteolytically cleaved enzyme, obtained as described above, was loaded onto affinity columns at lew ionic strength and elution effected f irst with decamethonium at low ionic strength followed by an increase in ionic strength and elution with decamethonium at high ion strength. The relative amounts of AChE eluted by deca-methonium at low and high ionic strength and by the intervening increase in ionic strength are shown in Table 4-V. - 77 -Table 4-IV. Recovery of AChE Activity from Affinity Columns by Sequential Elution with Increased Salt, Decamethonium and Guanidine-HCl . a Ligand Cone. Enzyme Eluted ( ^ A ^ min~^)^ (ymol/ml) Buffer Decamethonium Guanidine Total % of HC HC1 Load 0.22 4820 2260 80 7160 60 0.44 2140 4900 160 7200 60 0.98 0 6110 220 6330 53 1.55 0 2070 480 2550 21 "Affinity columns with the ligand concentrations shown were loaded, washed and eluted as described for Figure 4-2. bThe activities are the sum of all fractions collected during each elution procedure. cBuffer H has the composition 1 M NaCl, 20mM sodium phosphate pH 7.0. - 78 -Figure 4-2. Release of AChE Activity from 0.2 ymol/ml Affinity Resin by Salt Elution Followed by Decamethonium Elution: Sedi-mentation Composition of the Eluted Species. A, Affinity columns containing 5 ml of affinity resin were equilibrated with 0.1 M NaCl, 20 mM sodium phosphate, pH 7.0 (Buffer L) and loaded with 12,000 units of 86% globular and 14% asymmetric AChE in 118 ml of the same buffer. The columns were washed with an equal volume of Buffer 1, no AChE activity was present in the load or wash effluent. Elution was commenced with Buffer H. At hollow arrow (I) elution with 0.1 column volumes of 20 mM decamethonium in Buffer H was started and then followed by Buffer H. At hollow arrow (II) elution with a similar volume of 2M guanidine-HCl was commenced and followed by Buffer H. B, Sucrose gradient profile of the peak fraction eluted by Buffer H (arrow B, Fig. 2A). C, Sucrose gradient profile on peak fraction eluted by decamethonium (arrow C, Fig. 2A). The heavy arrows in B and C show the position of 3-galactosidase (15.9S) in the gradient. Acetylcholinesterase activity is depicted in arbitrary units scaled to the same maximum value for each profile. The actual peak activities were 83 units/ml for B and 42 units/ml for C. Direction of sedimentation was from right to left. Fraction Number - 80 -Elution with 20 mM decamethonium at low ionic strength re-leased AChE only from columns with ligand concentrations of 0.22 and 0.44 ymol/ml. Only small amounts of additional activity were eluted on increasing the salt concentration to 1M and subsequent with 20 mM de-camethonium in 1M salt (Table 4-V). Conversely, AChE was eluted from columns with ligand concentrations of 0.98 and 1.55 ymol/ml only by 20 mM decamethonium in 1M salt (Table 4-V). Total recovery showed a step-wise dependence on ligand concentration with equal amounts of AChE being eluted from the two lower ligand concentrations while reduced, but again similar, amounts were recovered from the two higher ligand concentrations. These results show the low ionic strength characteristics of the acridinium affinity resin to be analogous to those of the phenyl-trimethyl ammonium based resins (11). 4-111-IV. preparative affinity chromatography The performance of the N-methylacridinium affinity matrix at various ligand concentrations was investigated to determine optimum conditions for the purification of asymmetric AChE on a preparative scale. The results from experiments using high salt extracts showed that a ligand concentration of ca^  0.44 ymol/ml gel gave the most efficient results when both recovery and specific activity were con-sidered (see Table 4-III). In one preparative operation the selective purification of the asymmetric forms of the enzyme was suggested by the recovery of exclusively this form from an affinity column loaded with a high salt extract that was known to also contain the globular form of the enzyme. Figure 4-3A shows the AChE composition of the high salt - 81 -Table 4-V. Recovery of AChE Activity from Affinity Columns by Sequential Elution with Decamethonium under Low and High Salt Conditions.9 Ligand cone. Enzyme Eluted (AA 4 1 2 min' V, (ymol/ml) deca/Buffer c Buffer deca/Buffer Total % of L H H Load 0.22 11000 200 840 12040 86 0.44 10790 110 1080 11980 85 0.98 0 0 7220 7220 51 0.55 0 0 8330 8330 59 'Affinity columns containing 5ml of affinity resin at the ligand con-centrations shown were loaded with 14,000 units of 100% globular (IIS) enzyme in Buffer L and washed with a similar volume of Buffer L. No activity was present in the effluent from the load or buffer wash. Columns were eluted first with 20mM decamethonium in Buffer L (deca/ Buffer L) followed by Buffer H and finally by 20mM decamethonium in Buffer H (deca/Buffer H). 'The activities are the sum of all fractions collected during each elution procedure. cBuffer L has the composition 0.1 M NaCl., 20mM sodium phosphate pH 7.0. - 82 -extract and 4-3B the composition of the eluted enzyme. Selective puri-fication was also suggested by the low yields and preferential recovery of asymmetric enzyme that were obtained when tryptic digests of purified enzyme were re-chromatographed. Figure 4-3C shows the AChE composition of the tryptic digest and 4-3D the composition of the eluted enzyme. In contrast to the results shown in Figure 4-3, when chromatography of tryptic tryptic digests was done at lower loads (3500 units/ml) the eluate was enriched in globular enzyme ias shown in Figure 4-4B and this enrichment was enhanced with increased ligand concentration on the affinity matrix (Fig 4-4C). The performance of the N-methylacridinium affinity matrix was very reproducible when chromatography columns were operated under identical conditions in separate preparations. Affinity material re-generated by treatment with 5 M guanidine hydrochloride was not dis-tinguishable from new material except for a build up of material at the top of the gel bed. This build up did not affect the purification obtainable with regenerated columns but did cause a decrease in column flow rates. When columns were used with very high loads of high salt extract (ca_ 20,000 units/ml column volume) the build up of material at the top of the column bed was very rapid and flow rates declined markedly under these conditions. A single experiment was performed to test the properties of the N-methylacridinium affinity matrix under highly unfavourable con-ditions. The supernatant obtained in the first homogenization of 320 g of electric tissue in 5% sucrose was adjusted to a salt concentration of 0.1 M by addition of Buffer H, loaded onto a 0.44 ymol/ml affinity - 83 -Figure 4-3. Enzyme Composition on Sucrose Gradients Before and After Preparative Affinity Purification. Isokinetic sucrose gradients of AChE before and after affinity puri-fication on the acridinium ligand. The large solid arrows indicate the position of e-galactosidase (15.9S), the large hollow arrow the position of catalse (11.3S) and the dotted arrow the expected position of globular AChE. AChE activity is scaled to the same maximum value for comparison. A, High salt extract of AChE prior to affinity chromatography B, Acetylcholinesterase composition after affinity chromatography of high salt extract at a column load of 19,000 units per ml of column volume. C, Tryptic digest of purified AChE prior to affinity chromatography. D, Acetylcholinesterase composition after affinity chromatography of tryptic digest at a column load of 10,000 units per ml of column volume. Direction of sedimentation was from right to left and actual peak activities for the four gradients were; A, 119 units/ml; B, 222 units/ ml; C, 8 units/ml and D, 292 units/ml. F I G U R E 4 - 3 -84-- 85 -Figure 4-4. Enzyme Composition on Sucrose Gradients Before and After Preparative Affinity Purification at Low Column Load on Two Ligand Concentrations. A, Tryptic digest of AChE prior to affinity chromatography. B, Acetylcholinesterase composition after affinity chromatography of tryptic digest at a column load of 3500 units per ml of column on an affinity matrix with a ligand concentration of 0.44 ymol/ml. C, Acetylcholinesterase composition after affinity chromatography on an affinity matrix with a ligand concentration of 0.98 ymol/ml, other conditions identical to B. Isokinetic sucrose gradient sedimentation conditions were the same as for Figure 4-3. The actual peak activities for the three gradients were; A, 111 units/ml; B, 39 units/ml and C, 67 units/ml. - 87 -column and washed with Buffer L. Elution was carried out in a manner similar to that described for Table 4-V and the elution profile and composition of the eluted AChE are shown in Figure 4-5. The specific activity of the final product from the purification, and the yield obtained were comparable to preparations carried out with high salt extract. However, the affinity column could not be re-used due to a large quantity of material which adsorbed to the gel and did not release during treatment with guanidine hydrochloride. A selectivity between AChE forms was obtained during the different elution procedures with the 18S form of the enzyme, containing three tetramers, being the most firmly retarded of the different forms present. The performance of the N-methylacridinium affinity matrix changed markedly when columns were used with different enzyme loads. The retention of AChE during loading decreased with increasing column load and the recovery of enzyme was also lower at high column loads. However, the specific activity of the eluted AChE increased with column load to a maximum of about 9000 (umol min"1 mg"1). Table 4-VI shows the results obtained for several preparations. Optimum purification, yield and longevity of columns were obtained at column loads of around 9000 units (AA^-|2 min"1) per ml of column volume. The highest specific activity obtained was comparable to pub-lished values for AChE (30) and the performance of the affinity material was similar to that reported recently by other workers (Footnote to Table 4-VI). - 88 -Figure 4-5. Elution Profile and AChE Composition of Product Obtained by Different Elution Procedures Following Loading of Affinity Column With Crude Extract at Low Ionic Strength. A, An affinity column containing 10 ml of 0.44 yumol/ml affinity resin was equilibrated with Buffer L and loaded with 313,000 units of crude extract as described in the text and washed with 100 column volumes of Buffer L. Approximately 10% of the load activity was present in the effluent and buffer wash. Elution was commenced with 20 mM decamethonium in Buffer L followed by Buffer H at the hollow arrow marked |I), and finally 20 mM decamethonium in Buffer H at the hollow arrow marked |II). The AChE activity and A^QQ f ° r t n e fractions are shown. B, Sucrose gradient profile of the peak fraction eluted by 20 mM deca-methonium in Buffer L (arrow B, Fig 4-5A). C, Sucrose gradient profile of the peak fraction eluted by 20 mM deca-methonium in Buffer H (arrow D, Fig 4-5A). The solid arrow in B shows the position of 8-galactosidase (15.9S) in the gradient and the hollow arrows in C and D show the position of catalase (11.3S). Acetylcholinesterase activity is depicted in arbitrary units scaled to the same maximum value for each profile, actual activities were 49 units/ml for B, 345 units/ml for C, and 118 units/ml for D. The overall yield of AChE activity was 60% of the bound enzyme, dis-tributed 10% in the decamethonium eluate at low salt, 13% in the 1 M salt (Buffer H) eluate, and 37% in the decamethonium eluate in high salt. -89-F I G U R E 4 - 5 ro_14 o X 1 2 >-< UJ XZ o < E10 'C E 8 I CM 6 20 40 60 80 100130 240 60 280 E L U A T E V O L U M E (ml) MO-I— I — I I— o < 6 UJ > 4. B hi / \ UJ 0 0 10 15 0 5 10 15 0 5 F R A C T I O N NUMBER 10 15 - 90 -Table 4-VI. Column Load, Retention, AChE Recovery and Specific Activity of Purified AChE for Preparative Affinity Chromatography.3 Load Retention Recovery Specific Activity 1 _ i b .] _ i b 1 1 (AA»,9min ml ) (AA.T9min ml ) (% of retained AChE) (ymol min mg ) 4407 4217 77 5800 6159 5140 82 7300 5861 5193 67 6820 9697 8555 74C 9000 8600 73C 8637 18240 9284 54 8558 19665 16478 55 9200 19014 16914 38 The data shown were obtained from preparative procedures carried out over a 1 1/2 year period. Comparative values calculated from data given in ref 32 are; Load, 4472; Retention, 3448; Recovery, 60%; and Specific Acitivity, 5940. The units for load and retention are activity units (AA^jnin - ) per ml of column volume. These values were obtained during re-purification of the enzyme by a second affinity chromatography procedure as suggested by other workers (31). Acetylcholinesterase with a specific activity of less than 7000 was re-purified. - 91 -4-1V. Discussion The observed differences in the retention and release of the globular and asymmetric forms of AChE on the N-methylacridinium affinity matrix can be explained by postulating that the enzyme may simultaneously interact with more than one immobilized ligand. At high ionic strength reversible retention occurs with interaction at two-sites while irrever-sible retention (under decamethonium elution) occurs with interaction at more than two sites. Conversely, at low ionic strength, interaction at only one site is sufficient for retention of the enzyme within the affinity matrix. This postulate is based on the modulation of the affinity of AChE for quaternary nitrogen ligands by changes in the ionic strength of the medium and on the enhancement of binding which occurs when two species interact at two separate sites. Both of these phenomena can be i l l u -strated with reference to the literature on AChE as discussed below. The affinity of AChE for phenyltrimethylammonium under different buffer compositions varies considerably as measured by the inhibition constant, for this compound (73), and, in the case of 6-amino hex-anoyl-phenyl-trimethylammonium (the PTA ligand derivative shown in Fig 2-1) the affinity is tenfold lower at 1 M NaCl than at 0.1 M NaCl (30). This decrease in affinity, from = 6 uM to K. = 60 yM is sufficient to abolish retention of IIS AChE on affinity columns using this ligand. Acetylcholinesterase has a high affinity for N-methyl-acridinium, = 0.3 yM, in the presence of 1.0 M NaCl (31), but in-hibition constants for the 6-aminohexanoyl derivative under conditions of different ionic strength have not been reported. - 92 -The lack of retention of. l lS AChE on the N-methylacridinium affinity matrix at 1.0 M NaCl suggests that the affinity of AChE for the immobilized ligand is less than is indicated by the value deter-mined for the free ligand. Simultaneous interaction at multiple sites, however, results in the retention of the asymmetric forms of AChE despite the low affinity of the individual sites. Two-site interaction has been suggested by other workers to explain features observed during affinity chromatographic purification of multimeric enzymes (74, 75). The enhancement of binding between species which interact at two sites is exemplified by the affinity of AChE for mono- and bisquaternary ammonium compounds. Decamethonium, which can bind simultaneously to two distinct sites on the surface of the enzyme (see Fig 2-1) has an inhibition constant of 0.046 yM (76) for AChE from bovine erythrocytes while decyltrimethylammonium which can interact at only one site has an inhibition constant of 226 yM for the same enzyme (77). Similar, though less pronounced changes in inhibition constants are observed for mono- and di-pyridine derivatives (78, 79) and with bis-quaternary ammonium compounds in which the bridging methylene chain is too short to permit simultaneous interaction of both quaternary nitrogen functions with the binding sites (80, 81). - 93 -It must be emphasized that the two interacting sites in these examples are both on the same catalytic monomer of the enzyme. N-methylacridinium binds specifically at the substrate anionic site (Fig 2-1 and ref 82) and the sites at which individual immobilized ligands si-multaneously interact must be on separate monomers. The individual mono-mers need not be within the same tetrameric unit and i t is this fact which makes possible the retention of the asymmetric forms of AChE with simultaneous non-retention of the globular form of the enzyme. However, at the appropriate ligand concentration, two monomers within the same tetramer may interact simultaneously with immobilized ligands resulting in reversible retention of the US form of the enzyme and irreversible retention of the asymmetric forms as such a ligand concentration would result in more-than-two-site interaction for these forms of the enzyme. This postulate provides good explanation for several features of the experimental results. Acetylcholinesterase retained by two-site interaction will elute at the same decamethonium concentration, irres-pective of differences in overall immobilized ligand concentration. When chromatography is done in the presence of high ionic strength at low ligand concentration the amount of ligand suitably spaced for two-site interaction will be small and no retention of AChE will be observed. Conversely, at high ligand concentration the amount of ligand suitably spaced for more-than-two-site interaction will be appreciable and no recovery of enzyme will be obtained. . The preferential recovery of either globular or asymmetric AChE (Figures 4-3 and 4-4) when mixtures are - 94 -chromatographed at high ionic strength on an intermediate ligand con-centration can also be explained. At low column loads, asymmetric AChE will be irreversibly bound on that proportion of the ligand suitably spaced for more-than-two-site interaction while reversible retention of the globular form of the enzyme will take place on the portion of closely spaced ligand not occupied by asymmetric enzyme (Fig 4-4). At higher column loads, however, the globular enzyme will not be retained, as the closely spaced ligand, needed for two-site interaction with a single tetramer, will be saturated with irreversibly bound asymmetric enzyme (Fig 4-3). When mixtures are chromatographed and eluted under increas-ingly vigorous conditions the 18S form, consisting of three tetramers, will encounter greater retardation than the other forms which will tend to elute earlier as was observed for Figure 4-5. Two alternative explanations are possible for the observed differential retention of the different forms of AChE on the N-methyl-acridinium ligand. The first is that the ' t a i l ' of the asymmetric forms is involved in ionic interactions with the cationic ligand. There are two reasons for not favouring this explanation. Firstly, other workers have suggested that the amino group of lysine is involved in tai l-tai l interactions leading to aggregation of the asymmetric forms of the enzyme at low ionic strength (27) and this group would, i f anything, result in repulsion between the tail component of the asymmetric forms of AChE and the N-methylacridinium ligand. Secondly, Figure 4-5B shows an appreciable amount of AChE activity with a sedimentation coefficient of both IIS (fraction number 12) and 9S (fraction number 15) but neither Figure 4-5C nor Figure 4-5D show much enzyme activity with a sedimenta-tion coefficient of less than 14S. This indicates that the 9S form of - 95 -the enzyme, which consists of one tetramer plus the t a i l , is eluting in a manner similar to the single tetramer of the IIS form. The second possible explanation for the observed preferential retardation is that hydrophobic interactions between the enzyme and the immobilized ligand or the hydrocarbon spacer molecule result in a greater overall affinity between the ligand matrix and the asymmetric forms of the enzyme. A selective retardation of the asymmetric forms of the enzyme has been observed by workers using a Sepharose gel to which only 1-aminohexane had been attached (83). The workers did not specify the concentration of hexyl chain used but observed that the globular form of AChE was not retarded under conditions which did lead to retention of the asymmetric forms of the enzyme. It is possible that the hydrophobic interactions that these workers postulate to occur between the hexyl chain and the enzyme were insufficient to retain a single tetramer but the enhanced affinity arising from simultaneous interaction of two or three tetramers with the hexyl chain resulted in retention of the asymmetric forms of AChE. The selective recovery of either globular or asymmetric forms of AChE during affinity chromatographic purification has not been described previously. Other techniques such as gel chromatography and sucrose gradient sedimentation can be applied to the separation of the forms of AChE but these techniques are limited in capacity and always result in a dilution of the enzyme during separation. The affinity chromatographic procedure is not limited in capacity and can actually result in the concentration of enzyme protein during separation of the forms. A fractionation of the asymmetric and globular forms of the - 96 -enzyme, based on the preferential re-solubilization of the globular form, from an ammonium sulphate precipitate containing all the forms, has been reported recently (84). The separation of the 14S and 18S forms of the enzyme was not attempted. However, the results shown in Figure 4-5 suggest that i t may be possible to obtain a product greatly enriched in the 18S form of the enzyme by sequential operations similar to that described. It is also reasonable to expect that the 18S form, consisting of three tetramers, will encounter greater retardation than the 14S form, with only two tetramers, during passage down an affinity column containing a ligand concentration toolow to result in complete retention of the enzyme (ca_ 0.12 ymol/ml; see Fig 4-1). The properties of the N-methylacridinium ligand described in this thesis coupled with the revised synthesis (33) will make this valuable tool more useful for the purification of the asymmetric forms of AChE. The data presented here demonstrates that this ligand has a great versatility, possibly surpassing that of any other affinity puri-fication technique, in the separation of different forms of the same enzyme. The following chapter describes some investigations of the molecular properties of the asymmetric forms of AChE. This work was done using enzyme purified by the preparative affinity purification pro-cedures described in this chapter and summarized in Table 4-VI. - 97 -Chapter Five CHARACTERIZATION OF THE ASYMMETRIC FORMS OF ACETYLCHOLINESTERASE: SEDIMENTATION COMPOSI-TION, ACTIVITY ASSAYS* AMINO ACID COMPOSITION, SUB-UNIT COMPOSITION, ISOTOPIC LABELLING, PRO-TEOLYTIC DEGRADATION AND GEL CHROMATOGRAPHY. 5-1. Introduction This chapter describes two new forms of asymmetric AChE obtained by proteolytic modification of the 18S and 14S forms with the enzyme collagenase and identifies the molecular weight of the subunits of the tail component of the asymmetric forms. It describes a previously unnoticed effect of proteolytic cleavage with trypsin and extends the isotopic labelling of the globular form of the enzyme with radioactive iodine to the asymmetric forms. In addition i t confirms published reports of the hydroxyproline content and amino acid composition of the asymmetric forms of the enzyme and discusses some problems encountered during activity assays and the handling of the tail-containing forms of AChE. 5-1 I . Methods 5 - 1 1 - 1 . acetylcholinesterase purification and assays The asymmetric forms of AChE were purified as described under Methods in chapter four and enzymatic activity was routinely determined using the spectrophotometry assay described under Methods in chapter three. In addition AChE was assayed in the laboratory of Dr. B.D. - 98 -Roufogalis in the Faculty of Pharmaceutical Sciences by a titrimetric method which measures the rate of proton production from the hydrolysis of acetylcholine in an unbuffered medium as described below. Titrimetric assays were done at 25° and pH 7.5 in 40 ml of 0.10 M NaCl, 20 mM MgCl2, 2.5 mM acetylcholine chloride and 5 yl of AChE in 1 M NaCl, 20 mM sodium phosphate pH 7.0 (Buffer H). Folowing addition of the enzyme the stirred solution was maintained at pH 7.5 by the addi-tion of 10 mM NaOH in distilled water and the rate of acetylcholine hydrolysis was calculated from the rate of addition of sodium hydroxide solution. The rate of hydrolysis remained linear for at least one minute and non-enzymatic hydrolysis was negligible. The titrimetrically deter-mined activity is reported as ymol acetylcholine hydrolysed min ^. 3 5-11-11. active site labelling of acetylcholinesterase with H-diisopro-pylphosphof1uori date. 3 Acetycholinesterase was labelled with H-diisopropylphospho-3 fluoridate ( H-dipF) to permit the identification.of protein fragments containing the active site of the enzyme during SDS-PAGE analysis and to allow the detection of small quantities of enzyme following isokinetic sucrose gradient centrifugation and gel chromatography. Radioactivity in samples from SDS-PAGE analysis was determined as described under Methods in chapter three. Radioactivity in fractions from sucrose gradient centrifugation and gel chromatography was determined by counting 0.10 to 2.0 ml of the fractions in 10 ml of Scintiverse liquid scintillation fluid using a Nuclear Chicago Mk II liquid scintillation counter. Counting efficiency was monitored by the sample channels ratio method (85) but, - 99 -with the exception below, the data are reported as cpm, scaled to the same maximum value for comparative purposes, in sucrose gradient and chromatography profiles. 3 The init ial labelling of AChE with H-dipF followed a published procedure (86) with respect to the time of incubation of the enzyme in 3 the presence of the active site label. The stock solution of H-dipF, 3 as received from the supplier, contained H-dipF with a specific radio-activity of 0.9 Ci/mmol at a concentration of 1.1 ymol/ml in propylene glycol. The first labelling procedure used 1.5 ml of a solution of AChE at a concentration of 0.65 mg/ml in Buffer H. The purified enzyme was dialysed overnight against 500 volumes of Buffer H at 4° and the dialysis repeated twice with fresh buffer to remove decamethonium. Following 3 dialysis, 40 yl of the stock solution of H-dipF was added to give 3 approximately 3 equivalents of H-dipF per active site and the solution was maintained at 4° for 2 h. At the end of this time enzymatic activity 3 was s t i l l detectable so additional H-dipF was added to give a final amount of approximately 7 equivalents. After a further 2 h at 4° AChE activity was not detectable and the solution was dialysed as described above to remove all of the tritium not covalently attached to protein. 3 This labelling procedure yielded H-DIP-AChE with a specific radioactivity of ca_ 0.8 Ci/mmol of active site indicating that labelling was almost complete. The detectable radioactivity was 4.6 x 10^  cpm/mg of protein and, as this level of labelling was not necessary for the unequivocal detection of active site label on SDS-PAGE analysis, sub-sequent labelling procedures used only one equivalent of ^H-dipF to conserve the active site label. - 100 -5—TI-111. collagenase proteolytic cleavage of acetylcholinesterase Purified AChE was dialysed for 24 h at 4° against 200 volumes of 0.5 M NaCl, 5 mM CaCl 2, 50 mM tris(hydroxymethyl)aminomethanehydro-chloride pH 7.5 (Buffer C). Following dialysis AChE solutions were diluted with this buffer to give a final concentration of ca^  0.2 mg/ml estimated from the A 2 R Q of the original solution prior to dialysis. N-Ethylmaleimide at a concentration of 1 M in 95% ehtanol was then added to a final concentration of 5 mM followed by addition of the stock collagenase solution to a final concentration of 0.04 mg/ml., Incubation was carried out at 30° for up to 97 hours and proteolysis was terminated either by simply cooling samples to 4 ° , dialysing samples at 4° against 500 volumes of 1 M NaCl, 20 mM sodium phosphate, 5 mM ethylene-diamine-tetraacetic acid (EDTA) pH 7.0 or by addition of EDTA solution to a final concentration of 10 mM followed by storage at 4 ° . No difference was observed after sucrose gradient sedimentation of identical samples in which proteolysis was terminated in the three different ways. Acetylcholinesterase samples were incubated in the absence of collagenase to check for non-coilagenolytic proteolysis and samples containing collagenase and AChE were also incubated after the addition of EDTA to a final concentration of 10 mM to determine the need for calcium ion in the collagenolytic proteolysis. Control samples containing either calf skin collagen (0.2 mg/ml) or lysozyme (0.2 mg/ml) in the absence of AChE were incubated with collagenase as described above, but reactions were terminated by rapid freezing at - 2 0 ° . The calf skin collagen samples were dissolved init ia l ly in 6N HCl and titrated to the point of turbidity with 6N NaOH before dilution to the final incubation - 101 -concentration with 10 volumes of Buffer C. The progress of proteolysis in the lysozyme and calf skin collagen incubations was monitored by the release of primary amine detected by a published procedure (87). Acetylcholinesterase trace labelled at the active site by 3 3 reaction with H-dipF ( H-DIP-AChE) was also subjected to collagenase proteolysis as described above. 5-II-IV. trypsin proteolytic cleavage of acetylcholinesterase 3 • H-DIP-Acetylcholinesterase was dialysed against 200 volumes of Buffer C at 4° for 24 h. Volume after dialysis was 6 ml with a 3 H-DIP-AChE concentration of ca_ 0.25 mg/ml and to this was added 0.20 ml of 0.1 mg/ml trypsin in 0.001 N HC1. This solution was incubated at room temperature and 0.60 ml aliquots removed to tubes containing 0.040 ml of 0.1 mg/ml soybean trypsin inhibitor at intervals of 0.5, 5.0, 10, 40, 80, 160, and 320 minutes. Five minutes after addition of the aliquot to the soybean trypsin inhibitor solution the tube was placed in a cold box at 4 ° . These samples were all dialysed for 24 h against 2 L of 0.01% sodium dodecyl sulphate, 10 mM sodium phosphate pH 7.2 as were two addi-3 tional samples, one containing only H-DIP-AChE the other only trypsin and soybean trypsin inhibitor. After dialysis all samples were lyo-philized. The ratio of soybean trypsin inhibitor to trypsin used in this experiment totally inhibited enzymatic activity toward the synthetic substrate p-toluenesulphonyl-L-arginine methyl ester, with a lag period of about 2 min, under similar incubation conditions. Acetylcholinesterase was treated with trypsin on a preparative scale with similar concentrations of trypsin to that described above - 102 -and incubation was done for 15 min before terminating the reaction by the addition of soybean trypsin inhibitor. 125 5-II-V. radioactive labelling of acetylcholinesterase with Iodine Purified asymmetric AChE was isotopically labelled at tyrosine residues by the lactoperoxidase mediated procedure introduced in Figure 2-3A and at lysine residues by the incorporation of the previously iodinated TAGIT reagent shown in Figure 2-3B. 5-II-V-a. lactoperoxidase iodination The lactoperoxidase mediated radioiodination of purified enzyme was based on a published procedure (34) which was slightly modi-fied to improve the efficiency of iodine incorporation with minimal loss of AChE enzymatic activity. Iodinations were done at room tempera-ture in Buffer H for 30 min, nonradioactive iodide was present at 170 mM 125 concentration, approximately 25 yCi of iodide was used per iodination, the final concentration of lactoperoxidase was 8 yg/ml and iodination was initiated by the addition of hydrogen peroxide to a final concentration of 0.00012%. Iodination was terminated by the addition of sodium azide to a final concentration of 4 mM and free iodide was removed by dialysis 3 as described for H-dipF isotopic labelling. No significant change occured in the labelling obtained when the AChE concentration was varied from 0.2 to 0.8 mg/ml. 125 5-II-V-b. I-TAGIT labelling of acetylcholinesterase The radioiodination of AChE at lysine residues was done by a - 103 -published method (34). However, the iodinated reagent was dried from the benzene solution into four small tubes and 0.8 ml of AChE in Buffer H, within the concentration range given above, was sequentially added to and held for 30 min at 4° in each of the four tubes. Excess reagent was removed by dialysis as described above. 5-II-VI. amino acid composition of asymmetric acetylcholinesterase Amino acid analyses conducted by standard procedures were obtained from AAA Laboratories, 6206 89th Avenue Southeast, Mercer Island, Washington. The samples for amino acid analysis were passed through a Sephadex 6 25 gel chromatography column, equilibrated in de-ionized water, to remove buffer salts, lyophilized and sealed under nitrogen for shipment to the above address. Samples for hydroxyproline assay were dialysed for 24 h against 500 volumes of 0.1 M ammonium acetate pH 6.8 and then lyophilized. The residue was dissolved in ca_ 1 ml of water and re-lyophi 1 ized for acid hydrolysis. Hydrolysis was done for 24 h at 110° in double distilled 6 N HC1 in a sealed tube under nitrogen. The hydroxyproline content was assayed by a published procedure (88) with the modification that the hydroxyproline was calculated as the percentage of the primary amine released during the acid hydrolysis. The primary amine was determined by a published procedure (87) using glycine as the standard. 5-11-VII. characterization of intact and proteolytically modified forms of acetylcholinesterase The different forms of AChE were characterized by isokinetic sucrose gradient sedimentation, SDS-PAGE and Sepharose 4B gel chromatograhy - 104 -as described under Methods in chapter three. 5-111. Results 5 - 1 1 I - I . sedimentation composition of affinity purified acetylcholin-esterase A typical sucrose gradient profile is shown in Figure 5-1. Since the affinity purification procedure used in this work was opti-mized to preferentially purify the multi-tetrameric forms of AChE (see chapter four) l i t t l e , i f any, of the 9S and 11S forms of the enzyme were present in freshly purified preparations. Acetycholinesterase was routinely stored at 4° in plastic vials in 1 M NaCl, 20 mM sodium phos-phate pH 7.0 in the condition in which it was eluted from affinity columns, without any intervening dialysis to remove the decamethonium present from the elution operation. 5 - 1 1 1 - 1 1 . titrimetric assay, comparison with spectrophotometric assay One preparation of AChE was assayed by the titrimetric and spectrophotometric methods to obtain a conversion factor between the two assay procedures. An enzymatic rate of hydrolysis of 1 ymol acetylcholine per min was equivalent to a change in absorbance at 412nm of 3.92 per min under the conditions described for the two assays. This is almost identical to the value of 3.94 recently published for AChE purified by a similar procedure and assayed under similar conditions (32). 5 - 1 1 1 - 1 1 1 . hydroxyproline content and amino acid composition of affinity purified acetylcholinesterase - 105 -The percent hydroxyproline of AChE obtained in this work was 0.94 + 0.16 (8 assays), calf skin collagen gave a value of 21 + 3% (3 assays) and a value of 0.03% was obtained for bovine serum albumin. The amino acid composition for AChE is shown in Table 5-1. This analysis and the hydroxylproline assays were done on a preparation with a com-position of 60% 18S enzyme and 40% 14S enzyme as estimated by sucrose gradient sedimentation. 5—111-1V - subunit composition of affinity purified acetylcholinesterase Acetylcholinesterase purified to a specific activity of between 7000 and 9000 umol acetylthiocholine hydrolyzed min'^mg-^ typically gave the densitometric profile shown in Figure 5-2 when analysed by SDS-PAGE. Interpolation of the migration relative to the dye front onto the cali-bration curve shown in Figure 5-3 yielded an apparent molecular weight of 79,000 for the major peak of Figure 5-2 and this value corresponds closely to the published molecular weight for the catalytic monomer (10, 11,22, 23, 24). A very distinct and reproducible peak occured with a molecular weight of 275,000 . and smaller and variable peaks with molecular weight of ca_ 165,000 and 115,000 were also apparent. The small peaks in the region of 25,000 - 30,000 daltons and the peak at 55,000 daltons were consistently present while the small shoulder in the region of 45,000 daltons was a variable feature of the preparations. Analysis of 3H-DIP-AChE by SDS-PAGE showed that the loca-tion of catalytic site coincided with the 79,000 and 55,000 daltons peaks with a small and variable amount of label also present in the region of 275,000 daltons (ref. Figure 5-5A). To simplify comparison between - 106 -F I G U R E 5 - 1 +— 1 — i r 1 T-' 0 5 10 15 20 25 FRACTION NUMBER Figure 5-1. Sedimentation Profile of Affinity Purified AChE A typical sucrose gradient sedimentation profile for affinity purified enzyme is shown. The ratio of the 18S AChE peak to the 14S AChE peak varied from 4:1 to 1:1 for different preparations. The nominal sedi-mentation coefficients for AChE forms of 18S, 14S and IIS are plotted with the values for g-galactosidase (15.9S) and catalase (11.3S). - 107 -Table 5-1. Amino Acid Composition of Affinity Purified Acetylcholinesterase Amino Acid mol%a Hyp 0.8 Asp 11.9 Thr 3.8b Ser 7.2b Glu 10.1 Pro 7.2 Gly 11.3 Ala 5.6 Val 7.1C Met 3.0 He 3.8C Leu 8.3 Tyr 3.5 Phe 4.8 Hyl N.D. Lys 3.4 His 2.6 Arg 4.5 Half-Cys i . o d Trp N.D. 6N-HC1 hydrolysis at 110° , values are averages for 24hr, 48hr and 96hr hydrolysis time unless otherwise noted. 'Extrapolated to zero time hydrolysis Maximum value Performic acid oxidized prior to acid hydrolysis. Calculated from cysteic/alanine ratio, 24hr hydrolysis only. - 108 -preparations and to explain data obtained during experiments involving tryptic and collagenolytic proteolysis the migration of the peaks at 55,000 and 45,000 daltons was calculated with reference to the migration of the 79,000 daltons peak. In the experiment shown in Figure 5-2 the migration of the two smaller peaks was 1.25 and 1.33 relative to the major peak. The mean migration and standard deviation for 24 densitor metric profiles was 1.25 + 0.03 for the 55,000 daltons peak and a value of 1.33 + 0.01 was obtained for the lower molecular weight peak from 6 densitometric profiles. 125 5-III-V. isotopic labelling of acetylcholinesterase with iodine The distribution of iodine following the two methods of isotopic labelling is shown in Figure 5-4A and B. This distribution is similar to that obtained with the IIS form of AChE (34) with the ex-ception of the very noticeable labelling in the region of 45,000 daltons in the densitometric profile for AChE iodinated by the lactoperoxidase mediated procedure (Figure 5-4B). Acetylcholinesterase labelled at lysine residues suffered negligible loss of enzymatic activity, but the lactoperoxidase mediated iodination of tyrosine residues caused a 60 -100% loss of enzymatic activity. Minimal loss of activity was obtained with the conditions described under Methods and attempts to protect the active site of the enzyme by adding butyrylcholine at up to 20 mM con-centration in the iodination incubation mixture were not successful. 125 The amount of iodine incorporated into AChE protein by the two label-ling methods varied between preparations. The labelling levels were 3.4 x 10 cpm/mg protein for the chemical iodination procedure using - 109 -F I G U R E 5 - 2 Figure 5-2. SDS-PAGE of Fully Reduced AChE Components. The densitometric profile of a Coomassie blue stained gel showing the subunit composition of freshly purified AChE. The numerical values shown adjacent to each peak are the molecular weights x 10 obtained by interpolation onto the calibration curve shown in Figure 2. The parenthesized values are the mean relative migration and standard deviation calculated with reference to the major peak. - n o -F I G U R E 5 - 3 OAChE 0 0.1 0.2 0.3 OM 0.5 0.6 0.7 0.8 Rf Figure 5-3. Calibration Curve for SDS-PAGE. Calibration curve of log (molecular weight) versus migration relative to dye front for the standard proteins given in Table 3-1. The positions of peaks visible in Figure 3 are indicated by hollow diamonds. - m -125 7 I-TAGIT and 10 cpm/mg protein by the lactoperoxidase mediated pro-cedure for the samples shown in Figure 5-4. 5-III-VI. trypsin cleavage of affinity purified acetylcholinesterase Tryptic proteolysis of AChE was very rapid at room temperature in the presence of 5 mM Ca + + . Figure 5-5 shows densitometric profiles obtained for samples of AChE not exposed to trypsin and exposed to trypsin for 5 min and 40 min. Additional profiles were obtained for all the incubation times given under Methods. These profiles differed from Figure 5-5C only in that the shoulder with an average migration of 1.31 + 0.03 (vide infra) was not discernible. In the sample of AChE not exposed to trypsin (Fig 5-5A) the active-site fragment had a migra-tion of 1.25 which was identical to the average value given previously for this fragment (Fig 5-2). Following proteolysis with trypsin this fragment had a migration of 1.38 .+ 0.03 (calculated from all 7 densito-metric profiles) and this change in migration indicates that there is a site of trypsin cleavage that is distinct from the site which is cleaved during the endogenous proteolysis which gives rise to the fragment shown in Figures 5-2 and 5-5A. This distinction had not been noted prior to this work. In addition to the changes in migration of the active-site fragment during trypsin proteolysis a shoulder with a migration of 1.30 + 0.03 (calculated from the profiles for 0.5, 5.0, 10 and 40 min incuba-tion) gradually disappeared and was not discernible on densitometric pro-files after greater than 40 min incubation. The components with molecular weights of 275,000 and 165,000 also were not discernible after - 112 -F I G U R E 5 - 4 DISTANCE (cm) Figure 5-4. SDS-PAGE of.Fully Reduced AChE Following Isotopic Labelling With 1 2 5Iodine. 125 A Densitometric profile for I-TAGIT labelled AChE and distribution of radioactivity determined following slicing of polyacrylamide gel after scanning at 550 nm. B Densitometric profile and radioactivity distribution for AChE iodinated by the lactoperoxidase mediated method. - 113 -Figure 5-5. SDS-PAGE of Fully Reduced AChE Before arid After Trypsin Proteolysis. The solid line indicates the densitometric profile of the Coomassie blue stained gels and the fi l led circles the radioactivity due to active-site label. The dotted line in B is the profile obtained for a sample containing only trypsin and soybean trypsin inhibitor. The migration relative to the 79,000 dalton peak (Fig 5-2) is indicated. A, Control not exposed to trypsin. B, 5 min incubation with trypsin. C, 40 min incubation with trypsin. -114-FIGURE 5-5 ORIGIN DYE MIGRATION - 115 -exposure of AChE to trypsin. 5-III-VII. collagenase proteolysis of affinity purified acetylcholin-esterase Proteolysis by collagenase was relatively slow under the con-ditions used in these experiments and complete degradation by collagenase to a final stable form was not achieved. Larger amounts of collagenase could not be used because collagenase subunits migrate similarly to AChE fragments in SDS-PAGE (Fig 5-6A). Densitometric profiles obtained for samples analysed by SDS-PAGE after zero incubation time, 6 h and 97 h incubation at 30° in the presence of collagenase and 97 h incubation at 30° without collagenase are shown in Figure 5-6. The only distinct change with increased in-cubation time in the presence of collagenase was the complete dis-appearance of a shoulder migrating 1.31 times as far as the major peak. This shoulder also diminished in the sample incubated in the absence of collagenase (Fig 5-6D). Figure 5-7 shows the progress of conversion of AChE forms during incubation with collagenase for up to 96 h as determined by sucrose gradient sedimentation. The starting material had a composition similar to that shown in Figure 5-1. The 18S and 14S forms were con-verted to species with sedimentation coefficients of 21.1 +0.6 S and 17.3 +0.3 S respectively. With non-labelled AChE the conversion was complete in 24 h (Fig 5-7B) while 3H-DIP-AChE was more labile to col-lagenase and conversion was complete in less than 6 h. (This increased susceptibility to collagenase proteolysis is illustrated in Figures -AT3-A18 - 116 -Figure 5-6. SDS-PAGE of Fully Reduced AChE Before and After Collagenase Proteolysis. The solid lines indicate the densitometric profiles and the dotted line in A is the profile for collagenase alone under similar conditions; The migration relative to the major peak (79,000 daltons Fig. 5-2) is indicated in A. Samples were incubated with at 30° collagenase for Oh (A), 6 h (B) and 97 h (C) and without collagenase for 97 h (D). F IGURE 5 - 6 - 1 1 7 -1 . 2 5 B D O R I G I N D Y E ORIGIN MIGRATION D Y E - 118 -in the Appendix.) The 21S and 17S species did not aggregate during sucrose gradient sedimentation in the presence of 0.1 M NaCl (see Appendix Figure A16). No changes were observed when AChE was.incubated for 96 h at 30° in the absence of collagenase (Fig 5-7E) and addition of EDTA greatly reduced collagenase proteolysis (Fig 5-7F). (Active-site labelled enzyme did show some degradation during incubation in the absence of collagenase and again this is illustrated in the Appendix.) In addition to the changes in sedimentation coefficient observed during collagenase cleavage a gradual decrease in the 21S AChE content was observed with a concomitant increase in the IIS form while the 17S AChE content remained essentially constant. This point is i l l u -strated in Figure 5-8 where the relative proportions of the different forms (plus the species from which they were derived, ie^  18S in the case of the 21S form) are plotted for increasing incubation time. No proteolysis could be detected following incubation of ly-sozyme samples under conditions similar to those used for the collagenase treatment of AChE. Proteolysis of calf skin collagen under these con-ditions was rapid however, and, as judged by the production of primary amine, was complete within 12 h under the conditions described under Methods. These results were expected from the description of the acti-vity of the further purified collagenase (71) used in these experiments and serve to confirm that the proteolytic activity being observed was collagen specific. - 119 -Figure 5-7. Sucrose Gradient Sedimentation Profiles for Affinity Purified AChE Following Exposure to Collagenase A - D Acetylcholinesterase activity profile plotted as a percentage of total activity for AChE exposed to collagenase for 6, 24, 48 and 96 hrs at 30° prior to sucrose gradient sedimentation. The solid arrow indicates the position of s-galactosidase (15.9S) in the gradients. The nominal sedimentation coefficients for the AChE forms are shown. E Acetylcholinesterase sample incubated for 96 h at 30° without collagenase. F Acetylcholinesterase sample incubated for 96 h at 30° in the presence of collagenase and 10 mM EDTA. (The sedimentation co-efficients were not determined for this gradient profile). FIGURE 5-7 -120-\r C \ J o co . CD < \ J o (ID j o ; 10 o/o) A1IAI10V 3M0V FRACTION NUMBER - 123 -FIGURE 5 - 8 0 10 20 30 40 50 60 70 80 90 100 INCUBATION TIME (hours) Figure 5-8. Progress of Collagenase Cleavage The relative proportions of the three froms of AChE; 18(21 )S, 14(17)S and IIS were estimated from sucrose gradient profiles obtained following varying times of exposure to collagenase. - 124 -5-III-IIX. gel chromatography; Stokes radius and molecular weight of collagenase modified acetylcholinesterase Following proteolysis with collagenase the position of elution from Sepharose 4B was markedly changed for the multi-tetrameric forms of AChE. The elution profiles obtained for 18S, 14S, 21S, 17S and 11S AChE are shown in Figure 5-9. Calibration of the elution profile by 112. plotting (-log K )^ versus Stokes radius (Re) where was calculated according to the equation; KQ = (Ve-Vo)(Vf-Vo)"'' in which Ve is the elution volume for the protein, Vf is the elution volume of the small molecule Fe(CN)g . and Vo is the elution volume of Blue Dextran 2000, yielded a straight line for the standard proteins with a slope of 0.0272 + 0.0007 and an intercept of 0.259 + 0.0009 as shown in Figure 5-10. Interpolation of the value obtained for 21S AChE gave a Stokes radius of 12.9 +_ 0.5 nm and the corresponding value for the 17S form of the enzyme was 11.1 + 0.5 nm. These values are respectively 2.7 and 3.3 nm less than the values reported for the unmodified forms of the enzyme (21). The molecular weights for the collagenase modified forms of AChE were estimated by a graphical comparison of the product of the sedimentation coefficient and Stokes radius for these species with the same product and the molecular weights for the 18S and 14S forms. This graph is shown in Figure 5-11. . The same procedure, with correction for differences of partial specific volumes of the proteins, has been used previously to determine the molecular weight of the 18S and 14S forms of the enzyme by comparison with a range of known proteins (17). Sub-sequently, the molecular weights for the 18S, 14S and US forms were determined by sedimentation equilibrium and the graphical procedure was - 125 -FIGURE 5 - 9 ELUTION VOLUME (ml) Figure 5-9. Elution Profiles for AChE Forms when Chromatographed on Sepharose 4B. Individual forms of AChE were chromatographed following separation of the forms by sucrose gradient sedimentation. The void volume (hollow arrow V ) was determined using Blue Dextran 2000 and the included o 3 volume (hollow arrow V )^ was determined using potassium ferricyanide. No difference in the elution position of the US form of AChE was observed when samples obtained by trypsin proteolysis, collagenase proteolysis or resulting from spontaneous degradation were chromato-graphed. F IGURE 5-10 - 126 -Stokes Radius (nm) Figure 5-10. Calibration of Gel Chromatography; (-log KD)l/2 Versus Stokes Radius (Rg) for Standard Proteins. The calibration line of (-log K^)1/2 for the standard proteins listed in Table 3-1 is shown and the position of the collagenase modified forms of AChE is indicated by the two small arrows. The standard proteins are indicated by solid circles and the 18S, 14S and US forms of AChE are indicated by open circles. The errors in the values were not greater than the size of the symbols on the graph. - 127 -used to estimate the molecular weights for minor forms of AChE derived from the IIS form (21). The molecular weights obtained by this pro-cedure were 750,000 daltons for the 17S form of the enzyme and 1,080,000 daltons for the 21S species. The change in molecular weights with col-lagenase treatment for the four species was 70,000 daltons for the 18S - 21S conversion and 46,000 daltons for the 14S - 17S conversion. The level of precision in this determination is not high enough to attach any significance to the difference in molecular weights of the fragments removed by collagenase from the 18S and 14S forms but is sufficient to show that a very minor change in molecular weight has occurred in each case. 5-IV. Discussion This work was carried out with the intent of identifying and characterizing the tail component of the asymmetric forms of AChE. The presence of hydroxyproline in these forms, reported by other workers shortly after work was started on this thesis, was confirmed by the values obtained in this work. The amino acid analysis shown in Table 5-1 supported the data for hydroxyproline content that was obtained by specific assays. Amino acid analyses had been reported previously for the globular form of AChE (89) and more recently for the asymmetric forms of the enzyme (21). However, in both these reports no mention was made of the hydroxyproline content. The amino acid composition reported here was confirmed however, by a very recent report (32). The composition of AChE forms determined by sucrose gradient sedimentation, the SDS-PAGE migration pattern and the stability observed 128 -FIGURE 5-11 300 CO ZD MOLECULAR WEIGHT x10 r 5 Figure 5-11. Calibration Curve of the Product of Stokes Radius and Sedimentation Coefficient Versus Molecular Weight The values for the 18S and 14S forms of AChE are plotted and the positions of the 21S and 17S forms are indicated by the small arrows. - 129 -for purified enzyme observed during this work were also supported by the recent report (32). Some differences were observed in the stability of enzyme subjected to the prolonged dialyses used during active-site labelling. Enzyme stored for up to 3 months as described under Results showed negligible change in composition as determined by sucrose gradients and gel chromatography. However, preparations of H-DIP-AChE were less stable and showed some degradation during storage at 4 ° . Likewise, 3 H-DIP-AChE incubated for 96 h in the absence of collagenase did undergo some degradation to the IIS form of the enzyme and toward the latter part of this work i t also became apparent that AChE stored in plastic vials at 4° following dialysis into the calcium-ion containing buffer used for collagenase modifications was very labile to proteolytic degradation. These observations on the proteolytic instability of even highly purified AChE are illustrated in the Appendix to this thesis. An additional observation, made very early in the work for this thesis, was also confirmed in ref. 32. When the tail-containing forms of AChE were stored in glass containers serious loss of enzyme activity occurred. This loss was due to adsorption of these forms of the enzyme onto the walls of the containers. Similar adsorption took place during activity assays. This could be demonstrated by f i l l ing the cuvettes used for assays with a solution of 18S and 14S AChE in the 1 M NaCl buffer used for storing the enzyme, pouring out the solution, rinsing the cuvette with buffer and then f i l l ing the cuvette with the assay mixture containing acetylthiocholine and 5,5'-dithiobis(2-nitrobenzoic acid). The immediate generation of colour due to enzymatic hydrolysis of the acetylthiocholine could be observed adjacent to the walls of the cuvette. - 130 -Immersing the cuvettes in a 5:1 solution of ethanol and concentrated HCl removed the adsorbed enzymatic activity. This was done for spectro-photometric assays and the equipment used for the titrimetric assays was soaked for 5 minutes in a solution of proteolytic enzymes. This adsorption was also observed during dilution of the enzyme solutions before assay and dilution had to be done in plastic vessels for best reproducibility of results. The addition of bovine serum albumin to fractions from sucrose gradients and gel chromatography columns was done to prevent the loss of enzyme activity due to adsorption onto the glass tubes used for these fractions. This behaviour of the tail-containing forms of AChE is similar to the behaviour described for structural proteins isolated from central nervous system synapses (90). In addition, similar adsorption onto glass surfaces has been described for fragments of procollagen isolated from bone tissue (91). The SDS-PAGE densitometric profiles obtained for freshly puri-fied AChE (Figs. 5-2, 5-4, 5-5A, 5-6A) show that the well characterized endogenous proteolysis of the catalytic monomer of the enzyme (22,23) has not occured to any significant extent in these preparations. Because the purification procedure used for this work yields predominantly the multi-tetrameric forms of the enzyme (vide supra, Fig 5-1) SDS-PAGE analysis should display subunits which can be attributed to the tail protein of these forms. Four components, with molecular weights of 275,000, 165,000, 115,000 and 45,000 in Figure 5-2, are features which are not present in SDS-PAGE densitometric profiles for AChE purified by most other procedures (10, 11, 22). - 131 -The tail protein is believed to derive from the basement membrane collagen which forms the 'ectolemma' within the synaptic cleft (Fig 1-3). This suggestion arose from the observation that digestion of the ectolemma released AChE (6), from the solubilization of AChE by collagenase (13,26) and from the appearance of the tail in electron-micrographs (19,11). The presence of hydroxyproline in the asymmetric forms of the enzyme to a greater extent than the US form supported this suggestion (88) as did the simultaneous solubilization of hydroxyproline rich polypeptides with AChE during collagenase treatment (42). In addition, calculations based on the known hydroxyproline content of basement membrane collagen (92) and the proportion of the total mole-cular weight of the 18S and 14S forms of AChE that can be attributed to the tail protein (19,21) predicted a hydroxyproline content of 0.7-1 % for these forms of the enzyme (88). The measured values are within this range. Basement membrane collagen is reported to consist of polypep-tides ranging in size from 30,000 to 700,000 daltons (93). Therefore any of the four components referred to above could represent subunits of the tail protein. The expected molecular weight for the tail of AChE, however, is ca 100,000 daltons (Fig 1-5 and refs. 19,21) so the com-ponents with molecular weights of 165,000 daltons and 275,000 daltons are too large to be considered as possible tail subunits. In addition active-site label coincides with the 275,000 daltons component, and, to a lesser extent, with the 165,000 daltons species (Fig 5-5A and un-published data) suggesting that these features may represent incompletely denatured and reduced tetramer and dimer of catalytic protein. The 115,000 - 132 -daltons component cannot be positively identified at this time. However, the changes observed during proteolytic degradation of the asymmetric forms of AChE suggest that the 45,000 daltons component can be identified as a subunit of the tail protein as detailed below. Collagenase proteolysis of the asymmetric forms of AChE resulted in five distinct changes in the properties of the enzyme. The first three changes, an appreciable reduction in Stokes radius, a minor reduc-tion (less than 10%) in molecular weight and the loss of low ionic strength aggregation, indicate that a very asymmetric, but small mole-cular weight, component, responsible, for the aggregative properties of the 18S and 14S forms of the enzyme, is removed by collagenase. The fourth change, the conversion of the multi-tetrameric forms to the IIS form indicates that this component is a structural unit to which the in-dividual tetramers are attached in the multi-tetrameric forms of AChE. Finally, the disappearance of a component with a migration of 1.31 in SDS-PAGE densitometric profiles (Fig 5-6) identified this collagenase sensitive component as the 45,000 daltons species with a migration of 1.33 + 0.01 in Figure 5-2. A point to note is that the collagenase treatment did not alter the relative amounts of the 79,000 and 55,000 daltons components (Fig 5-6) indicating that the catalytic subunits are not susceptible to collagenase proteolysis and further indicating that no non-collagenase proteolytic enzymes were present. A component with a migration of 1.31 + 0.03 also disappeared from SDS-PAGE densitometric profiles during trypsin proteolysis (Fig 5-5) supporting the identification of the 45,000 daltons species as the tail component, which is absent in the trypsin degraded 11S form of the - 133 -enzyme. The relatively slow removal of the tail component by trypsin, relative to the rate of modification of catalytic protein, is understand-able if the tail protein is of a collagenous nature. The disappearance of the 275,000 and 165,000 daltons components from densitometric profiles during trypsin proteolysis is probably due to an increased susceptibility to denaturation and reduction in the trypsin degraded IIS form of the enzyme. The presence of protein fragments with a molecular weight greater than 79,000 daltons in SDS-PAGE densitometric profiles following trypsin proteolysis suggests that some trypsin resistant species exist in the multi-tetrameric forms of AChE (Fig 5-5). The 115,000 daltons component visible in Figure 5-2 and Figure 5-4 could represent intact tail protein which has an enhanced resistance to trypsin proteolysis. This suggestion is tentative as support cannot be adduced from the results obtained with collagenase proteolysis shown in Figure 5-6. While small changes are visible in the region of 115,000 daltons in the den-sitometric profiles in Figure 5-6B firm conclusions can not be made due to the presence of subunits of collagenase in this region of the profiles. The relatively high level of lactoperoxidase mediated iodina-tion of the component with a molecular weight of 45,000 daltons also differentiates this species from the 79,000 daltons subunit and the 55,000 daltons fragment (Fig 5-4B) and indicates that this species either contains an increased proportion of tyrosine residues or the tyrosine residues are more readily accessible to lactoperoxidase. It has been shown that different proteins iodinated under identical conditions by the lactoperoxidase mediated procedure show different levels of iodine - 134 -incorporation (35,94) and increased accessibility of tyrosine residues resulting from proteolytic degradation is probably responsible for the high level of labelling observed for the 25,000 - 30,000 daltons fragments (Fig 5-4B). With reference to the 115,000 daltons component visible in Figure 5-4B, this species does show slightly increased labelling relative to the two higher molecular weights species but again the data are not conclusive-. The tentative molecular weight of 45,000 for the sub-unit of the tail component of the asymmetric forms of AChE is close to the predicted subunit molecular weight (19,21) and was supported by recently reported value of 40,000 - 44,000 daltons (32). The small change in overall molecular weight of the multi-tetrameric forms of AChE following collagenase treatment also agrees with predictions for the total molecular weight of the tail protein (vide supra). The recent report (32) also suggests that the 115,000 daltons species can be attributed to a residual aggregate of catalytic subunit and tail subunit, citing unpublished observations of the location of active-site radiolabel. During the work for this thesis active-site label was not seen to coincide with this region of SDS-PAGE densitometric profiles (Fig 5-5A) and therefore further work is required for an un-ambiguous identification of the 115,000 daltons component. The variability of the appearance of the species with a mole-cular weight of 45,000 in SDS-PAGE densitometric profiles (see Results) was also observed by these.other workers (32). This change in the subunit composition observed with SDS-PAGE analysis did not correlate with any observable change in either the sedimentation properties of - 135 -the asymmetric forms of the enzyme or with the gel chromatography be-haviour. It is possible that a very slow proteolytic conversion does take.place during or after affinity purification. The effects of this conversion could remain unnoticed until the enzyme is denatured and reduced for SDS-PAGE analysis. The proteolytic degradation of the catalytic monomer which results in the generation of fragments with mole-cular weights of ca 55,000 and 25,000 - 30,000 remains undetected until the protein is denatured and reduced (22-24). It has been reported that purified or partially purified preparations of AChE did not show spontaneous degradation from the asymmetric forms to the globular form (95) and the possibility of autolytic degradation has been ruled out (11). However, spontaneous degradation does occur in some apparently pure preparations (22). Furthermore, AChE is capable of catalysing the hydrolysis of amide bonds (96). Inactivation of AChE with dipF did not prevent degradation of the asymmetric forms to the globular form (97). However the inactivated enzyme is subject to slow reactivation (8) and, in view of the reduced stability of enzyme subjected to prolonged dialyses as are used during active site labelling, these results cannot be considered conclusive. The work for this thesis shows that there is a minimum of three specificities for proteolytic degradation of the asymmetric forms of AChE. The endogenous degradation which leads to the small amount of 55,000 species daltons/present in freshly purified preparations arises from proteolysis at a site distinct from the site of trypsin proteolysis as shown by the data of Figure 5-5. In addition, collagenase proteolysis occurs at a site which permits loss of a portion of the tail of the asymmetric forms - 136 -without marked changes being observed in SDS-PAGE densitometric profiles as shown in Figure 5-6. An additional distinct site for collagenase proteolysis may also be present. Six hours of collagenase treatment generated species with apparent sedimentation coefficients of 19S and 16S (Fig 5-7A) while 24 hours of exposure produced the 21S and 17S forms (Fig 5-7B). This stepwise conversion suggests that the entire length of the tail protein may not be equally labile to collagenase proteolysis but certain regions may contain the three amino acid sequence for which collagenase is specific (98,99). The production of the faster sedimenting forms more rapidly than the conversion of these forms to the single tetramer of the IIS species does indicate that a site of collagenase proteolysis, close to the attachment point of the tail protein to the tetramers, is less accessible than other sites on the tail protein. The sedimentation coefficients obtained for the collagenase modified multi-tetrameric species were confirmed by an account which appeared in the literature after the work for this thesis was concluded (100) . An earlier account which appeared while the work was in progress reported that the collagenase modified species had sedimentation co-efficients similar to the values shown for Figure 5-7A. It is likely that this report was presenting the results of incomplete collagenase conversion as the incubation medium described did not contain calcium ion. Collagenase requires calcium ion for maximum proteolytic activity (101) and addition of a calcium ion chelator to the incubation medium used for this work greatly retarded collagenase proteolysis as shown by Figure 5-7F. The more recent report (100) utilized an incubation medium containing calcium ion. - 137 -The data presented in this chapter strongly'indicate that the tail component of the multi-tetrameric asymmetric forms of AChE has a collagenous nature. This supports the postulate that this protein component derives from the basement collagen within the synaptic gap. Some of the properties of basement membrane collagen will be discussed in the concluding remarks to this thesis in chapter seven. The following chapter presents in an abbreviated form some work designed to directly investigate the association between the 18S and T4S forms of AChE and membrane fragments derived from the electric tissue of the electic eel. - 138 -Chapter Six 'REASSOCIATION' OF PURIFIED ASYMMETRIC ACETYLCHOLINESTERASE WITH MEMBRANE FRAGMENTS DERIVED FROM ELECTRIC TISSUE. 6-I. Introduction This chapter describes the generation of two species of mem-brane fragments from the electric tissue of the electric eel, and some experiments to characterise the membrane fragments. It is shown that AChE activity is only loosely associated with the membrane fragments and that purified AChE does not appear to reassociate with the membrane fragments in a specific manner. The data presented in this chapter concur almost completely with thai presented in a report which appeared late in 1977 describing similar work (102). 6-II. Methods 6-II-I. purification of acetylcholinesterase and assays The asymmetric forms of AChE were purified as described for chapter four and AChE assays were done as described under Methods for chapter three. The activity of the sodium and potassium dependent adenosine triphosphatase was determined by the release of inorganic phosphate from the substrate adenosine triphosphate in the presence and absence of the specific inhibitor ouabain (103). Incubations were carried out at 37° and the enzymatic activity was terminated by the addition of perchloric acid to cause precipitation of all proteins. Following the addition of perchloric acid the incubation mixture was then assayed for inorganic phosphate by a procedure which generates a - 139 -chromophore with an absorbance at 650 nm proportional to the amount of inorganic phosphate present (103). Activities are presented as the difference in absorbance at 650 nm between incubations at 37° for the same time period in the presence and absence of the inhibitor. Protein was assayed by the Lowry method (104) using bovine serum albumin as the standard or was estimated by the absorbance of gradient fractions at 280 nm. No correction was done for non-protein absorbance or for turbidity. 6-1I-II. preparation of membrane fragments A sequential homogenization procedure was developed in which a sample of electric tissue was homogenized briefly and then centri-fuged at a low speed to sediment the large tissue fragments. The supernatant was carefully poured off and the pellet again homogenized and centrifuged. These operations were repeated on the pellet and with careful control of the homogenization conditions i t was possible to obtain the major portion of the total AChE activity associated with membrane fragments which remained in suspension during the low speed centrifugation but which would sediment when centrifuged at high speed on a sucrose gradient. All homogenizations and subsequent operations were done at 4°and a. model 6-105-AF Virtis blender with flask number 16 235 was used for all successful preparations. A typical preparation used 11 g of fresh or frozen electric tissue homogenized in 30 ml of unbuffered 5% sucrose solution for 45 sec at the 'High' setting. The tissue was finely diced (while frozen) before homogenization. Centrifugation was done for 5 min - 140 -in the SS34 rotor of the Sorval RC2B refrigerated centrifuge at a speed of 19,000 rpm (43,500 x g). The homogenization - centrifugation sequence was repeated up to 6 times and the point in the sequence at which membrane fragments remained in suspension after low speed centrifugation could be con-trolled by varying the homogenization time and volume for the individual steps. Yields of AChE activity in low speed supernatants obtained with two different homogenization sequences are given under Results. 6 - 1 1 - 1 1 1 . sucrose gradient sedimentation of membrane fragments Discontinuous sucrose gradients were prepared for the Beckman SW 27 rotor with the following volumes and concentrations; 4.5 ml - 50% sucrose, 10 ml - 35% sucrose, 10 ml - 15% sucrose. These gradients were loaded with 14 ml of selected supernatants from the homogenization sequence and centrifuged for 5 h at 25,000 rpm (112,000 x g at r max) in the Beckman L3-50 refrigerated ultracentrifuge. Following centri-fugation the gradients were fractionated into 26 tubes and assayed for AChE activity, sodium and potassium dependent adenosine triphosphatase activity (AtPase activity) and protein concentration. 6-II-IV. sucrose gradient sedimentation of membrane fragments in the presence of excess added acetylcholinesterase Selected supernatants from the homogenization procedure were diluted with an equal volume of 1 M NaCl, 20 mM sodim phosphate pH 7.0 containing 10 equivalents of AChE activity and the mixture dialysed at 4° overnight against 50 volumes of 5% sucrose. Following dialysis, - 141 -centrifugation on discontinuous sucrose gradients was carried out as described. Parallel gradients were loaded with samples of supernatant that had been diluted twofold with 5% sucrose and stored at 4° over-night but had not been exposed to a high concentration of salt. 6-II-V. sucrose gradient sedimentation of pure acetylcholinesterase in the absence -of membrane fragments Pure asymmetric AChE in 1M NaCl, 20 mM sodium phosphate pH 7.0 was dialysed overnight against 50 volumes of 5% sucrose and centrifuged on discontinuous sucrose gradients as described for membrane fragments. A parallel gradient was loaded with a sample of pure AChE which had been diluted to 0.1M NaCl by the addition of 5% sucrose solution. A control gradient containing 1M NaCl was loaded with a sample of pure AChE which had not been subjected to a reduction in ionic strength. 125 6-II-VI. labelling of membrane proteins with iodine The lactoperoxidase mediated iodination procedure was used to introduce radioactive iodine into the proteins associated with the membrane fragments. Because the membrane fragments agglutinated and precipitated upon addition of hydrogen peroxide solution the hydrogen peroxide was generated in situ by the action of glucose oxidase on glucose using a slight modification of a published method (105). Selected supernatants from the homogenization procedures were incubated for 18 h at 4° in the presence of 1 mg lactoperoxidase, 123 activity units (Sigma) of glucose oxidase, 100 ymol glucose and 25 yCi 125 -I in a final volume of 20 ml. Iodinated membrane fragments were - 142 -loaded directly onto discontinuous sucrose gradients in which the 15% sucrose solution was replaced by 10% sucrose, centrifuged and frac-tionated as before. Radioactive iodine was located in the gradient fractions by counting 100 yl of each fraction in 10 ml of Scintiverse liquid scintillation cocktail as described for chapter four. 6-11-VII. SDS-PAGE analysis of membrane fragments Following centrifugation on discontinuous sucrose gradients the fractions containing the two different species of membrane fragments were pooled. Samples were kept for SDS-PAGE analysis of the fragments and the balance of the two pools were made to a concentration of 2M NaCl by the addition of solid sodium chloride. The high ionic strength solutions were centrifuged at 180,000 x g for 4 h and the. high speed supernatant and the high speed pellet obtained were analysed by SDS-PAGE as described under Methods in chapter three. 6-111. Results 6-11I-I. preparation of membrane fragments Figure 6-1A shows the release of AChE in a particulate form form 11 g of electric tissue during sequential homogenization as described under Methods. Supernatants.3, 4 and 5 contained 71% of the total AChE and showed distinct turbidity. The effect of varying the volume and time of homogenization is shown in Figure 6-1B. Preparations using from 10 to 30 g of electric tissue yielded qualitatively identi-cal results to those shown in Figure 6-1B. - 143 -FIGURE 6 -1 1 6 Figure 6-1 2 3 * 5 6 ( 2 5 m | ; 2 0 s ) 1 2 3 4 5 (25ml : 20s)' (25ml;40s) ••-(25 ml; 40s) (30 ml; 45s) (25ml; 40s) Preparation of Membrane Fragments From Electric Tissue. A, Eleven grams of tissue was sequentially homogenized six times for 45 sec in 30 ml of 5% sucrose. The total AChE activity in the low speed supernatant is shown for each homogenization. B, Twenty three grams of tissue was homogenized in 5% sucrose. The volume of sucrose solution and the time of homogenization was varied as shown in parentheses for each homogenization. The total AChE activity in the low speed supernatant is shown. - 144 -6—111 — 11 . sucrose gradient separation of membrane fragments After centrifugation bands of turbidity were visible at the 15%-35% sucrose interface and the 35%-50% sucrose interface. The separation of AChE activity and Na+, K+-ATPase activity on different species of membrane fragments is shown in Figure 6-2A. This initial separation was lost with increasing homogenization as shown in Figures 6-2B and C. No difference was observed for the distribution of either enzyme when fresh or liquid nitrogen frozen tissue was used. When the time and volume of homogenization was modified (Figure 6-1B) the distribution of AChE activity always showed two peaks as in Figure 6-3A. Modified homogenizations were used in the experi-ment described below to investigate the re-association of pure AChE with the membrane fragments; Figure 6-3A is the control sucrose gradient from this experiment. 6—III-III. separation of membrane fragments in the presence of excess pure acetylcholinesterase Centrifugation of membrane fragments in the presence of pure AChE yielded the sucrose gradient profile shown in Figure 6-3B. Tur-bidity was visible at both the 50%/35% and 35%/l5% sucrose interfaces after centrifugation and AChE activity was present at both interfaces in the control gradient (Fig 6-3A). Virtually all of the AChE activity in the gradient containing both membrane fragments and added AChE however, was present at the upper interface. This distribution of activity was also observed when membrane fragments were exposed to high salt either by dialysis against 1 M NaCl or by the addition of an equal 145 -FIGURE 6 - 2 | l . 8 -t £CN1.4-o *-g1.2-CN ^ _ | ^ § 0 . 8 O °-p0.6 < O ^ Q 2 4 8 12 16 20 24 FRACTION NUMBER 0 Figure 6-2 A 4 8 12 16 18 0 4 8 FRACTION NUMBER 12 16 Sucrose Gradient Sedimentation of Membrane Fragments. B, C, The distribution of AChE activity and ATPase activity obtained with minimum homogenization 3, Figure 6-1 A. The large hollow arrows indicate the interfaces between the 50%, 35%,.and 15% sucrose solutions that formed the gradient and the membrane fragment load in 5% sucrose. Distribution of AChE activity with increasing homogenization. Distribution of ATPase activity with increasing homogenization. ( Homogenization 3; Homogenization 4; - • - Homogenization 5.) - .146. r, FRACTION NUMBER Figure 6-3. Sucrose Gradient Sedimentation of Membrane^ Fragments in the Presence of Excess Pure AChE. A, Control gradient containing only membrane fragments from homogeniza-tion 5 in Figure 6-1B. B, Gradient containing membrane fragments plus a tenfold excess of pure AChE. The AChE activity is scaled to the same maximum value for each profile, the peak activity in A, was 154 units/ml and B, was 2667 units/ml. - 147 -volume of 2 M NaCl followed by dialysis back to low ionic strength without addition of pure AChE. The endogenous AChE activity associated with membrane fragments was totally released by exposure of membrane fragments to high ionic strength solutions and when the ionic strength was reduced before centrifugation the AChE activity always occurred only at the 35%/l5% sucrose interface in the gradients. 6-111-IV. centrifugation of pure acetylcholinesterase under the con-ditions used for the separation of membrane fragments Figure 6-4 shows the sedimentation profiles obtained when pure AChE was centrifuged under the conditions used for the separation of membrane fragments and under similar conditions with the addition of 1 M NaCl to all sucrose solutions. At high ionic strength AChE does not sediment appreciably during the short centrifugation time used, but, when the pure enzyme is centrifuged at low ionic strength all of the AChE activity is localized at the 35%/l5% sucrose interface. This distribution is virtually identical to that observed in Figure 6-3B for pure AChE centrifuged in the presence of membrane fragments with the exception that no enzyme activity is present at the 50%/35% sucrose interface. 6-III-V. radioiodination of membrane fragment proteins During iodination the membrane fragment suspensions became heterogeneous; a small residue settled while some material formed a layer at the top of the suspension. Samples not subject to iodination showed no change. The sedimentation profile on a discontinuous sucrose - 148 -FIGURE 6 - 4 O 2 4 6 8 10 12 14 16 18 20 22 24 FRACTION NUMBER Figure 6-4. Centrifugation of Pure AChE Under The Conditions Used for Separation of Membrane Fragments. The sedimentation profiles for centrifugation at high ionic strength (-#-) and low ionic strength (-•-) are shown. The peak distribution of AChE activity at low ionic strength was identical when the ionic strength was reduced by dilution or by dialysis. The profile shown is that obtained using dialysis to reduce the ionic strength. The hollow arrows indicate the interfaces between the 50%, 35% and 10% sucrose solutions used for the gradient, and the load solution in 5% sucrose. - 149 -gradient for a successful iodination is shown in Figure 6-5. Incor-poration of radioactive iodine into membrane fragments was very low and attempts to increase incorporation either by longer incubation or incubation at higher temperatures failed due to agglutination of mem-brane fragments either during iodination or centrifugation. Large amounts of free radioactive iodide remained in the upper portions of the discontinuous sucrose gradients when samples were cen-trifuged without prior dialysis to remove this unreacted material. Such dialysis was attempted, however agglutination of membrane fragments always occured. Despite the free iodide a small shoulder of radio-activity can be observed in Figure 6-5 coinciding with the upper protein peak while a peak of radioactivity also coincides with the lower protein peak. The level of iodination, counts per minute per mg protein, was 1.6:1 for the upper to lower protein peak. 6-111-VI. SDS-PAGE analysis of membrane fragments Figure 6-6 shows the SDS-PAGE densitometric profiles obtained for membrane fragments before and after exposure to high salt and for the proteins solubilized by high salt treatment of the fragments. Figure 6-6A shows the results obtained for the membrane fragments sedimenting to the 50%/35% sucrose interface and Fig 6-6B is for the fragments with remain at the 35%/15% sucrose interface. Two features should be noted. Firstly, the densitometric profiles obtained with membrane fragments not exposed to high salt show that the most common subunit molecular weights are between 40,000 and 56,000 for both species of membrane fragments,. Secondly, a prominent feature of the 150 FIGURE 6 - 5 0 2 4 6 8 10 12 14 16 18 20 22 24 26 FRACTION NUMBER Figure 6-5. Distribution of Radioactivity, AChE Activity and Protein After Separation of Radiolabeled Membrane Fragments on Sucrose Gradient. The iodine radioactivity (-#-), AChE activity (-^ -) and protein concentra-tion ( ) are shown for the most successful membrane iodination. N , ATPase activity could be detected in the gradient fractions. Figure 6-6. SDS-PAGE Densitometric Profiles for Membrane Fragment Proteins. A, Faster sedimenting fragments at 50%/35% sucrose interface. B, Slower sedimenting fragments at 35%/15% sucrose interface. The three densitometric profiles in each figure are, from top to bottom; intact membrane fragments, membrane fragments after exposure to 2 M NaCl and proteins solubilized from membrane fragments by exposure to 2 M NcCl. The numbers adjacent to selected peaks in the profiles are the apparent _3 molecular weights x 10 + 10%. - 153 -proteins removed during the exposure of the membrane fragments to 2 M NaCl is the subunit molecular weight of 40,000 - 42,000. 6-IV. Discussion This work on the generation of membrane fragments from the electric tissue of the electric eel was started with the intent of identifying the ' tai l receptor' which was responsible fro the loca-lization of AChE activity within the synaptic cleft. However, as the results shown in Figures 6-3 and 6-4 indicate, i t was not possible to detect whether the interaction between the membrane fragments and the associated AChE activity was in any way specific. The lack of firm association between AChE and the membrane fragments was shown in some attempts that were made to concentrate AChE-rich membrane fragments. The fractions obtained after the separation of the two species of mem-brane fragments were pooled, diluted to reduce the sucrose concentration, and were then centrifuged for several hours to concentrate the fragments above a 50% sucrose cushion at the bottom of the centrifuge tube. Less than 40% of the AChE activity recovered was associated with the membrane fragments and the balance of the activity remained in the supernatant solution. This result was identical with the results recently reported by other workers (102). In addition, membrane fragments, which had been treated in this manner, agglutinated extensively and could not be re-suspended, suggesting some similarities in properties between these preparations and the previously mentioned synaptic complexes (90) which aggregate readily when treated in a similar manner. The most prominant subunit molecular weights of 40,000 - 56,000 obtained in this - 154 -work are also similar to the range of 50,000 - 55,000 reported for the subunits of the proteins comprising the synaptic complexes (106,107,108). The SDS-PAGE densitometric profiles in Figure 6-6 show l i t t l e difference between the protein composition of the two species of membrane fragments and this result was also recently re-ported (102). The results of the lactoperoxidase mediated iodination in Figure 6-5 do show a slight difference between the two species of membrane fragments. The slower sedimenting species has more tyrosine residues accessible to iodination than the faster sedimenting species suggesting that the slower species contains more surface associated membrane proteins. A similar difference in the level of iodine in-corporation per unit weight of protein has been reported for slow and fast sedimenting membrane fragments obtained from muscle tissue (109). The recent report on the properties of the membrane fragments obtained from electric eel tissue (102) shows that the major difference between the two species of membrane fragments lies in their protein to phos-pholipid ratios with the faster sedimenting fragments containing more protein, probably integral membrane protein, than the slower sedimenting species. -155 -Chapter Seven CONCLUDING REMARKS 7-1. Affinity Purification of Acetylcholinesterase The N-methylacridinium based affinity purification procedure characterized in chapter four was extremely important to the work described in this thesis. The rapid and complete purification possible with this ligand made isolation of sufficient enzyme for the structural investigations a simple routine. While the selective purification capabilities of the affinity resin were not fully exploited some bene-f i t was obtained in having only low levels of the 11S form of the enzyme present at the initiation of collagenase incubations. The multiple-site interaction hypothesis presented in chapter four provides a good explanation for the data obtained. No recent reports describing multiple-site interactions for other proteins during affinity chromatography were found in the literature and it is possible that the multi-tetrameric forms of AChE are unique in displaying this behaviour in such an obvious manner. 7-II. The Tail of Acetylcholinesterase as a Membrane Anchor Almost coincident with the discovery of the tail-containing forms of AChE another protein bearing a ' t a i l ' was reported. This was the protein cytochrome b^  which is now known to have a hydrophobic tail attached to a globular hydrophilic unit (110). Solubilization of this protein was first obtained by lytic procedures that removed the tail which forms a membrane anchor for this protein. Subsequently, other proteins with a similar amphipathic nature were discovered (111,112,113). - 156 -An analogy was drawn between the tail-containing forms of AChE and these other tail-containing proteins (97) but when work was started on this thesis a direct hydrophobic.interaction between the tail of AChE and the bilayer of the post-synaptic membrane had not been demonstrated. This possibility was discussed in two reports which appeared during the course of this work (21,42) and the data presented did not support the conclusion of hydrophobic interaction. The collagenase susceptibility of the tail component of AChE demonstrated in this thesis does not support the postulate that the tail is a hydro-phobic membrane anchor. Some workers have reported that the aggregative forms (18S and 14S) of AChE do form lipid containing complexes (114) which can be dissociated by treatment with lipase enzymes. The con^ elusion was made that the aggregative forms of the enzyme contained a feature responsible for hydrophobic interactions. However the data were. subsequently explained on the basis of proteolytic activity present in the lipase preparations used (115). Very recently however, i t has been shown, that the 18S and 14S forms of AChE bind to the lipid sphingo-myelin and that the binding ability is removed concurrently with the generation of the 21S and 17S species of AChE by treatment with col-lagenase (100). At the same time lack of association with phosphatidyl-choline was reported but no conclusive explanation for the results was given While i t is certain that the tail of AChE is the com-ponent responsible for. the immobilization of the enzyme in the synaptic gap the mechanism of this interaction between the - 157 -tail and other components within the gap is s t i l l unclear. Hydrophobic interactions between the tail and lipid bilayer of the postsynaptic membrane have not been unequivocally demonstrated and the insertion of a collagen like protein into a hydrophobic environment has never been suggested in the literature on AChE from the electric eel. An inter-action between the tail component and the polar heads of the phospho-lipids forming the membrane bilayer is a more tenable hypothesis and this could explain the association between sphingomyelin and the col-lagnease sensitive portion of the tail referred to above. The inter-vention of additional proteins in this association leading to the localization of AChE adjacent to the post-synaptic membrane, via an intermediary matrix of basement membrane, best reconciles the available data. 7-Itt. Basement Membranes In the discussion for chapters five and six brief reference was made to the nature of the components of basement membrane. The ectolemma, which f i l l s the synaptic cleft, is continuous with the base-ment membrane matrix that surrounds the cells (6). Digestion of this matrix with collagenase and other proteases results in the release of AChE and in the partial or complete break up of the structure of the synapse (6,116). The structure of basement membranes is complex and contains dissimilar protein subunits rich in carbohydrate. One of the protein components is a collagen which is unique in containing the amino acid cystine as well as different proportions of other amino acids compared with other types of collagens, and which also contains carbo-- 158 -hydrate. The carbohydrate component includes in particular the dis-accharide glucosyl-galactose attached to the modified amino acid hydro-xylysine. These properties of basement membranes have been studied for a variety of tissues and the data reviewed (117), but the form associated with the nerve.axon has not been described. The similarities in pro-perties between the basement membrane collagens isolated from different sources however, suggests that axonal and synaptic basement membrane collagen will probably be similar to the known forms of this collagen. The presence of both collagen-like proteins and non-collagen proteins in basement membranes make these structures susceptible to proteolysis by both collagenase enzymes and by proteases which do not hydrolyse collagenous proteins (118). One study has shown that the collagen of basement membranes is attached to the nonrcollagenous pro-teins by disulfide bonds and selective digestion with proteolytic enzymes can yield either the collagenous protein or the non-collagenous protein intact after proteolysis of the other component (119). The collagen of basement membranes has a triple helical conformation and is composed of three identical chains with molecular weights of 115,000 (117). These chains are covalently linked by disulfide bonds (120) with no free sulfhydyls (121). The apparent conflict between the data in-dicating the presence of disulfide bonds between collagenous and non-collagenous proteins (119) and the lack of free sulfhydryls in the collagen triple helix (121,119) was not commented on in the references consulted. The apparent subunit molecular weight of 45,000 that was obtained for the collagenase sensitive tail component of AChE in the - 159 -work for this thesis is difficult to reconcile with the subunit molecular weight of 115,000 reported for the collagen of basement mem-branes (117,120). It is unlikely that AChE is attached directly to the main collagenous component of basement membranes. The extent of pro-teolysis which occurs during isolation of the components of basement membrane has been assessed and the conclusion reached was that the smaller polypeptides did not arise from proteolytic cleavage of a high molecular weight component during basement membrane isolation (93). It was observed however, that the component most susceptible to proteolysis during isolation had a molecular weight of 50,000 . but i t was not stated whether or not this component was susceptible to collagenase proteolysis. 7-IV. Synaptic Complexes Synaptic complexes, briefly referred to in chapters five and six, in the central nervous system are structures containing both the pre and post-synaptic membrane joined by the synaptic cleft (122). These structures define the synapse and remain intact during purification. Reports on the isolation of similar complexes from the neuromuscular junction were not found in the literature during the writing of this thesis but the presence of similar complexes at the neuromuscular junction of the frog is apparent from the description of the "nerve/muscle cemen-ting substance" which can be digested by proteolytic enzymes including collagenase with the release of AChE and the concurrent disruption of the structure of the synapse (6,116). The central nervous system synaptic complexes are distinguishable as opaque "synaptic cleft material" - 160 -in electron microscopic studies. However neuromuscular synaptic cleft material is indistinguishable from the basement membrane surrounding other regions of the cells involved in the synapse (116). Studies on the central nervous system synaptic complexes have focussed primarily on the region of the post-synaptic membrane but the entire structure is reported to consist of a spectrum of polypeptides which range in molecular weight from 13,000 to 250,000 and which are extensively joined by disulfide bonds (123,107,108,106). The presence of collagen-like components has not been reported but a high proportion of the polypeptides associated with the complexes contain carbohydrate (108). In particular the presence of galactosyl residues was confirmed. While no direct connection between the proteins of synaptic complexes and the proteins of basement membrances or cell surface-associated proteins was found in the literature i t is obvious that all these proteins bear similarities in the disulfide bonded nature of the matrix and in the polypeptide molecular weights most prominent (90,106-108,122,123). However i t is not possible to determine the relevance of these similarities. Additional similarities can be found between the properties of the synaptic complexes and the membrane fragments which can be derived from the electrtc tissue of the electric eel. In the isolation of the post-synaptic membrane from synaptic complexes the membrane forms a closed vesicle just large enough to accomodate the protein matrix, known as the post synaptic density, associated with the membrane (122). This is similar to the closed vesicles which can be obtained from electric tissue (102) and, as - 161 -mentioned in chapter six, the vesicles display similar aggregative properties. The synaptic complexes have not been studied with respect to the catalytic or receptor functions of the proteins associated with the complex. This has been done for the vesicles obtained from electric tissue (37-41,102,103) but is obvious from the results presented in chapter six, and confirmed in reference 102, that the association be-tween AChE and these vesicles does not accurately reflect the native state of the enzyme in the synaptic gap. 7-V. Acetylcholinesterase at the Synapse While i t is clear that the tail component of AChE has some similarities to collagen and that AChE is localised within': the synaptic gap by interaction with a matrix of proteins the complete identity of the tail is not yet clear. It is unlikely, as mentioned above, that the tail of AChE is derived directly from the main collagenous component of basement membranes. While the aims of this thesis were in a large part realised much work s t i l l remains to be done before the manner in which AChE is located in the synaptic gap is clearly understood. Furthermore, once this aspect is clarified the question of how the enzyme is brought to synaptic location remains. The work presented in this thesis, and in particular the well characterized purification procedure described here, will form a useful background for further studies on this enzyme. -162-BIBLIOGRAPHY 1 Singer, S.J., and Nicolson, G.L., (1972) Science 175, 720-731. 2 Keynes, R.D. (1958) Sci. Am. 199, 83-90. 3 Ehrenstein, G. (1976) Physics Today, Oct., pp. 33-39. 4 Katz, B., Nerve, Muscle and Synapse, McGraw-Hill, New York, 1966. 5 Lester, H.A, (1977) Sci. Am., 236, 106-116. 6 Betz, W., and Sakmann, B. (1971) Nature New Biol . , 232, 94-95. 7 Chagas, C , and de Carvalho, A.P. , Bioelectrogenesis: A,Comparative Survey of its Mechanisms with Particular-Emphasis on Electric  Fishes. Elsevier, New York, 1961. 8 Froede, H.C., and Wilson, I.B. (1971) in The Enzymes (Boyer, P.D., ed.) vol. 5, pp. 87-114. 9 Nachmannson, D., (1970) Science 168, 1059-1066. 10 Rosenberry, T.L. (1975) Adv. Enzymol., 43, 103-218. 11 Rosenberry, T.L. (1976) Enz. Biol. Memb., 4, 331-363. 12 Nachmannson, D. (1971) i_n Principles of Receptor Physiology (Loewen-stein, W.R., ed.) chap. 2, Springer-Verlag, New York. 13 Dudai, Y., and Silman, I. (1974) J. Neurochem., 23, 1177-1187. 14 Powell, J .T. , Bon, S., Rieger, F., and Massoulie, J. (1973) FEBS Letters 36, 17- 32. 15 Massoulie, J . , and Rieger, F. (1969) Eur. J. Biochem., 11, 441-455. 16 Massoulie, J . , Rieger, F., and Bon, S. (1971) Eur. J. Biochem., 21, 542-551. -163-17 Bon, S., Rieger, F., and Massoulie, J. (1973) Eur. J. Biochem., 35, 372-379. 18 Rieger, F., Bon, S., Massoulie, F., and Cartaud, J. (1973) Eur. J. Bioc. 34, 539-547. 19 Cartaud, J . , Rieger, F., Bon, S., and Massoulie, J. (1975) Brain Res., 88, 127-130. 20 Bon, S., and Massoulie', J. (1976) FEBS Letters, 67, 99-103. 21 Bon, S., Huet, M., Lemmonier, M., Rieger, F., and Massoulie, J. (1976) Eur. J. Biochem., 68, 523-530. 22 Morrod, P.J., Marshall, A .G. , and Clark, D.G. (1975) Biochem. Biophys. Res. Commun., 63, 335-342. 23 Dudai, Y., and Silman, I. (1974) Biochem. Biophys. Res. Commun., 59, 117-124. 24 Chen, Y.T. , Rosenberry, T . L . , and Chang, H.W. (1974) Arch. Biochem. Biophys., 161, 479-487. 25 Chothia, C , and Leuzinger, W. (1975) J. Mol. Biol . , 97, 55-60. 26 Hall, Z.W., and Kelly, R.B. (1971) Nature New Biol . , 232, 62-63. 27 Duadi, Y., Herzberg, M., and Silman, I. (1973) Proc. Nat. Acad. Sci. U.S.A. , 70, 2473-2476. 28 Kalderon, N., Silman, I,, Blumberg, S., and Dudai, Y. (1970) Biochim. Biophys. Acta, 207, 560-562. 29 Cuatrecasas, P., and Anfinsen, C.B. (1971) Ann. Rev. Biochem., 40, 259-278. -164-30 Dudai, Y., Silman, I., Kalderon, N. , and Blumberg, S. (1972) Biochim. Biophys. Acta, 268, 138-157. 31 Dudai, Y. , Silman, I., Shinitzky,. M., and Blumberg, S. (1972) Proc. Nat. Acad. Sci. U.S.A., 69, 2400-2403. 32 Rosenberry, T . L . , and Richardson, O.M. (1977) Biochemistry 16, 3550-3558. 33 Webb, G., and Clark, D.G. (1978) (submitted for publication). 34 Morrod, P.O., and Clark, D.G. (1975) FEBS Letters 59, 316-319. 35 Marchalonis, 0.0. (1969) Biochem. 0., 113, 299-305. 36 Bolton, A . E . , and Hunter, W.M. (1973) Biochem. 0., 1.33, 529-539. 37 Kasai, M., and Changeux, 0-P. (1971) 0. Memb. Biol . , 6, 1-23. 38 ibid pp. 24-57. 39 ibid pp. 58-80. 40 Bauman, A . , Changeux, 0-P., Benda, P., and Huchet, M. (1970) FEBS Letters 8, 145-148. 41 Duguid, O.R., and Raftery, M.A. (1973) Biochemistry 12, 3593-3597. 42 Lwebuga-Mukasa, U.S., Lappi, S., and Taylor, P. (1976) Biochemistry 68, 531-539. 43 Phillips, D.R., and Morrison, M. (1970) Biochem. Biophys. Res. Commun., 40, 284-289. 44 Nairn, R., and Hughes, R.C. (1976) Biochem. Soc. Trans., 4, 165-167. 45 Teng, N.N.H., and Chen, L.B. (1976) Nature 259, 578-580. -165-46 Teng, N.N.H., and Chen, L.B. (1975) Proc. Nat. Acad. Sci. USA, 72, 413-417. 47 Svedberg, T . , and Pedersen, K.O., The Ultracentr.ifuge. Oxford Univ. Press, New York, 1940. 48 Van Holde, K.E., Physical Biochemistry, Prentice-Hall, New Jersey, 1971. 49 McCall, J.S., and Porter, B.J., Ultracentrifugation; Williams and Wilkins, Baltimore, 1973. 50 Meselson, M., Stahl, F.W., and Vinograd, J. (1957) Proc. Nat. Acad. Sci . , USA, 43, 581-588. 51 Martin, R.G., and Ames, B.N. (1961) J. Biol. Chem., 236, 1372-1379. 52 Noll, H.,(1967) Nature 215, 360-363. 53 Handbook of Biochemistry (Sober, H. A . , and Harte, R.A., eds.) 2 ed., Chemical Rubber Co., Cleveland, Ohio. 54 Ogston, A.G. (1966) Brit. Med. Bul l . , 22, 105-108. 55 Winzor, D.J. (1969) vn Physical Principles and Techniques of Protein Chemistry, (Leach, S.J., ed_.) Academic Press, New York. 56 Andrews, P. (1966) Brit. Med. Bul l . , 22, 109-114. 57 Ackers, G.K. (1964) Biochemistry 3, 723-730. 58 Siegel, L.M., and Monty, K.J. (1966) Biochim. Biophys. Acta, 112, 346-362. 59 Shapiro, A .L . , Vinuela, E . , and Maizel, J.V. (1967) Biochem. Biophys. Res. Commun., 28, 815-820. -166-60 Weber, K., and Osborn, M. (1969) J. Biol. Chem., 244, 4406-4412. 61 Reynolds, J .A., and Tanford, C. (1970) J. Biol. Chem., 245, 5161-5165. 62 Fish, W.W., Reynolds, J .A., and Tanford, C. (1970) J. Biol. Chem., 245, 5116-5168. 63 Rodbard, D., and Crambach, A. (1970) Proc. Nat. Acad. Sci. USA, 65, 970-977. 64 Banker, G.A., and Cotman, C.W. (1972) J. Biol. Chem., 247, 5856-5861. 65 Neville, D.M. (1971) J. Biol. Chem., 246, 6328-6334. 66 Hedrick, J .L . , and Smith, A.J. (1968) Arch. Biochem. Biophys., 126, 155-164. 67 Fisher, M.P., and Dingman, C.W. (1971) Biochemistry 10, 1895-1899. 68 Webb, G. (1978) Anal. Biochem. (in press). 69 Davies, G.E., and Stark, G.R. (1970) Proc. Nat. Acad. Sci. USA, 66, 651-656. 70 Ellman, G.L., Courtney, D.K., Andres, V. , and Featherstone, R.M. (1961) Biochem. Pharmacol., 7, 88-95. 71 Peterofsky, B., and Diegelmann, R. (1971) Biochemistry 10, 988-994. 72 March, S.C., Parikh, I., and Cuatrecasas, P. (1974) Anal. Biochem., 60, 149-152. 73 Changeux, J-P. (1966) Mol. Pharmacol. 2, 369-392. 74 Lowe, C.R., Harvey, M.J.., and Dean, P.D.G. (1974) Eur. J. Biochem., 42, 1-6. -167-75 Harvey, J .M., Lowe, C.R., Craven, D.B., and Dean, P.D.G. (1974) Eur. J. Biochem., 41, 335-340. 76 Roufogalis, B.D., and Quist, E.E. (1972) Mol. Pharmacol. 8, 41-49. 77 Belleaa,B.7 Tani, M., and Lie, F. (1965) J. Am. Chem. Soc. 87, 2283-2285. 78 Shinitzky, M., Dudai, Y., and Silman, I. (1973) FEBS Letters 30, 125-128. 79 Mooser, G.,.Schulman, H., and Sigman, D.S. (1972) Biochemistry 11, 1595-1602. 80 Roufogalis, B.D., and Wickson, V.M. (1975) Mol. Pharmacol. 11, 352-360. 81 Wee, V.T. , Sinha, B.K., Taylor, P.W., and Chignell, C F . (1976) Mol. Pharmacol. 12, 667-677. 82 Rosenberry, T . L . , and Neumann, E. (1977) Biochemistry 16, 3870-3878. 83 Massoulie, J . , and Bon, S. (1976) Eur. J. Biochem., 68, 531-539. 84 Johnson, C D . , Smith, S.D., and Russell, R.L. (1977) J. Neurochem., 28, 617-624. 85 Wang, C.H., and Willis, D.L., Radiotracer Methodology in the Biological, Environmental and Physical Sciences, Prentice-Hall, New Jersey, 1975. 86 Berman, J.D. (1973) Biochemistry 12, 1710-1715. 87 Snyder, S.L., and Sobocinski, P.Z. (1975) Anal. Biochem. 64, 284-288. - 1 6 8 -88 Anglister, L . , Rogozinski, S., and Silman, I. (1976) FEBS Letters 69, 129-132. 89 Rosenberry, T . L . , Chang, H.W., and Cheng, Y.T. (1972) J. Biol. Chem. 247, 1555-1565. 90 Cotman, C.W., and Taylor, D. (1972) J. Cell Biol. 55, 696-711. 91 Murphy, W.M., v.d. Mark, K., McEneany, L.S.6., and Bornstein, P. (1975) Biochemistry 14, 3243-3249. 92 Kefalides, N.A. (1968) Biochemistry 7, 3103-3112. 939:freytag, J.W., Ohno, M., and Hudson, B.G. (1976) Biochem. Biophys. Res. Commun. 72, 796-802. 94 Gow, J . , and Wardlaw, A.C. (1975) Biochem. Biophys. Res. Commun. 67, 43-49. 95 Rieger, F., Bon, S., and Massoulie, J. (1972) C.R. Acad. Sci. Ser. D 274, 1753-1756. 96 Moore, D.E., and Hess, G.P. (1975) Biochemistry 14, 2386-2388. 97 Morrod, P.J. (1975) Ph.D. Thesis, Dept. of Chemistry, University of British Columbia. 98 Bornstein, P. (1967) Biochemistry 6, 3082-3093. 99 Adams, E . , Antoine, S., and Goldstein, A. (1969) Biochim. Biophys. Acta 185, 251-254. lOOWatkins, M.S., Hitt, A.S. , and Bulger, J.A. (1977) Biochem. Biophys. Res. Commun., 79, 640-647. lOlGrant, N.H., and Alburn, H.E. (1959) Arch. Biochem. Biophys. 82, 245-255. -169-102 Rosenberg, P., Silman, I., Ben-David, E . , de Vries, A . , and Condrea, E. (1977) J. Neurochem., 29, 561-578. 103 Reed, J.K. and Raftery, M.A. (1976) Biochemistry 15, 944-953. 104 Lowry, O.H., Rosebrough, N.J., Far, A . L . , and Randall, R.J. (1951) J. Biol. Chem., 193, 265-275. 105' Hubbard, A . L . , and Cohn, Z.A. (1972) J. Cell Biol. 55, 390-405. 106 Feit, H. , Kelly, P., and Cotman, C.W. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 1047-1051. 107 Kelly, P.T., and Cotman, C.W. (1976) Biochem. Biophys. Res. Commun., 73, 858-864. 108 Kelly, P.T., and Cotman, C.W. (1977) J. Biol. Chem. 252, 786-793. 109 Barchi, R.L., BOnilla, E. , and Wong, M. (1977) Proc. Nat. Acad. Sci. U.S.A. 74, 34-38. 110 Spatz, L. , and Strittmatter, P. (1971) Proc. Nat. Acad..Sci. U.S.A. 68, 1042-1046. 111 Spatz, L . , and Strittmatter, P. (1973) J. Biol. Chem. 248, 793-799. 112 Sigrist, H. , Ronner, P., and Semenza, G. (1975) Biochim. Biophys. Acta. 406, 433-446. 113 Maroux, S., and Louvard, D. (1976) Biochim. Biophys. Acta. 419, 189-195. 114Grafius, M.A., Bond, H.E., and Millar, D.B. (1971) Eur. J. Biochem. 22, 382-390. 115 Rieger, F., Bon, S., and Massoulie, J. (1973) FEBS Letters 36, 12-16. -170-116 Betz, W., and Sakmann, B. (1973) J. Physiol. 230, 673-688. 117 Kefalides, N.A. (1973) Int. Rev. Connec. Tiss. Res. 6, 63-104. 118 Bardos, P., Lanson, M., Degand, P., Gutman, N. , Garrigue, M.A., and Muh, J.P. (1976) FEBS Letters 64, 385-390. 119 Olsen, B.R., Alper, R., and Kefalides, N.A. (1973) Eur. J. Biochem. 38, 220-228. 120 Williams, I .F. , Harwood, R., and Grant, M.E. (1976) Biochem. Biophys. Res. Commun. 70, 200-206. 121 Daniels, J.R., and Chu, G.H. (1975) J. Biol. Chem. 250, 3531-3537. 122 Cotman, C.W., and Taylor, D. (1972) J. Cell Biol. 55, 696-711. 123 cotman, C.W., Banker, G., Churchill, L. , and Taylor, D. (1974) J. Cell Biol. 63, 441-455. -171-APPENDIX A-1. Direct Identification of Hydroxyproline-Rich Subunits of Acetyl- cholinesterase This work investigated the individual hydroxyprol ine content of the subunits of AChE. Preparative SDS-PAGE was used to separate the subunits which were then acid hydrolysed and assayed for total primary amine and hydroxyproline as described in the main text of the thesis. It was expected that those subunits arising from the tail protein of the asymmetric forms of AChE would show higher levels of hydroxyproline than the subunits known to arise from the catalytic protein. This expecta-tion was not realised and this approach to the characterization of the tail component, of AChE was abandoned. Methods Preparative SDS-PAGE was done by a published procedure (Al) with the modification that AChE was fluorescently labelled with fluoe rescamine (Fluram) (A2) to visualize the protein subunits during electro-phoretic separation and elution. The protein was labelled with fluo-rescamine by adding 300 yl of a 10 mg/ml solution of fluorescamine in acetonitrile to 3 ml of AChE at a concentration of ca^  0.6 mg/ml in 1 Ml NaCl, 20 mM sodium phosphate pH 7.0. Electrophoretic separation of subunits was done in a manner similar to that described for analytical SDS-PAGE but the polyacrylamide gels were prepared in tubes with an internal diameter of 10 mm. The migration of the protein subunits was monitored using a short wave ultraviolet lamp. After electrophoresis the polyacrylamide gels were removed from the tubes and sliced into -172-sections containing the major fluorescent bands. These gel sections were then inserted into separate tubes which had been slightly con-stricted at one end to prevent the gel from passing right through. The protein subunits were then electrophoresed out of the end of the gel into a small sac made of dialysis membrane. The progress of elution and the retention of the fluorescently labelled protein within the dialysis membrane sacs was monitored under ultraviolet illumination. Samples of the eluted subunits were kept for analysis by SDS-PAGE and the balance was dialysed against 0.1 M ammonium acetate buffer pH 6.8, and lyophilized. Acid hydrolysis and hydroxyproline assays were done exactly as described under Methods for chapter five. Analytical SDS-PAGE was done on samples of the eluted protein subunits from preparative SDS-PAGE and also on samples of fluorescamine labelled and non-labelled AChE. Results Fluroescamine labelling of AChE did not substantially alter the SDS-PAGE migration pattern obtained with unlabel led enzyme. Figure A-1 shows the SDS-PAGE densitometric profiles for non-labelled (Fig A-1A) and fluorescently labelled (Fig A-1B) AChE. Figure A-2 shows the SDS-PAGE densitometric profiles obtained for the protein eluted from the polyacrylamide gels during preparative SDS-PAGE. The small arrows in Figure A-1B show the approximate places at which the preparative poly-acrylamide gels were sliced for elution and the designation a, b, c, and d, refers to the densitometric profiles labelled A, B, C, and D, in Figure A-2. FIGURE A1 - 1 7 3 -L U O ORIGIN DYE M I G R A T I O N ^ -175-The hydroxyproline content for the eluted proteins was; A, 1.4%; B, 0.7%; C, 0.3%; and D, 0.8%. Discussion The results shown here are the best obtained in three pre-parative SDS-PAGE experiments. The hydroxyproline content of the eluted proteins was low in all the experiments and the expected enrichment of hydroxyproline associated with a particular subunit or group of subunits was never observed. The SDS-PAGE densitometric profiles in Figure A-2 show that complete separation of catalytic protein (subunit molecular weight 79,000 ) from non-catalytic protein was never obtained. The failure to detect appreciable levels of hydroxyproline in the protein eluted from preparative SDS-PAGE could either be due to loss of the hydroxyproline-containing portion of fragments during the handling of the protein subsequent to elution or due to some interferences in the hydroxylproline assay procedure. This assay involves the acid hydrolysis of the protein, the oxidation of the hydroxyproline to pyrrole-2-carbo-xylic acid or to pyrrole in some procedures (A3) and the reaction of either of these species with p-dimethylaminobenzaldehyde to generate a chromophore with a distinct absorbance peak at 560 nm (A4). The assay is time consuming, a single experiment involving preparative SDS-PAGE and subsequent hydroxyproline assays involved 10 days of work with almost 5 days being taken up in the actual assay. In addition the assay is subject to interference by other amino acids in the oxidation step to convert the hydroxyproline in the acid hydrolysate to pyrrole-2-carbo-xylic acid (A3). While interference in this step of the assay could - 1 7 6 -have been responsible for the low levels of hydroxyproline detected the alternative explanation invoking loss of hydroxyproline-containing fragments is equally tenable. The analytical SDS-PAGE profiles shown in Figure A-2 were obtained with samples of eluted protein that were concentrated by lyophilization followed by solubilization in a reduced volume of buffer, with no other treatment. Profiles A, B, and D in Figure A-2 all show an appreciable peak immediately adjacent to the tracking dye. In order to migrate in this position the protein fragments giving rise to this peak must have molecular weights of considerably less than 17,200 the molecular weight of myglobin, which has a migration of less than 0.8 relative to the tracking dye (see Fig 5-3). Furthermore, these fragments were not observed in the analytical SDS-PAGE profiles obtained before preparative SDS-PAGE as shown in Figure A-1B„therefore the degradation leading to these fragments must have taken place during the preparative SDS-PAGE. The tail protein of AChE is more labile to proteolysis than the catalytic protein, as is evident from the data discussed in this thesis and the catalytic protein eluate from preparative SDS-PAGE shows the least amount of small molecular weight fragments (Figure A-2C). So i t is reasonable to conclude that these fragments originate from the tail components. The dialysis mem-branes (obtained from Fisher Scientific Company) used for this work did not retain protein molecules with a molecular weight of less than ca 15,000 as determined by dialysing the enzyme lysozyme (molecular weight 13,930 ) so the small molecular weight protein fragments would be lost during the dialysis against ammonium acetate before lyo-philization for acid hydrolysis. These small fragments could have arisen - 1 7 7 -from proteolytic degradation partially during the dialyses prior to preparative SDS-PAGE and also during the electrophoretic elution. All operations involving sodium dodecyl sulphate had to be performed at room temperature due to the low solubility of this detergent at low temperatures and microbial growth occurs in solutions containing up to 20% sodium dodecyl sulphate when stored for several days. Fresh buffer solutions were made but sufficient microbial contamination could have occured almost immediately to produce the very small amount of degrada-tion needed for the results observed. A-2. Proteolytic Instability of Acetylcholinesterase Subjected to Active-Site Labelling or Storage in Buffer Containing Calcium Ion. The following series of isokinetic sucrose gradient profiles were obtained during the work for this thesis. In most cases the ex-periments shown were for the purpose of obtaining individual forms of AChE for gel chromatography. Three sets of sucrose gradient profiles are shown which demonstrate the proteolytic degradation to which the enzyme was subject following active-site labelling. The final figure shows two gel chromatography elution profiles obtained for a simple of enzyme that had been stored for an extended period of time in a buffer containing calcium ion. Figure A3 shows intact AChE, this profile was obtained 8 days after the enzyme was eluted from the affinity column. This enzyme preparation was used for the experiments using collagenase for varying periods of time to observe the progress of collagenase modification. Figure A4shows the final sucrose gradient profile obtained for enzyme -178-FIGURE A3 -t 1 1 1 r-0 5 10 15 20 FRACTION NUMBER -179-from the above preparation. This profile was for a sample of enzyme that had been]incubated at 30° for 96 h without the addition of col-lagenase. The time period between this gradient and the gradient shown in Fig A3 was 41 days and it is clear that no proteolytic degradation had occurred during this time. Figure A5shows the gradient profile obtained 3 days after active-site labelling of an enzyme preparation that was 4 weeks old at the time of labelling. FigureA6shows the profile obtained with the same sample but at 15 days after labelling. These two gradients were used to obtain individual 18S and 14S enzyme for gel chromatography. A small amount of degradation can be seen in the different proportions of the 18S and 14S enzyme in FigA6and in the slight shoulder in FigA6 coinciding with the position of the 11.3S marker catalase. The labelled enzyme shown in Figures A5 and A6 was subjected to collagenase treatment to obtain the 17S and 21S forms of AChE for gel chromatography. FigureAT shows the sucrose gradient profile obtained after 30 h of incubation with collagenase and FigureAS shows the results of 52 h of incubation. These experiments were done 43 days after the enzyme was active-site labelled and considerable degradation had occurred by this time. This is shown in FigureA9which shows a sample incubated for 30 h without collagenase and FigureAlOfor a sample incubated for 52 h without collagenase. FigureAll shows the sucrose gradient profile obtained for a sample of the same enzyme which had been kept at 4° for the entire time. Very marked proteolytic degradation had occurred by this time compared with Figure A6which was obtained 29 days .earlier. Standard proteins were not assayed in the gradient fractions for Figures A8, AlOand All. -180-FIGURE A4 0 - 1 — 1 1 r -0 5 10 15 20 FRACTION NUMBER It' • i 1 r -0 5 10 15 20 FRACTION NUMBER FIGURE A6 -182-1 0 5 10 15 20 FRACTION NUMBER -183-F IGURE A7 0- - r 5 T — 20 0 10 15 FRACTION NUMBER -184-F I G U R E A 8 FIGURE A9 -185--186-FIGURE A10 0-1 . 1 1 — T -0 5 10 15 20 FRACTION NUMBER -187-F IGURE A11 FRACTION NUMBER -188-A s i m i l a r s e r i e s o f exper iments was c a r r i e d o u t u s i n g f r e s h l y p r e p a r e d enzyme. F i g u r e A 1 2 shows t h e s u c r o s e g r a d i e n t p r o f i l e o b t a i n e d i m m e d i a t e l y a f t e r p u r i f i c a t i o n and e b e f o r e a c t i v e - s i t e l a b e l l i n g . F i g u r e s A 1 3 r A i 4 andA15 show the p r o f i l e s o b t a i n e d a f t e r l e b e l l i n g and c o l l a g e n a s e t r e a t m e n t f o r 5 , 12 and 24 h r e s p e c t i v e l y . The sequence o f c o n v e r s i o n f rom 18S t o I I S enzyme i s q u a l i t a t i v e l y i d e n t i c a l t o t h a t d e s c r i b e d i n t h e t e x t o f t h e t h e s i s . A n o t a b l e d i f f e r e n c e though i s t h a t t h e c o n v e r s i o n t o the f a s t e r s e d i m e n t i n g (21S and 17S) forms i s complete w i t h i n 6 h as shown i n F i g u r e A l 3 . F i g u r e A 1 6 shows t h e s u c r o s e g r a d i e n t p r o f i l e o b t a i n e d w i t h a c t i v e - s i t e l a b e l l e d enzyme a f t e r 12 h o f c o l l a -genase t r e a t m e n t , when c e n t r i f u g a t i o n was done i n t h e p r e s e n c e o f 0 .1 M N a C l . F i g u r e A17 shows t h e c o n t r o l p r o f i l e f o r a sample i n c u b a t e d a t 30° w i t h o u t c o l l a g e n a s e and F i g u r e A 1 8 shows a c o n t r o l sample kept a t 4° i n t h e c a l c i u m i o n c o n t a i n i n g b u f f e r t h r o u g h o u t t h e e n t i r e e x p e r i m e n t . T h i s g r a d i e n t was o b t a i n e d 4 days a f t e r d i a l y s i s o f the a c t i v e - s i t e l a b e l l e d enzyme i n t o the b u f f e r w i t h c a l c i u m i o n . F r a c t i o n 15 i n both F i g u r e A17 and A18 c o n t a i n e d 70% o f the U S fo rm o f t h e enzyme as judged by g e l ch romatography . T h i s s e r i e s o f s u c r o s e g r a d i e n t p r o f i l e s shows the p r o t e o l y t i c i n s t a b i l i t y o f a c t i v e - s i t e l a b e l l e d enzyme compared w i t h t h e h i g h s t a b i l i t y o f i n t a c t enzyme. T h i s i n s t a b i l i t y t o p r o t e o l y t i c d e g r a d a t i o n c o u l d a l s o be observed i n enzyme samples s t o r e d f o r ex tended p e r i o d s o f t i m e . i n the p resence o f c a l c i u m i o n w i t h o u t a c t i v e s i t e l a b e l l i n g . F i g u r e A 1 9 shows t h e g e l chromatography e l u t i o n p r o f i l e s o b t a i n e d f o r a sample o f enzyme s t o r e d a t 4° i n t h e b u f f e r c o n t a i n i n g c a l c i u m i o n . -191-FIGURE AU FRACTION NUMBER -192-FIGURE A15 FRACTION NUMBER FIGURE A16 -193--194-FTGURE A17 -I r — — ' "TT 0 5 10 15 20 FRACTION NUMBER -195-FIGURE A18 FRACTION NUMBER •196-FIGURE A19 4 0 F i g u r e A19. 6 0 8 0 1 0 0 1 2 0 ELUTION VOLUME (ml) 1 4 0 1 6 0 Changes In The C o m p o s i t i o n o f AChE F o l l o w i n g D i a l y s i s I n t o a C a l c i u m I o n - C o n t a i n i n g B u f f e r . Gel chromatography e l u t i o n p r o f i l e o f i n t a c t 18S and 14S AChE shown f o r r e f e r e n c e p u r p o s e s . E l u t i o n p r o f i l e o f 18S and 14S AChE 66 days a f t e r p u r i f i c a t i o n and 8 days a f t e r d i a l y s i s i n t o a c a l c i u m i o n - c o n t a i n i n g b u f f e r . E l u t i o n p r o f i l e o f enzyme 76 days a f t e r d i a l y s i s i n t o t h e c a l c i u m i o n - c o n t a i n i n g b u f f e r . V Q and a r e t h e v o i d volume and i n c l u d e d volume o f t h e g e l chromatography column d e t e r m i n e d w i t h B l u e Dext ran 2000 and p o t a s s i u m f e r r i c y a n i d e r e s p e c t i v e l y . - 197 -REFERENCES Al Stephens, R. E. (1975) Analyt. Biochem., 65, 369-379. A2 . Weigele, M., DeBernardo, S. L . , Tengi, J. P., and Leimgruber, W. (1972) J. Am. Chem. Soc , 94, 5927-5928. A3 Prockop, D. J . , and Udenfriend, S. (1960) Analyt. Biochem., 1, 228-239. A4 Bergman, I., and Loxley, R. (1963) Analyt. Chem., 12, 1961-1965. PUBLICATIONS Webb, 6., and Clark, D.G. (1976) Analytical Biochemistry 71, 629-631. Chamber Seal for Condensation Free Load-ing and Unloading of Beckman Zonal Rotors at Low Temperatures, (short communication). Mansfield, D.H., Webb, G., Clark, D.G., and Taylor, I . E.P. (1978) Biochemical Journal, accepted for publi-cation. Partial Purification and Some Properties of a Cholinesterase from Bush Bean (Phaseolus vul- garis L.) Roots. Webb, G. (1978) Analytical Biochemistry, accepted for publication. Multiple Fraction Collection Using a Peristaltic Pump and a Fraction Collector Modified for Multiple Channel Operation, (short communication). Webb, G., and Clark, D.G., (1978) Archives of Biochem-istry and Biophysics, accepted for publication. Ace-tylcholinesterase: Differential Affinity Chromato-graphic Purification of IIS and 18S plus 14S Forms; the Importance of Multiple-site Interactions and Salt Concentration. Webb, G. , (1978) Canadian Journal of Biochemistry, accept-ed for publication. Acetylcholinesterase: Character-ization of Native and Proteolytically Derived Forms and Identification of Structural Protein Components. 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0059491/manifest

Comment

Related Items