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Protein chemistry of acetylcholinesterase Morrod, Peter John 1976

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PROTEIN CHEMISTRY OF ACETYLCHOLINESTERASE by PETER JOHN MORROD B . S c , U n i v e r s i t y of Sussex, 1971 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n The Department of Chemistry We accept t h i s t h e s i s as conformi/hg. t,o the required standard THE UNIVERSITY OF BRITISH COLUMBIA DECEMBER, 1975 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree t h a t permission for e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of C4t&n<U£7l&-/ The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date %2ye-c ;9?5 ABSTRACT The p r o t e i n a c e t y l c h o l i n e s t e r a s e (AChE) has been i s o -l a t e d from the e l e c t r o p l a x t i s s u e of the e l e c t r i c e e l (Electrophorus e l e c t r i c u s ) and p u r i f i e d by a f f i n i t y chroma-tography. Working with f r e s h t i s s u e , the s t r u c t u r a l s t a b i l i t y of t h i s kind of preparation towards p r o t e o l y s i s and/or a u t o l y s i s has been i n v e s t i g a t e d . Gel e l e c t r o p h o r e s i s of the p u r i f i e d enzyme, i n the presence of sodium dodecyl-sulphate and d i t h i o t h r e i t o l , shows predominantly one component at 80,000 molecular weight. However, gels run at various times a f t e r p u r i f i c a t i o n demonstrate that the 80,000 polypeptide i s s u s c e p t i b l e to cleavage generating peptides of 55,000, 28,000 and 25,000 molecular weight. Evidence i s presented to show that AChE i s composed of four i d e n t i c a l subunits arranged as a dimer of dimers ( c ^ ^ -Incubation of the f r e s h l y a f f i n i t y p u r i f i e d AChE with t r y p s i n i s shown to mimic the cleavage of the 80,000 sub-u n i t by endogeneous protease. Sucrose gradient c e n t r i f u -gation of p u r i f i e d AChE shows i t to be composed of two forms c h a r a c t e r i s e d by t h e i r sedimentation c o e f f i c i e n t s of 18S and 14S which upon p r o t e o l y s i s convert to a g l o b u l a r 11S form. Furthermore conversion«of the 'native' molecular forms to the g l o b u l a r form occurs f a s t e r than p r o t e o l y t i c cleavage of the c a t a l y t i c subunit. i i i Some chemical m o d i f i c a t i o n of the p r o t e i n i s described i n the l a s t s e c t i o n of the t h e s i s . The. enzyme has been l a b e l l e d , by two d i f f e r e n t and complementary methods, so 1 2 5 as to incorporate r a d i o a c t i v e i o d i d e , I. Of p a r t i c u l a r i n t e r e s t i s the r e s u l t observed with an enzymatic, l a c t o -peroxidase i o d i n a t i o n of the 11S form of the enzyme which shows that greater than 9 0% of the l a b e l i s incorporated i n t o the low molecular weight components of the subunit. The other r e s u l t s of the two i o d i n a t i o n methods are described and discussed. F i n a l l y , an appendix d e s c r i b i n g the c h a r a c t e r i s a t i o n of AChE v i a i s o k i n e t i c sucrose gradients i n i n c l u d e d . i v TABLE OF CONTENTS CHAPTER pAGE 1. GENERAL INTRODUCTION 1 1.1 C e l l Membranes 1 1.2 Propagation and transmission of the nerve impulse 14 1.3 A c e t y l c h o l i n e s t e r a s e 24 2. THE ISOLATION AND PURIFICATION OF ACETYL-CHOLINESTERASE FROM THE ELECTRIC ORGAN OF THE ELECTRIC EEL (ELECTROPHORUS ELECTRICUS) 33 2.1 Introduction 33 2.2 Experimental-Materials and Methods 44 2.3 Results and Discussion 64 3. THE MOLECULAR CHARACTERISATION OF AFFINITY PURIFIED ACETYLCHOLINESTERASE 74 3.1 Introduction 74 3.2 Experimental-Material and Methods 83 3.3 Results and Discussion 102 4. CHEMICAL MODIFICATION STUDIES OF AFFINITY PURIFIED ACETYLCHOLINESTERASE 129 4.1 Introduction 129 4.2 Experimental-Materials and Methods 133 4.3 Results and Discussion 136 V CHAPTER P A G E 5. CONCLUSIONS 14 9 REFERENCES 161 APPENDIX:THE CHARACTERISATION OF THE MOLECULAR FORMS OF AChE 17 2 APPENDIX: REFERENCES 1 8 0 v i LIST OF FIGURES FIGURE PAGE 1 I n i t i a t i o n and Propagation of the Nerve Impulse ....... 18 2 The Synapse 21 3 Molecular Forms of AChE 31 4 Chemistry used to couple ligands to Sepharose 3 8 5 Synthesis of A f f i n i t y Ligands 49 6 The E l e c t r i c Organs of Electrophorus E l e c t r i c u s 55 7 Flow chart f o r e x t r a c t i o n and i s o l a t i o n of AChE from t i s s u e 56 8 Chemistry of the Ellman AChE a c t i v i t y assay 58 9 A c e t y l t h i o c h o l i n e assay f o r AChE a c t i v i t y 61 10 Standardisation curve f o r the Lowry determination of P r o t e i n 63 11 E f f i c i e n c y of high s a l t e x t r a c t i o n of AChE from the E l e c t r o p l a x t i s s u e 66 12 E l u t i o n p r o f i l e of a f f i n i t y column 68 13 E l u t i o n of Con A - Sepharose column 72 14 E l e c t r o p h o r e s i s apparatus 84 15 Molecular weight c a l i b r a t i o n curve f o r SDS g e l el e c t r o p h o r e s i s 8 9 16 C a l i b r a t i o n curve f o r I s o k i n e t i c sucrose gradients .... 92 17 A Sketch of the I s o e l e c t r i c focussing column 94 18 The molecular forms and subunit composition of AChE p u r i f i e d from f r e s h e l e c t r o p l a x t i s s u e 104 19 Gel e l e c t r o p h o r e s i s of p u r i f i e d AChE as a fun c t i o n of time 106 v i i FIGURE PAGE 20 Trypsin treated AChE samples 110 21 Molecular forms and subunit composition of AChE before and a f t e r mild t r y p s i n treatment I l l 22 I s o e l e c t r i c focussing of the high s a l t e x t r a c t of e l e c t r o p l a x t i s s u e 116 23 I s o e l e c t r i c f o c u s s i n g of the high s a l t e x t r a c t of e l e c t r o p l a x t i s s u e - 2% Ampholines 117 24 I s o e l e c t r i c focussing of A f f i n i t y p u r i f i e d AChE 118 25 Sepharose 2B e l u t i o n of a f f i n i t y p u r i f i e d AChE 124 26 Sepharose 4B e l u t i o n of a f f i n i t y p u r i f i e d AChE 125 27 Sepharose 4B - column c a l i b r a t i o n 126 28 C a l i b r a t i o n of Sepharose 4B column with standard pr o t e i n s 127 29 P l o t of Stokes Radius versus /-logK^ 128 30 The molecular forms and subunit composition of a sample of p u r i f i e d AChE a f t e r 8 weeks stored at 4°C ... 138 31 The Io d i n a t i o n of the 11S form of AChE under reducing conditions 145 32 The Io d i n a t i o n of the 11S form of AChE without p r i o r reduction 146 33 The Iodination of the 18,14S forms of AChE under reducing conditions 147 3 4 The Io d i n a t i o n of the 18,14S forms of AChE without p r i o r reduction 148 35 Shape of the i s o k i n e t i c gradients 169 36 I s o k i n e t i c gradients i n buffers* 170 TABLE II I LIST OF TABLES I E f f i c i e n c y of e x t r a c t i o n of AchE from the e l e c t r o p l a x I s o e l e c t r i c focussing s o l u t i o n s f o r column . IV I s o e l e c t r i c focussing s o l u t i o n s f o r gels ... V l i l PAGE 60 II Stock s o l u t i o n s and b u f f e r s f o r SDS g e l ^ e l e c t r o p h o r e s i s 99 100 V i o d i n a t i o n r a t i o s f o r lactoperoxidase and ester i o d i n a t i o n of AChE i x ACKNOWLEDGEMENTS I would l i k e to express my sincere a p p r e c i a t i o n to Dr. D.G. Clark whose h e l p f u l advice and o p t i m i s t i c approach provided e x c e l l e n t s u p e r v i s i o n of my work. I am al s o indebted to Dr. A.G. Marshall f o r h i s support and considera-t i o n during my stay at U.B.C. May I al s o express my gr a t i t u d e to Dr. T.L. Rosenberry f o r the kind g i f t of 2 0 mg of compound V I I . I would l i k e to thank my wife Michele,: Monica E. Rosenberg and Luanna Larusson f o r t h e i r help i n the production of t h i s t h e s i s . F i n a l l y I am very g r a t e f u l f o r the generous support of a Canadian Commonwealth Scholarship to study at U.B.C. (1971-1975). 1. CHAPTER ONE GENERAL INTRODUCTION 1. Points of departure The work presented i n t h i s t h e s i s describes some chemistry of the p r o t e i n a c e t y l c h o l i n e s t e r a s e ( a c e t y l c h o l i n e hydrolase, E.C. 3.1.1.7). However, i t would seem appropriate to begin with a general i n t r o d u c t i o n and o u t l i n e some of the motiva-t i o n f o r undertaking the work and provide a perspective f o r the reader who may question i t s relevance when seen i n the i s o l a t e d format of a d o c t o r a l t h e s i s . Such a task w i l l i n v o l v e some d i s c u s s i o n of c e l l mem-branes and the r o l e of pro t e i n s t h e r e i n ; a b r i e f o u t l i n e of the current theory concerning the transmission of nerve impulses and f i n a l l y an h i s t o r i c a l i n t r o d u c t i o n to the study of the p r o t e i n a c e t y l c h o l i n e s t e r a s e . At best then t h i s i n t r o d u c t i o n w i l l expose the reader to the many e x c i t i n g p o i n t s of entry i n t o the work which i s described here. At l e a s t i t w i l l provide a l i s t of references which defend t h i s study. 1.1 C e l l Membranes During the l a s t decade the p r o p e r t i e s and fu n c t i o n of c e l l membranes have been one of the most a c t i v e l y explored 2. f i e l d s i n the b i o l o g i c a l sciences; much information has been obtained from e l e c t r o n microscopy combined with biochemical and b i o p h y s i c a l analyses. Membrane s t r u c t u r e has thus evolved from one popular model to another as more information has become a v a i l a b l e ; no attempt w i l l be made here to fo l l o w t h i s e v o l u t i o n but rather draw from i t some important f a c t s and concepts that have a r i s e n . I t i s now w e l l e s t a b l i s h e d that c e l l membranes are hi g h l y organised and dynamic s t r u c t u r e s i n which many pro-t e i n s and enzymes are lo c a t e d and form an e s s e n t i a l p a r t of the st r u c t u r e and c o n t r o l mechanisms e f f e c t e d by mem-branes. I t i s the d i v e r s i t y of i t s p r o t e i n a c t i v i t y that gives each p a r t i c u l a r membrane i t s d i s t i n c t i v e character. Membranes are a l s o composed of l i p i d s , which are them-selves complex, amphipathic molecules. As such, they are organised i n a b i l a y e r ; two l a y e r s back to back so that t h e i r h y d r o p h i l i c heads c o n s t i t u t e the top and bottom sur-faces of the membrane and t h e i r hydrophobic, hydrocarbon t a i l s are buried i n t o the membrane b i l a y e r (1,2). 0 HYDROCARBON TAILS C H - 0 - C - ( C H 2 ) - ( C H = C H ) - ( G H ^ - C H g 0 C H - 0 - C - ( C H 2 ) - ( C H = C H ) - ( C H J - C H , <Chp3 M - C H j C H - 0 - P - 0 - C H 2 0 POLAR HEAD GROUP D i o l e y l L e c i t h i n 3. In view of the c r u c i a l r o l e of p r o t e i n s i n membrane fun c t i o n , there has been great impetus to study the s t r u c -ture and f u n c t i o n of a l a r g e v a r i e t y of membrane bound p r o t e i n s . These p r o t e i n s can be c l a s s i f i e d i n t o two broad categories, which d i f f e r , q uite simply, by t h e i r p o s i t i o n with respect to the l i p i d b i l a y e r and therefore i n t h e i r mode of i n t e r a c t i o n with the l i p i d . I n t r i n s i c (3) or i n t e g r a l (4) membrane prot e i n s pene-t r a t e i n t o and sometimes completely through the i n t e r i o r of the b i l a y e r and have, t h e r e f o r e , a predominantly hydro-phobic i n t e r a c t i o n with l i p i d . However, according to the thermodynamic arguments which l e d to the concept of amphi-pa t h i c i n t e g r a l p r o t e i n s (5), f o r completely buried p r o t e i n s to e x i s t they would have to contain very few i o n i c amino a c i d residues. Although there i s a tendency f o r i n t e g r a l membrane proteins to contain a l a r g e r f r a c t i o n of hydro-phobic amino a c i d residues than do s o l u b l e p r o t e i n s (6) those so f a r analysed a l s o contain a s u b s t a n t i a l number of i o n i c residues. One of the most hydrophobic i n t e g r a l p roteins known i s the C^^-isoprenoid a l c o h o l phosphokinase of Staphylococcus aureus (7) and even i t has between 16 and 32 i o n i c residues (depending on o x i d a t i o n of a s p a r t i c and glutamic a c i d moieties) per molecule of 17,000 daltons. The t o t a l free energy required to embed a l l or most of these residues within the hydrophobic i n t e r i o r of the membrane 4. could be as high as s e v e r a l hundred k i l o c a l o r i e s per mole (5). Even though larg e p o r t i o n s of the i n t e g r a l p r o t e i n s may be embedded i n the i n t e r i o r of the membrane there i s , as yet, no t h e o r e t i c a l or experimental reason to expect any known p r o t e i n to be completely embedded. The major g l y c o p r o t e i n of the human erythrocyte mem-brane has been i s o l a t e d and c h a r a c t e r i s e d (8,9); i t i s composed of 60% carbohydrate and 4 0% peptide with a t o t a l molecular weight of about 55,000 and i s c a l l e d glycophorin. Various e l e c t r o n microscope experiments with f e r r i t i n -l a b e l l e d l e c t i n s (10) and other chemical evidence (11,12) has shown that the carbohydrate p o r t i o n i s on the e x t e r i o r surface of the membrane, present as short o l i g o s a c c h a r i d e chains confined to the -NE^ terminal h a l f of the polypeptide chain. The -COOH terminal fragments have been shown to be exposed to the cytoplasmic surface and thus the i n t e r -vening sequence of 23 amino acids i s probably hydrophobic i n nature and spans the b i l a y e r i n a continuous a - h e l i c a l arrangement. Cytochrome b^ of microsomal membranes was f i r s t extracted from the membrane by enzyme treatment of the i n t a c t mem-branes (13)and as such was found to contain 100 amino a c i d residues. However, recent experiments (14,15) i n two la b o r a t o r i e s have shown that the enzyme extracted by detergent treatment of the membranes i s l a r g e r than that previously-p u r i f i e d . In f a c t i t contained 4 0 a d d i t i o n a l amine a c i d r e s i d u e s , which are predominantly hydrophobic and are attached to the carboxyl end of a t r y p t i c fragment (16) (see t a b l e below). Further, the e n t i r e cytochrome b,- mole-cul e s could bind spontaneously t o the mitochondria and microsomal membranes but i f the hydrophobic fragment was f i r s t removed the p r o t e i n would not bind to the membrane. Almost i d e n t i c a l r e s u l t s have been found f o r the NAD-cytochrome b,- reductase of microsomal membranes (17). When s o l u b i l i s e d by p r o t e o l y s i s i t has a molecular weight of 33,000. Upon e x t r a c t i o n with the detergent T r i t o n X-100, however, i t has a molecular weight of 43,000. The d i f f e r e n c e , again, i s a membrane-bound fragment with a corresponding larg e f r a c t i o n (65%) of non-polar amino a c i d residues (see tabl e below). This fragment must be present f o r the pro-t e i n to bind to the membranes. cytochrome b g cytochrome b 5 reductase u Ltpase-extracted cyt ki* Determent-extracted cyt i>,1 Hydro-phobic (b — o) peptide! Amino add A D t l r i « n t -< M I U I « t <>•!,* h,..m. ) t r c i l i K l a j c B f x t i a * led «> l<« Siomc 1, irrfuda't A — B !'>•» . 10 11 1 1 25 20 5 lib 7 7 0 0 .12 9 3 Arg 3 4 1 1 20 10 4 Asp Thr 10 7 16 10 6 3 4 3 34 2S C Scr 7 10 3 3 10 14 2 Clu 14 15 1 1 19 13 0 Pro Gly 3 6 5 7 2 1 2 1 (Ihttamatc 3S 33 29 29 9 4 Ala S 10 5 4 27 20 -Cys 0 0 0 0 21 15 Val 4 7 3 3 7 C 1 Met 1 3 2 2 28 10 12 He 4 S 4 4 10 S 2 Leu 9 IS 6 4 24 19 5 Tyr 3 6 2 2 40 27 13 Plus Tip 3 1 4 4 1 3 1 3 Tyrosine 13 IS 13 5 ' 5 Total 07 141 • 44 40 G 2 4 Molecular Weight 11,079 10,072 4,003 4,579 391 292 99 Amino ac i d composition of 43,320 33,000 10,110 cytochrome b5 and b^ reductase 6. These i n t e g r a l p r o t e i n s and the others can only be l i b e r a t e d from t h e i r r e s p e c t i v e membranes by reagents which d i s r u p t hydrophobic i n t e r a c t i o n . They are i n s o l u b l e or aggregated i n aqueous s o l u t i o n i n the absence of deter-gent of other s o l u b i l i s i n g agent. Further, they are us u a l l y associated with some l i p i d when s o l u b i l i s e d and have the unique a b i l i t y to recombine with the membrane. In c o n t r a s t to i n t r i n s i c membrane p r o t e i n s is the c l a s s of pr o t e i n s which do not penetrate the l i p i d b i l a y e r but are held at the surface of the membrane by predominantly e l e c t r o s t a t i c i n t e r a c t i o n s . These molecules have been c a l l e d e x t r i n s i c (3) or p e r i p h e r a l (4) membrane p r o t e i n s . Examples of t h i s kind of p r o t e i n include cytochrome c of the mitochondrial inner membrane (17) and the ba s i c p r o t e i n of myelin (18), s p e c t r i n of the red c e l l membrane (19) and o<-lactalbumin, a p e r i p h e r a l p r o t e i n of the membranes wi t h i n the mammary gland (20). A l l of these p r o t e i n s can be removed from t h e i r r e s p e c t i v e membranes by reagents which d i s r u p t i o n i c , e l e c t r o s t a t i c i n t e r a c t i o n s . They are soluble i n aqueous s o l u t i o n and once s o l u b i l i s e d are very hard to r e c o n s t i t u t e . I t has been suggested that p e r i p h e r a l proteins w i l l g e n e r a l l y be attached to membranes Toy binding to exposed h y d r o p h i l i c ends of amphipathic i n t e g r a l p r o t e i n s and i t i s t h i s feature which makes them a s p e c i a l c l a s s s e r v i n g to modulate and regulate s p e c i f i c membrane functions (21). To pause f o r breath and complicate the p i c t u r e f u r t h e r i t i s perhaps important to mention that l i p i d s and pro-t e i n s may be c o v a l e n t l y attached to carbohydrate. Thus glyc o p r o t e i n s and g l y c o l i p i d s are al s o an important c l a s s of molecules being i n v e s t i g a t e d w i t h i n the context of a n t i -body-antigen i n t e r a c t i o n s and other c e l l u l a r communication occ u r r i n g at the membrane surface. The carbohydrate ex-tending i n t o the aqueous environment around the c e l l may provide a d e t a i l e d ' f i n g e r - p r i n t ' f o r r e c o g n i t i o n purposes. As one delves i n t o the many fa c e t s of membrane s t r u c -ture and f u n c t i o n one becomes aware very q u i c k l y of the s o p h i s t i c a t i o n and complexity of what one i s studying. Three major and chemically d i s t i n c t c l a s s e s of molecules: phospholipids, carbohydrates and p r o t e i n i n t e r a c t i n g to form very s p e c i f i c and h i g h l y organised molecular s t r u c t u r e s . The p i c t u r e of membrane str u c t u r e presented so f a r though, i s e s s e n t i a l l y a s t a t i c one and i n t h i s respect i t i s inaccurate. One of the major advances i n c e l l studies during the past few years has been to r e a l i s e that both l i p i d s and proteins have considerable freedom of movement wi t h i n the b i l a y e r s t r u c t u r e . The f l u i d i t y of the phospholipid molecules depends on two f a c t o r s : f i r s t , the degree of unsaturation of t h e i r long hydrophobic t a i l s and second the ambient temperature. Much work has been done to study the temperature e f f e c t s on l i p i d b i l a y e r systems w i t h i n both model and n a t u r a l membrane systems (22). I t has been shown that there i s a c h a r a c t e r i s t i c t r a n s i t i o n temperature f o r each b i l a y e r at which the r i g i d l y 'frozen' phase of the phospholipids becomes l i q u i d c r y s t a l l i n e and more mobile. Not only do these phase changes and the r e s u l t i n g l i p i d f l u i d i t y e f f e c t the mechanical p r o p e r t i e s of the membrane (23,24) but they i n f l u e n c e membrane transport, (25-30), enzyme a c t i v i t y (31-33) and the d i s t r i b u t i o n of p r o t e i n s (34,35) . F l u i d i t y and m o b i l i t y w i t h i n the membrane s t r u c -ture are of prime importance i n membrane biology, and a great deal of a t t e n t i o n i s thus being given to understand the nature and mechanism of these phenomena. The freedom of movement of l i p i d s has been studied e x t e n s i v e l y by the groups of H.M. McConnell and O.H. G r i f f i t h both using the technique of e l e c t r o n spin resonance spec-troscopy (E.S.R.). This method involves the attachment of a "reporter" group, u s u a l l y a n i t r o x i d e which contains an unpaired e l e c t r o n , to one of the carbon atoms of the f a t t y a c i d chain i n a phospholipid or i n s t e a r i c a c i d . The information contained w i t h i n the E..S.R. spectrum due to t h i s unpaired e l e c t r o n can be analysed to gain information about the degree of motion i n the system. Hence a n i t r o x i d e was attached v i a some elegant synthesis (36) to e i t h e r the f i f t h , t w e l f t h or s i x t e e n t h carbon atom i n the ph o s p h o l i p i d or s t e a r i c a c i d hydrocarbon ' t a i l ' . S e veral l a b e l l e d molecules were then i n s e r t e d i n t o a model membrane system and the E.S.R. spectrum recorded. From the shape of these spectra i t was p o s s i b l e to gain information concerning the degree of m o b i l i t y e x i s t i n g w i t h i n the b i l a y e r . Both McConnell and G r i f f i t h were able to show that there i s i n f a c t a m o b i l i t y gradient w i t h i n the b i l a y e r ; f l e x i b i l i t y i n c r e a s i n g towards the centre of the hydrophobic r e g i o n . These r e s u l t s have been v e r i f i e d by Metcalfe et a l . using another resonance technique; nuclear magnetic resonance spectroscopy (N.M.R.) (37a) to study the r e l a x a t i o n times 13 (T-^) of the n a t u r a l abundance C n u c l e i contained i n an a r t i f i c i a l d i p a l m i t o y l l e c i t h i n (C-^g) b i l a y e r . The T^ values are c h a r a c t e r i s t i c o f both the chemical s t r u c t u r e of the l e c i t h i n and the s t e r i c i n t e r a c t i o n s between molecules i n a given solvent. The values above the thermal t r a n s i t i o n temperature (mentioned above) showed that the molecular motion increases from the g l y c e r o l carbons towards both of + the terminal methyl groups of the chain and the -NMe^ p o l a r head group. „ - 1 L Relaxation Timoa In seca. \n DjO at 52°C 3-3 1-8 M 0-6 0-2 0-1 23 01 C H 3 C H 2 C H 2 < C H 2 ) w C H 2 C H 2 C O C H 2 O I | 0-1 O I 0 H 2 C O P O C H 2 C H 2 N ( C H 3 ) 3 ir 0-1 0-3 0-3 0-7 10. This work was then extended to study the microsomal membrane of the sarcoplasmic r e t i c u l u m (3 8) and i n v e s i c l e s prepared from the extracted l i p i d s . I t was found that the 13 C chain resonances have very s i m i l a r values i n both + membrane and v e s i c l e preparation but the -NMe^ T^ value was s i g n i f i c a n t l y shorter i n the membrane. This was t e n t a t i v e l y a t t r i b u t e d to i n t e r a c t i o n with membrane p r o t e i n s , an idea substantiated by the f a c t that only 2 0% of the f a t t y a c i d chain protons appear i n the sharp components of the ~*"H membrane spectrum. The major f r a c t i o n of the l i p i d being broadened e i t h e r as a r e s u l t of being t i g h t l y packed or by d i r e c t intermolecular i n t e r a c t i o n with membrane p r o t e i n s . Recently deuterium magnetic resonance has been used to study molecular motion i n d i p a l m i t o y l l e c i t h i u m b i l a y e r s (37b). The l i p i d molecules are deuterated at nine d i f f e r e n t carbon atoms of the f a t t y a c y l chains. Of p a r t i c u l a r s i g n i f i c a n c e i s the conclusion that there i s i n f a c t very l i t t l e freedom of motion i n the f i r s t nine carbon atoms of the chains and that the ordered s t r u c t u r e decreases only i n the c e n t r a l region of the b i l a y e r . The deuterium data d i f f e r then from the e l e c t r o n spin resonance experiments and t h i s the authors claim may be a t t r i b u t e d to a perturbation of the b i l a y e r by the s p i n - l a b e l group. The r e s u l t s are thus con-t r o v e r s i a l and the q u a n t i t a t i v e data which can be obtained from t h i s kind of work w i l l be h e l p f u l i n probing the molecu-l a r s t r u c t u r e and motion of the membrane b i l a y e r . To extend the s p i n - l a b e l l i n g work and gain information on the movement of phospholipids w i t h i n the b i l a y e r , McConnell and Devaux studied the a b i l i t y of these molecules to d i f f u s e l a t e r a l l y (34) and move from one side o f a b i -l a y e r to the other (40). V e s i c l e s of egg l e c i t h i n c o n t a i n -ing paramagnetic phosphatidylcholine molecules only on the i n s i d e of the b i l a y e r are prepared by reduction of the S p i n - l a b e l l e d phosphatidylcholine external spin l a b e l with a s c o r b i c a c i d (ascorbic a c i d does not penetrate the v e s i c l e s at 0°). I t was found that at 30° there was a slow exchange ( f l i p - f l o p ) between the inner and outer b i l a y e r surfaces occuring with a half-time of 6.5 hours between i n s i d e and outside. The authors suggest that t h i s elegant method should be s e n s i t i v e to f a c t o r s which may acc e l e r a t e the rate of exchange and i n f a c t r e c e n t l y extended t h e i r study to ion movements across b i l a y e r s (41). Here the exchange i s an order of magnitude f a s t e r than i n the synthetic l e c i t h i n b i l a y e r (half-time of 4-7 minutes) C H 2 — 0 — C O — (CH2) 1 4 — CH 3 C H 0 — (CH„) , ,—CO—0—CH J Z ± 4 I 0 II CH„-0-P—0-CH 0 ~ 12 . and a s p i n - l a b e l assay f o r d i f f u s i o n p o t e n t i a l s i n e x c i t a b l e membrane v e s i c l e s was developed (41,42). In c o n t r a s t to movement across the b i l a y e r , r a p i d l a t e r a l d i f f u s i o n of l i p i d s has been shown to occur w i t h i n both model and n a t u r a l membranes (39,43) with a c h a r a c t e r i s t i c — 8 2 d i f f u s i o n constant of about 10 cm /sec (37°). With t h i s constant a phospholipid can d i f f u s e a distance of the order of 500 Angstroms i n one second. Since f o r a glo b u l a r p r o t e i n i n water the d i f f u s i o n constant v a r i e s as the r e c i p r o c a l of the cube root of the protein's molecular weight the l a t e r a l d i f f u s i o n f o r some pro t e i n s i n a membrane may be j u s t as r a p i d . Indeed estimates f o r surface antigens i n mouse-human c e l l hybrids (44) are compatible with the d i f f u s i o n constants that are found f o r phospholipids. Not every l i p i d molecule however has the p o t e n t i a l f o r such m o b i l i t y . In f a c t , ' i n t r i n s i c ' p r o t e i n s have been shown to severely r e s t r i c t the l i p i d molecules which are nearest to them. G r i f f i t h and Capaldi have used spin l a b e l l e d s t e a r i c acids to probe the l i p i d movement adjacent to cytochrome oxidase i n a model system (45). CH 3 S p i n - l a b e l l e d s t e a r i c a c i d They found that the p r o t e i n had e f f e c t i v e l y immobilised s u f f i c i e n t l i p i d to coat i t s e l f with a monomolecular l a y e r . This t i g h t l y associated l a y e r has been c a l l e d "boundary l i p i d " . I t i s i n t e r e s t i n g too, that the amount of t h i s l i p i d i s j u s t that which i s required f o r the f u l l a c t i v i t y of t h i s enzyme, and indeed there are other reports i n the l i t e r a t u r e to i n d i c a t e that somemembrane l i p i d s are i n t i -mately involved i n i n t e r a c t i o n with i n t r i n s i c p r o t e i n s (46-49) The "purple membrane1 of the Halobacterium halobium i s an example of a membrane containing only one p r o t e i n which i s arranged i n a r i g i d c r y s t a l l i n e array l i k e the cytochrome oxidase model membrane. 'Gap junctions' which are thought to be used f o r the t r a n s f e r of substances from one c e l l to another contain only one p r o t e i n and one kind of phospho-l i p i d arranged i n an hexagonal l a t t i c e which i s c l e a r l y v i s i b l e i n the e l e c t r o n microscope. The membranes of c e l l s that l i n e the inte.stine d i s p l a y a d e f i n i t e arrangement of pr o t e i n s : the glycoproteins are concentrated at the i n t e s -t i n a l surface and the sodium-potassium ATP'ase (sodium pump) i s located at the other end. In a s i m i l a r way the c e l l s studied i n t h i s t h e s i s ; c h o l i n e r g i c nerve c e l l s , are high l y organised i n t o an innervated face, containing a c e t y l c h o l i n e s t e r a s e and a receptor p r o t e i n involved i n transmission of the nerve impulse and a non-innervated face containing the sodium-potassium ATP ' ase which r e s t o r e s the e l e c t r i c a l imbalance a f t e r a c e l l has transmitted the a c t i o n p o t e n t i a l . As more information on the s t r u c t u r e of various c e l l membranes i s revealed i t i s becoming evident that there i s no o v e r a l l simple model to account f o r the d i s t r i b u t i o n of the various components. What seems to be important i s understanding the p r i n c i p l e s involved i n the r e l a t i o n s h i p between the molecular array and the membrane f u n c t i o n of the c e l l . The problem then i s how to study such a complex system and i t i s perhaps t h i s very complexity which has a t t r a c t e d a m u l t i - d i s c i p l i n a r y approach ranging from gross p h y s i c a l techniques to the molecular probing of the b i o l o g i c a l chemist. As a f i r s t step i n studying membrane s t r u c t u r e , one approach of the p r o t e i n chemist has been to i s o l a t e and s e l e c t i v e l y p u r i f y a p r o t e i n of i n t e r e s t so that i t s mole-c u l a r s t r u c t u r e and p r o p e r t i e s can be probed. This t h e s i s i s i n p a r t concerned with such a p u r i f i c a t i o n and more w i l l be s a i d l a t e r about t h i s approach i n the l i g h t of some of the observations that have been made. (50-5 1.2 Propagation and transmission of- the nerve impulse: How do c e l l s communicate with one another? Nature has evolved two very basic coordinating mechanisms. One depends 15. on the r e l e a s e and c i r c u l a t i o n of 'chemical messengers', the hormones that are manufactured by c e r t a i n s p e c i a l i s e d c e l l s and that are able to r e g u l a t e the a c t i v i t y of c e l l s i n other parts of the organism. The second mechanism which i s i n general f a r s u p e r i o r i n speed and s e l e c t i v i t y depends on a s p e c i a l i s e d system of nerve c e l l s or neurons whose f u n c t i o n i t i s to r e c e i v e and give information by means of e l e c t r i c a l impulses d i r e c t e d over s p e c i f i c pathways. How then, w i t h i n the context of c e l l u l a r communication, does a nerve c e l l transmit i t s i n f o r -m a t i o n / i n s t r u c t i o n s to another c e l l ? We w i l l be concerned here to d i s c u s s the motor neurons, which are the c l a s s of nerve c e l l s which c a r r y impulses from the b r a i n to the 'working' c e l l s and i n so doing provide the organism with a means to respond to changes i n i t s e x t e r n a l and i n t e r n a l environment. How, f o r example i s information propagated from the neuronal c e l l body to a muscle c e l l and once a t the synapse with the muscle, how i s the e s s e n t i a l communication made between these two d i f f e r e n t kinds of c e l l s ? L ike other c e l l s each nerve c e l l has a nucleus and a surrounding cytoplasm. However, i t s outer surface c o n s i s t s of numerous f i n e branches c a l l e d dendrites which r e c e i v e impulses from other nerve c e l l s and one long f i b e r , the axon which transmits them. When the c e l l s d i f f e r e n t i a t e i n the embryo, the c e l l body sends out t h i s axon which then grows towards i t s ' t a r g e t ' muscle or s k i n c e l l . I t thus forms a kind o f cable f o r conduc-t i n g information from the periphery and the p r o t e c t e d c e l l body i n the s p i n a l canal or the b r a i n to a neural or neuro-na l synapse. The nerve impulse t h a t i s propagated along the axon ceases abruptly when i t comes to the 1 end p l a t e ' regions where contact i s made with another nerve c e l l or a muscle c e l l i n the case of a motor neuron. The cytoplasms of the adjacent c e l l s are separated by d i s t i n c t membranes and moreover there i s a small e x t r a -o c e l l u l a r gap u s u a l l y of some 1 0 0 - 2 0 0 A between adjacent membranes. These junc-t i o n p o i n t s are c a l l e d "synapses" and i f the nerve impulse i s to continue i t must be regenerated a f r e s h on the other s i d e , i n a neighbouring c e l l . The axon membrane separates two aqueous s o l u t i o n s that are almost e q u a l l y e l e c t r o c o n d u c t i v e and that contain approximately the same number of e l e c -t r i c a l l y charged p a r t i c l e s or ions. 1 7 . But the chemical composition o f the two s o l u t i o n s i s q u i t e d i f f e r e n t . In f a c t the concentration of sodium i s about 10 times higher outside the axon and that of potassium i s about 30 times higher i n s i d e the axon. This e l e c t r i c a l imbalance r e s u l t s i n a voltage drop of some 60 to 90 m i l l i -v o l t s across the membrane with the i n s i d e of the c e l l being negative with respect to the outside (see f i g u r e one). To maintain these d i f f e r e n c e s i n ion concentration the nerve axon contains w i t h i n i t s membrane a sodium pump which seems to f u n c t i o n v i a an energy y i e l d i n g metabolic r e a c t i o n i n which breakdown of ATP i s involved. Indeed enzymes which hydrolyse ATP and possess some of the c h a r a c t e r i s t i c ' d i r e c t i o n a l ' p r o p e r t i e s ascribed to a c t i v e i on transport are now known. These ATP'ases are locat e d at c e l l sur-faces and are a c t i v a t e d by exte r n a l potassium and i n t e r n a l sodium ions. Furthermore, they are i n h i b i t e d by low con-cent r a t i o n s of car d i a c glycosides (e.g. ouabain or d i g i t o x i n ) which are known to be potent i n h i b i t o r s of the ' u p h i l l ' t ransport of sodium and potassium i n nerve and muscle (53). The mechanism of transmission of the nerve impulse evolves around the i o n imbalance described above. When the voltage d i f f e r e n c e across the membrane i s lowered the immediate e f f e c t i s to increase the axon's permeability to the i n f l u x of sodium. Why t h i s happens i s s t i l l not c l e a r , but i t i s a f a c t of greatest importance. As sodium ions leak 18. FIGURE 1: INITIATION AND PROPAGATION OF THE NERVE IMPULSES A. Propagation of Nerve Impulse c o i n c i d e s with changes i n the pe r m e a b i l i t y o f the axon membrane. When a nerve impulse a r i s e s sodium ions pour i n t o the axon making the axon i n t e r i o r l o c a l l y p o s i t i v e . A f t e r the impulse potassium ions flow out r e s t o r i n g the normal negative p o t e n t i a l . 2 msec pulse B. The i n i t i a t i o n of an impulse by l o c a l d e p o l a r i s a t i o n . Current pulses of f i x e d duration but v a r i a b l e s i z e cause v a r i a t i o n s of membrane p o t e n t i a l shown by the family of curves. through the membrane they lower the p o t e n t i a l d i f f e r e n c e and make i t e a s i e r f o r more ions to flow i n t o the axon. This then i s a regenerative process and eve n t u a l l y so many ions flow i n that the i n t e r n a l p o t e n t i a l of the c e l l changes from negative to p o s i t i v e ; the process has thus ' f i r e d ' to create the nerve impulse or a c t i o n p o t e n t i a l . This i n turn changes the permeability of the axonal membrane immediately ahead of i t and sets up the conditions f o r a progressive wave t r a v e l l i n g down the axon. Meanwhile as t h i s spike peaks other events are taking place at the molecular l e v e l . The sodium channels are clos e d and now b r i e f l y , potassium rushes out of the axon to res t o r e the o r i g i n a l negative charge of the i n t e r i o r of the axon. The axon has been l i k e n e d to an e l e c t r i c cable and though there i s some analogy between the two, there i s a l s o some inaccuracy. The e l e c t r i c a l r e s i s t a n c e of the axon's f l u i d core i s about 10 0 m i l l i o n times greater than that of a copper wire and the axon membrane i s about a m i l l i o n times l e a k i e r to e l e c t r i c current than a good cable! Indeed a pulse of e l e c t r i c i t y too weak to t r i g g e r the nerve impulse fades out i n only a few m i l l i m e t e r s . Thus i t i s important to remember than the axon functions to amplify the input and regenerate i t s e l f so there i s no los s as the s i g n a l migrates down the length of an axon. This i t does v i a the i n f l u x of sodium and concomitant e f f l u x of potassium ions. 20. B r i e f pulses of constant amplitude f o l l o w i n g each other at i n t e r v a l s l i m i t e d by the r e f r a c t o r y p e r i o d of each c e l l provide the long-distance communication needs of our nervous system. This simple coding system r e l i e s f o r i t s e f f e c t i v e n e s s on lar g e numbers of axon channels each from a separate nerve c e l l , to be arranged i n p a r a l l e l . For example, i n the o p t i c nerve trunk emerging from the eye there are more than a m i l l i o n channels running c l o s e t o -gether a l l capable of t r a n s m i t t i n g separate s i g n a l s to higher centers i n the b r a i n . Let us now look at what happens at the synapse, the poin t at which the impulse reaches the end of one nerve c e f l and must be relayed on to the next. There i s not abundant p h y s i o l o g i c a l evidence that transmission i s achieved by the release of s p e c i f i c chemical substances from the end of the axons which then d i f f u s e across the small ext r a -c e l l u l a r gap (100-200 angstroms). These molecules then com-bine with receptors i n the postsynaptic membrane and opening the ion channels f o r the i n f l u x of sodium allows the nerve impulse to be t r i g g e r e d once again (see f i g u r e 2) . The neuromuscular j u n c t i o n has been studied i n great d e t a i l and much information has been obtained from i t which w i l l be h e l p f u l i n understanding the process of chemical transmission of other neuronal synapses. The chemical transmitter f o r t h i s j u n c t i o n has been i d e n t i f i e d as FIGURE 2: THE SYNAPSE DENDRITE S Y N A P T I C K N O B S a r e d e s i g n e d to d e l i v e r s h o r t b u r s t s o f a c h e m i c a l t r a n s m i t t e r sub-stance i n t o t h e s y n a p t i c c le f t , w h e r e i t c a n act o n t h e s u r f a c e o f t h e n e r v e - c e l l m e m b r a n e b e l o w . B e f o r e r e l e a s e , m o l e c u l e s of t h e c h e m i c a l t r a n s m i t t e r a r e s t o r e d i n n u m e r o u s v e s i c l e s , o r sacs. M i t o c h o n d r i a a r e s p e c i a l i z e d s t r u c t u r e s that h e l p to s u p p l y t h e c e l l w i t h e n e r g y . SYNAPTIC VESICLES PRESYNAPTIC MEM3R/-NE SYNAPTIC CLEFT TRANSMITTER . MOLCCULES SUBSYNA?T|C MEM3RANL S Y N A P T I C V E S I C L E S c o n t a i n i n g a c h e m i c a l t r a n s m i t t e r a r e di;-tri !>uled t h r o u g h o u t t h e s y n a p t i c k n o b . T h e y are a r r a n g e d h e r e i n a p r o b a b l e s e q u e n c e , s h o w i n g b o w t h e y m o v e u p to the s y n a p t i c c l i - f l , d i s c h a r g e t h e i r c o n t e n t s a n d r e t u r n to t h e i n t e r i o r f o r r e c h a r g i n g . a c e t y l c h o l i n e and motor nerve f i b e r s are known to contain c h o l i n e a c e t y l t r a n s f e r a s e , the enzyme needed to synthesise t h i s substance. I t should perhaps be emphasised here that a c e t y l c h o l i n e i s only one of many t r a n s m i t t e r s . The t a b l e below shows some of the others that are known; and t h e i r p h y s i o l o g i c a l f u n c t i o n : a c e t y l c h o l i n e - e x c i t a t o r y and i n h i b i t o r y ( c h o l i n e r g i c ) noradrenaline - e x c i t a t o r y and i n h i b i t o r y (adrenergic) 4-aminobutyric a c i d - i n h i b i t o r y (crustacean synapses) L-glutamate - e x c i t a t o r y (crustacean and i n s e c t neuro-muscular junction) g l y c i n e - i n h i b i t o r y (strychnine synapses) E l e c t r o n microscopy has been used to v i s u a l i s e several i n t e r -e s t i n g features of the presynaptic area. C h a r a c t e r i s t i c v e s i c l e s about 50 0 angstroms i n diameter p o s s i b l y containing a c e t y l c h o l i n e are seen c l o s e to the presynaptic membrane adjacent to the synaptic c l e f t . Even i n the absence of a nerve impulse these v e s i c l e s i n t e r e a c t with the axonal mem-brane and discharge d i s c r e t e numbers of a c e t y l c h o l i n e molecules. Each time one of these quanta of transmitter molecules i s l i b e r a t e d i t i s p o s s i b l e to detect minute l o c a l response i n the muscle or nerve c e l l on the post-synaptic s i d e . How-ever, i t has been p o s s i b l e to show that the b r i e f 120 m i l l i -v o l t change i n p o t e n t i a l associated with the d e p o l a r i s a t i o n of the axon causes the frequency of release of v e s i c l e s of a c e t y l c h o l i n e to r i s e by a f a c t o r of nearly one m i l l i o n ; hundreds of v e s i c l e s being discharged i n a f r a c t i o n of a m i l l i s e c o n d . Much work has been done to i d e n t i f y the a c e t y l c h o l i n e receptor s i t e on the postsynaptic membrane. In recent years a so c a l l e d receptor p r o t e i n has been i s o l a t e d from the c h o l i n e r g i c neuromuscular j u n c t i o n (54) using some of the detergent s o l u b i l i s i n g techniques described i n part one of t h i s i n t r o d u c t i o n . This i n t r i n s i c p r o t e i n i s at present being c h a r a c t e r i s e d and studied by a v a r i e t y of chemical and p h y s i c a l methods to probe i t s s t r u c t u r e and f u n c t i o n i n t r a n s m i t t i n g the nerve impulse. The same t i s s u e a l s o contains a powerful enzyme which hydrolyses a c e t y l c h o l i n e . This a c e t y l c h o l i n e s t e r a s e i s h i g h l y concentrated at the neuromuscular j u n c t i o n e s p e c i a l l y at the post-synaptic surface where i t s presence can be demonstrated by histochemical methods (55). A c e t y l c h o l i n e -sterase functions very e f f e c t i v e l y to r a p i d l y remove the a c e t y l c h o l i n e molecules from the synapse a f t e r the nerve impulse has been transmitted, and so begins the process by which the c e l l can regain i t s r e s t i n g p o t e n t i a l . I t i s a study of t h i s p r o t e i n a c e t y l c h o l i n e s t e r a s e that t h i s t h e s i s i s concerned with. 1.3 A c e t y l c h o l i n e s t e r a s e : (AChE, E.C. 3.1.1.1.7) A c e t y l c h o l i n e s t e r a s e , the enzyme which c a t a l y s e s the h y d r o l y s i s of the c h o l i n e r g i c t r a n s m i t t e r of the nerve impulse, a c e t y l c h o l i n e , i s widely d i s t r i b u t e d i n e x c i t a b l e membranes of nerve and muscle (56). CH CH > A C h E . +\ CH 0- N-(CH„) „-0-C0-CH o + H„0 * CH 0- N-(CH o)„-0H + CH-COOH 3 j A I 3 2 3 j 2 2 3 CH 3 CH 3 a c e t y l c h o l i n e c h o l i n e Most of the work on the molecular s t r u c t u r e of AChE, i n c l u d i n g that described i n t h i s t h e s i s , has u t i l i s e d enzyme p u r i f i e d from the e l e c t r i c organs of the e l e c t r i c e e l , Electrophorus e l e c t r i u s . However other sources have been studied, and these include another e l e c t r i c organ present i n the ray f i s h , Torpedo mamorata and Torpedo c a l i f o r n i c u s , a l s o b r a i n and red blood c e l l s have been shown to possess esterase a c t i v i t y . In blood i t has been shown that there are at l e a s t two d i s t i n c t enzymes which are present; one i n the c e l l s themselves and one i n the serum (57). The red c e l l type, c a l l e d a c e t y l c h o l i n e s t e r a s e hydrolyses a c e t y l c h o l i n e f a r more r a p i d l y than butyrylcholine, whereas the serum type, c a l l e d b u t y r y l c h o l i n e s t e r a s e hydrolyses b u t y r y l c h o l i n e about four times more r a p i d l y than a c e t y l -c holine (58). Within the c l a s s i f i c a t i o n f o r membrane prot e i n s described e a r l i e r i n t h i s i n t r o d u c t i o n AChE would almost c e r t a i n l y be described as an e x t r i n s i c or p e r i p h e r a l p r o t e i n . However, i t s exact mode of attachment to the membrane i s not yet w e l l understood. Study of the p r o t e i n i s o l a t e d from these widely d i f f e r e n t sources w i l l help to c l a r i f y t h i s and be a valuable a i d i n understanding the forces involved i n the i n t e r a c t i o n s of pro t e i n s and membranes i n general. The organs of the e l e c t r i c e e l are embryologically evolved muscle c e l l s and i n these organs the enzyme i s present i n the membrane of the nerve terminal and i n the synaptic and conducting membranes (59). The concentration of AChE i n the Electrophorus i s very high; one kilogram of t i s s u e hydrolyses three to four kilograms of a c e t y l c h o l i n e per hour i n s p i t e of the low p r o t e i n (3%) and the high water content (92%) of these organs. Because v i r t u a l l y a l l of the enzyme i s l o c a l i s e d i n the e x c i t a b l e membrane a more c o r r e c t value of t h i s extraordinary enzyme a c t i v i t y i s obtained when i t i s expressed as the amount of a c e t y l c h o l i n e hydrolysed per gram of membrane and not per gram of whole - 4 t i s s u e . Since the membrane forms only 10 or l e s s of the whole c e l l mass one gram of e x c i t a b l e e l e c t r o p l a x membrane hydrolyses many kilograms of a c e t y l c h o l i n e per hour. The a c t i v e s i t e of a c e t y l c h o l i n e s t e r a s e c o n s i s t s of two s u b s i t e s , an a n i o n i c s i t e and an e s t e r a t i c s i t e (60,61) The a n i o n i c s i t e determines s p e c i f i c i t y with respect to the c h o l i n e moiety and the e s t e r a t i c s i t e i s i n v o l v e d i n the a c t u a l c a t a l y t i c process. Anionic Esteratic site site . T h e e n z y m e - s u b s t r a t e c o m p l e x a n d o n e o f t h e m o r e c o m m o n l y s u g g e s t e d i : i i c l i : i n i s m s w h e r e b y t h e h y d r o x y l g r o u p o f s e r i n e f u n c t i o n s as a n u c l e o p h i l e t o >li>l>liici> c h o l i n e a n d f o r m a n a c e t y l e n z y m e . T h e i m i d a z o l e o f h i s t i d i n e s e r v e s as :t i i c n o r a l b a s e , a n d a p r o t o n i s s h o w n a t t h e e s t e r a t i c s i t e t o e n v i s a g e t h e p o s s i b i l i t y t h a t a p r o t o n m a y b e i n v o l v e d i n t h e c a t a l y s i s , p e r h a p s b y t r a n s f e r t o m a k e c h o l i n e ( r a t h e r t h a n t h e c h o l i n e d i p o l a r i o n ) t h e l e a v i n g g r o u p . As i t s name implies the an i o n i c s i t e i s the locus of an e l e c t r i c a l l y negative p o t e n t i a l which a t t r a c t s the quaternary ammonium group of a c e t y l c h o l i n e . This i n t e r -a c t i o n c o n t r i b u t e s about 2 kcal/mole of free energy c o r r e s -ponding to a measured d i f f e r e n c e of a f a c t o r of t h i r t y i n the bind i n g of a charged molecule as compared to i t s un-charged analog (60,61). Changes i n pH may a f f e c t a p r o t e i n i n many ways and i t has been suggested that the b e l l shaped pH curve f o r enzymic hydrolyses might r e f l e c t the i o n i s a t i o n of two e s s e n t i a l groups i n the e s t e r a t i c s i t e with pKa values of about 6.5 and 10.5 (62,63). In the present view i t i s the 2 7 . E H + t E H E +H + -H + i n a c t i v e a c t i v e i n a c t i v e h y d r o x y l g r o u p o f s e r i n e t h a t i s a c e t y l a t e d w i t h a n i m i -d a z o l e e n v i s a g e d a s p l a y i n g a n a u x i l i a r y r o l e i n p r o m o t i n g t h e n u c l e o p h i l i c i t y o f t h e s e r i n e : a n u m b e r o f s i m i l a r p r o p o s a l s h a v e b e e n m a d e f o r t h e s e r i n e p r o t e a s e s ( 6 4 - 6 6 ) . T h e g e n e r a l m e c h a n i s t i c s c h e m e i s g i v e n b y : E + S E - S ± E 1 • P . E = e n z y m e E ' = a c e t y l e n z y m e S = s u b s t r a t e , a c e t y l c h o l i n e P-^= c h o l i n e * E ' + P , « 1 + H 2 ° E - P , ir E + P , P2 = a c e t i c a c i d S t u d i e s o n t h e p r o p e r t i e s o f a c e t y l c h o l i n e s t e r a s e s t a r t e d i n 1 9 3 8 w h e n t h e e n z y m e w a s f i r s t o b t a i n e d i n a h i g h l y a c t i v e s o l u t i o n b y s a l t e x t r a c t i o n f r o m e l e c t r i c t i s s u e ( 6 7 ) . I n t h e e a r l y 1 9 4 0 ' s a f i v e h u n d r e d f o l d p u r i f i c a t i o n w a s o b t a i n e d b u t t h e a m o u n t o f p r o t e i n a v a i l -a b l e w a s v e r y s m a l l ( 6 8 ) . T h e p r e p a r a t i o n w a s u s e f u l f o r k i n e t i c s t u d i e s a n d f o r a n a l y s i n g m a n y r e a c t i o n s o f l i g a n d s w i t h m o l e c u l a r g r o u p s i n t h e a c t i v e s i t e a n d t h e m e c h a n i s m 2 8 . i n t h e h y d r o l y t i c p r o c e s s ( 6 9 ) . T h e e f f e c t s o f p o t e n t c o m p e t i t i v e i n h i b i t o r s w i d e l y u s e d i n n e u r o p h a r m a c o l o g y a n d m e d i c i n e w e r e e x p l a i n e d i n t e r m s o f t h e i r i n t e r a c t i o n w i t h t h e m o l e c u l a r g r o u p s i n t h e c a t a l y t i c s i t e o f t h e e n z y m e . O f t h e o r e t i c a l a s w e l l a s p r a c t i c a l i n t e r e s t w e r e t h e s t u d i e s w i t h o r g a n o p h o s p h a t e s w h i c h a r e p o t e n t i a l i n h i b i t o r s o f a c e t y l c h o l i n e s t e r a s e a s w e l l a s o t h e r e s t e r -s p l i t t i n g e n z y m e s . S o m e o r g a n o p h o s p h a t e s a r e p o t e n t i a l c h e m i c a l w a r f a r e a g e n t s a n d m a n y a r e w i d e l y u s e d a s i n s e c -t i c i d e s . T h e f a t a l e f f e c t s a r e d u e t o t h e r e a c t i o n w i t h A C h E . T h e p h o s p h o r o u s a t o m o f t h e o r g a n o p h o s p h a t e f o r m s a c o v a l e n t b o n d w i t h a n o x y g e n a t o m o f a s e r i n e r e s i d u e i n t h e a c t i v e s i t e o f t h e e n z y m e . T h e r e s u l t i n g p h o s p h o r y -l a t e d e n z y m e i s m u c h m o r e s t a b l e t h a n t h e a c e t y l a t e d e n z y m e . T h e r e a c t i o n w a s i n f a c t t h o u g h t a t f i r s t t o b e i r r e v e r s i b l e b u t e l u c i d a t i o n o f t h e r e a c t i o n m e c h a n i s m ( 7 0 ) h a s n o w p e r m i t t e d t h e d e v e l o p m e n t o f a h i g h l y e f f i c i e n t a n d w i d e l y u s e d a n t i d o t e a g a i n s t p o i s o n i n g b y o r g a n o p h o s p h a t e s . T h e m o l e c u l e p y r i d i n e 2 - a l d o x i m e m e t h i o d i d e ( P A M ) r a p i d l y a n d q u i t e s p e c i f i c a l l y r e a c t i v a t e d t h e e n z y m e b y r e m o v i n g t h e p h o s p h o r y l g r o u p f r o m t h e s e r i n e i n a d i s p l a c e m e n t r e a c t i o n ( 7 1 ) . P y r i d i n e 2 - a l d o x i m e m e t h i o d i d e i s m u c h m o r e e f f i c i e n t a n d l e s s h a r m f u l t h a n a t r o p i n e a n d t h u s p r o v i d e s a g o o d a l t e r n a t i v e f o r p h y s i c i a n s t r e a t i n g o r g a n o - p h o s p h a t e p o i s o n i n g . 29. C H 3 I P y r i d i n e - 2 - a l d o x i m e m e t h i o d i d e T h e a n a l y s i s o f p r o t e i n p r o p e r t i e s a t a m o l e c u l a r l e v e l r e q u i r e s c o n s i d e r a b l e a m o u n t s o f h o m o g e n e o u s , a n d f o r X - r a y c r y s t a l l o g r a p h y , c r y s t a l l i s e d p r o t e i n . D e s p i t e t h e e n z y m e s h i g h a c t i v i t y i n t h e e l e c t r i c o r g a n o f t h e e l e c t r i c e e l t h e r e a r e o n l y a b o u t 5 0 - 7 0 m g s o f e n z y m e p e r k i l o g r a m o f w e t t i s s u e . C o n s e q u e n t l y , p r o g r e s s i n t h e s t u d y o f t h i s p r o t e i n h a s a l w a y s b e e n h i n d e r e d b y a l a c k o f a d e q u a t e a m o u n t s o f m a t e r i a l . I t i s o n l y w i t h i n t h e l a s t f i v e o r s i x y e a r s w i t h t h e a d v e n t o f n e w c h r o m a t o -g r a p h i c t e c h n i q u e s t h a t t h e p r o t e i n h a s b e c o m e e a s i l y i s o -l a t a b l e o n a c o m p a r a t i v e l y l a r g e s c a l e . P a r t o f t h i s t h e s i s w i l l d e s c r i b e s u c h a n i s o l a t i o n p r o c e d u r e a n d p u r i f i c a t i o n v i a w h a t i s r e f e r r e d t o a s a f f i n i t y c h r o m a t o g r a p h y . I t i s n o t p o s s i b l e o r n e c e s s a r y t o r e v i e w h e r e a l l o f t h e p e r t i n e n t l i t e r a t u r e t h a t h a s a p p e a r e d w i t h i n t h e l a s t f e w y e a r s c o n c e r n i n g a c e t y l c h o l i n e s t e r a s e . I n f a c t m o r e s p e c i f i c r e f e r e n c e s w i l l b e m a d e i n t h e c h a p t e r s w h i c h f o l l o w t o t h e w o r k t h a t h a s b e e n r e l e v a n t a n d h e l p f u l t o w h a t i s d e s c r i b e d . H o w e v e r i t d o e s s e e m i m p o r t a n t , a t t h i s 30. stage to introduce the reader to some information discovered r e c e n t l y concerning the molecular form of AChE which i s extracted from the membrane. The enzyme when i s o l a t e d from the membrane has i n f a c t been shown to e x i s t i n s e v e r a l molecular forms c h a r a c t e r i s e d by t h e i r sedimentation co-e f f i c i e n t ('S' number) (72). V i s u a l i s a t i o n of these s t r u c -tures by e l e c t r o n microscopy (73) has revealed that three 'native' elongated forms composed of a r o d - l i k e s t r u c t u r e c a l l e d the " t a i l " and one to three c l u s t e r s of tetramers c a l l e d "head" groups attached are present i n any t i s s u e e x t r a c t of f r e s h e l e c t r i c organ (see f i g u r e 3_) . The simplest form then, with one head group on the t a i l has a sedimentation c o e f f i c i e n t of 9S and when one and two more groups are added these c o e f f i c i e n t s change to 14S and 18S r e s p e c t i v e l y . In a d d i t i o n to t h i s , and perhaps h i s t o r i -c a l l y more s i g n i f i c a n t i s the observation that a l l of these native forms can be converted to a fourth form v i a proteo-l y t i c and/or a u t o l y t i c degradation. This form of the enzyme i s globular and has a c h a r a c t e r i s t i c sedimentation c o e f f i c i e n t of about IIS. I t i s formed then as a r e s u l t of cleavage of the long asymmetric forms r e s u l t i n g i n lo s s of the ' t a i l ' l i k e s t r u c t u r e . I t was t h i s form, the globular 1'lS s t r u c t u r e , that was o r i g i n a l l y p u r i f i e d and associated with the enzyme AChE. In f a c t , the e l e c t r i c organ t i s s u e had been stored under toluene f o r s e v e r a l years as a routine procedure and f i r s t step i n p u r i f i c a t i o n intended as a means of removing mucins (68). This r e s u l t e d i n the a u t o l y s i s described above and a l l the p u r i f i e d AChE was i n the g l o b u l a r 11S form. I t i s i n t e r e s t i n g that l o s s of ' t a i l ' and conversion to a g l o b u l a r form does not seem to a l t e r the c a t a l y t i c p r o p e r t i e s of the enzyme; c e r t a i n l y there i s no l o s s i n a c t i v i t y as a r e s u l t of these p h y s i c a l changes. However, considering that AChE i s a membrane-bound p r o t e i n one can speculate as to the s t r u c t u r a l r e l a t i o n s h i p of the 'native' forms to the phospholipid b i l a y e r . More w i l l be s a i d l a t e r about these molecular forms of the enzyme and t h e i r s t a b i l i t y . The observations discussed above i l l u s t r a t e a major theme for the work described i n t h i s t h e s i s ; namely a concern to always r e l a t e to the f a c t that AChE i s membrane bound and that i n order to study i t the native membrane environment has been removed. Thus i s was hoped to be able to p u r i f y the p r o t e i n v i a a f f i n i t y chromatography and study i t s s t r u c t u r a l s t a b i l i t y a f t e r r e -moval of the membrane environment. This was p a r t i c u l a r l y important i n view of the c o n f l i c t i n g reports i n the l i t e r a -ture at the time t h i s work began. CHAPTER 2 THE ISOLATION AND PURIFICATION OF ACETYLCHOLINESTERASE FROM THE ELECTRIC ORGAN OF THE ELECTRIC EEL (ELECTROPHORUS ELECTRICUS) 2 .1 Introduction The most important t o p i c to be discussed i n t h i s chapter i s the use of a comparatively new chromatographic technique f o r p r o t e i n p u r i f i c a t i o n . Recent advances i n biochemical separation methods and enzymology have centred around the preparation of s o l i d phases i n which a p r o t e i n or other molecule has been immobilised by i t s attachment to an i n -soluble matrix. This has l e d to the development of s o l i d s t a t e enzymology and separations based on the s p e c i f i c adsorption of b i o l o g i c a l l y important molecules to a r e s i n . The technique i s c a l l e d a f f i n i t y chromatography and the ideas inherent i n the technique are not new. Twenty, years ago a method of a f f i n i t y chromatography of antihapten a n t i -bodies was described (74) and following that a system was developed f o r the p u r i f i c a t i o n of the enzyme tyrosinase based on e s s e n t i a l l y the same p r i n c i p l e s as are used today. The problem then, i n t h i s chromatographic technique was not i n the development of the basic idea but i n the production of s u i t a b l e s o l i d matrices and recog n i t i o n of the a v a i l a b l e chemistry f o r the attachment of ligands to them. Once these developments had been made, the r a p i d progress and widescale use of the techniques which followed i s r e f l e c t e d i n the exponential growth of l i t e r a t u r e i n t h i s area which has taken place i n j u s t the l a s t f i v e years. The i n t e r e s t and enthusiasm f o r t h i s technique f o r p u r i f y i n g macromolecules i s not s u r p r i s i n g . I t provides an 'easy way' which c a p i t a l i z e s on,and e x p l o i t s the f u n c t i o n a l design of b i o l o g i c a l systems. Such unique s p e c i f i c i t y of b i o l o g i c a l i n t e r a c t i o n s as antigen-antibody, enzyme-inhibitor and hormone-carrier provide an opportunity f o r achieving simple and s e n s i t i v e separations using t h i s technique. A f f i n i t y chromatography then, d i f f e r s r a d i c a l l y from con-v e n t i o n a l chromatographic techniques i n which separation depends on p h y s i c a l and chemical d i f f e r e n c e s between the substances. I t i s quite simply a type of adsorption chromatography, i n which the bed m a t e r i a l has b i o l o g i c a l a f f i n i t y f o r the substance to be i s o l a t e d . These s p e c i f i c adsorptive p r o p e r t i e s of the bed m a t e r i a l are obtained by c o v a l e n t l y coupling an appropriate binding li g a n d to an i n s o l u b l e matrix. The binding l i g a n d i s then able to adsorb from s o l u t i o n the substance which i s to be p u r i f i e d and desorption can subsequently be achieved by a change i n the experimental conditions (buffer, pH, tempera-ture, etc.) a f t e r unbound substances have been washed away. Separations are r a p i d since i n most cases only small bed volumes are required. As w i l l be described l a t e r i n t h i s chapter, o f t e n a t i s s u e e x t r a c t can be a p p l i e d s t r a i g h t onto an a f f i n i t y column and with one e l u t i o n a homogeneous p r o t e i n , i n t h i s case AChE, can be obtained. The nature of the matrix to which the ligands are attached i s important i n several respects. I t must be p h y s i c a l l y and chemically s t a b l e under the experimental conditions used and columns packed with the matrix must have s a t i s f a c t o r y flow p r o p e r t i e s . I d e a l l y , the matrix must be f r e e from non-s p e c i f i c adsorption e f f e c t s which could obscure the d e s i r e d separation or i n t e r f e r e with the a c t i o n of the coupled l i g a n d . A range of r e s i n s produced by Pharmacia under the name of Sepharose and which are a beaded form of an agarose g e l d i s p l a y most of the required p r o p e r t i e s and are quite commonly used for the preparation of a f f i n i t y r e s i n s . The open pore st r u c t u r e of Sepharose 4B f o r example, allows i t to be used f o r g e l f i l t r a t i o n ( i t s p r i n c i p a l use) of proteins up to 20 m i l l i o n dalton and thus molecules can be coupled to t h i s r e s i n and t h e i r binding a c t i v i t y to large macromolecules i s ensured. The small pore stru c t u r e s of c e l l u l o s e and conven-t i o n a l polyacrylamide gels i s a d i s t i n c t disadvantage i n t h i s respect (75). By f a r the most widely used method f o r coupling ligands to the Sepharose matrix involves the use of a cyanogen bromide (CNBr) a c t i v a t i o n of hydroxy1 groups (76). By t h i s method, almost any molecule containing amino groups can then be coupled. Although t h i s r e a c t i o n has been i n use f o r some years the exact nature of the r e a c t i o n products i s s t i l l i n question. I t i s thought that the CNBr reacts with s p a c i a l l y adjacent hydroxyl groups on the Sepharose to form c y c l i c and a c y c l i c imidocarbonates. The a c y c l i c imidocarbonates serve to c r o s s - l i n k the r e s i n and probably account f o r the a c t i v a t e d Sepharose having an increased heat s t a b i l i t y (77). NH C y c l i c and A c y c l i c imidocarbonates However, i t seems that these groups are u n l i k e l y to a i d i n coupling due to t h e i r obvious s t e r i c hindrance. In f a c t , during coupling the a v a i l a b l e a c t i v e imidocarbonate groups react with amino groups on a l i g a n d or p r o t e i n to form stable covalent linkages. Based on studies of model reactions with methyl 4,6-0-benzylidene-a-D-glucopyranoside i t seems that the major product of the coupling r e a c t i o n i s an isourea d e r i v a t i v e (78). N H — o — c — N H R — 0 it— O H C = N H + H- N R — < _ 0 " — O H — 0 — C — N H R II 0 Coupling r e a c t i o n between primary amino group and imidocarbonate So that t h i s i n t r o d u c t i o n to a f f i n i t y chromatography i s r e l a t i v e l y complete f o r the reader f i g u r e 4_ summarises some of the chemistry that has been done to extend co u p l i n g through many other f u n c t i o n a l groups. Another recent method used to a c t i v a t e Sepharose in v o l v e s r e a c t i o n with a b i s -epoxide; 1,4-bis-(2,3 epoxypropoxy-)butane. A s t a b l e un-charged ether linkage i s formed between the long spacer molecule and the matrix. Free r e a c t i v e groups are then a v a i l a b l e to form other ether linkages with hydroxyl groups i n molecules l i k e sugars, alkylamine l i n k s with primary amine groups and t h i o - e t h e r linkages with ligands containing t h i o l groups. FIGURE 4: CHEMISTRY USED TO COUPLE LIGANDS TO SEPHAROSE : — N H ( C H , ) s C O O H ;CH-Sepharose 48 carbodi imido RNH , -NH (CH , ) s CONHR NH; ( CH j ) s COOH A -A H 2 C — C H — C H 2 — 0 — C H 2 — CH 2 — CH 2 CH 2 — 0 — C H 2 — C H — C H 2 1,4-bis-(2,3 epoxypropoxy-)butane The concept of a spacer group as mentioned above i s an obvious and simple one. I t i s merely an unreactive chemical arm which removes the r e a c t i v e l i g a n d from the matrix back-bone and ensures that s t e r i c hindrance between the l i g a n d and p r o t e i n i s minimised (79). Often t h i s spacer group i s incorporated i n t o the synthesised molecule p r i o r to coupling, but r e c e n t l y several r e s i n s have been synthesised to incorporate s i x carbon atom chains i n them (see f i g u r e 4_) . In the case of the epoxide coupling too, the 1,4-bis-(2,3-epoxypropoxy)butane introduces a long h y d r o p h i l i c spacer arm equivalent to twelve carbon atoms and also c r o s s - l i n k s the carbohydrate chains of the gel matrix to give enhanced s t a b i l i t y to the bed m a t e r i a l . With reference to some work presented at the end of the chapter i t should perhaps be mentioned that a f f i n i t y tech-niques have been used to immobilise adsorbent ligands which recognise a group of substances rather than one s p e c i f i c molecule. The s p e c i f i c case to be described i s that of immobilised concanavalin A which i s a plant m e t a l l o p r o t e i n which agglutinates erythrocytes and stimulates the t r a n s f o r -40 . mation of lymphocytes. I t has a binding s i t e f o r ot-D-mannosyl and a-D-glycosyl residues and hence i n t e r a c t s with sugars, polysaccharides and gly c o p r o t e i n s containing these groups. I t was f o r the l a t t e r kind of i n t e r a c t i o n that i t was used to i n v e s t i g a t e some of the p r o p e r t i e s of a c e t y l c h o l i n e s t e r a s e . The use of a f f i n i t y chromatography i n the p u r i f i c a t i o n of AChE was introduced by Kalderon et a l (80). An i n h i b i t o r -Sepharose conjugate was prepared by the synthesis of the i n h i b i t o r [N-(e-amino-caproyl)-p-aminophenyl]trimethyl-ammonium bromide hydrobromide which was c o v a l e n t l y l i n k e d to Sepharose a c t i v a t e d with cyanogen bromide. P.T.A. inhibitor-Sepharose conjugate This compound was chosen since the phenyltrimethyl-ammonium moiety i s a good competitive i n h i b i t o r of AChE (81) and the e-aminocaproyl group can adequately serve as a 'spacer' arm (79). Rosenberry et a l . (82) showed that an important co n s i d e r a t i o n was the distance that the i n h i b i t o r , i n t h i s case phenyltrimethylammonium (P.T.A.) was extended from the gel matrix v i a the spacer arm. Monocaproyl and d i c a p r o y l d e r i v a t i v e s o f P.T.A. were compared and t h e l a t t e r was shown t o have a b e t t e r c o m p e t i t i v e i n h i b i t i o n c o n s t a n t (14 uM v s 3 0 uM) when a t t a c h e d t o t h e S e p h a r o s e and a l s o r e -s u l t e d i n b e t t e r l e v e l s o f p u r i f i c a t i o n o f AChE. A f f i n i t y r e s i n s c o n t a i n i n g e i t h e r t h e P.T.A. l i g a n d o r an N - m e t h y p y r i d i n i u m l i g a n d (83) have t h u s , been u s e d t o p u r i f y e e l AChE f r o m p a r t i a l l y p u r i f i e d c o m m e r c i a l p r e p a r a -t i o n s o r f r o m t o l u e n e t r e a t e d e l e c t r i c o r g a n t i s s u e , and hence i n b o t h c a s e s t h e enzyme o b t a i n e d was i n t h e g l o b u l a r 11S f o r m . As m e n t i o n e d i n c h a p t e r one, AChE has been shown t o be e x t r a c t e d f r o m f r e s h e l e c t r i c o r g a n t i s s u e as t h r e e m a i n components w h i c h c a n be d i s t i n g u i s h e d by t h e i r s e d i m e n -t a t i o n c o e f f i c i e n t s (18,14 and 8S) on s u c r o s e g r a d i e n t c e n t r i f u g a t i o n a t h i g h i o n i c s t r e n g t h ( 7 2 ) . I n o r d e r t o p u r i f y t h e s e forms d i r e c t l y f o r a h i g h - s a l t e x t r a c t o f t h e t i s s u e , D u d a i and S i l m a n made use o f t h e o b s e r v a t i o n t h a t N - m e t h y l a c r i d i n i u m i o n i s a p o w e r f u l AChE i n h i b i t o r w i t h a o f a b o u t 0.3 yM even i n 1M N a C l , and s y n t h e s i s e d an a p p r o p r i a t e a f f i n i t y column t h a t w o u l d a b s o r b AChE even a t h i g h i o n i c s t r e n g t h . T h i s p r o c e d u r e was t h e f i r s t t o d e s c r i b e t h e p u r i f i c a t i o n o f t h e n a t i v e forms o f t h e enzyme ( 8 4 ) . Br H 0 N — ( C H » ) C — C O N H J A D I NH 1-methyl-9- [N -(e-aminocaproyl) -7-aminopropylamino]acridinium N bromide hydrobromide Br • CH 3 The nature of the a c t i v e s i t e of AChE, suggests two p o s s i b l e approaches that can be taken i n designing an a f f i n i t y column. The f i r s t makes use of the anionic sub-s i t e and i s based on r e v e r s i b l e i n h i b i t o r s as described above. A second approach however involves the other region of the a c t i v e s i t e ; the e s t e r a t i c s u b s i t e . This type of p u r i f i c a t i o n i s discussed by Wilson et a l . (85) and i s based on ligands which c o v a l e n t l y react with the enzyme. The molecules chosen are based on the w e l l known reactions of serine esterases with organo-phosphate and phosphonate esters that contain a good leaving group (86) The l i g a n d used i s 2-aminoethyl, p-nitrophenyl methyl-phosphonate which i n t e r e s t i n g l y i s not a potent i n h i b i t o r of the enzyme u n t i l i t becomes acylated by attachment to a Sepharose g e l which has an N-succinyl 1,5-diaminopentane arm 43. 2 - a m i n o e t h y l p - n i t r o p h e n y l m e t h y l p h o s p h o n a t e a t t a c h e d t o i t . The c o v a l e n t a f f i n i t y column o b t a i n e d i n t h i s way has been u s e d t o t r a p and p u r i f y t h e n a t i v e m o l e c u l a r forms o f AChE e v e n t h o u g h some p r o b l e m s i n r e m o v i n g t h e bound enzyme f r o m t h e g e l have been o b s e r v e d ( 8 5 ) . F i n a l l y i t s h o u l d be m e n t i o n e d t h a t a f f i n i t y c h r o m a t o g r a p h y p r o c e d u r e s have been d e s c r i b e d i n t h e l i t e r a t u r e f o r p u r i -f i c a t i o n o f AChE f r o m o t h e r s o u r c e s . P,T.A.-Sepharose c o n j u g a t e s have been u s e d t o p u r i f y AChE f r o m b o v i n e e r y t h r o c y t e s (87) , mouse n e u r o b l a s t o m a c e l l (88) and b o v i n e b r a i n ( 8 9 ) . 44. 2.2 EXPERIMENTAL - MATERIALS AND METHODS (a) Synthesis of the A c e t y l c h o l i n e s t e r a s e I n h i b i t o r : [N-(e-Aminocaproyl-p-aminophenyl]Trimethylammonium Bromide Hydrobromide (IV). The synthesis was e s s e n t i a l l y that of Rosenberry et a l . (82), and i s summarised i n f i g u r e 5. (1) Synthesis of 6-Carbobenzoxyaminocaproic a c i d ( I ) . A s o l u t i o n of 19.7 g (0.15 mole) of 6-aminocaproic a c i d i n 75 ml of 2N NaOH was c h i l l e d to 0° i n an i c e - s a l t bath. Carbobenzoxy-chloride, 28.75 g (0.16 mole) and 37 ml of 4N NaOH were added simultaneously, with good s t i r r i n g over a period of 50 minutes keeping the r e a c t i o n temperature between 0°-7°C. The r e a c t i o n was then s t i r r e d f o r a f u r t h e r 30 minutes and extracted with 10 0 ml of ether to remove the excess carbobenzoxychloride. The aqueous s o l u t i o n was a c i d i f i e d to pH 3 with 6N HC1. The product was extracted three times with 2 50 ml of ether and the combined ether l a y e r s were washed with brine s o l u t i o n and d r i e d over anhy-drous Na^So^. The ether was then evaporated o f f to give an o i l which could be c r y s t a l l i s e d from benzene and hexane on c h i l l i n g at 4°C overnight. The crude product was then r e -c r y s t a l l i s e d from benzene to y i e l d 25.0 g (64% y i e l d ) of white c r y s t a l s . Melting point, (m.p.) 59°-60°C. I n f r a red (IR) absorption spectrum ex h i b i t e d major peaks at 3320, 1710 45. 1685, 1540 cm . E l e m e n t a l a n a l y s i s : c a l c u l a t e d f o r C 1 4 H i g 0 4 N C, 63.38; H, 7.22; N, 5.28, Found: C, 63.21; H, 7.19; N, 5.51. (2) S y n t h e s i s o f N - ( 6 - C a r b o b e n z o x y a m i n o c a p r o y l ) - p - N , N -d i m e t h y l p h e n y l e n e d i a m i n e ( I I ) . 5.75 g (0.03 m oles) o f N , N ' D i c y c l o h e x y l c a r b o d i i m i d e was added t o a s o l u t i o n o f 3.5 g (0.03 moles) d i m e t h y l p h e n y l -e n e d i a m i n e and 7.0 g (0.03 moles) o f compound I i n 150 ml o f a c e t o n i t r i l e ( p r e v i o u s l y d r i e d o v e r CaEL^) and s t i r r e d a t room t e m p e r a t u r e o v e r n i g h t . The w h i t e , d i c y c l o h e x y l u r e a (4.4 g) p r e c i p i t a t e was f i l t e r e d away and t h e f i l t r a t e e v a p o r a t e d and r e p l a c e d w i t h b o i l i n g benzene (100 m l ) . The s o l u t i o n was t r e a t e d w i t h N o r i t , f i l t e r e d and t h e s l i g h t l y c o l o u r e d c r y s t a l l i n e p r o d u c t was o b t a i n e d upon c o o l i n g . R e c r y s t a l l i s a t i o n f r o m benzene/hexane gave w h i t e c r y s t a l s , 4.4 g (50%) M.P. 113°-114°C. I.R. A b s o r p t i o n s p e c t r u m e x h i b i t e d m a j o r peaks a t 3320, 1700, 1660, 1520 cm \ E l e m e n t a l a n a l y s i s : c a l c u l a t e d f o r C 2 2 H 2 8 N 3 ° 3 : C ' ^8.90; H, 7.62; N, 10.96; f o u n d : C, 68.87; H, 7.85; N, 11.04. (3) S y n t h e s i s o f [ N ' - 6 - C a r b o b e n z o x y a m i n o c a p r o y l ) - p - a m i n o -p h e n y l ] t r i m e t h y l a m m o n i u m i o d i d e ( I I I ) . An e x c e s s o f m e t h y l - i o d i d e (6-7 ml) w a s 1 a d d e d t o a s o l u -t i o n o f 2.2 g (0.01 mole) o f compound I I i n 6 ml o f dimethylformamide. The s o l u t i o n was then heated f o r 20-30 minutes on a steam bath. At the end of t h i s time, e t h y l acetate was added dropwise to c r y s t a l l i s e out the product. This was r e c r y s t a l l i s e d from ethanol to y i e l d 2.2 g (75%) of m a t e r i a l with a melting point of 148°-149°C. I.R. shows -1 major absorptions at 3320, 3270, 1705, 1670 cm . Elemental Anal y s i s c a l c u l a t e d f o r C 2 3 H 3 2 N 3 ° 3 I : C ' 5 2 • 5 8 ' H ' 6.14; N, 7.99; found, C, 52.50; H, 6.40; N, 7.70. (4) Synthesis of [N-(6-Aminocaproyl)-p-aminophenyl]Trimethyl ammonium Bromide Hydrobromide (IV). G l a c i a l a c e t i c a c i d which had been saturated with an-hydrous HBr was added to 1 g (0.002 mole) compound I I I . There was an immediate e v o l u t i o n of CC^ and when there was no f u r t h e r s i g n of decarboxylation 25 ml of anhydrous e t h y l ether were added with s w i r l i n g . A gummy product was ob-served to separate out, the supernatant was decanted and the p r e c i p i t a t e was washed twice with e t h y l ether and re-c r y s t a l l i s e d from methanol and e t h y l acetate, to y i e l d 0.5 g (65%) of product. Decomposition point 184-185°C. I.R. shows major peaks at 1690, 1610, 1540 cm"1. N.M.R. (D 20) showed f o l l o w i n g peaks: T 7.6 (m, 4, -Ph-N), 3.5 (s, 9, + -N(CH,)_) 1.0 to 3.0 (m, 10, - (CH-_) K-C-) . • U l t r a - v i o l e t 3 3 lb absorption spectrum had a A 245 nm (e 15,500) i n a 0.1 M 47 . sodium b o r a t e b u f f e r , pH 9.3. E l e m e n t a l A n a l y s i s c a l c u l a t e d : f o r C 1 5 H 2 7 N 3 O B r 2 : C, 42.37; H, 6.40; N, 9.88; f o u n d , C, 42.54; H, 6.24; N, 9.42. B. P r e p a r a t i o n o f t h e A c e t y l c h o l i n e s t e r a s e I n h i b i t o r : [ N - ( 6 - A m i n o c a p r o y l - 6 ' - a m i n o c a p r o y l ) - p - a m i n o p h e n y l ] T r i m e t h y l -ammonium Bro m i d e H y d r o b r o m i d e ( V I I ) . (1) S y n t h e s i s o f [ N ' - ( 6 - A m i n o c a p r o y l ) - p - N , N - d i m e t h y l p h e n y l e n e -d i a m i n e ] D i h y d r o b r o m i d e (V) 2.2 g (0.01 mole) o f compound I I were added t o a s a t u r a t e d s o l u t i o n o f a n h y d r o u s HBr i n g l a c i a l a c e t i c a c i d and t h i s was s t i r r e d u n t i l CC>2 c e a s e d e v o l v i n g (15 m i n u t e s ) . The 50 m l o f an h y d r o u s e t h e r was added t o p r e c i p i t a t e t h e s a l t w h i c h i s f o r m e d . The gummy p r e c i p i t a t e was washed w i t h 20 ml o f e t h e r and t h e p r o d u c t r e c r y s t a l l i s e d f r o m a b s o l u t e e t h a n o l and e t h e r . Y i e l d 2.8 g (75%) M.P. 181°-182°C. I.R. (KBr p e l l e t ) shows m a j o r peaks a t 3070, 2980, 1680, 1542, 1505 cm" 1. N.M.R. (D2<D) 7.3 ( s , 4, HN-Ph-NH-C-) 3.0 ( s , 6, - N H ( C H 3 ) 2 ) , 1.2 t o 2.8 (m, 10, - ( C H 2 ) 5 " N H 3 ) . E l e m e n t a l a n a l y s i s c a l c u l a t e d f o r C 1 4 H 2 5 N 3 O B r 2 : C, 40.98; H, 6.14; N, 10.24; f o u n d C, 41.17; H, 5.93; N, 10.10. 48. (2) Synthesis of N-(6-carbobenzoxyaminocaproyl)-6'-amino-caproyl-p-N,N-dimethylphenylenediamine (VI) . Two grams (0.005 moles) of compound V and 1.0 g (0.01 mole) of tr i e t h y l a m i n e were s t i r r e d i n 100 ml of a c e t o n i t r i l e f o r a few minutes. Then 1.3 g (0.005 mole) compound I and 1.07 g (0.005 mole) of dicyclohexylcarbodiimide were added. The mixture was s t i r r e d at room temperature over-night and the p r e c i p i t a t e (dicyclohexylurea and some t r i -ethylamine hydrobromide) was f i l t e r e d o f f . A f t e r the f i l t r a t e had been evaporated the residue was treat e d with 10% Na2CC>3 (20 ml) and extracted three times with c h l o r o -form (80 ml). The CHCl^ la y e r s were dri e d over anhydrous Na^SO^, evaporated and the residue was c r y s t a l l i s e d from b o i l i n g a c e t o n i t r i l e . The r e s u l t i n g 150 mg (15%) of white c r y s t a l s had a m.p. 135-136°C. I.R. displayed major peaks at 3290, 1683, 1640, 1636, 1525, 1260 cm"1. Elemental a n a l y s i s c a l c u l a t e d f o r C ^ H ^ O ^ : C, 67.74; H, 8.06; N, 11.29; found C, 67.77; H, 8.33; N, 11.45. (3) Synthesis of [N-(6-Aminocaproyl-6'-Aminocaproyl)-p-aminophenyl]Trimethy1ammonium Bromide Hydrobromide (VII). 130 mg of compound VI was methylated and deblocked as was described f o r compounds III and IV to y i e l d 3 0 mg of coloured c r y s t a l l i n e m a t e r i a l . M.P. 173-175°C. I.R. (KBr p e l l e t ) has major peaks at 2950, 1695, 1640, 1540 cm - 1. FIGURE 5 SYNTHESIS OF AChE AFFINITY LIGAND 1. ^ ^ C H 2 0 C ^ c i + H2N-(CH2)5C02H > Z-N-(CH2)5C02H Z = < ^ ) - CH,OC-DCC 2. I H2N N(CH3) 3'2 -> Z-N-(CH2)5CON-H H \^5^N(CH 3) 2 II CH3I 3. II Z-N-(CH2)5-CON H H + N(CH3) 3^3 III HBr/HOAc 4. I l l Br" H3N-(CH2)5C0N-<(^|) + IV 3 / 3 OH r-OH CNBr IV s=NH NaHCO-N~(CH2)5CON r-OH H H + N(CH3) 3' 3 4^  WD C. Preparation of A c e t y l c h o l i n e s t e r a s e I n h i b i t o r : [N-6-Aminocaproyl-m-aminophenyl] Trimethylammonium Bromide Hydrobromide V I I I . This compound was synthesised i n e s s e n t i a l l y the same manner as that described f o r compound IV, r e p l a c i n g the para-aminophenyl group with i t s meta-isomer. The compound VIII, m.p. 190-192°C (Decomposes), N.M.R. shows major peaks + at x 7.2 (m, 4, -NH-Ph-N), 3.1 (s, 9, -N(CH 3) 3), 0.4-2.8 x (m, 10, -(CH0),--) U.V. absorption spectrum had a X z D max 242 nm (e 15,200). Elemental a n a l y s i s c a l c u l a t e d f o r C, c.H„_N o0Br o: C, 42.37; H, 6.40; N, 9.88; found C, 41.54; lb ZI 5 a H, 6.24; N, 9.42. D. C r o s s - l i n k i n g and Removal of ion-exchange groups of Sepharose r e s i n f o r use i n a f f i n i t y chromatography. The method used r e s u l t s i n an epichl o r o h y d r i n cross-l i n k e d and desulphated r e s i n as described i n reference (92) and i s c a r r i e d out as fo l l o w s : 2 l i t e r s of Sepharose 2B was washed extensively with d i s t i l l e d water. I t was then mixed with 2 l i t e r s of 1M NaOH containing 40 ml of epichlorohydrin and 10 g NaBH^. The mixture was s t i r r e d w ell and heated to 60°C and held at t h i s temperature f o r 1 hour. 51. The c r o s s - l i n k e d g e l was then washed with hot d i s t i l l e d water to n e u t r a l pH. Then 2 l i t e r s of 2M NaOH and a f u r t h e r 10 g of NaBH 4 were added and the mixture was put i n t o c o n i c a l f l a s k s sealed with f o i l and autoclaved (120°) f o r 1 hour. The g e l was then washed with 1M NaOH containing 0.5% (5g/l) of NaBH 4; i n the f i r s t instance with a hot s o l u t i o n and then with a room temperature one. The g e l was again t r a n s f e r r e d to a beaker containing crushed i c e and the cooled mixture was q u i c k l y t i t r a t e d to pH 4.0 with g l a c i a l a c e t i c a c i d . The g e l i s again t r a n s f e r r e d to a s i n t e r e d glass funnel and washed with hot d i s t i l l e d water to remove b o r i c a c i d and f i n a l l y with ice-water. This g e l was then stored i n 0.02% sodium azide to prevent m i c r o b i a l growth. The net y i e l d was 1500 ml of r e s i n . I t should be noted that t h i s treatment enhances the r e s i n beads'physical s t a b i l i t y and ensures that p o t e n t i a l ion exchange groups are removed. This then makes the modified Sepharose an i d e a l r e s i n to use i n the production of a f f i n i t y r e s i n s . E. Cyanogen Bromide a c t i v a t i o n of the modified Sepharose 2B, and the coupling of the a f f i n i t y l i g a n d . The l i g a n d was coupled to the Sepharose as follows, (93) 100 ml of the modified Sepharose 2B was well washed with approximately 2 l i t e r s of d i s t i l l e d water to remove the sodium azide and was then t r a n s f e r r e d to a beaker and an equal volume of water added. The beaker was placed i n the c o l d box and cooled to 4°C. The coupling with cyanogen bromide (CNBr) was c a r r i e d out i n the fume hood. A pH electrode and thermometer were suspended i n the r e s i n and the weighed amount of CNBr, d i s s o l v e d i n a minimum of dioxane was added and the pH maintained at 10.5±0.1 with 4N NaOH. A micrometer syringe was used to add small amounts of base and the r e s i n was constantly s t i r r e d . A f t e r 10 to 15 minutes there was only a s l i g h t change i n pH and the r e a c t i o n was thus taken to be e s s e n t i a l l y complete. The r e s i n was q u i c k l y t r a n s f e r r e d to a s i n t e r e d glass funnel and washed ex t e n s i v e l y with cold d i s t i l l e d water and then with 1 l i t e r of 0.05 M carbonate-bicarbonate b u f f e r (pH=9.8). The a c t i v a t e d Sepharose was suspended i n 25 ml of water and the weighed amount of li g a n d was d i s s o l v e d and added to the r e s i n . The r e a c t i o n was allowed to continue with s t i r r i n g at 4°C overnight. A f t e r t h i s time, the r e s i n was f i n a l l y washed with a l i t e r of 0.2 M NaCl and 1 l i t e r of d i s t i l l e d water. A l l the washings were c o l l e c t e d and from the absorbance at 242 nm, associated with the l i g a n d ' s t r u c t u r e , i t was p o s s i b l e to a s c e r t a i n the amount of ligand which had coupled to the r e s i n . 53 . F. Preparation of an a f f i n i t y r e s i n containing Concanavalin A The procedure found to be most su c c e s s f u l was that which Steinemann and Stryer have used i n t h e i r i s o l a t i o n of rhodopsin (94). CNBr a c t i v a t i o n was as described above and then a f t e r washing w e l l , a s o l u t i o n of 150 mgs of concanavalin A (Sigma) (Con A) i n 75 ml of pH 6.8 b u f f e r was added to the r e s i n . The mixture was then gently s t i r r e d overnight at 4°C and then the unattached Con A was removed by washing the r e s i n with 0.5 M NaCl with 10~3M C a C l 2 and MnCl 2 added, and assayed f o r by measuring i t s absorbance at 280 nm. Hence the concentration of the coupled Con A could be expressed i n mgs Con A coupled per ml of r e s i n . The coupled r e s i n was then washed and stored i n 0.1 M acetate pH = 6.0 with 1M NaCl and 10~3M, C a + 2 , Mn + 2 and Mg + 2 added (95). Stryer's method of coupling at pH = 6.8 i s pr e f e r r e d over others at pH = 8.0 as i t i s known that Con A i s un-+2 sta b l e at pH = 8.0 l o s i n g Mn and i t s dimerxc s t r u c t u r e d i s s o c i a t i n g (96,97). G. E x t r a c t i o n and I s o l a t i o n of Ac e t y l c h o l i n e s t e r a s e The e l e c t r i c e e l s , E1ectrophorus e l e c t r i c u s , were ob-tained from Paramount Research Supply Co., Ardsley, N.Y., or Paramount Aquarium Tampa, F l o r i d a , and were kept a l i v e i n an aquarium i n the laboratory p r i o r to use. Thus f r e s h e l e c t r i c organ t i s s u e was used i n a l l the experiments to be described here. The y i e l d of the t i s s u e v a r i e d between 2 00 gms and 1 kilogram depending on the s i z e of the animal, the l a r g e s t being of the order of 4 fee t i n length. The d i s t r i b u t i o n of the three e l e c t r i c organs are as shown i n f i g u r e 6_. A l l three could be removed by a simple 'blunt' d i s s e c t i o n a f t e r the surface s k i n had been removed. A l l these operations and concomitant homogenisation of the t i s s u e i n the various media employed was c a r r i e d out at 4°C. A l l b u f f e r s were made up i n d i s t i l l e d water j u s t before use. The methods used to s o l u b i l i s e and separate the r e s u l t -ing homogenate v i a c e n t r i f u g a t i o n and the use of appropriate b u f f e r s were developed i n the laboratory so as to optimise both the y i e l d and s p e c i f i c a c t i v i t y of the a c e t y l c h o l i n e -sterase p r i o r to loading the extract onto the a f f i n i t y column (see f i g u r e 1) . In a t y p i c a l experiment the i s o l a t e d e l e c t r o p l a x was cut i n t o small cubes and minced i n a Waring Blender or V i r t i s Homogeniser (depending on the amount of tissue) with a volume (ml) of buf f e r equivalent' to i t s weight (gms) . This f i r s t b u f f e r was 50 mM T r i s , 100 mM NaCl and 10 mM EDTA at pH = 7.40. In l a t e r experiments 0.2 mg/ml of 55. FIGURE 6 THE ELECTRIC ORGANS OF ELECTROPHORUS ELECTRICUS ELECTRIC ORGANS A Main B Sachs C Hunter A t B C FIGURE 7 FLOW CHART FOR EXTRACTION AND ISOLATION OF A C h E FROM TISSUE ELECTRIC ORGAN TISSUE HOMOGENISE BUFFER l) Low speed 2rtiins 4°C P - Pellet S — Supernatant BUFFER 1 - 50mM TRIS 100 M NaCl 10mM EDTA pH 7-4 BUFFER 2 • 50mM TRIS^  2M NaCl 10 mM EDTA pH 7-4 SS34 19,000 rpm 40mins SI RE- HOMOGENISE (BUFFER 2) 4°C 42-1 41,000 rpm 45mins S3 S2 P-discard SS 34 19,000rpm 40mins 42-1 41,000 rpm 45mins S4 P-discard R E - H O M O G E N I S E ( B U F F E R 2) 4°C \ S S 34 19,000 rpm 40mins S5 P —lyophilise 42-1 41,000 rpm 45mins S6 soyabean t r y p s i n i n h i b i t o r was added to the bu f f e r s i n an attempt to prevent any p r o t e o l y t i c degradation of AChE, during the i s o l a t i o n procedure. The t i s s u e was homogenised f o r 2 minutes and the r e s u l t i n g s o l u t i o n was spun at 46,000 x g f o r 40 minutes i n a S o r v a l l RC2B c e n t r i f u g e . The supernatant was discarded and the p e l l e t was then resus-pended i n a 50 mM T r i s , 2M NaCl and 10 mM E.D.T.A. b u f f e r a t pH = 7.40 (+0.2 mg/ml soyabean t r y p s i n i n h i b i t o r ) . This homogenate was spun a t 46,000 x g f o r 4 0 minutes and then the supernatant was removed and spun at 195,000 x g i n a Beckman L3-50 u l t r a c e n t r i f u g e f o r 45 minutes to remove the microsomal and membrane fragments. I t was found to be worthwhile to resuspend the p e l l e t by rehomogenisation i n the high i o n i c strength (2M NaCl) b u f f e r and thus e x t r a c t the AChE a second time from the t i s s u e . The c e n t r i f u g a t i o n s a t 46,000 g and 195,000 g were repeated and the r e s u l t i n g supernatants, which were very r i c h i n AChE a c t i v i t y were d i a l y s e d versus 20 mM phosphate b u f f e r (pH = 6.90) f o r 24 hours with at l e a s t two changes p r i o r to loading onto the a f f i n i t y column which had al s o been e q u i l i b r a t e d i n the phosphate b u f f e r . The d i a l y s i s into what i s a low i o n i c strength b u f f e r caused some p r o t e i n p r e c i p i t a t i o n and t h i s was r o u t i n e l y removed by c e n t r i f u g a t i o n at 46 ,000 x g f o r 20 minutes before the s o l u t i o n was loaded onto the columns. 58. A 1.5 x 3 0 cm chromatography column (Pharmacia) was packed with 10-30 ml of a f f i n i t y r e s i n . The volume of r e s i n needed depends on the volume of e x t r a c t to be loaded onto the column and the amount of AchE which i s present. This could be estimated from the p r o t e i n concentration and the s p e c i f i c a c t i v i t y of the sample. The r e s i n was e q u i l i b r a t e d with 20 mM phosphate b u f f e r u n t i l the pH of the eluant r e -mained constant and then the AchE e x t r a c t was appl i e d to the column at a flow rate of approximately 4 0 ml/hr. A f t e r a p p l i c a t i o n the r e s i n was exte n s i v e l y washed with phosphate b u f f e r u n t i l the e f f l u e n t had an absorbance at 280 nm which was l e s s than 0.02. Then, i n order to elute the AChE, a gradient of 0-5 mM decamethonium bromide i n 2 0 mM phosphate b u f f e r was a p p l i e d at a flow rate of about 30 ml/hr. Three m i l l i l i t e r f r a c t i o n s (90 drops) were c o l l e c t e d on a G i l s o n MF-80 f r a c t i o n c o l l e c t o r . These f r a c t i o n s were then assayed f o r p r o t e i n and enzyme a c t i v i t y and the AChE pooled and stored i n 20 mM phosphate bu f f e r at 4°C. H. A c e t y l c h o l i n e s t e r a s e a c t i v i t y assay The AChE a c t i v i t y was r o u t i n e l y assayed by the use of the d i t h i o c h o l i n e photometric method developed by Ellman et a l . (90), and summarised i n f i g u r e 8_. The p r i n c i p l e of t h i s method i s the measurement of the rate of h y d r o l y s i s of 5 9 . t h e p s e u d o s u b s t r a t e , a c e t y l t h i o c h o l i n e b y A C h E . T h i s i s e f f e c t e d b y t h e c o n t i n u o u s r e a c t i o n o f t h e t h i o l a n i o n t h u s f o r m e d w i t h d i t h i o b i s n i t r o b e n z o i c a c i d ( D T N B ) c r e a t i n g t h e y e l l o w 5 - t h i o - 2 - n i t r o - b e n z o i c a c i d a n i o n , t h e r a t e o f p r o d u c t i o n o f w h i c h m a y b e f o l l o w e d s p e c t r o p h o t o m e t r i c a l l y a t 4 1 2 n m . I t h a s b e e n s h o w n t h a t t h i s r e a c t i o n w i t h D T N B i s s u f f i c i e n t l y f a s t s o a s n o t t o b e r a t e l i m i t i n g i n t h e m e a s u r e m e n t o f t h i o l a n i o n p r o d u c t i o n a n d i n t h e c o n c e n -t r a t i o n s u s e d i t d o e s n o t i n h i b i t t h e e n z y m i c r e a c t i o n i t s e l f . T h e i n c r e a s e i n a b s o r b a n c e w i t h t i m e i s l i n e a r w i t h r e s p e c t t o c o n c e n t r a t i o n p r o v i d e d t h e r e a c t i o n t i m e i s l i m i t e d s o a s t o e n s u r e t h a t a l l t h e s u b s t r a t e i s n o t u s e d u p . T h e r a t e o f h y d r o l y s i s c a n t h e n b e d i r e c t l y e x t r a p o l a t e d f r o m t h e s l o p e o f t h e a b s o r b a n c e v e r s u s t i m e p l o t . U s i n g t h e e x t i n c t i o n c o e f f i c i e n t f o r t h e y e l l o w 4 - 1 - 1 a n i o n p r o d u c e d , 1 . 3 6 x 1 0 M c m ( 9 0 ) , t h e r a t e o f t h e e n z y m e w a s s u b s e q u e n t l y d e t e r m i n e d . I n m a n y c a s e s , t h e a c t i -v i t y o f t h e e n z y m e w h i c h h a s b e e n p u r i f i e d i s s o h i g h t h a t d i l u t i o n s o f a t l e a s t 1 0 0 f o l d a r e n e e d e d t o e n s u r e t h a t t h e r a t e i s l i n e a r w i t h r e s p e c t t o t i m e . T h e f o l l o w i n g s t o c k s o l u t i o n s w e r e p r e p a r e d f o r t h e a s s a y : ( F r e s h e v e r y t w o w e e k s ) 1 ) b u f f e r : 0 . 1 M p h o s p h a t e , p H 8 . 0 2 ) s u b s t r a t e : a c e t y l t h i o c h o l i n e h a l i d e 0 . 0 7 5 M 3 ) r e a g e n t : 0 . 0 1 M D T N B ; i n 1 0 m l 0 . 1 M p h o s p h a t e b u f f e r , p H = 7 . 0 w i t h 1 5 m g s o d i u m b i c a r b o n a t e a d d e d . FIGURE 8: Chemistry of the Ellman AChE a c t i v i t y assay (enzyme) a c e t y l t h i o c h o l i n e > t h i o c h o l i n e + acetate t h i o c h o l i n e + d i t h i o b i s n i t r o b e n z o a t e > yellow colour CH, J ' (enzyme) H 20 + CH 3—N—CH 2CH 2SCOCH 3 CH3-N—CH 2CH 2S + CH. CH. CH CH. 3 CH, CH3COO + 2 H + CH_—N—CH„CH„ — S + RSSR > CH,—N—CH„CH„SSR + RS 3 1 2 2 3 I 2 2 CH. CH. R = 02N. COO 60. The t y p i c a l procedure was to p i p e t t e the fo l l o w i n g i n t o a cuvette and mix thoroughly; 3.0 ml b u f f e r ; 25 J J I substrate, 100 u1 reagent. The c e l l was then placed i n the spectro-photometer and zeroed at 412 ran, against a blank containing a l l of the above. Then at zero time a 5-50 y l sample of enzyme s o l u t i o n was added with a Lang-Levy micropipette to i n i t i a t e the r e a c t i o n . A time was s t a r t e d and the cuvette shaken and replaced i n the holder. The absorbance was noted then every 15 seconds f o r a t o t a l of 1.5 minutes. A Zeiss PMQ II (UV-visible op G i l f o r d spectrophotometer) were used. The absorbance was then p l o t t e d against time and the slope obtained i n u n i t s of change i n o p t i c a l density u n i t s per minute (OD/min). The t h i o c h o l i n e assay i s extremely s e n s i t i v e and i s thus a p p l i c a b l e to very low concentrations of the enzyme. I t makes d e t a i l e d k i n e t i c studies o f AChE a c t i v i t y r o u t i n e l y f e a s i b l e . The r e s u l t s of a t y p i c a l assay are shown i n f i g u r e 9_ to i l l u s t r a t e the l i n e a r i t y of the rate of h y d r o l y s i s with time. Enzyme a c t i v i t i e s are expressed e i t h e r i n OD/min per enzyme a l i q u o t or i n the u n i t s of ymolar a c e t y l t h i o c h o l i n e hydrolysed per min per enzyme a l i q u o t . S p e c i f i c a c t i v i t i e s are given i n the un i t s of ymoles of a c e t y l t h i o c h o l i n e hydrolysed per min per mg of p r o t e i n (units/mg) where the 1% pro t e i n concentration i s estimated using £280nm = 1 8 - 0 ^ 8 2 ) • FIGURE 9 ACETYLTHIOCHOLINE ASSAY FOR AChE ACTIVITY - ELLMAN PROCEDURE 30ml pH = 80 buffer 25 0y\ substrate 100 0 pi DTNB 5ul enzyme-SIGMA (02mg/ml) 4——+- ~4—~—* „*____ 4 _ _ „ — j • f-1 2 3 4 5 10 15 20 TIME (mins) I. P r o t e i n concentrations P r o t e i n concentrations were obtained from the absorbance of a s u i t a b l y d i l u t e d sample of enzyme s o l u t i o n at 280 nm 1% using the e x t i n c t i o n c o e f f i c i e n t , e280 = (82). When the amount of p r o t e i n present was small, or where other m a t e r i a l s present (e.g. detergents) absorbed at 280 nm the sample were assayed f o r p r o t e i n by the method of Lowry et (91). P r o t e i n concentrations were then extrapolated o f f a standard curve prepared using crude AChE (Sigma) (See f i g u r e 10) . FIGURE 10 STANDARDISATION CURVE FOR LOWRY DETERMINATION OF PROTEIN 64. 2.3 RESULTS AND DISCUSSION a) Preparation of A f f i n i t y r e s i n s Three d i f f e r e n t ligands were synthesised f o r use i n the a f f i n i t y chromatography of AChE: 9, sCE? , | / — V , / 3 H 3 N- (CH2) 5-C-NH- <^  0 ^ - N—CH 3 2Br~ (IV) X C H 3 para'-isomer .CHo 0 , + / 3 II / ^ N - C H 3 H 3 N-(CH 2) 5-C-NH-«f 0 > C H 3 2 B r (VIII) 'meta 1-isomer 0 0 , , CH H 3 +N-(CH 2) 5-C-NH-(CH2) 5-C-NH-<^ 0 ^-'N^-CH3 2Br (VII) 'CH, 'para'-isomer, lenghthened spacer arm 6 and each of these was then coupled to Sepharose 4B (modified) as described under materials and methods. The r e s u l t of a set of t y p i c a l coupling experiments f o r the 'meta'-ligand i s o u t l i n e d below: Volume of [CNBr] Weight of T h e o r e t i c a l Attached % r e s i n added l i g a n d added coupling ligand coupling l-imole/ml ymole/ml 100 ml 50 mg/ml 212.5mg 5.0 3.5 70 100 ml 25 mg/ml 42.5mg 1.0 0.85 '85 In t h i s way r e s i n s were prepared with various concentrations of l i g a n d attached. I t was found that f o r the p u r i f i c a t i o n of the enzyme l e s s that 1 umole/ml coupled was much bet t e r than r e s i n s with higher concentrations of attached l i g a n d s . Concanavalin A a f f i n i t y r e s i n The p r o t e i n Con A, was coupled to Sepharose 4B as de-s c r i b e d i n the methods s e c t i o n of t h i s chapter and the con-c e n t r a t i o n of attached p r o t e i n determined as described above, but now u t i l i s i n g the absorption of the p r o t e i n at 280 nm. The concentration of Con A attached to the Sepharose was found to be 1.8 mg/ml of r e s i n . G. E x t r a c t i o n and I s o l a t i o n of AChE The e x t r a c t i o n and i s o l a t i o n procedure f o r AChE from the e l e c t r o p l a x t i s s u e i s summarised on page (56) and described i n some d e t a i l i n the experimental s e c t i o n of t h i s chapter. The r e s u l t s of a t y p i c a l preparation are shown on page 66 (Table 1). I t can be seen from t h i s data that the supernatant extracts S4 and S6 from the f r a c t i o n s which are eventually loaded onto the a f f i n i t y column, both have a high s p e c i f i c a c t i v i t y compared to the f i r s t e x t r a c t of the t i s s u e , S i . Further, f i g u r e 11 shows that any TABLE I: T y p i c a l r e s u l t s f o r the E x t r a c t i o n and I s o l a t i o n of AChE from E l e c t r o p l a x Tissue 0 D412/min AChE a c t i v i t y P r o t e i n S p e c i f i c a c t i v i t y uM/min-u.l mg/ml umoles/min-mg SI 0.08 11.8 8.4 4.4 53 1.40 206.0 14.5 43.5 54 1.00 147.0 9.9 46.5 55 0.48 70.4 8.5 26.0 56 0.40 58.8 5.8 31.8 f u r t h e r e x t r a c t i o n of the t i s s u e does not y i e l d very a c t i v e enzyme. Hence r o u t i n e l y S4 and S6, the f i r s t two high s a l t e x t racts of the e l e c t r o p l a x t i s s u e were used as the source of AChE f o r a f f i n i t y p u r ' i f i c a t i o n . The d i a l y s i s of these s o l u t i o n s against a low i o n i c strength b u f f e r was needed so as to ensure that the enzyme would bind s u c c e s s f u l l y to the a f f i n i t y r e s i n . The need f o r a low i o n i c strength i s probably due to the combined e f f e c t of decreased a f f i n i t y of AChE f o r the quaternary l i g a n d with i n c r e a s i n g i o n i c strength (98,99) together with decreased n o n - s p e c i f i c e l e c t r o s t a t i c i n t e r a c t i o n between the enzyme and the r e s i n . D i a l y s i s i n f a c t r e s u l t s i n an increase of AChE s p e c i f i c a c t i v i t y ; however some AChE i s l o s t i n the r e s u l t i n g pre-c i p i t a t e , which i s obviously not very d e s i r a b l e . A t y p i c a l e l u t i o n p r o f i l e of the a f f i n i t y column i s shown i n f i g u r e 12_. As can be seen from the p r o t e i n pro-f i l e , the vast majority of the extract passes s t r a i g h t through the r e s i n and does not bind to the l i g a n d . A f t e r e x t e n s i v e l y washing the r e s i n the p u r i f i e d AChE i s eluted from the column by applying a gradient of decamethonium (DECA) which competes with the binding of the a f f i n i t y l igand CE 3 N CH 2Br (CH,) 2' 10 3 CH CH 3 3 Decamethonium bromide (DECA) FRACTION # - 3ml 69. to AChE. The p u r i f i e d AChE has a s p e c i f i c a c t i v i t y i n the region of 4,000-6,000 units/mg and t h i s implies that t h i s one step chromatographic procedure r e s u l t s i n a p u r i f i c a t i o n of around 100 f o l d ; thus i l l u s t r a t i n g the great s p e c i f i c i t y of t h i s r e s i n and providing an example of the technique of a f f i n i t y chromatography. I t i s i n t e r e s t i n g to note that when the column was f u r t h e r treated with a gradient of i n c r e a s i n g i o n i c strength both n o n - s p e c i f i c a l l y adsorbed proteins and a f u r t h e r amount of AChE was removed from the column. This has been commented on by other authors, (82,98) and i n f a c t i l l u s -t r a t e s a problem i n the use of t h i s kind of a f f i n i t y column. The f a c t that n o n - s p e c i f i c a l l y absorbed pr o t e i n s are released with i n c r e a s i n g i o n i c strength implies that even when a gradient of decamethonium i s used some other p r o t e i n s w i l l be released with the AChE. This i s r e f l e c t e d i n the observed f a c t that the s p e c i f i c a c t i v i t y of the eluted AChE was not homogeneous throughout the peak, but rather slowly decreased as the concentration of decame-thonium increased i n e l u t i n g b u f f e r . The p o s i t i o n of the enzyme a c t i v i t y peak r e l a t i v e to the pr o t e i n impurity i n d i c a t e d that the column was functioning as a true a f f i n i t y support. When the column i s eluted with a s a l t gradient the enzyme a c t i v i t y i s eluted a f t e r the p r o t e i n impurities because i t i s bound more strongly by v i r t u e of i t s s p e c i f i c , 70. a c t i v e - s i t e binding. When e l u t i o n i s c a r r i e d out with decamethonium on the other hand, the enzyme a c t i v i t y pre-cedes any p r o t e i n impurity because the i n h i b i t o r competes f o r the s p e c i f i c a c t i v e - s i t e binding and at lower concen-t r a t i o n s c o n t r i b u t e s very l i t t l e to n o n - s p e c i f i c , i o n -exchange type e l u t i o n . Three a f f i n i t y columns were prepared as has been de-scri b e d e a r l i e r . The s p e c i f i c a c t i v i t y of AChE p u r i f i e d from each of them was found to be very comparable and r e -producible. As reported by Rosenberry et a l . (82), however, i t was observed that the di-(-6-aminocaproyl) (VII) arm attached to the i n h i b i t o r r e s u l t e d i n a somewhat increased a f f i n i t y f o r the AChE. Resins with IV and VIII attached d i f f e r i n being meta- and para-isomers of each other. Of some i n t e r e s t i n the use of these r e s i n s was the observation that the meta—ligand, retained less contaminating pr o t e i n i n d i c a t i n g a s l i g h t l y higher s p e c i f i c i t y f o r the AChE. This had already been commented on i n the p u r i f i c a t i o n of the enzyme from guinea :pig b r a i n (99). A p a r t i c u l a r l y u s e f u l feature of these and other a f f i n i t y r e s i n s i s that they can be regenerated and used many times to p u r i f y the p r o t e i n of i n t e r e s t . This was achieved by washing the r e s i n with 6M guanidine hydrochloride to remove any adsorbing p r o t e i n and then e q u i l i b r a t i n g with b u f f e r . The r e s i n s could then be stored at 4° i n the presence of 0.02% sodium azide u n t i l required. 71. Concanavalin A - A f f i n i t y Chromatography Carbohydrate residues have been shown to be c o v a l e n t l y joined to AChE i s o l a t e d from the e l e c t r i c e e l (100,101) and i t was of i n t e r e s t to a s c e r t a i n whether the presence of these residues would y i e l d a s p e c i f i c i n t e r a c t i o n between the p l a n t l e c t i n concanavalin A and AChE. Thus an a f f i n i t y column with Con A attached was prepared as described e a r l i e r i n t h i s chapter and a f f i n i t y p u r i f i e d AChE was passed through i t . I t was found that 95% of the AChE bound to the column. The 5% that d i d not may j u s t r e f l e c t overloading. No a c t i -v i t y could be detected i n the washing of the r e s i n . I t i s important to note that the Con A column and the AChE were e q u i l i b r a t e d i n a b u f f e r which contained 1 M NaCl. Hence the f a c t that AChE bound at a l l i n d i c a t e s a very d i f f e r e n t i n t e r a c t i o n than that of the previous ' a c t i v e - s i t e ' a f f i n i t y ligands and i s evidence of the gly c o p r o t e i n nature of t h i s enzyme. The e l u t i o n p r o f i l e of the column i s shown i n f i g u r e 13, which i n d i c a t e s that the AChE would be eluted from the r e s i n upon a p p l i c a t i o n of 0.1 m a-methyl-D-mannoside, a very strong i n h i b i t o r and haptenic determinant of concanavalin A. However, t h i s peak d i d not account f o r a l l of the enzyme , that had been applied ( 50% of load) and f u r t h e r work with t h i s r e s i n showed that AChE was very slowly leaking from the 72. FIGURE 13 ELUTION OF C o n A - S E P H A R O S E C O L U M N FRACTION f* - 3 ml column when the mannoside was a p p l i e d . This i n d i c a t e s that the enzyme i s very s t r o n g l y bound to the Con A a f f i n i t y column with a binding constant i n the region of 10 M. No p u r i f i c a t i o n of the enzyme was achieved and as the p r o t e i n was so hard to elute the column was d e f i n i t e l y i n f e r i o r to the others mentioned f o r the p u r i f i c a t i o n of AChE. During the course of t h i s work, s i m i l a r observations were published on the i n t e r a c t i o n of Con A with AChE from p l a i c e body muscle, human erythrocytes and e l e c t r i c e e l (102) . AChE p u r i f i e d from Torpedo C a l i f o r n i c a with mild p r o t e o l y s i s has been found to contain 7.9% carbohydrate present as hexoses, hexosamixes and s i a l i c a c i d residues (103) . This enzyme also binds to Con A and fu r t h e r i t has been shown that a d d i t i o n of Con A to e l e c t r o p l a x membranes markedly i n h i b i t s t r y p s i n induced a c e t y l c h o l i n e s t e r a s e release from the membrane surface (103). 74. CHAPTER 3 The Molecular C h a r a c t e r i s a t i o n of A f f i n i t y P u r i f i e d A c e t y l c h o l i n e s t e r a s e 3.1 Introduction This chapter on the molecular c h a r a c t e r i s a t i o n of AChE w i l l be p r i m a r i l y concerned with a d i s c u s s i o n of the s t r u c -t u r a l s t a b i l i t y and composition of the enzyme p u r i f i e d by a f f i n i t y chromatography from fre s h e l e c t r o p l a x t i s s u e of Electrophorus e l e c t r i c u s . The work i s p a r t i c u l a r l y r e l e -vant to a study of what happens to an ' e x t r i n s i c ' membrane bound p r o t e i n when i t i s removed from i t s membrane environment. As was mentioned i n the i n t r o d u c t i o n to t h i s t h e s i s , i t has been shown that AChE i s present i n extracts of e l e c t r i c organ t i s s u e i n three p r i n c i p a l s t r u c t u r a l forms which can be d i s t i n g u i s h e d by t h e i r sedimentation c o e f f i -c i e n t s (8S, 14S and 18S). Another globular 11S form of the enzyme can be derived from these 'native' forms by pro-t e o l y s i s but t h i s must now be thought of as a degradation product since i t has been shown to have l o s t a long asymmetric structure c a l l e d the ' t a i l ' which though i t has no c a t a l y t i c a c t i v i t y may be s t r u c t u r a l l y important p a r t i -c u l a r l y as AChE i s a membrane bound p r o t e i n . H i s t o r i c a l l y , i t was t h i s l i s form of the enzyme which was f i r s t p u r i f i e d by a f f i n i t y chromatography. In studying the subunit s t r u c t u r e of the p r o t e i n , SDS e l e c t r o p h o r e s i s (with reduction) always displayed two p r i n c i p a l bands at about 8 0,000 and 60,00 0 molecular weight and hence i t was thought that the p r o t e i n contained two kinds of subunit (104,105). The molecular weight of t h i s form of AChE ob-tain e d i n s e v e r a l l a b o r a t o r i e s was found to be 230-260,000 (106-108) and i t was ge n e r a l l y assumed therefore that AChE could be cl a s s e d as an ®2®2 P ^ ^ e i n . I n a n attempt to gain information about these subunits, the a c t i v e s i t e o f the enzyme was l a b e l l e d with r a d i o a c t i v e d i i s o p r o p y l -fluorophosphate (D.F.P.) and t h i s work showed that indeed both types of the subunits contained aa a c t i v e - s i t e ; the 3 H-DFP d i s t r i b u t i o n e s s e n t i a l l y mimicing the AChE p r o t e i n pattern (10 9). Also the enzyme was subjected to treatment with 5M guanidine and 5M guanidine plus 2-mercaptoethanol. The guanidine alone d i s s o c i a t e d the enzyme i n t o p a r t i c l e s of 6.2S (120,000 M.wt.) and i n the presence of 2-mercap-toethanol caused a f u r t h e r d i s s o c i a t i o n of the enzyme i n t o p a r t i c l e s of 3.8S (40,000 M.wt.). Thus i t was suggested that the four subunits were arranged as p a i r s of dimers held together by disulphide bonds (-110) . In the l a s t year, the f a c t that t h i s l i s form of AChE i s a degradation product has become very apparent. Two 7 6 . l a b o r a t o r i e s h a v e r e p o r t e d i n v e s t i g a t i o n s o n t h e s u b u n i t h e t e r o g e n e i t y o f v a r i o u s p r e p a r a t i o n s o f t h e g l o b u l a r e n z y m e o b t a i n e d f r o m t o l u e n e s t o r e d t i s s u e ( 1 1 1 - 1 1 3 ) . B r i e f l y , d i f f e r e n t s a m p l e s o f A C h E d i s p l a y e d d i f f e r e n t S D S g e l e l e c t r o p h o r e t i c p a t t e r n s d e p e n d i n g o n h o w l o n g t h e t i s s u e h a d b e e n s t o r e d u n d e r t o l u e n e a n d t h e m e t h o d o f e n z y m e p u r i f i c a t i o n w h i c h h a d b e e n u s e d . P r o t e o l y s i s a n d / o r a u t o l y s i s h a s t h u s b e e n s h o w n t o e f f e c t m a j o r d i f f e r e n c e s i n t h e c o m p o s i t i o n o f t h e d i s u l p h i d e r e d u c e d p o l y p e p t i d e s o f t h e c a t a l y t i c s u b u n i t s o f t h i s g l o b u l a r f o r m o f t h e e n z y m e . M o r e w i l l b e s a i d a b o u t t h i s a t t h e e n d o f t h e c h a p t e r . I n t h e w o r k p r e s e n t e d i n t h i s s e c t i o n o f t h e t h e s i s , f r e s h e l e c t r o p l a x t i s s u e h a s b e e n u s e d e x c l u s i v e l y i n a n a t t e m p t t o p u r i f y t h e m o s t n a t i v e f o r m s o f t h e e n z y m e . T h e s t a b i l i t y o f t h i s k i n d o f p r e p a r a t i o n t o w a r d s p r o t e o -l y s i s b y c o n t a m i n a t i n g e n d o g e n e o u s p r o t e a s e w h i c h i s p r e s e n t e v e n a f t e r a f f i n i t y c h r o m a t o g r a p h y h a s b e e n i n v e s t i g a t e d . I n o r d e r t o u n d e r s t a n d t h e c o n s t i t u t i o n a n d a s s e m b l y o f t h e s u b u n i t s o f A C h E a n d g a i n i n f o r m a t i o n a b o u t t h e m o l e -c u l a r e v e n t s o c c u r i n g u p o n p r o t e o l y s i s w e h a v e t r e a t e d t h e p u r i f i e d A C h E w i t h t r y p s i n u n d e r v a r i o u s c o n d i t i o n s . S D S g e l e l e c t r o p h o r e s i s a n d i s o k i n e t i c s u c r o s e g r a d i e n t s w e r e u s e d t o m o n i t o r t h e r e s u l t i n g c h a n g e i n e n z y m e s t r u c t u r e . T h e r e m a i n d e r o f t h i s i n t r o d u c t i o n w i l l d e s c r i b e f o u r i m -p o r t a n t p h y s i c a l m e t h o d s f o r t h e s e p a r a t i o n a n d c h a r a c t e r i -s a t i o n o f p r o t e i n s . Sucrose Gradient C e n t r i f u g a t i o n I s o k i n e t i c sucrose gradients are a s e n s i t i v e and con-venient method f o r the c h a r a c t e r i s a t i o n and i d e n t i f i c a t i o n of p r oteins by t h e i r hydrodynamic behaviour. In general, molecules layered on top of a sucrose gradient move down-ward under the f o r c e of a c e n t r i f u g a l f i e l d at a r a t e which depends on both t h e i r s i z e and shape. The v e l o c i t y of sedimentation of each molecule i s r e l a t e d to the speed of c e n t r i f u g a t i o n by a constant, the sedimentation constant S, u s u a l l y expressed i n Svedberg u n i t s f o r convenience (see appendix f o r d e t a i l s ) . With conventional gradients, the a n a l y t i c a l determina-t i o n of S numbers i s a procedure complicated by the f a c t that the v i s c o s i t y of the medium increases more r a p i d l y than the c e n t r i f u g a l force as a molecule moves down the gradient, and consequently the r a t e of sedimentation de-creases with time of spinning. This makes the determination of sedimentation constants too d i f f i c u l t f o r r o u t i n e c a l -c u l a t i o n , and computers must be employed. Also, the non-l i n e a r rate of sedimentation creates another problem which s e r i o u s l y l i m i t s the e f f e c t i v e n e s s of the method. The tendency f o r molecules of d i f f e r e n t sedimentation numbers, to congregate near the bottom of the tube d r a s t i c a l l y l i m i t s the r e s o l u t i o n and accuracy as i n most cases maximum reso-l u t i o n i s achieved i n the f i r s t t h i r d or h a l f of the gradient. 78. These shortcomings can be eliminated, however, by the use of gradients designed such that p a r t i c l e s of a given density range e x h i b i t a constant v e l o c i t y ( i s o k i n e t i c ) of sedimen-t a t i o n throughout the e n t i r e length of the tube. Such i s o k i n e t i c sucrose gradients have been developed by N o l l (114). The p r o f i l e of the gradient i s s p e c i f i c f o r the d e n s i t y range of the p a r t i c l e s under study, the ambient temperature and the r o t o r type used. In such gradients, the increase i n c e n t r i f u g a l d r i v i n g force with i n c r e a s i n g r a d i a l distance of the p a r t i c l e i s compensated f o r by an equal increase i n the opposing forces of v i s -c o s i t y and bouyant density and therefore sedimentation occurs at a constant speed. In contrast with other gradients, r e s o l u t i o n of components increases with time over the e n t i r e length of the tube, g r e a t l y improving s e n s i t i v i t y . Perhaps more important though, these gradients can be accurately c a l i b r a t e d by the presence of standard proteins of known sedimentation values which span the desired density range, and a n a l y t i c a l determination of unknowns i s achieved simply by e x t r a p o l a t i o n o f f a l i n e a r p l o t . I s o k i n e t i c gradients were thus r o u t i n e l y employed i n t h i s work as a method for the determination of the mole-c u l a r forms of AChE present and t h e i r r e l a t i v e abundance i n s o l u t i o n during any molecular c h a r a c t e r i s a t i o n of the p u r i f i e d enzyme. SDS gels SDS polyacrylamide g e l e l e c t r o p h o r e s i s i s a s e n s i t i v e method f o r the determination of both the subunit molecular weights and the homogeneity of the p u r i f i e d p r o t e i n prepara-t i o n . In the presence of SDS the non-covalent bonds of a p r o t e i n a>re broken causing polypeptide chains to u n f o l d with p o s s i b l e subunit separation. In some cases, however, subunits are held together by covalent d i s u l p h i d e bonds and the presence of a reducing agent such as d i t h i o t h r e i t o l i s r equired f o r complete separation. The SDS also coats the denatured polypeptide chains masking the l o c a l charges of the amino a c i d residues, g i v i n g the p r o t e i n a net nega-t i v e charge i n proportion to the amount of SDS bound. Therefore, i n c o n t r a s t with conventional e l e c t r o p h o r e s i s , separation i s achieved e n t i r e l y on the basis of molecular weight. However, because c r i t i c a l v a r i a b l e s i n g e l and sample preparation and i n the e l e c t r o p h o r e s i s procedure contr i b u t e to poor r e p r o d u c i b i l i t y between experiments i t i s necessary to include standard proteins of known mole-c u l a r weight i n each experiment as a c a l i b r a t i o n f o r the sample Rf values (distance moved by the sample band over the distance t r a v e l l e d by the solvent front) i n order f o r accurate r e s u l t s to be obtained. 80. I s o e l e c t r i c focussing of Proteins The method of i s o e l e c t r i c focussing has found many a p p l i c a t i o n s i n the study of proteins from various sources (115-117). The method requires the establishment of a st a b l e pH gradient between the anode and cathode o f an e l e c t r o l y s i s c e l l . This i s achieved by the e l e c t r o l y s i s of a water s o l u t i o n of a mixture of s u i t a b l e low molecular weight ampholytes, c a l l e d c a r r i e r ampholytes. When proteins are put i n t o a system such as t h i s each p r o t e i n w i l l migrate to, and focus at, i t s i s o e l e c t r i c p o int, p i . CH„ — N — (CH„) — N — CH...... 2 j 2 x | 2 ( C H 2 ) x R NR2 R = H or — (CH„) — COOH 2 x x = 2 or 3 Ampholyte structure The net charge of a p r o t e i n molecule i n an a c i d i c s o l u -t i o n i s p o s i t i v e because most amino groups carry a net p o s i -t i v e charge and most ca r b o x y l i c groups are protonated and hence e l e c t r i c a l l y n e u t r a l . I f the pH i s gradually increased the number of c a r b o x y l i c groups which gain a negative charge w i l l increase and the number of p o s i t i v e l y charged groups w i l l decrease. At a c e r t a i n pH value, the i s o i o n i c point, the net charge of the pr o t e i n molecule 81. i s zero. The i s o i o n i c point of a molecule i s thus determined by the number and types of i o n i z a b l e groups and t h e i r d i s s o c i a t i o n constants. In conventional e l e c t r o p h o r e s i s , there i s a constant pH between anode and cathode so that p o s i t i v e l y charged ions e.g. p r o t e i n , migrate to the cathode while negatively charged ions migrate to the anode. In e l e c t r o f o c u s i n g a stable pH gradient i s arranged: the pH i n c r e a s i n g from anode to cathode. I f a p r o t e i n i s put i n t o t h i s system at a pH lower than the i s o i o n i c point the net charge of the molecule w i l l be p o s i t i v e and i t w i l l migrate i n the d i r e c -t i o n of the cathode. Due to the presence of the pH gradient, the p r o t e i n w i l l migrate to an environment of succ e s s i v e l y higher pH values which i n turn, w i l l i n f l u e n c e the i o n i s a t i o n and net charge of the molecule. The p r o t e i n w i l l eventually reach a pH where i t s net charge i s zero and i t w i l l stop migrating. This i s the i s o e l e c t r i c point of the p r o t e i n , and i n a stable pH gradient generated i n a viscous medium l i k e sucrose, the focusing e f f e c t works against d i f f u s i o n and r e s o l u t i o n can be retained f o r a long period of time. What then are the a p p l i c a t i o n s of such a technique? From what has been s a i d i t i s understandable that the e l e c t r o f o c u s s i n g experiment can be used as a powerful separation method f o r a n a l y t i c a l as well as f o r preparative purposes. Proteins d i f f e r i n g i n i s o e l e c t r i c point by as l i t t l e as 0.01 of a pH u n i t may be separated (118,119). The p i values are e a s i l y determined by measurement of pH at the maximum concentration of the p r o t e i n a f t e r focussing and f r a c t i o n a t i n g the column contents. The numbers them-selves measure the i n t r i n s i c a c i d i t y of the p r o t e i n and are important f o r c h a r a c t e r i s a t i o n and comparative purposes. Gel f i l t r a t i o n of Proteins Gel f i l t r a t i o n i s a chromatographic procedure that i s used both i n a n a l y t i c a l and preparative work. I t r e l i e s on a chromatographic m a t e r i a l which i s capable of separating substances according to molecular s i z e and shape. Mole-cules that are l a r g e r than the pores of the g e l m a t e r i a l to be used (i.e. above exclusion l i m i t ) c a n n o t penetrate the pores and thus they pass through the bed i n the c a r r i e r l i q u i d phase. They are eluted f i r s t . Smaller molecules, however, penetrate the pores of the g e l to a varying extent depending on t h e i r s i z e and shape. Molecules are therefore eluted from a column i n order of decreasing molecular s i z e . Various types of g e l supports are commercially a v a i l a b l e and each has a c h a r a c t e r i s t i c range of molecular weights which i t can s u c c e s s f u l l y f r a c t i o n a t e , as determined by the pore s i z e and the degree of swelling of the g e l . By appropriate choice of g e l , peptides and proteins can be f r a c t i o n a t e d w i t h i n a very broad molecular weight range: 1 x 10 3 - 40 x 10 6. Thus on a preparative s c a l e g e l f i l t r a t i o n can be used to 'desalt'; r e f e r r i n g not only to the removal of s a l t s but a l s o to the removal of other low molecular weight compounds from s o l u t i o n s of macromolecules, and also by c a r e f u l choice of the most s u i t a b l e g e l type and column conditions molecules d i f f e r i n g very l i t t l e i n molecular weight can be f r a c t i o n a t e d and i s o l a t e d . 3.2 EXPERIMENTAL - MATERIALS AND METHODS SDS Gel E l e c t r o p h o r e s i s A s l i g h t l y modified procedure of Fairbanks et a l . (120) was used. The experiment was c a r r i e d out i n a v e r t i c a l e l e c t r o p h o r e s i s apparatus containing 8 00 ml buffer s o l u -t i o n i n each electrode compartment and a maximum of 12 g e l tubes ( f i g . 14). The gels were prepared so as to con-t a i n 1% SDS and e i t h e r 7.0% or 5.6% acrylamide. To f a c i l i t a t e the preparation of b u f f e r s , g e l s , etc., concentrated stock s o l u t i o n s when f i r s t made up and sub-sequently used to prepare the various solutions used i n the e l e c t r o p h o r e s i s experiment. These are l i s t e d i n Table Gels were made by f i r s t combining 1.4 ml concentrated Acbis (Table I I ) , 1.0 ml 10 x b u f f e r , and 5.6 ml of water. 85. This s o l u t i o n was then degassed and to i t was added 0.5 ml 20% SDS, 1 ml of ammonium persulphate (15 mg/ml) and 0.5 ml of 0.5% TEMED (N,N,N*,N'-tetramethylenediamine). This mixture was used to f i l l four 0.5 x 11 cm pyrex tubes (previously cleaned i n a cone HC1 bath and coated with Photoflo s o l u t i o n ) to w i t h i n 1 cm of the top. The top of each g e l was then c a r e f u l l y covered with overlay s o l u t i o n (to prevent d r y i n g out) and l e f t standing overnight to allow complete p o l y m e r i s a t i o n . A small amount of the denaturing s o l u t i o n (Table II) was e i t h e r made 80 mM i n d i t h i o t h r e i t o l or used d i r e c t and combined with an equal volume of p r o t e i n s o l u t i o n and heated f o r 15-20 minutes at 37°C to completely denature the p r o t e i n . Molecular weight standards were prepared i n the same way and denatured at 60°C. A f t e r c o o l i n g , a maximum volume of 100 u l of the p r o t e i n sample was care-f u l l y a p p l i e d to the top of each g e l (now contained i n the e l e c t r o p h o r e s i s apparatus and covered with b u f f e r s o l u t i o n ) with an appropriate s i z e d micropipet. The concentration of the p r o t e i n s o l u t i o n was such that a maximum of 100 yg p r o t e i n was loaded onto each g e l , thus avoiding e x c e s s i v e l y broad bands. Where the concentration o f the p r o t e i n sample was low, Amicon CF-25 cones were used i n a S o r v a l l r o t o r , SS 34 a t 2,000 rpm to concentrate the AChE to around 2 mg/ml. E l e c t r o p h o r e s i s was c a r r i e d out at a current of 5 mA/gel and required approximately 3 1/2 hours under these condi-t i o n s . In a l l cases gels were prerun f o r 1 hour p r i o r to sample a p p l i c a t i o n to ensure the removal of any excess ammonium persulphate. Molecular weight markers were run together i n separate tubes from the sample. The fo l l o w i n g proteins (Sigma) were used as standards; Phosphorylase b. (subunit M.wt. 94,000) bovine serum albumin (68,000), Ovalbumin (45,000), Pepsin (35,500) and Myoglobin (17, 000). A f t e r removal of the gels from the tubes, the p o s i t i o n of the t r a c k i n g dye was marked by notching the gels with a needle dipped i n India ink. The gels were then trans-f e r r e d to stoppered glasstubes and ag i t a t e d s e q u e n t i a l l y with the fo l l o w i n g p r o t e i n s t a i n i n g s o l u t i o n s (120). 1) 25% isopropanol, 10% a c e t i c a c i d , 0.025% coomassie blue (overnight). 2) 10% isopropanol, 10% a c e t i c a c i d , 0.0025% coomassie ... blue (6-9 hours) . 3) 10% a c e t i c a c i d , 0.0013% coomassie blue (overnight). 4) 10% a c e t i c a c i d (overnight). A f t e r t h i s time, the proteins were apparent as dark blue bands. The subunit molecular weight of AChE was e s t i -mated by p l o t t i n g l og (M.wt.) against Rf values measured r e l a t i v e to the t r a c k i n g dye. The points f o r the standard proteins f e l l on a s t r a i g h t l i n e from which any unknown subunit molecular weight could be obtained by i n t e r p o l a t i o n (Fig. 15). In some experiments the gels were stained f o r the pre-sence of carbohydrate. In t h i s case the procedure was as f o l l o w s : 1) Use the procedure described above (1-4) but i n the absence of coomassie blue. 2) 0.5% p e r i o d i c a c i d (2 hours). 3) 0.5% sodium a r s e n i t e , 5% a c e t i c a c i d (30-60 min). 4) 0.1% sodium a r s e n i t e , 5% a c e t i c a c i d (20 mins, with three changes). 5) A c e t i c a c i d (10-20 min). 6) S c h i f f reagent (overnight). 7) 0.1% N a 2S 20 4, 0.01 NHCl (several hours with changes. Test s o l u t i o n with formaldehyde - i t should not turn p i n k ) . S c h i f f reagent: 2.5 g basic fuchsin i n 500 ml H 20 5 g sodium metabisulphite 50 ml IN HC1 S t i r s o l u t i o n f o r several hours and d e c o l o r i z e with 2 g of a c t i v a t e d charcoal. 88. A f t e r t h i s , the p r o t e i n bands containing any carbohy-drate appear pale pink; the i n t e n s i t y of the colour being p r o p o r t i o n a l to the amount of carbohydrate that i s present. 89. Figure 15 Molecular weight calibration curve for S.D.S. gel e lectrophoresis 100-8 0 -to I 2 6 C H X •&40-CD o CD o 20-10 0.0 02 Phosphorylase B ( 9 4 , 0 0 0 ) Bovine serum albumin ( 6 8 , 0 0 0 ) Ovalbumin ( 4 5 , 0 0 0 ) Pepsin ( 3 4 , 0 0 0 ) Myoglobin (17,000) \ 0.4 0.6 0.8 .0 R f value 90. TABLE I I : Stock s o l u t i o n s and buffe r s f o r SDS g e l ele c t r o -phoresis A. Stock s o l u t i o n s 1) Concentrated AcBis 40 g acrylamide 1.5 g N,N 1-methylenebisacrylamide H 20 to 100 ml 2) 10X Buffer 0.4 M T r i s 0.2 M sodium acetate 0.02 M EDTA pH = 7.4 with a c e t i c a c i d 3) 20% SDS (W/W) B. E l e c t r o p h o r e s i s b u f f e r (per l i t r e ) 100 ml of 10X bu f f e r 50 ml 20% SDS H„0 to 1 l i t r e C. Denaturing S o l u t i o n 2g SDS lOg sucrose 74.5 mg EDTA 2 mg pyronin y 0.242 g T r i s H o0 to 100 ml, pH 8 with HCl D. Overlay s o l u t i o n 0.1% SDS 0.15% Ammonium persulphate 0.05% TEMED 91. I s o k i n e t i c sucrose gradients Gradients were c a l c u l a t e d f o r a Beckman SW 41 T i r o t o r by the method of N o l l (114) and a d e t a i l e d d e s c r i p t i o n of t h i s work i s provided i n an accompanying appendix (page 163) . The experiments were run at 42,000 rpm. f o r 12-14 hours and the gradients were c a l i b r a t e d with standard proteins of known sedimentation c o e f f i c i e n t s centrifuged i n the same tube as the sample: 3-galactosidase (15.9S), catalase (11,4S) and a l c o h o l dehydrogenase (7.6S). The i s o k i n e t i c gradients were prepared as follows: 23.4 ml of 10% sucrose i n 20 mM phosphate -0.5 M NaCl (pH 6.90) was p i p e t t e d i n t o a constant volume mixing r e s e r v o i r and s t i r r e d continuously while a 29.3% sucrose s o l u t i o n (in same buffer) was added at a flow rate of about 0.5 ml/ml, using a multi-channel p e r i s t a l t i c pump (Desaga) . At the same time as the dense s o l u t i o n was being added the mixed s o l u t i o n was pumped out of the r e s e r v o i r (at the same flow rate) and i n t o two 8.5 x 1 cm n i t r o c e l l u l o s e tubes v i a c a p i l l a r y tubes. This continued u n t i l the gradient was 8.25 cm up the tube, a t o t a l volume of close to 13.2 ml then having been added. The c a p i l l a r i e s were slowly and c a r e f u l l y removed from the tubes and the gradients loaded i n t o the S.W. 41 r o t o r buckets and cooled to 5°C; the tem-perature of the c e n t r i f u g a t i o n . A t o t a l of s i x gradients 92. Figurel6 Sedimentation coefficient calibration curve for isokinetic sucrose gradients /3-ga!actosidase (15.9 s) catalase (11.4s) A.D.H. ( 7 . 6 s ) 4 6 8 10 12 14 Gradient vo lume (ml) could be prepared i n t h i s way: the maximum capacity f o r the SW 41 r o t o r . Meanwhile AChE samples and the standard proteins were made up i n or d i a l y s e d against the 20 mM phosphate -0.5 M NaCl (pH 6.9) b u f f e r and 100 y l of each was layered care-f u l l y onto the gradients with a micropipet. I f required, b u f f e r was then layered on top to bring the gradients to w i t h i n 2 mm of the top of each tube. This i s a precaution so as to ensure that the tubes do not c o l l a p s e during c e n t r i f u g a t i o n (121) . A f t e r c e n t r i f u g a t i o n the gradients were pumped out of the tubes by i n s e r t i n g a f i n e c a p i l l a r y i n t o each tube and using the p e r i s t a l t i c pump at a low flow r a t e (0.25 ml/min). The gradients could then be f r a c t i o n a t e d i n a Gi l s o n MF-8 0 m i c r o f r a c t i o n a t o r and the samples assayed f o r AChE and the standards as described elsewhere. The sedimentation constants f o r the standard proteins p l o t t e d against the volume i n the gradient at which they appear provides a s t r a i g h t l i n e from which the unknown AChE value can be obtained by i n t e r p o l a t i o n (figure 16). I s o e l e c t r i c focussing A diagram of the e l e c t r o f o c u s s i n g column used i n these experiments to be described i s shown i n f i g u r e 17_. A de-t a i l e d d e s c r i p t i o n of the experimental procedure can be found i n s e v e r a l references (112,123) but a t y p i c a l e x p e r i -ment would be as f o l l o w s : The valve 12 i s opened and c o o l i n g water passed through the compartments (18 and 16) u n t i l the apparatus i s Figure 17 - A Sketch of the I s o e l e c t r i c Focussing Column F i g . 17: E l e c t r o f o c u s s i n g column of 110 ml capacity. The outer c o o l i n g jacket (18) has an i n l e t at 14, and an o u t l e t at 5. From the outer jacket the water flows through a tube i n t o the c e n t r a l c o o l i n g jacket at 4 and leaves the column at 3. Two platinum electrodes are used. One electrode 13, i s i n contact with the plug 7, i s i n the upper part of the column. The gas formed at t h i s electrode escapes at 2. The other i s wound on a T e f l o n bar 11, and gas escapes at 1. Before d r a i n i n g the column the c e n t r a l tube i s closed by l i f t i n g the plug 12 which has a rubber gasket on the upper surface and seals at 15. I s o e l e c t r i c focussing takes place i n compartment 16, which i s f i l l e d through n i p p l e 2. At the bottom of the column there i s a plug 18, with an a t t a c h -ment for^ a c a p i l l i a r y tube to enable the column to be f r a c t i o n a t e d . e q u i l i b r a t e d at 5°C. In t h i s case, the cathode s o l u t i o n i s then added v i a the n i p p l e (1) with the a i d of a p e r i s t a l t i c pump so as to f i l l up the bottom of the column. The p r o t e i n sample, the Ampholine c a r r i e r ampholytes and the sucrose s o l u t i o n are then mixed by means of a gradient mixer and compartment 17 i s f i l l e d so as to achieve a density gradient. The f i l l i n g i s done v i a n i p p l e 2 again using the p e r i -s t a l t i c pump. The anode s o l u t i o n i s then added on top of the s o l u t i o n to be elec t r o f o c u s s e d , and the experiment i s st a r t e d . To determine whether the e l e c t r o f o c u s s i n g procedure i s completed or not, the current passing through the s o l u t i o n i s checked from time to time. When the current (at constant voltage) has decreased to a constant value most of the c a r r i e r ampholytes i n the system are focussed at or near t h e i r i s o e l e c t r i c p o i n t s . The sample p r o t e i n w i l l also then be l a r g e l y focussed but generally a further 8-10 hours i s allowed so as to ensure good focussing. An e l e c t r o -focussing experiment normally takes from 24-7 2 hours de-pending on the pH range used; the narrower the pH range the longer the focussing time. When t h i s procedure i s complete the valve 12 i s closed to prevent the c e n t r a l electrode s o l u t i o n from mixing with the e f f l u e n t . The clamp on the c a p i l l a r y tube, 20 i s opened and the column emptied at a flow rate of about 1 ml/min. The f r a c t i o n s are c o l l e c t e d on a G i l s o n f r a c -t i o n a t o r and t h e i r pH determined at 5°C; the temperature of the experiment. P r o t e i n i s measured by i t s absorption at 280 nm and AChE can be assayed f o r by i t s a c e t y l t h i o c h o l i n e a c t i v i t y . F i n a l l y , i t should be mentioned that i s o e l e c t r i c focussing can a l s o be c a r r i e d out i n polyacrylamide g e l s . The apparatus f o r running t h i s experiment i s the same as t h a t used f o r d i s c or SDS g e l e l e c t r o p h o r e s i s . The gels can be e i t h e r photopolymerised or chemically prepared, and serve as a replacement f o r the sucrose density gradient used i n the column. Photopolymerisation i s to be pre-f e r r e d and i s g e n e r a l l y s u i t a b l e over the range pH 5-8 or pH 3-10. The disadvantages of the production of a r t i f a c t s due to the presence of persulphate i n chemically polymerised gels has been commented on by several authors (124-126) . Gel e l e c t r o f o c u s s i n g i s f a s t , requires small amounts of p r o t e i n and uses much l e s s ampholines than the column technique. I t i s however, l i m i t e d i n determining an accurate value f o r the p i of a p r o t e i n as the g e l i s short and must be s l i c e d and soaked i n order to a s c e r t a i n the pH. Also the gel p o r o s i t y prevents the technique being used f o r large, asymnetric p r o t e i n s . The solutions f o r t h i s method are given i n t a b l e IV. Gel f i l t r a t i o n AChE s o l u t i o n s were concentrated on an Amicon u l t r a -f i l t r a t i o n apparatus equipped with a PM-3 0 membrane and appl i e d to a Sepharose 2B or 4B column (1.5 x 80 cm) pre v i o u s l y e q u i l i b r a t e d i n 0.02M phosphate, -2mM decamethonium, pH 6.90. Four ml f r a c t i o n were c o l l e c t e d at a flow rate of 10 ml/hour and the p r o t e i n 0 D „ o n monitored. AChE a c t i v i t y was determined as pre v i o u s l y described. The Sepharose 4B column was also c a l i b r a t e d with standard p r o t e i n s of known Stoke's radius,(Re). Fibrinogen, 125 g-galactosidase, c a t a l a s e , a l c o h o l dehydrogenase and I-bovine serum albumin were d i s s o l v e d i n column buffer and di a l y s e d f o r several hours p r i o r to loading onto the column. A 1 ml s o l u t i o n of these standard proteins was loaded onto the column and 1 ml (30 drop) f r a c t i o n s were c o l l e c t e d and the flow rat e slowed to 4 ml/hr to improve the column r e s o l u t i o n . The pr o t e i n , OD 2 8 Q p r o f i l e was recorded and the i n d i v i d u a l proteins assayed f o r where p o s s i b l e . B-galactosidase, catalase and ADH assays are 125 described at the end of t h i s s e c t i o n . I-BSA was detected by i t s r a d i o a c t i v i t y and f o r fi b r i n o g e n alone the OD 2 8 Q was used. The experiment was repeated several times to ensure the accuracy of the p r o f i l e s and using the data obtained 98. values f o r each standard were c a l c u l a t e d . The e l u t i o n parameter i s defined as: V - V D V , . - V f o where V i s the p r o t e i n e l u t i o n volume, V i s the e x c l u s i o n e o volume and i s the e l u t i o n volume of small molecules. K D i s thus a q u a n t i t a t i v e measure of the proteins movement through the g e l column. V q has been determined using Blue Dextran (Pharmacia) and by the e l u t i o n of potassium f e r r i c y a n i d e . These r e s u l t s are shown i n f i g u r e 27. The experimental parameter K , can be r e l a t e d to the Stokes radius (Re) by (132) Re = f (/log K D) A p l o t of R g versus /-logK^ was a s t r a i g h t l i n e and from t h i s values f o r AChE, could be obtained by i n t e r p o l a t i o n ( f i g . 29). 99. TABLE I I I - I s o e l e c t r i c focussing s o l u t i o n s f o r column. Column - LKB. 8101 with a capacity of 110 ml Electrode s o l u t i o n s -Cathode Ethanolamine 0.4 ml H 20, d i s t i l l e d 14.0 ml Sucrose 12.0 g Anode H 2 S O 4 100 y l H 20, d i s t i l l e d 10 ml Gradient s o l u t i o n s -Dense 1.9 ml Ampolines (40% or 25%) H 20 to 42 ml 28 g sucrose L i g h t 0.6 ml Ampholines P r o t e i n sample (dialysed) H o0 to 56 ml Ampholyte pH ranges, a v a i l a b l e from L.K.B. include: 3.5-10 5-8 2.5-4 6-8 3.5-5 7-8 4- 6 8-8.5 5- 7 9-11 100. TABLE IV: I s o e l e c t r i c focussing s o l u t i o n s f o r gels Photopolymerisation - Stock s o l u t i o n s kept at 5°C and i n the dark have a u s e f u l l i f e of about one month. A. C a t a l y s t 1.0 ml TEMED 14 mg r i b o f l a v i n H 20 to 100 ml B. Acrylamide 30 g acrylamide 0.8 g methylene-bis-acrylamide H 20 to 100 ml Gel mixture Mix 0.8 ml of A with 3.0 ml B and add 0.3 ml 40% Ampholine. For each 10 cm tube take 1.0 ml of t h i s mixture and add 30-300 ug p r o t e i n sample and water to 3.0 ml. Poly-merisation i s complete a f t e r 1 hour exposed to b r i g h t l i g h t . Chemical polymerisation A. C a t a l y s t 1.0 ml TEMED H 20 to 100 ml B. Acrylamide 30 g acrylamide 0.8 g methylene-bis-acrylamide H 20 to 100 ml C. 1% aqueous potassium persulphate ( f r e s h ) . Gel mixture For 4 gels mix 8.0 ml d i s t i l l e d water, 3.0 ml B, 0.3 ml 40% Ampholine and add 0.7 ml C.~ Polymerisation u s u a l l y takes 1 hour a f t e r a d d i t i o n of C. 10.1. Enzyme Assays a) g-galactosidase S o l n A: 10 mM o-nitrophenylgalactoside i n 0.2 M phosphate (pH 7.0). S o l n B: 1.5 mM MgCl 2 0.3 mM MnCl 2 0.9 m l / l i t r e 3-mercapto-ethanol S o l n C: 1M Na 2C0 3 Procedure: 0.5 ml of s o l u t i o n A i s mixed with 1.0 ml of s o l u t i o n B and a s u i t a b l e a l i q u o t of the gradient f r a c t i o n to be assayed i s added with mixing. A f t e r a few minutes an intense yellow colour of o-nitrophenol becomes apparent i n the a c t i v e f r a c t i o n s . The r e a c t i o n i s quenched by adding 1.5 ml of s o l u t i o n C and the absorption at 420 nm i s determined, at w i l l . b) Alcoho l Dehydrogenase assay medium: 35 ml H 20 35 mg NAD 50 y l ethanol 2 ml TRIS, 1M (pH 7.6) Procedure: 3 ml of assay medium i s added to a cuvette and an a l i q u o t of the gradient i s added at time zero. The i n -crease i n absorbance at 34 0 nm i s followed with time f o r the a c t i v e f r a c t i o n s . c) Catalase - Measured d i r e c t l y by measuring the absorbance at 405 nm of the f r a c t i o n * d) AChE - Method of Ellman, et a l . as described elsewhere. 102. 3.3 Results and Discussion a) S t r u c t u r a l S t a b i l i t y and Composition Having p u r i f i e d AChE by a f f i n i t y chromatography the preparation was c h a r a c t e r i s e d v i a i s o k i n e t i c sucrose gradient c e n t r i f u g a t i o n and polyacrylamide g e l e l e c t r o p h o r e s i s i n the presence of SDS and d i t h i o t h r e i t o l . The r e s u l t s f o r a t y p i c a l preparation are shown i n f i g u r e 1_8. This data i n d i c a t e s that we are d e a l i n g with the large asymmetric forms (18S,14S) composed of a long ' t a i l ' s t r u c t u r e and s ev eral groups of c a t a l y t i c , tetrameric 'head' groups. Since the SDS g e l c o n s i s t s p r i m a r i l y of one component, each subunit w i t h i n the c a t a l y t i c tetramer i s i d e n t i c a l and of molecular weight approximately 80,000. Samples pre-pared fo r SDS e l e c t r o p h o r e s i s i n the absence of reducing agents d i s p l a y no band at 80,000 molecular weight at a l l . In f a c t a s i n g l e band i s observed with a molecular weight w e l l i n excess of 100,000. Although the 5.6% SDS a c r y l a -mide gels were not c a l i b r a t e d under non-reducing conditions f o r molecular weights i n t h i s region, t h i s component most probably corresponds to the dimeric structure of subunits (160, 000) .connected by i n t e r s u b u n i t disulphide bonds as reported by others f o r the l i s form of the enzyme (111-113). SDS gels of AChE, with reduction, run as a function of time a f t e r p u r i f i c a t i o n i n d i c a t e that the 80,000 subunit 103 . Figure 18: The molecular forms and subunit composition of AChE p u r i f i e d by a f f i n i t y chromatography from f r e s h e l e c t r o -plax t i s s u e . I s o k i n e t i c sucrose gradients done i n 0.5 M NaCl - 0.02 M phosphate b u f f e r (pH = 6.9) were c a l i b r a t e d with standard p r o t e i n s as described i n the te x t . Acrylamide g e l e l e c t r o p h o r e s i s was done i n 10 cm, 5.6% gels i n the presence of SDS and d i t h i o t h r e i t o l . 104 . F I G U R E 18 S U C R O S E G R A D I E N T P R O F I L E O F A F F I N I T Y P U R I F I E D A . C h . E . S.D.S.gel w i t h r e d u c t i o n 80 K 55 K N o . of m i s . of gradient 105. i s s u s c e p t i b l e to p r o t e o l y t i c cleavage, by endogeneous protease which i s present even a f t e r a f f i n i t y chromatography. Figure 19 summarises a t y p i c a l experiment and shows g e l scans at 550 nm, the absorption of the p r o t e i n s t a i n coo-massie blue, from one to four weeks a f t e r p u r i f i c a t i o n of the enzyme. The time taken to bring about the observed changes i n the AChE polypeptide d i s t r i b u t i o n v a r i e d as a fun c t i o n of the s p e c i f i c a c t i v i t y of the preparation and conditions of storage. The r e s u l t s from a s i n g l e sample show c l e a r l y that the 80,000 subunit i s cleaved i n a w e l l defined way to generate a peptide of molecular weight 55,000 and two of 28,000 and 25,000 r e s p e c t i v e l y . P a r a l l e l g e l s stained f o r carbohydrate with the S c h i f f reagent procedure described under methods show an 80,000 to 55,000 peptide conversion and i n d i c a t e that these are glycopeptides. The 28,000 and 25,000 bands however, do not s t a i n f o r car-bohydrate. These experiments which probe the subunit composition of the enzyme i n d i c a t e that the tetramers of the 'native' forms are composed of c a t a l y t i c subunits which undergo a w e l l ordered p r o t e o l y t i c degradation. Using p-t o s y l - L - a r g i n i n e methyl ester (TAME) to assay the p u r i f i e d AChE f o r the presence of any t r y p s i n l i k e protease was negative. A standard assay and a Very s e n s i t i v e one showed no change i n o p t i c a l density at 247 nm even a f t e r an incubation period of 15 minutes. 106. Figure 19: SDS-acrylamide g e l e l e c t r o p h o r e s i s of the p u r i -f i e d 18S, 14S forms of AChE under reducing c o n d i t i o n s . Gel scanned from cathode ( l e f t ) to anode (right) at 550 nm. A, AChE sample 1 week a f t e r a f f i n i t y p u r i f i c a t i o n . B, 2 weeks C, 3 weeks. D, 4 weeks. 107 . Various attempts have been made to prevent t h i s degra-dation. The AChE was o r i g i n a l l y e l uted from the a f f i n i t y r e s i n with 0-10 mM gradient of decamethonium and so as to t r y and remove the impurity t h i s gradient was made s l i g h t l y shallower 0-5 mM. This seemed to slow down the conversion but not prevent i t . Two other attempts involved f i r s t , the treatment of the AChE with diisopropylfluorophosphate (D.F.P.), a potent i n h i b i t o r which binds s p e c i f i c a l l y at the a c t i v e s i t e of the enzyme and second storage of the AChE i n the presence of a protease i n h i b i t o r benzethonium c h l o r i d e . Both were unsuccessful; even a f t e r treatment with D.F.P. (Sigma) degradation of the c a t a l y t i c subunit was found to occur and i n the one case where p u r i f i e d AChE was stored i n the presence of 0.1 mM benzethonium c h l o r i d e the p r o t e i n pre-c i p i t a t e d , and AChE a c t i v i t y was l o s t . In a d d i t i o n to t h i s , a sample of f r e s h l y p u r i f i e d enzyme was immediately a p p l i e d to the concanavalin A a f f i n i t y column described i n chapter two. Even though a l l the AChE binds and hence the column could be washed ex-t e n s i v e l y with high i o n i c strength b u f f e r before e l u t i n g the AChE with mannoside, s t i l l the p r o t e i n was observed to undergo the conversion described above. E v i d e n t l y any impurity present must adhere to t h i s Con A column as w e l l . 108 . T r y p s i n i n high concentration has been used to e x t r a c t the g l o b u l a r 11S form of AChE from the e l e c t r o p l a x t i s s u e of both Electrophorus elect'ricus and Torpedo c a l i f o r n i c u s (127,103). Using these same conditions on the already p u r i f i e d native forms of the enzyme we have found that i t i s p o s s i b l e to b r i n g about marked changes i n the subunit composition. Figure 2_0 shows g e l scans of SDS gels where AChE has been incubated with two d i f f e r e n t concentrations of t r y p s i n f o r 16 hours. A c o n t r o l sample with no t r y p s i n added shows only the component at 80,000 but i t can be seen that when t r y p s i n i s added and the concentration v a r i e d t h i s 80,000 peptide i s cleaved to generate the 55,000 peptide and bands around 25,000. Indeed, i t i s p o s s i b l e using high concentrations of t r y p s i n to mimic the endo-geneous p r o t e o l y t i c cleavage of the native subunit observed to occur slowly a f t e r p u r i f i c a t i o n by a f f i n i t y chromatography. V a r i a t i o n of the p r o t e o l y t i c conditions reveals i n t e r -e s t i n g d i f f e r e n c e s i n the rates of p r o t e o l y s i s of the d i f f e r e n t elements of the 18S, 14S AChE s t r u c t u r e . The e f f e c t of incubations of AChE with 0.01 mg/ml t r y p s i n f o r 15 minutes as monitored by SDS g e l el e c t r o p h o r e s i s and i s o k i n e t i c sucrose gradients are shown i n f i g u r e 21. I t can be seen that before and a f t e r t r y p s i n treatment there i s no great change i n the d i s p o s i t i o n of the 80,000 and 55,000 polypeptides. However, t r y p s i n i s known to convert 109. Figure 20: SDS-Acrylamide g e l e l e c t r o p h o r e s i s i n the presence of d i t h i o t h r e i t o l of t r y p s i n treated AChE samples. (A) and (B) represent incubations of the 18S, 14S forms of AChE with t r y p s i n f o r 16 hours, 4°C, at the concentrations of t r y p s i n noted i n the f i g u r e . Gels scanned as i n F i g . 19. Figure 21: The molecular forms and subunit composition of AChE before (A) and a f t e r (B) mild t r y p s i n treatment. Samples were incubated with 0.01 mg/ml t r y p s i n f o r 15 minutes p r i o r to quenching with 0.2 mg/ml soyabean t r y p s i n i n h i b i t o r . I s o k i n e t i c sucrose gradients i n 0.5 M NaCl - 0.02 M phosphate b u f f e r (pH = 6.9). Gels scanned as i n F i g . 19. T R Y P S I N FIGURE 21 S U C R O S E G R A D I E N T P R O F I L E S.D.S. G E L S C A N Distance (cm) No. of m is . 112. the native forms of the enzyme to the globular ( l i s ) form and indeed even t h i s short incubation i s s u f f i c i e n t to convert the vast majority of the enzyme to the l i s form. Some 18S and 14S i s however s t i l l present. Thus the k i n e t i c data seem to i n d i c a t e that cleavage of the long asymmetric t a i l from the p u r i f i e d 18S and 14S forms of the enzyme occurs f a s t e r than degradation of the c a t a l y t i c subunit. I t should be noted that the r e s u l t s on the subunit heterogeneity and s t a b i l i t y of the native (18S,14S) enzyme are i n agreement with two recent papers on the l i s form of the enzyme (111,112). Thus i t now appears that 'native' AChE i s composed of i d e n t i c a l subunits of molecular weight 80,000 and that i n each tetramer these subunits are arranged as a dimer of dimers, (a^)^. The r e s u l t s thus support an i n t e r s u b u n i t d i s u l p h i d e model of the quaternary structure of the enzyme (model 2) proposed by Dudai and Silman (111). In t h i s context, i t should be noted that SDS gels run without reduction do not show any band at 80,000 when the enzyme i s p u r i f i e d from f r e s h t i s s u e . Rather, t h i s band appears as a r e s u l t of t r y p s i n treatment or p r o t e o l y s i s / a u t o l y s i s which occurs with storage at 4°C. I t i s perhaps s i g n i f i c a n t and,interesting to note here that a l l of the molecular changes observed i n the r e s u l t s described so f a r occur without any detectable loss i n a c t i v i t y of the enzyme. 113 . b) I s o e l e c t r i c focussing The r e s u l t s of several attempts to carry out e l e c t r o -focussing on AChE to determine the i s o e l e c t r i c point of t h i s membrane bound p r o t e i n have been hampered by the pro-t e i n s i n s o l u b i l i t y and i n s t a b i l i t y at i t s p i . In numerous experiments i n the i s o e l e c t r i c focussing column, p r e c i p i -t a t i o n was evident a f t e r only a few hours of running. However f a i n t , t h i s p r e c i p i t a t e tended to sink i n the sucrose gradient and also i n some cases adhere to the glass walls of the column when the contents were being emptied and f r a c t i o n a t e d . Consequently, anomalous p i values were a l l too e a s i l y obtained. I n i t i a l i s o e l e c t r i c focussing experiments were c a r r i e d out on the crude high s a l t e x tract of the e l e c t r o p l a x t i s s u e which i s very r i c h i n AChE a c t i v i t y and becomes the load (a f t e r d i a l y s i s ) f o r the a f f i n i t y columns. The r e s u l t s of two of these experiments are shown i n f i g u r e s 2J2 and 23. In f i g u r e 2_2 the a c t i v i t y p r o f i l e shows three major peaks of p i ; 4.8, 4.1 and 3.9. The p r o t e i n d i s t r i b u t i o n follows t h i s p r o f i l e , however i t should be noted that pre-c i p i t a t i o n was observed i n t h i s experiment. Decreasing the amount of p r o t e i n and r a i s i n g the ampholine concentra-t i o n to 2% d i d r e s u l t i n now only very f a i n t p r e c i p i t a t i o n . In t h i s experiment (figure 2_3) one major AChE a c t i v i t y peak i s observed with a p i of 4.9 and a shoulder at 4.5 i s also 114. present. From the observations of Massoulie et a l . i t was i n t e r e s t i n g to ask i f any one AChE peak corresponded to a s i n g l e molecular form of the enzyme. When, however the peak i n f i g u r e 2_3 ( f r a c t i o n 70) was d i a l y s e d f r e e of ampho-l i n e s and i n t o high i o n i c strength buff e r , a sucrose gradient showed i t to be composed of a l l three forms, 18,14 and 8S. I t fehould a l s o be commented that as these native forms are known to aggregate i n low i o n i c strength {12); i t i s hard to estimate what the s i t u a t i o n would be i n the i s o e l e c t r i c focussing column. The a f f i n i t y p u r i f i e d p r o t e i n which had been stored at 4°C f o r sometime, was s u c c e s s f u l l y focussed i n a pH 3-10 gradient, however on e l u t i o n of the column the AChE a c t i v i t y was d r a s t i c a l l y reduced by more than 50%. One major peak of enzyme a c t i v i t y was observed with a p i of about 3.6 (see f i g . 2_4) . I t should be noted however that here again two f a i n t bands of p r e c i p i t a t i o n , c l o s e together, were evident a f t e r 24 hours of focussing. An SDS g e l of the focussed p r o t e i n with reduction showed i t to be composed p r i m a r i l y of the i n t a c t 80,000 subunit but a trace of the 55,000 polypeptide was also present. Several experiment were c a r r i e d out with ampholines of a narrower pH r a n g e , t y p i c a l l y , pH 3-8, 3-6 and 3-5. In each experiment with the p u r i f i e d enzyme though,focussing r e s u l t e d i n a good pH gradient within 72 hours but no Figures 22-24: Column i s o e l e c t r i c focussing of AChE i n a pH gradient of 3-10. F i g s . 22 and 23 show r e s u l t s f o r the crude high s a l t e xtract of the e l e c t r o p l a x t i s s u e . F i g . 24 i s the focussing of the f r e s h l y p u r i f i e d enzyme. FIGURE 22 <=T 0 2 4 6 8 10 20 30 40 c n A r r i r \ M -H -119. AChE a c t i v i t y could be detected i n the eluted f r a c t i o n s . S i m i l a r r e s u l t s have been commented on i n the l i t e r a t u r e (112) but here a s u c c e s s f u l experiment was c a r r i e d out using a discontinuous sucrose gradient of 22 steps between 5% and 45% sucrose to e s t a b l i s h a pH 3-10 p r o f i l e . The AChE i n the 11S molecular form was introduced i n t o the centre of the column and focussed to give a p i of 4.5 (112). Some g e l i s o e l e c t r i c focussing was also attempted using the methods described by Wrigley (128) . Freshly a f f i n i t y p u r i f i e d enzyme was focussed i n both chemically polymerised and photopolymerised gels with a pH 3-10 gradients a f t e r running f o r seven hours at one watt. In the former case, the p r o t e i n was layered onto the g e l under an ampholine la y e r i n sucrose and i n the l a t t e r 75 \ig of AChE was i n c o r -porated i n t o each g e l . The photopolymerised gels a f t e r s t a i n i n g and s l i c i n g to determine the pH gradient showed reproducibly one major band at pH 4.3 and another f a i n t band at pH 3.8. The chemically polymerised gels showed the same two bands but now another major band at pH 4.7 and another f a i n t band at 3.6 were also observed. In summary then, i t can be seen that no d e f i n i t e p i numbers can be assigned to AChE as a r e s u l t of t h i s work alone. Rather, the r e s u l t s i n d i c a t e some of the d i f f i c u l t i e s found i n c a r r y i n g out e l e c t r o f o c u s s i n g work with t h i s 120. p r o t e i n , when i t has been i s o l a t e d from i t s membrane environment. Other values of the i s o e l e c t r i c point have been reported i n the l i t e r a t u r e ; p i 5.3 5 f o r an autolysed preparation (129) and p i 4.0 f o r the enzyme extracted with detergent from the e l e c t r o p l a x (130) . The r e s u l t s obtained here i n d i c a t e a somewhat lower i s o e l e c t r i c point and are more i n agreement with the r e s u l t s of Rosenberry et a l . (112) f o r the l i s form of the enzyme; p i = 4.5. In any case, the values obtained are t y p i c a l f o r a membrane bound p r o t e i n . The receptor p r o t e i n extracted from the e l e c t r o -plax with detergent and focussed as an AChR-bungarotoxin complex has been shown to have a p i of 5.2 (131). AChE i s known to be a gl y c o p r o t e i n and hence i t i s quite probable that negatively charged s i a l i c a c i d residues contribute to the low i s o e l e c t r i c p o i n t . I f accurate numbers could be obtained f o r the d i f f e r e n t molecular forms as well as the polypeptides making up the subunits some i n t e r e s t i n g comparisons could be made between the d i f f e r e n t structures which are incorporated into t h i s p r o t e i n . Gel F i l t r a t i o n The e l u t i o n p r o f i l e s of the a f f i n i t y p u r i f i e d 11S form of AChE which i s obtained upon storage of the f r e s h l y p u r i -f i e d enzyme at 4°C, on Sepharose 2B and 4B are shown i n f i g u r e s 25 and 26_ r e s p e c t i v e l y . 121. On Sepharose 2B two p r o t e i n peaks (one at the v o i d volume) were observed with only one showing AChE a c t i v i t y (see f i g . 25). B e t t e r r e s o l u t i o n of t h i s l a t t e r peak was obtained when the same m a t e r i a l was chromatographed on Sepharose 4B ( f i g . 26). Now the peak i s resolved i n t o two components with only the f i r s t showing any enzyme a c t i v i t y . This peak when pooled and concentrated r o u t i n e l y had a high s p e c i f i c a c t i v i t y between 8,000 and 10,000 units/mg. Only small amounts of m a t e r i a l are being chromatographed and i t has been found d i f f i c u l t to pool and concentrate the peak appearing at the void volume. In f a c t , attempts to do t h i s have shown that t h i s m a t e r i a l r e a d i l y adheres to the PM-3 0 membrane and C e n t r i f l o cones (CF-25, Amicon) which are r o u t i n e l y used to concentrate the p r o t e i n samples. The Sepharose 4B column was c a l i b r a t e d with standard proteins of known Stokes radius ( f i g s . 27_ and 2_8) and the s t r a i g h t l i n e obtained upon p l o t t i n g Re versus /-logK can be used to obtain values f o r the AChE samples ( f i g . 29). The Re value of 5.6 nm f o r the eluted AChE corresponds well with the IIS globular form of the enzyme where the asymmetric forms of the enzyme have been shown to have R& values be-tween 12 and 15 nm (132). Using t h i s 5.6 nm value f o r the R of the 11S form and assuming a value f o r the s p e c i f i c e volume, v of 0.77 (132) y i e l d s a molecular weight of 310,000 for t h i s species. One experiment where f r e s h l y a f f i n i t y 122. p u r i f i e d 18, 14S m a t e r i a l was applied to t h i s Sepharose 2B column confirmed the aggregation pr o p e r t i e s of those 'native' forms, as the AChE eluted completely i n the void volume; hence no R& data could be obtained i n t h i s case. I t w i l l be i n t e r e s t i n g to continue these experiments and attempt to c h a r a c t e r i s e the eluted p r o t e i n f u r t h e r . In p a r t i c u l a r , the p r o t e i n which appears at the void volume of the column but does not have enzyme a c t i v i t y must be large, aggregates of high molecular weight and i t i s tempting to speculate as to whether t h i s corresponds to p r o t e i n from the degradation of the native molecular forms. 123. Figures 25 and 26: Gel f i l t r a t i o n of a f f i n i t y p u r i f i e d AChE i n Sepharose 2B and 4B (1.5 x 8 0 cm). Figures 27-29: C a l i b r a t i o n of Sepharose 4B column with standard p r o t e i n s of known Re (see t e x t ) . FIGURE 25 SEPHAROSE 2B elution of AChE after affinity chromatography ( 1 • 5 x 80cm) 20mM Phosphate - 2mM Deca ( pH = 6 • 90) Load: 1-5ml, OD280 = 1-1 Q O 0 05--0-04--0-03--0 02--001-000. 10 20 30 40 50 60 70 FRACTION n (4ml) FIGURE 27 SEPHAROSE 4B - Column calibration FRACTION # (?mO FIGURE 28 CALIBRATION OF SEPHAROSE 4B COLUMN WITH STANDARD PROTEINS 60 70 80 90 100 110 120 1 30 140 150 160 170 18C ' FRACTION #Oml) CHAPTER 4 CHEMICAL' MODIFICATION STUDIES OF AFFINITY PURIFIED ACETYLCHOLINESTERASE 4.1 Introduction P r o t e i n chemists have long been i n t e r e s t e d i n a l t e r i n g the chemical, p h y s i c a l and b i o l o g i c a l p r o p e r t i e s of proteins by chemically changing t h e i r s t r u c t u r e . One of the f i r s t things that was discovered about proteins was how e a s i l y they were changed upon treatment with chemical reagents. The a p p l i c a t i o n of modern knowledge of proteins, new chemi-c a l reagents and s o p h i s t i c a t e d a n a l y t i c a l techniques has made chemical m o d i f i c a t i o n of proteins one of the most u s e f u l approaches to the study of many of t h e i r p r o p e r t i e s . The u t i l i t y of chemical m o d i f i c a t i o n i s g r e a t l y extended by i t s use i n conjunction with s o p h i s t i c a t e d p h y s i c a l methods l i k e X-ray cr y s t a l l o g r a p h y , N.M.R. and E.S.R. spectroscopy. In a s i m i l a r way r e s u l t s from many other procedures such as absorption spectra or pH t i t r a t i o n s may sometimes be better i n t e r p r e t e d i f chemical m o d i f i c a t i o n data are a v a i l a b l e . The chemical r e a c t i v i t y of proteins towards modifying reagents a r i s e s from the r e a c t i v i t y of the side-chains of the sulphur containing amino-acids (cysteine and methionine), the basic amino acids (aspartic 130. and glutamic acids) and the a c t i v a t e d aromatic amino acids (tyrosine and tryptophan). These side chains, with the exception of the side chain of tryptophan can f u n c t i o n as sulphur, nitrogen or oxygen nucleophiles i n a d d i t i o n and displacement r e a c t i o n s i n v o l v i n g various reagents; sulphur i s , i n general, more r e a c t i v e as a nucleophile than nitrogen and nitrogen more r e a c t i v e than oxygen. The hydroxy side chains of s e r i n e and threonine are on the whole so un-r e a c t i v e as not to be of use i n chemical m o d i f i c a t i o n but they sometimes lead to unwelcome side reactions during chemical m o d i f i c a t i o n of (more n u c l e o p h i l i c groups). Nucleo-p h i l e r e a c t i o n of amino-acid side chains are u s u a l l y strongly pH dependent. The r e a c t i v e species i s unprotonated (uncharged i n the case of basic amino acids and methionine; anioni c i n the case of cysteine, t y r o s i n e and the a c i d i c amino acids) and pH thus determines the amounts of the r e a c t i v e form that i s present. Appropriate c o n t r o l of pH can thus r e s t r i c t n u c l e o p h i l i c attack to one p a r t i c u l a r amino a c i d side chain. N u c l e o p h i l i c side-chains are often i n v e s t i g a t e d using a c y l a t i n g or a l k y l a t i n g reagents such as a c y l - and a l k y l -h a l i d e s or v i a a d d i t i o n across a double bond present i n the reagent. Some side chains a r e s u s c e p t i b l e to oxidation; those of cysteine and methionine are oxidised to c y s t i n e or c y s t e i c a c i d and methionine sulphoxide and tyrosine and 1 3 1 . t r y p t o p h a n a r e o x i d i s e d i n t h e r i n g s y s t e m . P h o t o o x i d a t i o n c a n a l s o b e u s e d t o m o d i f y t h i s k i n d o f r e s i d u e . T h e w o r k d e s c r i b e d i n t h i s c h a p t e r h a s i n v o l v e d i o d i n a -t i o n o f A C h E . T w o m e t h o d s h a v e b e e n u s e d , o n e e n z y m a t i c a n d t h e o t h e r e s t e r a t i c . T h e e n z y m a t i c m a k e s u s e o f t h e f a c t t h a t i n t h e p r e s e n c e o f h y d r o g e n p e r o x i d e , i o d i d e a n d a n u c l e o p h i l i c a c c e p t o r , l a c t o p e r o x i d a s e w i l l c a t a l y s e t h e f o r m a t i o n o f a c a r b o n h a l o g e n b o n d ( 1 3 3 , 1 3 4 ) . T n t h e e x p e r i m e n t s h e r e t h i s i s a p p l i c a b l e p r i m a r i l y t o t h e e x p o s e d t y r o s i n e r e s i d u e s o f A C h E . T h i s l a c t o -p e r o x i d a s e m e t h o d t h e n i s q u i t e g e n t l e , e m p l o y i n g l o w l e v e l s o f e n z y m e a n d s m a l l a m o u n t s o f w e a k o x i d i s i n g a g e n t s . I t i s s h o w n a l s o t o y i e l d p r o d u c t s w i t h a h i g h s p e c i f i c a c t i -v i t y ( 1 0 6 c p m / m g ) . T h e e s t e r a t i c m e t h o d o f B o l t o n a n d H u n t e r ( 1 3 5 ) h a s b e e n u s e d b y t h e s e a u t h o r s t o l a b e l p r o t e i n h o r m o n e s a n d i n v e s t i g a t e N H 2 I o d i n a t i o n o f t y r o s i n e 132. t h e i n t e r a c t i o n o f t h e s e h o r m o n e s w i t h s p e c i f i c a n t i s e r u m . T h e p r o c e d u r e i n v o l v e s r e a c t i n g t h e p r o t e i n u n d e r m i l d c o n -d i t i o n s w i t h t h e N - h y d r o x y s u c c i n i m i d e e s t e r o f 3-(4-h y d r o x y p h e n y l ) ' p r o p i o n i c a c i d (136) t h a t h a s b e e n p r e v i o u s l y 125 l a b e l l e d w i t h I. " ^ " " i - a C y l a t i n g a g e n t T h e e s t e r w i l l r e a c t w i t h f r e e a m i n o g r o u p s i n t h e 125 p r o t e i n a n d h e n c e t h e I i s i n t r o d u c e d i n t o a g r o u p o t h e r t h a n t h e t y r o s i n e r e s i d u e i n v o l v e d i n t h e e n z y m a t i c m e t h o d . H e n c e a n a l t e r n a t i v e i o d i n a t i o n m e t h o d b e c o m e s a v a i l a b l e . T h i s s e c t i o n o f t h e t h e s i s t h e n d e s c r i b e s t h e s e t w o m e t h o d s w h i c h h a v e b e e n d e v e l o p e d t o r a d i o a c t i v e l y l a b e l t h e p r o t e i n i n a n a t t e m p t t o f u r t h e r p r o b e i t s m o l e c u l a r a n d s u b u n i t c o m p o s i t i o n . R a d i o a c t i v e l y l a b e l l e d p r o t e i n 133. also provides an a l t e r n a t i v e method of d e t e c t i o n to the a c t i v i t y assay and t h i s could be u t i l i s e d f o r example i n the i s o e l e c t r i c focussing experiments described e a r l i e r . 4.2 Experimental - M a t e r i a l s and Methods Iod i n a t i o n techniques Several l i t e r a t u r e methods (137-139) on the enzymatic io.dination of membranes and proteins were modified so as 125 to s u c c e s s f u l l y incorporate l a b e l l e d I i nto bovine serum albumin and myoglobin to a high a c t i v i t y (10^ cmp/mg). In a t y p i c a l enzymatic experient with AChE, 10 y l of l a c t o -peroxidase (Sigma), (2 mg/ml) was added to 200 y l of the buffered AChE pH = 6.90 ( 0 D o o n = 0.80) i n the presence of 5 y l of Nal (10 _ 5M) and 10-100 yCi of c a r r i e r f r e e Na 1 2 5 I (New England Nuclear). The i o d i n a t i o n was then i n i t i a t e d by adding 10 y l of 0.03% hydrogen peroxide and allowed to continue f o r 30 minutes at room temperature before being quenched by the a d d i t i o n of 25 y l of sodium azide (0.025M). The samples were then d i a l y s e d overnight at 4°C against 125 20 mM phosphate (pH = 6.90) to remove the excess I. 125 The preparation of the iodinated ester, I - l a b e l l e d 3-(4-hydroxyphenyl) propionic a c i d N-hydroxysuccinimide es t e r was as described by Bolton and Hunter (135) with the 125 following m o d i f i c a t i o n s : only 10-100 yCi of Na I was 134 . used i n each preparation. A c e t o n i t r i l e was used as the solvent f o r the s t a r t i n g m a t e r i a l N-succinimidyl-3-(4-hydroxyphenyl) propionate and as a r e s u l t no dimethyl-formamide was needed f o r q u a n t i t a t i v e e x t r a c t i o n of the ester i n t o benzene. The small r e a c t i o n v e s s e l was s i l y l a t e d to ensure complete separation of the benzene/water l a y e r s . Two 300 y l a l i q u o t s of benzene were used i n the e x t r a c t i o n and these f r a c t i o n s were evaporated o f f i n a fume hood by blowing dry nitrogen gas over the s o l u t i o n . Complete re -moval of the benzene was achieved i n about f i v e minutes. The i o d i n a t e d m a t e r i a l was subsequently d r i e d under vacuum f o r 2-3 hours and used immediately f o r i o d i n a t i o n . The AChE, 200 y l buffered now at pH = 8.2 was added to the s o l i d 125 . . 1-containing a c y l a t i n g agent and mxxed by vortexing. The r e a c t i o n was allowed to continue at room temperature f o r 10-15 minutes and the pH was then adjusted to 7.2. The samples were then d i a l y s e d against 2 0 mM phosphate (pH 6.9 0) p r i o r to running on SDS g e l s . Iodinated AChE was r o u t i n e l y concentrated on CF-25 Amicon c e n t r i f l o membrane cones to a concentration of 1 mg/ml p r i o r to loading on to SDS g e l s . 125 SDS gels which contained I - r a d i o a c t i v e l y l a b e l l e d AChE were cut l a t e r a l l y i n 2 mm s l i c e s and incubated f o r 16 hours at room temperature with 1 ml of NCS t i s s u e s o l u -b i l i s e r p r i o r to the a d d i t i o n of 5 ml of dioxane based 135. s c i n t i l l a t i o n f l u i d . Samples were then counted on a Nuclear Chicago Mark II s c i n t i l l a t i o n counter and the r e s u l t s are reported as uncorrected counts per minute. Enzyme Assays AChE assays were performed at pH 8.0 using the method of Ellman et a l . (90). Lactoperoxidase a c t i v i t y was measured according to the Worthington Biochemical catalogue (Worthington Biochemical corporation, Freehold, N.J.) assay f o r horseradish p e r o x i -dase at pH 7.2 instead of 6.0 (139). 4.3 RESULTS AND DISCUSSION T h e g l o b u l a r l i s f o r m o f A C h E d e r i v e d f r o m t h e 18S a n d 14S m o l e c u l a r f o r m s w a s u s e d i n i t i a l l y i n t h e i o d i n a t i o n e x p e r i m e n t s . I n f a c t , c h a r a c t e r i s a t i o n o f t h e a f f i n i t y p u r i f i e d m a t e r i a l b y i s o k i n e t i c s u c r o s e g r a d i e n t s a n d SDS g e l e l e c t r o p h o r e s i s , a s s h o w n i n f i g u r e 3_0, d e m o n s t r a t e d t h a t t h e p r e p a r a t i o n s w e r e f r e e o f t h e 18S a n d 14S f o r m s o f t h e e n z y m e a n d t h a t p r o t e o l y t i c a n d / o r a u t o l y t i c c l e a v a g e o f t h e 80,000 m o l e c u l a r w e i g h t c a t a l y t i c s u b u n i t h a d o c c u r r e d y i e l d i n g a m a j o r c o m p o n e n t a t 55,000 m o l e c u l a r w e i g h t . T h e c o m p l e x i t y o f t h i s c a t a l y t i c s u b u n i t c o m p o s i t i o n h a s b e e n d i s c u s s e d e l s e w h e r e (111,112, 140) a n d t h e i o d i n a t i o n e x p e r i m e n t s d e s c r i b e d h e r e h a v e a t t e m p t e d t o p r o b e t h e s u b u n i t S t r u c t u r e f u r t h e r . T w o m e t h o d s h a v e b e e n u s e d t o c o v a l e n t l y l a b e l t h e 125 p r o t e i n w i t h r a d i o a c t i v e i o d i n e ( I ) ; t h e f i r s t b e i n g a n e n z y m a t i c i o d i n a t i o n u s i n g l a c t o p e r o x i d a s e w h o s e s p e c i -f i c i t y i s f o r e x p o s e d t y r o s i n e r e s i d u e s , a n d t h e s e c o n d m e t h o d b e i n g a n e s t e r a t i c m e t h o d d e s c r i b e d b y B o l t o n a n d 125 H u n t e r (135) u s i n g I l a b e l l e d 3 - ( 4 - h y d r o x y p h e n y l ) p r o p i o n i c a c i d N - h y d r o x y s u c c i n i m i d e e s t e r , w h o s e s i t e o f a c t i o n i s t h e e x p o s e d a m i n o g r o u p s o f l y s i n e a n d N - t e r m i n a l a m i n o a c i d s . T h e t w o m e t h o d s c o m p l e m e n t o n e a n o t h e r b y v i r t u e o f t h e i r d i f f e r e n t c h e m i c a l s p e c i f i c i t y . T h e u s e o f 137 . Figure 30: The molecular forms and subunit composition of a sample of AChE p u r i f i e d by a f f i n i t y chromatography from f r e s h e l e c t r o p l a x t i s s u e and stored at 4°C f o r 8 weeks i n 20 mM phosphate b u f f e r (pH = 6.90). I s o k i n e t i c sucrose gradients were done i n 0.5 M NaCl - 0.02 M phosphate b u f f e r (pH = 6.90) and were c a l i b r a t e d with standard proteins (see f i g . 16). Acrylamide g e l el e c t r o p h o r e s i s was done i n 10 cm, 5.6% gels i n the presence of SDS and d i t h i o t h r e i t o l . F I G U R E 3 0 Sucrose gradient profile of affinity purified A.Ch.E - after 8 weeks stored at 4° C 0.6 A ^ 0.5-O | 0.4H Q O "> a D LU 6 < 0.3H 0.2-0.H 0 2 11.6s o a a> a cn i o a a P\ c o 6 8 Vo l (ml) 12 S.D.S.gel with reduction 28 K 25 K DYE 00 1 139. an i ^ J I - a c y l a t i n g agent i s p a r t i c u l a r l y a t t r a c t i v e as the p r o t e i n i s never exposed d i r e c t l y to r a d i o a c t i v e i o d i n e . Neither of the procedures r e s u l t e d i n any l o s s of AChE a c t i v i t y . The r e s u l t s of SDS g e l e l e c t r o p h o r e s i s , with reduction, of the i o d i n a t e d AChE are summarised i n f i g u r e 31.. The g e l scans of the coomassie blue stained gels (figures 31. (c) and 31 (d)) emphasise that f o r both of the i o d i n a t i o n methods the c a t a l y t i c subunit c o n s i s t s of the three 55,000, 28, 000 and 25, 000 molecular weight components. Figures 3JL (a) and _3_1 (b) however demonstrate that these compounds behaved d i f f e r e n t l y towards the two i o d i n a t i o n techniques; s p e c i -f i c a l l y lactoperoxidase c a t a l y s e d i o d i n a t i o n r e s u l t e d i n greater than 90% of the l a b e l entering the low molecular weight 28,000 and 25,000 components while the e s t e r a t i c i o d i n a t i o n r e s u l t s i n a uniform d i s t r i b u t i o n of the co-valent l a b e l between a l l three subunit components. This i s emphasised i n Table V on page 141 which shows the r a t i o s 125 which are obtained when the I counts per mxnute i n each g e l band i s d i v i d e d by the corresponding absorbance at 550 nm of the g e l scan. This then, i s a normalised measure IOC , of the J - ^ - ' i incorporated i n t o each polypeptide per u n i t weight of p r o t e i n . I t should be noted that f i g u r e s 31, 32 and 33, 34 represent d i f f e r e n t sets of i o d i n a t i o n experi-ments and hence cannot be compared. From the data, however, 140. i t can r e a d i l y be seen how s p e c i f i c the lactoperoxidase, enzymatic i o d i n a t i o n method i s f o r the 28, 24K polypeptide components of the l i s form of AChE, with reduction (55 0 x 1 0 + 3 cpm/OD 5 5 Q). Con t r o l experiments i n which AChE i s exposed only to 125 I i n the absence of e i t h e r lactoperoxidase or a c y l a t i n g agent show a completely n o n - s p e c i f i c low l e v e l of i n c o r -125 p o r a t i o n of I thoughout the SDS g e l (less than 1000 cpm, t o t a l ) . Hence, i t appears that whereas a l l the components of the AChE subunit contain amino groups a v a i l a b l e f o r i o d i n a t i o n by the e s t e r a t i c method of Bolton and Hunter, only the low molecular weight components (28,000 and 25,000) possess the necessary number of exposed t y r o s i n e residues f o r e f f e c t i v e lactoperoxidase catalysed i o d i n a t i o n . Figure 32_ summarises the r e s u l t s obtained when the iodinated AChE samples are run on SDS gels without p r i o r r eduction with d i t h i o t h r e i t o l . The g e l scans (3_2 (c) and 32 (d)) show major bands at 80,000 molecular weight and greater than 100,000 molecular weight. The acrylamide g e l s were not c a l i b r a t e d under non-reducing conditions f o r components greater than 100,000 molecular weight but t h i s band has been observed by others and i s thought to be the dimeric s t r u c t u r e of subunits connected v i a i n t e r s u b u n i t d i s u l p h i d e bonds discussed elsewhere i n t h i s t h e s i s (page 102). 141. TABLE V: I o d i n a t i o n Ratios f o r Lactoperoxidase (enzymatic) and E s t e r i o d i n a t i o n (see text) Figure D e s c r i p t i o n Band Ratio (c.p.m/ODrl.n x 10 J) enzymatic ester 31 11S, with 55 K 25 24 reduction 28,25 K 550 58 32 US, without >100 K 154 23 reduction 80 K 120 28 33 18,14S with >100 K 31 70 reduction 80 K 27 26 55 K 7.5 22 28,25 K 77 33 34 18,14S without >100 K 48 29 reduction 8 0 K 10 142. The i o d i n a t i o n p r o f i l e s f i g u r e s 32 (a) and 32^  (b) mimic f o r both i o d i n a t i o n methods the p r o f i l e obtained by g e l scanning the coomassie blue stained g e l s ; the extent of the i o d i n a t i o n depending only on the amount of p r o t e i n present. Hence from non-reduced samples, no great d i f f e r e n c e s 125 m the I d i s t r i b u t i o n i n AChE are revealed (Table V). Further experiments of t h i s kind have been c a r r i e d out on samples of 18,14S AChE which show the f u l l spectrum of polypeptide components; 100,000, 80,000, 55,000 and 28,000 and 25,000 molecular weight, displayed upon reduction of the p r o t e i n . Figure 3_4 shows the r e s u l t s obtained when the sample i s not reduced; both enzymatic and e s t e r a t i c i o d i n a -t i o n techniques again g i v i n g r e s u l t s which resemble the g e l scans of the stained SDS g e l s . When these same samples are reduced with d i t h i o t h r e i t o l the SDS g e l saan shows the usual breakdown of the 8 0,000 polypeptide observed before, but also a band at greater than 100,000 molecular weight which accounts f o r a very small amount of p r o t e i n but i s always present i n f r e s h l y p u r i f i e d AChE samples. This band does not correspond to the Rf of the dimer of subunits but rather some other component which disappears as the enzyme i s degraded by p r o t e o l y s i s and/or a u t o l y s i s . I t i s not seen i n gels of the 11S form of AChE. This band does iodinate with both techniques but somewhat more a c t i v i t y i s observed with the e s t e r a t i c 14 3 . method. The r a t i o s f o r f i g u r e 3 3 shown i n Table V i n d i c a t e i n f a c t that t h i s band i o d i n a t e s more s p e c i f i c a l l y than the other polypeptide components of thes molecular forms (70 x 1 0 + 3 cpm/OD^.) . The other polypeptides provide r e s u l t s which confirm the previous experiments on the f u l l y degraded l i s form and i n t h i s context i t should be noticed that as before the 55,000 molecular weight band i s not l a b e l l e d to any great extent by the lactoperoxidase method of i o d i n a t i o n ; the i o d i n a t i o n r a t i o i s only one tenth of that f o r the 28,25K component (Table V). The r e s u l t s are summarised i n f i g u r e 33_ and the normalised r a t i o s f o r the polypeptides i s shown i n table V. In conclusion, i t has been demonstrated that AChE can 125 be chemically modified to c o v a l e n t l y incorporate I. Of p a r t i c u l a r i n t e r e s t i s the s e l e c t i v i t y shown by l a c t o p e r o x i -dase catalysed l a b e l l i n g towards the low molecular weight components of the subunit (141) . Also i t w i l l be i n t e r e s t i n g to i n v e s t i g a t e the i o d i n a t i o n of the 100K component which appears i n the g e l p r o f i l e of the native molecular forms but not the g l o b u l a r , 11S form of AChE. These i o d i n a t i o n methods can i n future, then be used to study the 'native' molecular forms of the solubilised„and membrane-bound enzyme. 144. Figure 31: SDS-acrylamide g e l e l e c t r o p h o r e s i s of the 11S form of AChE under reducing conditions, (a) and (b) : The d i s t r i b u t i o n of l ^ i a f t e r lactoperoxidase and e s t e r a t i c i o d i n a t i o n (see t e x t ) . (c) and (d) corresponding g e l scans at 550 nm from cathode ( l e f t ) to anode ( r i g h t ) . Figure 32: SDS-acrylamide g e l e l e c t r o p h o r e s i s of the l i s form of AChE without p r i o r reduction. (a), (b), (c) and (d) as above. Figure 33: SDS-acrylamide g e l e l e c t r o p h o r e s i s of the l i 14S forms of AChE under reducing conditions (a), (b), (c) and (d) as above. Figure 34: SDS-acrylamide g e l el e c t r o p h o r e s i s of the 18,14S forms of AChE without p r i o r reduction, (a), (b), (c), (d) as above. 145. FIGURE 31 enzymatic iodination' ester iodination i 1 1 1 1 1 r~-1 r 8 16 24 32 SLICE # "i 1 1 1 1 1 1 1 r DISTANCE (cm) 146 . FIGURE 32 enzymatic iodination ester iodination SLICE # c O LO LO CD O c o _Q i _ O CO Xi O 0 " c) 0 .6 - >I00K/ >IOOK - 80 K - I 0.4-H 8 0 K - r — T - i v i i T i UJ >-Q 1/ i i 0 . 2 -J i \i l i i UJ >-Q 8 8 DISTANCE (cm) FIGURE 33 ENZYMATIC ESTERATIC DISTANCE (cm) 148. FIGURE 34 ENZYMATIC ESTERATIC E d. o 24 32 >-Q i— i— i— i— i— i— i— i— i— 16 24 32 40 SLICE # 8 - i 2 DISTANCE (cm) 149 CHAPTER 5 Conclusions I t would seem important to end t h i s t h e s i s with a chapter concerned s o l e l y with a summary of the conclusions that can be drawn from the experimental work which has been pre-sented and discussed. Further i t would not be amiss to fu r t h e r appraise these r e s u l t s i n r e l a t i o n to what i s already known about the p r o t e i n a c e t y l c h o l i n e s t e r a s e . The i n i t i a l aim of the work described i n t h i s t h e s i s was to be able to i s o l a t e and p u r i f y the membrane-bound p r o t e i n a c e t y l c h o l i n e s t e r a s e . This has been s u c c e s s f u l l y achieved using a f f i n i t y chromatography which has r e s u l t e d i n a p u r i f i c a t i o n f a c t o r of greater than one hundred v i a a s i n g l e chromatographic step, thus i l l u s t r a t i n g the great u t i l i t y of the technique. I t has been p o s s i b l e to p u r i f y the enzyme to a very high s p e c i f i c a c t i v i t y . Though d i f f e r e n t workers use d i f f e r e n t assay, eitheft the ACh pH-stat t i t r a t i o n or the coupled a c e t y l t h i o c h o l i n e -DTNB assay , the r e s u l t s can be converted so as to be comparable with each other and t h i s i s shown i n the ta b l e below, (over page). In the l a s t two years exciting- developments have appeared i n the l i t e r a t u r e on AChE. Of p a r t i c u l a r s i g n i f i c a n c e i s 150. Workers Massoulie e t . a l . Dudai and Silman Wilson e t . a l . Taylor e t . a l . Rosenberry e t . a l . T h i s work Molecular form 18S,14S,9S 18S,14S U S 18S,14S US** U S 18S,14S Sp A c t i v i t y units/mg* 250 350 280 330 360 550 420 Ref. (101) (84) (80) (85) (103) (82) units/mg r e f e r s to mmoles a c e t y l c h o l i n e / t h i o c h o l i n e hydrolysed hour - mg ** This work i s on the i s o l a t i o n of AChE from Torpedo c a l i f o r n i c a some elegant e l e c t r o n microscopy (BUM.) work by Cartaud e t . a l . (73) which has l e d to the discovery that the 18S, 14S and 4S 'native' molecular forms of AChE a l l possess a long asymmetric structure 40-50 nm long, r e f e r r e d to i n the l i t e r a t u r e as a ' t a i l ' (en grappe). This structure does not possess any c a t a l y t i c a c t i v i t y but i t may be of great s t r u c t u r a l s i g n i f i c a n c e p a r t i c u l a r l y as AChE i s an e x t r i n s i c membrane-bound p r o t e i n . Recently f u r t h e r E.M. p i c t u r e s studying the f i n e structure of the 18S form of AChE have shown that e i t h e r the t a i l structure i s an a s s o c i a t i o n of three a - h e l i c e s or i t i s organised i n t o a c o l l a g e n - l i k e three stranded h e l i c a l conformation (142). C l e a r l y work towards understanding the molecular structure of t h i s t a i l i s very valuable and e x c i t i n g at present and i t i s p a r t l y to t h i s end that the radi o a c t i v e l a b e l l i n g studies have been undertaken, and discussed at the end of t h i s t h e s i s . 151. H i s t o r i c a l l y , much work has been d i r e c t e d towards p u r i f y i n g the globular l i s form of AChE as t h i s was the only form known to e x i s t . However, t h i s l i s form must now be looked upon as a degradation product r e s u l t i n g from pro-t e o l y t i c cleavage of the 'native' asymmetric molecular forms mentioned above. The work described i n t h i s t h e s i s has been d i r e c t e d towards p u r i f y i n g these native forms of AChE and has shown c l e a r l y that the a f f i n i t y technique w i l l i s o l a t e 18S and 14S AChE. The enzyme must i d e a l l y be i s o l a t e d from 'fresh' t i s s u e as even a f t e r p u r i f i c a t i o n i t i s shown to be s u s c e p t i b l e to p r o t e o l y t i c cleavage by an endogeneous protease. In view of t h i s observation i t would be an improvement i f the high s a l t e x t r a c t of the t i s s u e , which i s r i c h i n AChE a c t i v i t y , could be applied d i r e c t l y to the a f f i n i t y column so as to ensure a minimum of p r o t e o l y s i s taking place. Such a system has r e c e n t l y been described i n the l i t e r a t u r e (84) and involves the i n c o r p o r a t i o n of an a c r i d i n e d e r i v a t i v e (K^ = 0.3 yM) i n t o a l i g a n d attached to the r e s i n . This or a system l i k e i t would seem very worthwhile. Chapter three of the t h e s i s was concerned with the mole-c u l a r c h a r a c t e r i s a t i o n of the p r o t e i n and hopefully t h i s work has contributed to s e t t l i n g -some of the controversy concerning the quaternary structure and subunit composition of the enzyme, which has appeared i n the l i t e r a t u r e i n recent years (105, 109-113). In p a r t i c u l a r , published SDS g e l e l e c t r o p h o r e s i s patterns .have shown the presence of two d i s t i n c t bands at around 80,000 and 60,000 molecular weight. Thus AChE i n i t s 11S form with four subunits was assumed to be i n an « 2 6 2 a r r a n < 3 ' e m e n ' t * This work however has shown not only that f r e s h l y p u r i f i e d enzyme c o n s i s t s of the 18S and 14S asymmetric forms but that g e l e l e c t r o p h o r e s i s i n the presence of SDS and reducing agents confirms that the native forms of AChE c o n s i s t of only a s i n g l e subunit of molecular weight 80,000. Further, gels run i n the absence of reducing agents i n d i c a t e that two of these subunits are l i n k e d v i a int e r s u b u n i t d i s u l p h i d e bonds. Hence, the globular 11S form of AChE would be com-posed of four subunits arranged as a dimer of dimers, ( a 2 ) 2 . The 'native' 9S, 14S and 18S would then correspond to one, two and three groups of these c a t a l y t i c tetramers r e s p e c t i v e l y attached to the long asymmetric t a i l . A l so, by following the SDS g e l pattern of AChE as a fun c t i o n of time a f t e r p u r i f i c a t i o n i t has been shown here that the 8 0,000 subunit i s susceptible to p r o t e o l y t i c cleavage by an impurity which i s s t i l l present a f t e r a f f i n i t y chromatography. I t i s cleaved indeed to y i e l d a 55,0 00 component which has been shown v i a D.F.P. l a b e l l i n g 153. of the a c t i v e centre to contain the a c t i v e s i t e (109). As a r e s u l t of t h i s degradation two polypeptides of molecular weight 28,000 and 25,000 are generated as a fu n c t i o n of time. Hence i t has been p o s s i b l e to under-stand the confusing r e s u l t s that have been obtained by others f o r the subunit composition of the enzyme, where obviously d i f f e r e n t g e l patterns w i l l be obtained as the enzyme i s cleaved with time. The r e s u l t s f u r t h e r empha-s i s e the f a c t that the 11S form of AChE i s a degradation product of what i s o r i g i n a l l y i s o l a t e d from the membrane, as has been commented on by Massoulie et. a l . who have been i n t e r e s t e d i n studying the various molecular forms contained i n high s a l t e x t r a c t s of the t i s s u e and t h e i r i n t e r c o n v e r s i o n (72). The r e s u l t s on the degradation of the subunit of AChE complement three recent l i t e r a t u r e r eports on the subunit heterogeneity of the l i s form of the enzyme (111-113) by providing information as to the p r o t e o l y t i c degradation of the same p u r i f i e d AChE sample as a f u n c t i o n of time. I t i s important to r e a l i s e that the polypeptides which are formed by cleavage of the 80,000 subunit are not r e -leased from the molecule unless i t i s denatured and i n the case of the 28,000 and 25,000 components reduced as w e l l . The presence of two small polypeptides i s somewhat con-fu s i n g and may i n d i c a t e that there are two s i t e s of cleavage on the 80,000 subunit or that the 28,000 component i s i t s e l f s u s c e p t i b l e to breaking down. This l a t t e r idea i s presented i n the l i t e r a t u r e (113), but i t should be noted that i n t h i s work the 28,000 component i s never observed i n the absence of a 25,000 component even when degradation of the 80,000 subunit has j u s t begun. Another i n t e r e s t i n g p o s s i b i l i t y i s that one or other of these components a r i s e s from degradation not of the c a t a l y t i c subunit but of the long asymmetric ' t a i l ' . C e r t a i n l y i n gels of f r e s h l y p u r i f i e d enzyme a d i s t i n c t band at >100,000 i s observed to be present, which has been postulated to correspond to the t a i l (105), and t h i s band q u i c k l y d i s -sappears as the degradation with time begins to take place. I t may be p o s s i b l e then that these smaller polypeptide components do not a r i s e s o l e l y from cleavage of the cata-l y t i c subunit, and experiments with l a b e l l e d p r o t e i n may help to c l a r i f y t h i s f u r t h e r . Some i n s i g h t has been gained i n t o the mechanism of f i r s t , c o n v e r s i o n of the 'native' 18,14S forms i n t o a g l o b u l a r 11S form and second,the cleavage of the 80,000 subunit v i a the work that has been done using t r y p s i n to mimic these changes. Trypsin has been shown, depending on the length of i t s incubation with AChE and also i t s concentration to both cause conversion to the U S form 155. and also to r e s u l t i n a very s i m i l a r breakdown of the c a t a l y t i c subunit to that observed to occur with time... Further the r e s u l t s i n d i c a t e that l o s s of t a i l occurs at a f a s t e r r a t e than cleavage of the 80,000 subunit. I t i s p o s s i b l e then that these two events are l i n k e d i n some way. Dudai et. a l . (8 0) had p r e v i o u s l y shown that expo-sure of soluble e x t r a c t s of crude U S AChE to t r y p s i n r e -s u l t e d i n no s i g n i f i c a n t change i n the d i s t r i b u t i o n of the polypeptides i n the SDS g e l pattern. I t was t h i s evidence that caused d i r e c t p r o t e o l y t i c a c t i o n to be ignored as a p o s s i b i l i t y f o r understanding the breakdwon of the sub-u n i t s of AChE (112). Working with the p u r i f i e d enzyme i t has been p o s s i b l e to obtain the r e s u l t s mentioned above which h o p e f u l l y c l a r i f y t h i s issue (140). From the r e s u l t s which have been discussed here and i n the l i t e r a t u r e i t i s f e a s i b l e to propose a model f o r the quaternary s t r u c t u r e of AChE (111). Two simple models are summarised i n the diagram shown on the next page. Both models show p a i r s of subunits l i n k e d by d i sulphide bonds. Model one has the polypeptide fragments l i n k e d w i t h i n each subunit by d i s u l p h i d e bonds and the subunits arranged i n p a r a l l e l , (the s i t e of cleavage i s shown as a serrated l i n e ) . The r e s u l t s i n t h i s t h e s i s favour model two where the subunits are i n an a n t i p a r a l l e l arrangement • 6 | O CO TJ O 0 s 1 ' C O O H C i A A v> 1 "A V I vt -s-s-M if MW= 3 o x —1 1— • V T M 2 20-350,0 C O O H C O O H A A _ , v • v V _JP - s - s -NHj N H 2 00 n MW = M ft V Y c _s_s--s—s-O G H C A V T 3 XH M J 20-350.C N 4/ V, Y C ) C H ? C O O H A. -s-s •w -s-s-0 CO o CO ( it MW= C O O H c A A -s-s-~I65,00C 3 O H —i\ V ) MW = N I V Y C - S - S --s-s--I65.C c A V t M >0 O O H H 2 0 M [i V COOH ' 80,000 8 D JC 1 D . O O «> E i * tn a CO + C O O H C O O H M S - A ( V s " H S - " S H - S H H S " w w f*+2 N H 2 MW= ~80,000;~60POO;~25,000 C O O H C O O H H S - H S - 4 H S - - S H Ifl It1 MW= — 80,000 i~60,000;~ 25,000 Schematic model f o r l i s form of AChE and there i s no i n t r a s u b u n i t d i s u l p h i d e bonds. This model would p r e d i c t the appearance of an 80,000 species without any p r i o r reduction of the sample and t h i s i s indeed ob-served to be the case f o r samples that have undergone some subunit p r o t e o l y t i c cleavage upon storage a f t e r p u r i f i c a -t i o n . Both models account f o r the appearance of the 8 0,000, 55,000 and smaller polypeptides around 25,000 appearing when cleaved samples are denatured and reduced. The models make no comment on how the t a i l and c a t a l y t i c subunits can i n t e r a c t but although t h i s problem i s l a r g e l y uninvestigated there i s some evidence from e l e c t r o n micro-scopy (142) that each tetramer i s l i n k e d to i n d i v i d u a l 157. filaments which are a - h e l i c a l and themselves i n t e r a c t or int e r t w i n e to form the t a i l s t r u c t u r e . I t i s p o s s i b l e then that some covalent attachment of these filaments to the tetrameric subunits e x i s t s and that t h i s can be cleaved upon p r o t e o l y s i s , to r e s u l t eventually i n complete l o s s of the t a i l and conversion of AChE i n t o a globular U S form. The mechanism f o r t h i s conversion should be studied f u r t h e r as i t may have important consequences i n the way AChE can i n t e r a c t with i t s membrane environment. I t would be i n t e r e s t i n g to see i f the U S enzyme w i l l r e a s s o c i a t e with a model membrane and also i f phospholipids and/or other components of the membrane w i l l s t a b i l i s e the degra-da t i o n of the native forms. I t w i l l a l so be of i n t e r e s t to t r y and i s o l a t e the t a i l s t r u c t u r e so that i t s molecular s t r u c t u r e and composition can be studied. I t w i l l be informative to discover i t s amino a c i d composition, i t s i s o e l e c t r i c point and using o p t i c a l r o t a r y d i s p e r s i o n and c i r c u l a r dichroism study i t s secondary s t r u c t u r e . Any fu r t h e r informatin concerning t h i s s tructure w i l l a i d i n understanding how the p r o t e i n i n t e r a c t s with the membrane. However as the t a i l has no enzymatic a c t i v i t y i n i t s own r i g h t i t i s important to t r y and l a b e l i t i n some way. In the l a s t s e c t i o n of t h i s t h e s i s , then, some chemi-c a l modifications of AChE have been described to r a d i o a c t i v e l y 158 l a b e l the p r o t e i n which has been p u r i f i e d by a f f i n i t y chroma tography. This work i s only p r e l i m i n a r y but i t i s hoped that i t w i l l f u r t h e r probe the molecular and subunit composition and a l s o provide an a l t e r n a t i v e method f o r d e t e c t i o n of the p r o t e i n . L a b e l l e d p r o t e i n could help to c l a r i f y some of the i s o e l e c t r i c focussing r e s u l t s and a l s o be used i n any f u r t h e r g e l f i l t r a t i o n of the enzyme which i s c a r r i e d out. Two i o d i n a t i o n methods have been used s u c c e s s f u l l y ; the one an enzymatic method u t i l i s i n g lactoperoxidase and the other applying an iodinated hydroxysuccinimide ester that has been used to l a b e l p r o t e i n hormones (135). These two methods, provide d i f f e r e n t i a l l a b e l l i n g of the enzyme. Of p a r t i c u l a r s i g n i f i c a n c e amongst the r e s u l t s that have been discussed i s that whereas a l l the poly-peptide components contain amino groups a v a i l a b l e f o r i o d i n a t i o n only the low molecular weight components (28,000 and 25,000) which are postulated to be derived from cleavage of the native 80,000 subunit possess the necessary number of exposed tyrosine residues f o r e f f e c t i v e l a c t o -peroxidase catalysed i o d i n a t i o n . I t i s also i n t e r e s t i n g and e x c i t i n g f o r future work that the f a i n t band at greater than 100,000 molecular weight thought to cor r e s -pond to the t a i l of the 18S,14S forms of the enzyme (105) has been shown to be l a b e l l e d by both i o d i n a t i o n techniques 159. but p a r t i c u l a r l y w e l l by the e s t e r a t i c method of Bolton and Hunter (table 5). These r e s u l t s can now be a p p l i e d to study both the s o l u b i l i s e d and membrane bound forms of the enzyme. I t w i l l be i n t e r e s t i n g to attempt to i o d i n a t e the p r o t e i n p r i o r to e x t r a c t i o n from the membrane and compare the r e s u l t s with those that have been described here.' This w i l l h o p e f u l l y provide i n t e r e s t i n g information concerning the extent to which AChE may be considered to be an ' e x t r i n s i c ' membrane p r o t e i n . With a view to membrane work, experiments w i l l undoubtedly be c a r r i e d out on the r e i n c o r p o r a t i o n of t h i s enzyme i n t o model phospholipid systems that are simple enough to be studied by gross p h y s i c a l techniques. I t would be i n t e r -e s t i n g to discover to what degree the presence of the long asymmetric t a i l determines the success of any such r e -i n c o r p o r a t i o n . In t h i s context i t i s important to be aware of the work of Changeux et. a l . (143) i n which innervated and non-innervated microsacs are prepared. This l i t e r a t u r e i n d i c a t e s that the b e a u t i f u l s t r u c t u r a l o r g a n i s a t i o n of these e l e c t r o p l a x c e l l s i s such that closed v e s i c l e s can be s e l e c t i v e l y prepared to contain e i t h e r receptor and AChE proteins or the Na +, K + a c t i v a t e d ATP'ase. In p a r t i c u l a r these v e s i c l e s are i d e a l model systems f o r l a b e l l i n g of the AChE i n s i t u with the tech-niques described here. A study of these systems, w i l l 160. surel y help to extend the knowledge and information about the membrane environment of AChE and probe i t s r o l e i n the transmission of the nerve impulse. 161. References (1) J.M. S t e i n i n : Molecular A s s o c i a t i o n s i n B i o l o g i c a l and Related Systems. Adv. i n Chem. 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Clark, FEBS L e t t s , (in p r e s s ) . (142) J . Cartaud, F. Rieger, S. Bon, J . Massoulie, Brain Research 88, 127-130" (1975). (143) M. Kasai and J.P. Changeux, J . Membrane. B i o l . 6_, 1-23 (1971). 17 APPENDIX THE CHARACTERISATION OF THE MOLECULAR FORMS OF AChE This appendix i s included to explain and expand upon some of the work discussed i n the t h e s i s , concerning the p h y s i c a l p r o p e r t i e s of AChE extracted from the e l e c t r o p l a x membrane. The background to the work done with sucrose gradients to c h a r a c t e r i s e the molecular forms of the enzyme i n terms of t h e i r sedimentation c o e f f i c i e n t s w i l l be ex-pl a i n e d f u r t h e r . In p a r t i c u l a r , the preparation of i s o -k i n e t i c gradients v i a the method of N o l l (1) w i l l be o u t l i n e d . The information w i l l be u s e f u l f o r other workers entering t h i s f i e l d . Sedimentation c o e f f i c i e n t s I f we consider a macromolecule moving down a ce n t r i f u g e tube, then f o r sedimentation to occur there must be a density d i f f e r e n c e between the p a r t i c l e and the solvent. The e f f e c t i v e mass of the p a r t i c l e i s the true mass (m) l e s s the mass of the solvent displaced m e f f ~~ m m s o l v e n t displaced This may be rewr i t t e n as, m „ = m-mvp = m(l-vp) ef f 173. where p i s the density of the solvent and v i s the p a r t i a l s p e c i f i c volume. v i s the increase i n volume that occurs as a r e s u l t of the a d d i t i o n of one kilogram of the macro-molecule to an i n f i n i t e volume of water. In a c e n t r i f u g a l f i e l d the a c c e l e r a t i o n applied to the 2 molecule i s = co x Therefore the force = m x a c c e l e r a t i o n ef f _ 2 = m (1-vp ) co x However as the p a r t i c l e sediments, f r i c t i o n opposes the motion down the tube. This f r i c t i o n a l force i s propor-t i o n a l to the molecule's v e l o c i t y F = where f i s a constant c a l l e d the f r i c t i o n a l c o e f f i c i e n t . I t i s r e l a t e d to the d i f f u s i o n c o e f f i c i e n t (D) by where k i s the Boltzmann constant and T i s the temperature (°K) . In an i s o k i n e t i c gradient the two opposing forces be-come equal so that the p a r t i c l e moves at a uniform speed through the medium. ,-, - \ 2 kT dx Then, m(l-vp)co x = • 174. If.'.we m u l t i p l y both sides by the Avogadro number (N) mN = M, the molecular weight and kN = R (gas constant) then the equation becomes M = ^ J L . 1 ) (l-vp)D d t' " X dx 1 This i s the Svedberg equation and (^- . ^2 X) J-s t n e r a t e of sedimentation i n a u n i t f i e l d . This i s what i s r e f e r r e d to as the sedimentation c o e f f i c i e n t (S) and i t has the u n i t s of seconds. However since i t u s u a l l y has a value -13 i n the region of 10 s e c , the Svedberg u n i t (S) was -13 defined as S = 1 x 10 sec. Thus the equation may be rew r i t t e n as RT M = (1-vp) ' <§> The sedimentation c o e f f i c i e n t i s often expressed as an S „ A where the sub s c r i p t s 20 and u r e f e r to the measure-20, to ment being corrected to 20°C and a water medium. I s o k i n e t i c sucrose gradients f o r Beckmann r o t o r , SW4lTi In reference (1) the t h e o r e t i c a l equation (5) reduces to nm(r) = mr (D - D m(r) ) nm(r) = v i s c o s i t y , f ( r ) D (r) = density of medium m J 175. where m = n t / r t ( D -Dt) T) - v i s c o s i t y at the top of the tube I) = density at top of tube r t = radius of ro t o r to top of tube Hence choosing a concentration C^, of sucrose at the top of the gradient = 10% sucrose Dp = 1.343 gms/cc 2 Cross S e c t i o n a l Area of tube = 1.605 cm Given the concentration of sucrose, the v i s c o s i t y and density are obtained from t a b l e (3) T, m(r) = mr (D p - Dm<r)> m 711 2.073 7.00(1.343-1.041) 0.982 C = 10% n = 2.073 r t - 7.00 D = 1.343 P D^ = 1.041 t Eqt— becomes n (r) = 0.982 (1.343-Dm(r)) • r (1) m 176. Hence, by choosing various sucrose concentrations, equation (1) can be used to generate the distance down the gradient at which t h i s sucrose concentration w i l l appear. Thus the data below can be c a l c u l a t e d and p l o t t e d as shown i n f i g u r e 35 to dep i c t the shape of the t h e o r e t i c a l gradient. % sucrose n (r) m D (r) m r (cm) r-r^(cm) Vol(ml) Vol from eqtE (2) 10 2. 073 1. 041 7.00 0.00 0 0 11 2.150 1. 045 7.44 0.44 0.71 0.60 13 2.319 1. 053 8.14 1.14 1.83 1.97 15 2.513 1. 062 9.20 2.20 3.54 3.51 17 2.736 1. 071 10.25 3.25 5.23 5.23 19 2. 992 1. 080 11. 60 4.60 7.40 7.09 21 3.290 1. 089 13 .20 6.20 9.96 9.86 23 3.636 1.098 15.1 8.10 13.00 13.00 A constant v e l o c i t y gradient such as that shown i n f i g u r e 3 5 can be generate such that the sucrose concentra-t i o n C increases as a function of the volume V according V to i r ~ \ \ -V/Vm , 0 S C = C - (C - C )e ' (2) v R R t Choosing two points from the table above, eqt- (2) can be solved f o r V and Q.-, the r e s e r v o i r concentration and the m R 177. mixing volume (ref. 1), and t h i s eqt- then becomes V = 11.7 loge ( 2 9 * 3 _ C v ) C = 29.3% 19.3 V = 11.7 ml m and C"v versus V can be p l o t t e d . This data i s also p l o t t e d i n f i g u r e 35. In order to substantiate the v a l i d i t y of the method of producing i s o k i n e t i c gradients, the gradients were pre-pared using the parameter C T T and V discussed above i n V m s e v e r a l d i f f e r e n t b u f f e r s , spun i n the c e n t r i f u g e and then f r a c t i o n a t e d . The r e f r a a t i v e index of each f r a c t i o n was then measured i n an Abbe refractometer as t h i s i s d i r e c t l y r e l a t e d to the concentration of sucrose. The r e s u l t s f o r a t y p i c a l experiment are shown i n f i g u r e 3 5 and compare well with the t h e o r e t i c a l gradient. Perhaps a more c r u c i a l t e s t of the gradients i s obtained when three standard p r o t e i n s , 3 g a l a c t (15.9S), catalase (11.4S) and a l c o h o l dehydrogenase (7.6S) are centrifuged i n a gradient and t h e i r d i s t r i b u t i o n assayed f o r . The r e s u l t s f o r t h i s were shown i n f i g u r e 16 and the s t r a i g h t l i n e obtained from p l o t t i n g sedimentation c o e f f i c i e n t aqainst volume i s i n d i c a t i v e of an i s o k i n e t i c gradient. 178. Figure 35Shape of isokinetic gradient 1 1 1 1 1 r — i r i i i I 5 10 15 Gradient volume (ml) 179. F igure36Grad ients in bu f fe rs , Isokinetic 25 CD \_ o C L C L a »«—* V o 20-0 ^ c O — o CD O a o 15-o CD CO O V X.— O 13 CO 10-Gradient vol (ml) 180. Appendix references: (1) H. N o l l , Nature, 215, 360 (1967). (2) Campbell and Sargent i n Techniques i n P r o t e i n Bio-synthesis, V o l . 2. (3) C.R.C. Handbook, J288. 

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