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Studies on acetylcholinesterase and cell wall proteins in Phaseolus vulgaris L. Mansfield, Donald Holmes 1977

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STUDIES ON ACETYLCHOLINESTERASE AND CELL WALL PROTEINS IN PHASEOLUS VULGARIS L. by DONALD HOLMES MANSFIELD B.A., Colorado College, 1973 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (Botany Department) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 © Donald Holmes Mansfield, 1977 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 fo r an advanced d e g r e e at t h e U n i v e r s i t y o f B r i t i s h Columbia, I agree that t h e 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 a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Depar tment o f T > 0 1 A^ / The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date September 15, 1977 ABSTRACT Acetylcholinesterase (AchE) activity r was identified in roots and hypocotyls of etiolated Phaseolus vulgaris L. by means of a colorimetric assay which included the cholinesterase inhibitor neostigmine as a control. An inhibitor of this activity was observed in tissue homogenates but was removed by dialysis. Greater than 95% of the activity in the hypocotyl was localized in the c e l l walls. The enzyme was extracted from the buffer-insoluble residue of roots with 5% (NHp2 S 04 a n d purified by (NH^^SO^ precipitation, gel f i l t r a t i o n on Sepharose 6B and chromatography on N-methylacridinium-Sepharose 4B. Purified preparations had a specific activity of 210 ± 20 units -mg—L_protein and contained one active protein and one major inactive protein as determined by polyaerylamide gel electrophoresis and thin layer isoelectric focusing. The AchE had at least 3-fold greater activity against acetylthiocholine than,butyl- or propionylthiocholine. The of AchE for acetylthiocholine was 56 uM. The enzyme was stimulated by choline (0.5-50 mMO and totally inhibited by -4 diisopropylf luorophosphate (DIFP., 10 M) and decamethonium (60 mM.) . The catalytic center activity determined by DIFP ti t r a t i o n was 197 ± 5 mol substrate min ^ mol ^ active center. The isoelectric point of AchE was 5.3 ± 0.1, the sedimentation coefficient (S„_ ) was 4.2 ± 0.1 S, zu ,w and the Stokes radius was 4.00 nm. The mol. wt. calculated from sedimentation and gel f i l t r a t i o n data was 76 000 ± 2 000. The mol. wt. determined by SDS-gel electrophoresis was 77 000 ± 2 000. Subunit mol. wts. of 61 000 ± 2 000 (2 x 30 000) and 26 000 ± 2 000 were observed. The enzyme had a f r i c t i o n a l ratio (f/fo) of 1.37. A theoretical model i i of the quaternary structure of AchE was presented. M u l t i p l e forms of AchE a c t i v i t y were observed following g e l f i l t r a t i o n i n low i o n i c strength and ion exchange chromatography of preparations having low s p e c i f i c a c t i v i t y . I t was suggested that an i o n i c strength dependent equilibrium existed between aggregates of the 77 000 mol. wt. species. Properties of the bean root AchE were compared with the AchEs from e e l and other animal t i s s u e s . Though large differences existed, i n 'catalytic" cent er z'a'bt i v i t i e s V substrate 'hydrolysis rates, and behavior on N-methylacridinium-Sepharose 4B, the AchE from the d i f f e r e n t sources wereosimilariin^manyrespects. The s p e c i f i c a c t i v i t y of hypocotyl hook AchE was unaffected by exposure of e t i o l a t e d seedlings to l i g h t or an ethylene source f o r 3 days. _3 The s p e c i f i c a c t i v i t y of hook AchE increased a f t e r 3 days i n 10 M--4 g i b b e r e l l i n - t r e a t e d plants and decreased a f t e r 4 days i n 10 M-kinetin-treated plants. These r e s u l t s were interpreted i n terms of a possible r o l e of AchE i n hypocotyl aging. In a second study, walls of e t i o l a t e d P_. v u l g a r i s hypocotyls were 32 treated with P-DIFP under conditions which corrected for adsorption of the radioisotope, to determine the number of n u c l e o p h i l i c s i t e s i n c e l l walls from regions having d i f f e r e n t elongation rates. Alpha-chymotrypsin and serine were treated s i m i l a r l y to e s t a b l i s h optimum 32 conditions f o r diisopropylphosphorylation. The P-phosphoserine content of p a r t i a l l y hydrolyzed c e l l walls was determined. C e l l walls from 32 regions of act i v e c e l l elongation contained 2.71 pmol P-phosphoserine mg * c e l l w a l l and those from regions i n which c e l l elongation had i i i terminated contained no s i g n i f i c a n t P-phosphoserine. From these re s u l t s I concluded that the few n u c l e o p h i l i c s i t e s present' were, not gl y c o s y l a t i o n s i t e s i n the c e l l w a l l but rather were s i t e s i n active centers of enzymes which were bound to c e l l w a l l preparations. iv V TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES - CHAPTER I . . . . . . . . . . . . . . . . . . . . i x LIST OF FIGURES - CHAPTER I . . . . . . . . x LIST OF TABLES - CHAPTER II • x i i i LIST OF FIGURES - CHAPTER I I x i v ACKNOWLEDGEMENTS xv CHAPTER I. STUDIES ON THE ACETYLCHOLINESTERASE OF 'RHASEOLUS VULGARIS L . . 1 • A. INTRODUCTION . . . 1 B. MATERIALS AND METHODS 6 1. Chemicals . . 6 2. Plant M a t e r i a l 7 3. Assay of Acetylcholinesterase (AchE) 7 A c t i v i t y • 4. Protein Determination 10 5. P u r i f i c a t i o n of AchE from P. v u l g a r i s . . . . 10 a) E x t r a c t i o n and (NH^^SO^ P r e c i p i t a t i o n . . 10 b) Gel F i l t r a t i o n on Sepharose 6B 11 c) Chromatography on MAC-Sepharose 4B . . . . 11 6. L a b e l l i n g With DIFP 12 7. Determination of R a d i o a c t i v i t y . . . . . . . . 13 8. Polyacrylamide Disc Gel Electrophoresis . . . 13 9. SDS Gel Electrophoresis 14 10. Sedimentation i n Is o k i n e t i c Sucrose Gradients . . . . . . . . . . . . . . . . . 15 Chapter I. Page 11. I s o e l e c t r i c Focusing . . . . . . 15 12. Determination of Stokes Radius and Molecular Weight . . . . • 17 13. Ion Exchange Chromatography on DEAE-Sepharose CL-6B 18 14. C e l l Wall E x t r a c t i o n 19 15. Growth Regulator Experiments 20 16. Hypocotyl Hook Angle Measurements 20 17. Elongation Measurements 21 C. RESULTS . . . . . . . . 21 1. The Assay Methods 21 a) The e f f e c t of neostigmine on AchE a c t i v i t y , . . 21 b) The e f f e c t of assay time on AchE a c t i v i t y . . 23 c) The e f f e c t of enzyme quantity on AchE a c t i v i t y . . . . . . . 23 2. Ex t r a c t i o n and (NH,) 2S0, P r e c i p i t a t i o n of AchE . . . . . . . . . . . . 23 3.- L o c a l i z a t i o n of AchE i n P_. v u l g a r i s 27 a) AchE a c t i v i t y i n excised pieces of root and hypocotyl- 27 b) A c t i v i t y of AchE i n the c e l l w a l l . . . . 30 4. P u r i f i c a t i o n of Root AchE 30 5. Characterization of P u r i f i e d AchE 34 a) DIFP Labeling 34 b) Disc Gel Electrophoresis . . . . . . . . 34 c) . SDS Gel Electrophoresis ,. . 38 v i Chapter I. Page d) Sedimentation i n i s o k i n e t i c gradients . . . . 42 e) I s o e l e c t r i c focusing . 42 f ) Stokes radius and molecular weight 47 g) Substrate a f f i n i t i e s and the e f f e c t of various substances on AchE a c t i v i t y . . . . 47 6. Behavior of Low S p e c i f i c A c t i v i t y AchE on Chromatographic Media 58 a) Chromatography on MAC-Sepharose 4B 58 b) Gel f i l t r a t i o n on Sepharose 6B 68 c) Ion exchange chromatography 68 7. P h y s i o l o g i c a l Role of AchE i n the Hypocotyl . . . 71 a) E f f e c t of growth regulators on AchE a c t i v i t y i n the hypocotyl hooks 71 b) The e f f e c t of acetylcholine on the hypocotyl . . . . . . . . 71 D. DISCUSSION 75 1. Identity of AchE i n P_. v u l g a r i s 75 2. Extraction and L o c a l i z a t i o n of AchE 77 3. P u r i f i c a t i o n and Characterization 80 4. P h y s i o l o g i c a l role of the A c e t y l c h o l i n e / Acetylcholinesterase System . . . 95 E. SUMMARY 97 BIBLIOGRAPHY - CHAPTER I . . . • 101 CHAPTER I I . THE ABSENCE OF NUCLEOPHILIC SITES IN THE CELL WALLS OF ETIOLATED PHASEOLUS VULGARIS L. HYP0C0TYLS AND ITS RELATION TO CELL ELONGATION 110 A. INTRODUCTION ' . 1 1 0 B. MATERIALS AND METHODS • 114 1. Chemicals 114 v i i Chapter I I . Page 2. Plant M a t e r i a l 115 3. C e l l Wall Extraction . U5 4. Reaction of Serine with DIFP 117 5. Reaction of Alpha-chymotrypsin with DIFP . . . . . 117 6. Reaction of C e l l Walls with DIFP 1 1 8 7. Recovery of Phosphoserine . . . . . . . . . . . . . H 8 8. Determination of R a d i o a c t i v i t y . . . . 120 9. Calculations . . . . . 120 10. Protein Determination . . . . . . . . 1 2 1 11. Assay of Alpha-chymotrypsin A c t i v i t y . . . . . . . 121 12. Spin Labeling 121 C. RESULTS 122 1. M o d i f i c a t i o n of Serine with DIFP 122 2. Mod i f i c a t i o n of Alpha-chymotrypsin with DIFP . . . 124 32 3. • Phosphoserine Recovery from P-DIP-S-chymotrypsin . . . . . . . . 127 4. M o d i f i c a t i o n of C e l l Walls with DIFP 127 5. Phosphoserine Recovery from C e l l Walls . . . . . . 130 6. Spin Labeling 130 D. DISCUSSION .133 BIBLIOGRAPHY - CHAPTER II 141 v i i i i x LIST OF TABLES CHAPTER I Table Page I. The e f f e c t of d i a l y s i s and the rein t r o d u c t i o n of the di f f u s a t e to the non-dialyzable homogenate on the AchE a c t i v i t y i n ]?. v u l g a r i s root and hypocotyl 26 I I ; Recovery of AchE from P_. v u l g a r i s roots and hypocotyls extracted and fr a c t i o n a t e d with (NH 4) 2S0 4 , . 28 I I I . A c t i v i t y of AchE i n excised segments of roots and regions of the hypocotyls of P_. v u l g a r i s shown i n Figure 1 . . . . . . . . . . . 29 IV. P u r i f i c a t i o n of AchE from P. v u l g a r i s roots . . . . . . . . 33 3 V. Incorporation of H-DIFP into AchE p u r i f i e d by chromatography on MAC-Sepharose 4B 37 VI. Substrate s p e c i f i c i t y of AchE prepared by chromatography on MAC-Sepharose 4B for three choline esters . . . 52 VII. Recovery of AchE a c t i v i t y from MAC-Sepharose 4B having a ligand concentration of 0.4 pmol ml . . . . . . . . . 60 VIII. Recovery of AchE a c t i v i t y i n successive b u f f e r , decamethonium , and.NaCl .gradient eluates as a; function of bed volume of ,:MAC-Sepharose 4B 66 IX. Summary of ph y s i c a l properties of P_. v u l g a r i s AchE . . . . 99 X LIST OF FIGURES CHAPTER I Figure Page 1. Diagram of an e t i o l a t e d bean seedling showing the regions used i n d e t a i l e d studies of enzyme . . . . . d i s t r i b u t i o n 8 2. The e f f e c t of neostigmine on AchE a c t i v i t y 22 3. Neostigmine-inhibitable hydrolysis of AchE as a function of time . ' 24 4. The e f f e c t of enzyme quantity on the AchE a c t i v i t y . . . . 25 5. E l u t i o n p r o f i l e obtained a f t e r gel f i l t r a t i o n of AchE on Sepharose 6B • •• . . 31 6. E l u t i o n p r o f i l e obtained a f t e r chromatography of AchE on MAC-Sepharose 4B . . . . . . . 32 7. The e f f e c t of DIFP on the AchE a c t i v i t y p u r i f i e d by chromatography on MAC-Sepharose 4B . . . . . 35 8. Polyacrylamide gel electrophoresis of AchE . . 36 9. Mo b i l i t y of standard proteins r e l a t i v e to the tracking dye i n polyacrylamide gels containing 1% SDS . . . . . . 39 10. D i s t r i b u t i o n of protein and t r i t i u m a f t e r SDS-acrylamide gel electrophoresis of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and labeled with H-DIFP 40 11. D i s t r i b u t i o n of protein and t r i t i u m a f t e r SDS-acrylamide g e l electrophoresis of reduced AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and labeled with H-DIFP • 41 12. E l u t i o n p r o f i l e s obtained a f t e r i s o k i n e t i c sedimentation of standard proteins and AchE • 43 13. AchE a c t i v i t y and pH gradient a f t e r t h i n layer - . J i s o e l e c t r i c focusing of enzyme p u r i f i e d by chromatography on MAC-Sepharose 4B >. . . 46 Figure Page 14. E l u t i o n p r o f i l e a f t e r column i s o e l e c t r i c focusing of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B . . . 48 15. Stokes radius c a l i b r a t i o n curve . . . . . . . . . . . . . . 49 16. Globular protein molecular weight c a l i b r a t i o n curve . . . . 50 17. The e f f e c t of substrate concentration on AchE a c t i v i t y of MACjSepharose 4B-purified preparations . 51 18. A Lineweaver-B'urke plot for- a c e t y l t h i o c h o l i n e 53 19. The e f f e c t of choline on MAC-Sepharose 4B-purified AchE . . 55 20. The e f f e c t of decamethonium on. AchE a c t i v i t y . . . . . . . 5 6 21. The e f f e c t of NaCl on AchE a c t i v i t y 57 22. The e f f e c t of (NH^SO^ on AchE a c t i v i t y . . 59 23. AchE a c t i v i t y recovered from four MAC-Sepharose 4B columns of d i f f e r i n g ligand concentration . 61 24. Recovery of AchE from MAC-Sepharose 4B columns eq u i l i b r a t e d at d i f f e r e n t i o n i c strengths. . . . . . . . 63 25. Recovery of AchE from MAC-Sepharose 4B columns by e l u t i o n with a NaCl gradient, a c e t y l c h o l i n e , or a decamethonium gradient . . . . . . . . . . . . . . . . . 64 26. Recovery of AchE a c t i v i t y following delayed NaCl e l u t i o n on MAC-Sepharose 4B 67 27. E l u t i o n of AchE, prepared by method B, from Sepharose CL-6B 69 28. E l u t i o n p r o f i l e of AchE eluted from DEAE-Sepharose CL-6B ••. 70 29. The e f f e c t s of l i g h t and e t h r e l on s p e c i f i c a c t i v i t y of AchE i n hypocotyl hooks of 5-day old e l i o l a t e d P_. v u l g a r i s . 72 30. The e f f e c t s of g i b b e r e l l i n and k i n e t i n on s p e c i f i c a c t i v i t y of AchE i n hypocotyl hooks of 5-day old e t i o l a t e d P. v u l g a r i s . . 73 31. The e f f e c t of acetyl c h o l i n e on the hook angle of 20 h excised hypocotyl hooks of P_. v u l g a r i s 74 x i Figure Page 32. Postulated structure of P_. v u l g a r i s AchE'. . . . . . . . . 90a 33. Schematic model for the 11 S form of e e l AchE from Dudai and Silman (1974) . . 92 x i i x i i i LIST OF TABLES CHAPTER I I Table Page i I. Phosphoserine recovered following the reaction between serine and DIFP and s e r i a l hydrolysis with 2, 3, or 6 M-HCl 123 I I . A c t i v i t y of native and DIFP-inhibited a-chymotrypsin measured by hydrolysis of the synthetic substrate, N-acetyl tyrosine e t h y l ester . . . 125 32 I I I . Recovery of P a c t i v i t y from DIFP pretreated and non-pretreated P-DIP-a-chymotrypsin 126 32 IV. Recovery of P from electrophoregrams a f t e r s e r i a l h ydrolysis of DIP-a-chymotrypsin. . . 128 32 V. Phosphorus-32 recovery from P-DIFP treated c e l l walls i s o l a t e d from e n t i r e hypocotyls and regions of hypocotyls of e t i o l a t e d _P. v u l g a r i s shown i n Figure 1 . . 129 32 • VI. Phosphorus-32-phosphoserine recovered from P-DIFP treated c e l l walls i s o l a t e d from regions of hypocotyls of e l i o l a t e d P_. v u l g a r i s shown i n Figure 1 . . . . . 131 x i v LIST OF FIGURES CHAPTER.II Figure Page 1. Diagram of an e t i o l a t e d bean hypocotyl i l l u s t r a t i n g the regions from which c e l l walls were extracted . . . . 116 2. Electron spin resonance spectra of a-chymotrypsin labeled with HTMEP, e n t i r e hypocotyl c e l l walls labeled with HTMFP, and HTMFP alone 132 \ X V ACKNOWLEDGEMENT S D r . I a i n E . P . T a y l o r has p r o v i d e d r e s p o n s i v e , o p t i m i s t i c ' , and e n c o u r a g i n g s u p e r v i s i o n . D r . D o n a l d G . C l a r k and M r . G e o f f r e y Webb h a v e t a u g h t me a v a r i e t y o f t e c h n i q u e s and o f f e r e d numerous h e l p f u l s u g g e s t i o n s . A l l h a v e b e e n i n s t r u m e n t a l i n my d e v e l o p m e n t as a s c i e n t i s t . D r s . T a y l o r and C l a r k a r e a c k n o w l e d g e d f o r p r o v i d i n g s u p p l i e s and e q u i p m e n t . Thanks a r e e x t e n d e d to D r . B . D . R o u f o g a l i s , D r . G . H . N . T o w e r s , and D r . P . W, H o c h a c h k a f o r l o a n i n g e q u i p m e n t . I am g r a t e f u l t o D r s . T a y l o r , C l a r k , T o w e r s , B . A . Bohm, E . Camm and A . D . M . G l a s s f o r r e a d i n g and c r i t i c i z i n g t h e t h e s i s . I h a v e a p p r e c i a t e d d i s c u s s i o n s w i t h I l l i m a a r A l t o s a a r , Guy B e a u r e g a r d , M a r k Denny , Sue K r e p p , and A d r i a n n e R o s s . I am a l s o g r a t e f u l t o M r . Denny f o r h i s d r a f t i n g e x c e l l e n c e . Thanks a r e a l s o e x t e n d e d t o f r i e n d s who h e l p e d me m a i n t a i n s a n i t y d u r i n g t h e s e c o n d and t h i r d y e a r s , n o t a b l y D r . C h r i s t o p h e r F r e n c h , N i c h o l a s and D i a n n e R o e , D r . M i c h a e l S w i f t and Anne W a l t e r . G a r y C o u r t has h e l p e d i n c o u n t l e s s t i m e s o f n e e d . Thanks a r e a l s o e x t e n d e d t o C a r r o l Hudson f o r t y p i n g t h i s t h e s i s . iTjaeJcnowledg.e:^ -Research C o u n c i l o f Ganad_a ;^4dfeiye£S j f c t36 t °£ . J ^ i f i§frj Qj&ifflbia grants t o D r s . T a y l o r and C l a r k and a U n i v e r s i t y l o f B r i t i s h C o l u m b i a Summer (1974) R e s e a r c h S c h o l a r s h i p . F i n a l l y , I w i s h t o t h a n k my f a m i l y and. Nancy S c h e r e r f o r e n c o u r a g e m e n t , u n d e r s t a n d i n g and l o v e . CHAPTER I. STUDIES ON THE ACETYLCHOLINESTERASE OF PHASEOLUS VULGARIS L. A. INTRODUCTION Acetylcholine mediates b i o e l e c t r i c responses i n a number of animal tissues (Hebb and Krnjveic, 1962; Ruch and Patton, 1965). Regulation of acetylcholine l e v e l s i s of paramount importance i n the control of the information conveyed.by t h i s substance. Biosynthesis i s completed by the choline acetyltransterase reaction (Nachmansohn and Machado, 1943). Degradation involves e i t h e r an acetylcholinesterase (acetylcholine hydrolase EC 3.1.1.7) or a pseudocholinesterase, (acylcholine acylhydrolase EC 3.1.1.8). Acetylcholinesterases (AchEs) are membrane bound enzymes located i n nervous t i s s u e , e f f e c t o r organs innervated by c h o l i n e r g i c neurons, and erythrocytes. Their s p e c i f i c i t y i s f o r the acylcarboxylic a c i d of the ester (Cohen and Oosterbahn, 1963). Pseudocholinesterases are soluble enzymes located i n serum. Their s p e c i f i c i t y i s f o r the choline of the ester (Augustinsson, 1963). Although i t appears that both enzymes are s i m i l a r i n some respects, t h e i r t i s s u e l o c a l i z a t i o n , substrate s p e c i f i c i t i e s , and responses to i n h i b i t o r s , modulators and excess substrate d i f f e r (Rosenberry, 1977). The properties of these enzymes have been reviewed (Froede and Wilson, 1971; O'Brien, 1971; Rosenberry, 1975; Rosenberry, 1977). AchE i s involved i n regulation of acetylcholine l e v e l s i n  vivo (Nachmansohn, 1959). I t i s i n h i b i t e d by organophosphates, such as diisopropylfluorophosphate (DIFP), and carbamates, such as neostigmine 1 2 or eserine. I t i s f i v e times more act i v e against a c e t y l c h o l i n e than against propionylcholine and at le a s t 100 times more active against acetylcholine than butylcholine. Phenylacetate i s hydrolyzed at a rate s i m i l a r to that f o r acetylcholine. The enzyme i s i n h i b i t e d by choline and by excess substrate. Acetylcholine i s present i n a v a r i e t y of plant tissues (Fluck and J a f f e , 1974a). •Exogenous ap p l i c a t i o n s of acetylcholine to mung bean roots have e f f e c t s s i m i l a r to those of red l i g h t . I t i n h i b i t s secondary root formation and stimulates e f f l u x i n roots ( J a f f e , 1970); i t promotes the adhesion of root t i p s to negatively charged glass surfaces (Tanada, 1972); and i t increases oxygen uptake and decreases ATP l e v e l s i n roots (Junghans and J a f f e , 1972). Applications of acetylc h o l i n e promote flowering of .Lemna p e r p u s i l l a (a short day plant) under continuous i l l u m i n a t i o n but prevent flowering of Lemna gibba (a long day plant) under the same conditions (Kandler, 1972), and have the same e f f e c t as l i g h t i n increasing seed germination of at l e a s t four species (Holm and M i l l e r , 1972). Both acetylcholine and inductive photoperiods a l t e r the action spectrum of b i o e l e c t r i c responses i n spinach (Grerrin, et a l . , 1973). Red i r r a d i a t i o n r e s u l t s i n an increase i n endogenous acetylcholine l e v e l s i n mung bean roots ( J a f f e , 1970) and other plants (Hartmann and K i l b i n g e r , 1974). Acetylcholine a f f e c t s growth responses of wheat seedlings (Dekhuijzen, 1973) and Avena c o l e o p t i l e s (Evans, 1972), as w e l l as mung bean roots. I t i n h i b i t s i n d o l e a c e t i c a c i d (IAAT)-induced ethylenerproduction .and p a r t i a l l y prevents the lAA-promoted delay of hook opening i n Phaseolus  vu l g a r i s (Parups, 1976) yet mimics the e f f e c t of IAA on elongation and peroxidase a c t i v i t y patterns i n l e n t i l roots (Penel, et a l . , 1976). 3 On the basis of these observations, i t may be that acetylcholine mediates the effect of light on bioelectric responses in mung bean roots or other plant tissues through the involvement of phytochrome (Jaffe, 1972). Few studies of enzymes involved with the regulation of acetylcholine levels in plants have been undertaken. The presence of a choline acetyltransferase has been demonstrated only i n one plant species — Urtica dioica (Barlow and Dixon, 1973). In plant tissues, acetylcholine is hydrolyzed by a variety of esterases including citrus acetylesterase (Jansen, jet a l . , 1947; Schwartz, et a l . , 1964), wheat germ esterase (Jansen, et a l . , 1948; Mounter and Mounter, 1962), cucurbitacin esterase (Schwartz, et a l . , 1964), sinapine esterase (Tsagolbff, 1963), and a cholinesterase (Schwartz, 1967; Riov and Jaffe, 1973; Kasturi and Vasantharajan, 1976). The citrus acetylesterase and wheat germ esterase have K^s of 1.6 and 1.0 M, respectively, for acetylcholine but have not been tested for DIFP or carbamate inhibition. The cucurbitacin esterase activity is unaffected by 10 "*M- DIFP and is slightly stimulated by 10 ^M-eserine. The sinapine esterase has a lower (660yM) Km for acetylcholine but -3 is only marginally inhibited by 10 M-eserine. However, the cholinesterase, partially purified from mung bean roots and subsequently from a variety of other plant tissues (Riov and Jaffe, 1973; Fluck and Jaffe, 1974d; Kasturi and Vasantharajan, 1976) , resembles animal AchE in having inhibition by neostigmine and eserine, maximal activity against acetylesters, a K of less than m 200uM for acetylcholine or acetylthiocholine, and substrate inhibition. 4 I n h i b i t i o n of the pea root enzyme by an organophosphate, Fensulfothion, r e s u l t s i n an increase i n the endogenous acetylcholine l e v e l s over control plants with concomitant i n h i b i t i o n of l a t e r a l root formation (Kasturi and Vasantharajan, 1976). A rigorous p u r i f i c a t i o n of t h i s enzyme has not been achieved, though a v a r i e t y of chromatographic procedures have been applied (R. A. Fluck, personal communication). Few AchE assays have been s u i t a b l e f o r a p p l i c a t i o n to plant tissues or extracts because plants contain s u b s t a n t i a l l y lower cholinesterase a c t i v i t i e s (Fluck and J a f f e , 1974b; Rosenberry, 1977). Even the most s e n s i t i v e assay which uses the substrate analogue ac e t y l t h i o c h o l i n e (Ellman, et a l . , 1961) requires an incubation period of several minutes to detect enzyme a c t i v i t y i n plant extracts rather than the seconds required i n the animal enzyme assay. Spontaneous or non-specific hydrolysis of acetylcholine during the long incubation period i s c o n t r o l l e d by the addition of 10 ^M-neostigmine to assay mixtures (Fluck and J a f f e , 1974b). P u r i f i c a t i o n of AchE to homogeneity from various animal tissues has involved the use of ei t h e r the combination of (NH^^SO^ p r e c i p i t a t i o n , gel f i l t r a t i o n and ion exchange chromatography i n many media (Kremzner and Wilson, 1963; Leuzinger and Baker, 1967) or a f f i n i t y chromatography (Kalderon, et a l . , 1970; Berman and Young, 1971; Dudai, et a l . , 1972a; Rosenberry, et a l . , 1972). A v a r i e t y of a f f i n i t y chromatography matricies have been used to p u r i f y e e l AchE. The one most s u i t a b l e f o r p u r i f i c a t i o n of the native molecular form of the enzyme consists of the N-methyl acridinium d e r i v a t i v e : l-methyl-9-^N^-(e-aminocaproyl)-Y-aminopropylaminoj acridinium bromide hydrobromide; covalently linked to CNBr-activated Sepharose 4B (MAC-Sepharose 4B) (Dudai, et a l , , 1972a). MAC-Sepharose 4B: Sepharose 4B — NH 2(CH 2) 5 CONH ( C H 2 ) 3 C H 3 BP T h i s s t u d y was u n d e r t a k e n t o examine t h e p o s s i b i l i t y t h a t a n A c h E e x i s t s w h i c h r e g u l a t e s t h e l e v e l o f a c e t y l c h o l i n e i n p l a n t t i s s u e s . The o b j e c t i v e s o f t h e s t u d y w e r e : (1) To d e t e r m i n e t h e f e a s i b i l i t y o f u s i n g a c o l o r i m e t r i c enzyme a s s a y ( E l l m a n , e_t a l . , 1961) i n w h i c h n e o s t i g m i n e i n c u b a t i o n a c t s as a c o n t r o l f o r s p o n t a n e o u s o r n o n - s p e c i f i c h y d r o l y s i s o f s u b s t r a t e . (2) To i d e n t i f y AchE a c t i v i t y i n P h a s e o l u s v u l g a r i s L . t i s s u e segments and e x t r a c t s . (3) To p u r i f y t h i s A c h E by c h r o m a t o g r a p h y on M A C - S e p h a r o s e 4B. (4) To d e t e r m i n e some p h y s i c a l and c h e m i c a l p r o p e r t i e s o f t h i s enzyme and t o compare t h e s e p r o p e r t i e s w i t h t h o s e o f o t h e r A c h E s . (5) To i n v e s t i g a t e t h e r o l e o f A c h E a c t i v i t y i n h y p o c o t y l h o o k s o f P_. v u l g a r i s . 6 B. MATERIALS AND METHODS 1. Chemicals Supplies were obtained from sources as in d i c a t e d : acrylamide, N, N, N', N'-tetramethylethlylene diamine, and N, N'-methylene b i s acrylamide: Eastman Kodak Co., Rochester, N.Y.; t r i c h l o r a c e t i c acid, etheylene diamine, p-terphenyl, and 1,4-bis-2-(5-phenyloxazolyl)-benzene: Fisher S c i e n t i f i c Co., Pittsburgh, Pa.; isopropanol, dioxane and naphthalene: Mallinckrodt Chemical Works, St. Louis, Mo.; guanidinium chloride and sodium l a u r y l sulphate (SDS): B r i t i s h Drug House Chemicals, Toronto, Ont.; H-DIFP: Amersham/Searle, A r l i n g t o n Heights, 111.: DIFP: A l d r i c h Chemical Co., Milwaukee, Wn.; pHisolytes: Brinkman Instruments, Wesbury, N.Y.; a c e t y l c h o l i n e c h l o r i d e , a c e t y l t h i o c h o l i n e i o d i d e , arginine, bovine serum albumin (BSA), butylcholine c h l o r i d e , b u t y l t h i o -choline iodide, catalase, choline c h l o r i d e , cytochrome c, decamethylenebis-(trimethyl-ammonium bromide) decamethonium bromide), diethylaminoethyl-(DEAE-) Sepharose CL-6B, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), • eserine, 6-galactosidase, ;,gibberellic'.acid<j' glycera'ldehyde-3-phosphate dehydrogenase (G-3-PDH) (m-hydroxy phenyl)-trimethyl ammonium bromide dimethylcarbamate (neostigmine), k i n e t i n , l y s i n e , myoglobin, ovalbumin and propionylthiocholine iodide: Sigma Chemical Co., St. Louis Mo.; Sepharose 6B, and Sephadex G-75: Pharmacia Fine Chemicals A.Bi, Uppsala, Sweden; E t h r e l : Amehem Products Inc., Ambler, Pa. A l l other chemicals were obtained l o c a l l y . "Baker Analyzed" grade ( J . T. Baker Chemical Co., P h i l l i p s b u r g , N.J.) was used when a v a i l a b l e . Electrophorus e l e c t r i c u s AchE and MAC-Sepharose 4B were generously donated by Dr. D. G. Clark, Department of Chemistry, Un i v e r s i t y of B r i t i s h Columbia. 7 2. Plant M a t e r i a l Bush bean (Phaseolus v u l g a r i s L. var. Top Crop Green Pod) seeds were surface s t e r i l i z e d with 0.5% (v/v) sodium hypochlorite or 10% (v/v) commercial bleach f o r 15 min, rinsed four times with water, and grown i n verm i c u l i t e i n p l a s t i c trays (McConkey and Co., Sumner, Wn.) f o r 9 days i n a dark cabinet at room temperature (24 ± 2°C). Roots were separated and washed with cold tap water to remove v e r m i c u l i t e . Hypocotyls were separated from cotyledons and rins e d with cold tap water. In one experiment, the hypocotyl was further divided into the regions depicted i n Figure 1. 3. Assay of Acetylcholinesterase (AchE) A c t i v i t y The a c t i v i t y of AchE was•determined by the photometric method of Ellman, et a l . , (1961) as modified by Riov and J a f f e (1973). Assay mixtures i n f i n a l volume of 1.62 ml contained 1.00-1.48 ml of 0.5 M-potassium phosphate b u f f e r , pH 8.0, 60 V l of 2.6 mM-DTNB prepared i n the same buf f e r containing 4.5 mM-NaHCO^, and 0.02-0.5 ml of the sample to be assayed. For each sample, a second tube was prepared i n which 30 V l of 1.35 mM-neostigmine bromide replaced 30 u l of bu f f e r . The assay mixtures were incubated for 15 min at 37°C; then 60 ul of 12.5 mM-acetylthiocholine iodide was added. The reaction was allowed to proceed f o r 10-20 min and was terminated by c h i l l i n g to 0°C i n an i c e bath. Absorbance of clear assay mixtures was determined d i r e c t l y at 4.12 nm. P a r t i c u l a t e assay mixtures were allowed to s e t t l e f o r 10 min and the upper layer was removed with a Pasteur p i p e t t e . This suspension was centrifuged at 15,000 g_ f o r 15 min and the absorbance of the supernatant was recorded. 8 F i g u r e 1. D i a g r a m o f an e t i o l a t e d b e a n s e e d l i n g s h o w i n g t h e r e g i o n s u s e d i n d e t a i l e d s t u d i e s o f enzyme d i s t r i b u t i o n . A — a p i c a l , H — p l u m u l a r h o o k , S A — s u b a p i c a l , B b a s a l . 9 Enzyme a c t i v i t i e s were determined by the following c a l c u l a t i o n : One unit = AA.,„ v t * e * 412 where, AA412 = of. the mixture lacking neostigmine minus °f the - • mixture containing neostigmine v = volume of the assay mixture (ul) t = assay time (min) E = e x t i n c t i o n c o e f f i c i e n t of 2-nitro-5-thiobenzoate (1.36 x 10 4nl nmol" 1) With the exception of column or gradient e f f l u e n t f r a c t i o n s , a l l enzyme assays were performed i n duplicate or t r i p l i c a t e . In the experiment designed to determine the e f f e c t of neostigmine on enzyme a c t i v i t y , enzyme s o l u t i o n was b o i l e d f o r 30 min and cleared by ce n t r i f u g a t i o n at 15 000 g_ f o r 10 min. The AA^.^ value was the difference between absorbances of corresponding assay mixtures containing b o i l e d and unboiled enzyme solutions. In experiments designed to examine the e f f e c t of a given substance on AchE a c t i v i t y , 30-60 ul of stock s o l u t i o n of the substance was prepared i n water (or isopropanol i n the case of DIFP) and t h i s replaced the same volume of b u f f e r i n the assay mixtures. The method described by Fluck and J a f f e (1974b) was used f o r the assay of excised t i s s u e . Assay mixtures having a f i n a l volume 20.25 ml contained 18.75 ml of 0.5 M-potassium phosphate buffer, pH 8.0, and 0.75 ml DTNB s o l u t i o n . These were incubated f o r 15 min at 37°C with 1-3 g of uniformly excised t i s s u e ; 0.75 ml of substrate was added and a f t e r exactly 8 min, 0.375 ml of neostigmine s o l u t i o n ( f i n a l concentration of 25 UM) was added. Aliquots (1 ml) were taken at 2 min i n t e r v a l s , the absorbance 10 was recorded, and the aliquot was poured back into the reaction medium to maintain a constant volume. The absorbances versus time were pl o t t e d and the difference between the slopes of the regression l i n e s obtained before and a f t e r the addition of neostigmine was used as ^A^^ t ^ i n the c a l c u l a t i o n of enzyme a c t i v i t y . 4. P r o t e i n Determination The protein i n p a r t i c u l a t e f r a c t i o n s was s o l u b i l i z e d by b o i l i n g with one volume of 2 M- KOH f o r 30 min. The s o l u t i o n was d i l u t e d t e n - f o l d , shaken, and allowed to s e t t l e . The protein i n these and a l l soluble f r a c t i o n s was determined by the method of Lowry, et a l . , (1951) as modified by Eggstein and Kreutz (1955). The residue of extracted p a r t i c u l a t e f r a c t i o n s was washed with water and contained no protein detectable by the q u a l i t a t i v e microbiuret assay (Goa, 1953). The p r o t e i n content of chromatography column and density gradient eluates- was estimated by absorbance at 280 nm. 5. P u r i f i c a t i o n of AchE From P_. Vulgaris a) Extraction and (NH^^SO^ P r e c i p i t a t i o n METHOD A: Roots (344 g) were added to 788 ml of 20 mM-potassium phosphate bu f f e r , pH 7.0, i n a Waring blendor and homogenized at 20 000 rpm for 3 min. The homogenate was centrifuged at 4800 £ i n a BR 6000 centrifuge (International Equipment Co.) f o r 20 min at -4°C. The supernatant was f i l t e r e d through Whatman #1 f i l t e r paper and the residue was resuspended i n 788 ml of the extraction b u f f e r containing 5% (w/v) (NH^^SO^. The s l u r r y was s t i r r e d f o r 30 min at 4°C and centrifuged at 4800 j» for 20 min. The supernatant was f i l t e r e d through two layers of Whatman #1 f i l t e r paper, brought to 80% saturation with solid (NH^J^SO^ at 4°C and centrifuged at 4800 g_ for 20 min. The pellet was resuspended in 30-100 ml of 20 mM-potassium phosphate buffer, pH 7.0, containing 0.2 M-NaCl and dialyzed overnight against the same buffer at 4°C. The non-diffusible material was c l a r i f i e d by centrifugation at 15 000 £ for 10 min. The supernatant could be used immediately or stored at 0°C for up to one month without loss of activity. METHOD B: Root or hypocotyl tissue (200 g) was extracted with 400 ml of 10 mM-potassium phosphate buffer, pH 7.0, containing 5% (w/v) (NH^^SO^ by the procedures described above. The extract was precipitated in two steps with solid (NH^^SO^ to 40% and 70% saturation. The f i n a l pellet was resuspended with 10 mM-potassium phosphate buffer, pH 7.0, and dialyzed overnight against the same buffer, at 4°C. The non-diffusible material was treated the same as in method A. A l l fractions were dialyzed against buffer before being assayed. b) Gel F i l t r a t i o n on Sepharose.6B Extract (5-30 ml) prepared by method A was applied to a 3.5 X 95 cm Sepharose 6B column equilibrated with 20 mM-potassium phosphate buffer, pH 7.0, containing 0.2 M-NaCl. Gel f i l t r a t i o n was performed at a flow rate of 50-60 ml h. , Fractions were collected and assayed for AchE activity and protein; the active fractions were pooled. c) Chromatography on MAC-Sepharose 4B The AchE fraction (87 ml) from gel f i l t r a t i o n was applied to a 1.5 X 2.5 cm column of MAC-Sepharose 4B equilibrated with 20 mM-potassium phosphate buffer, pH 7.0, containing 0.2-M NaCl at a flow rate of 6-8 -1 -1 ml in ; the ligand concentration was 2.0 umol ml . After the entire 12 sample had entered, the column was washed with approximately 3 column volumes of e q u i l i b r a t i o n b u f f e r and then eluted with approximately 5 column volumes of e q u i l i b r a t i o n buffer containing 1 M-NaCl. Fractions were co l l e c t e d throughout the loading, washing and e l u t i n g procedures, and assayed f or AchE a c t i v i t y and protein. The t o t a l eluted a c t i v i t y was compared with that of the loaded sample to determine the recovery of AchE. The 1 M-NaCl eluate (approximately 30 ml) was concentrated to 2-3 ml by u l t r a f i l t r a t i o n through an Amicon XM50 membrane f i l t e r and stored at O C f o r no longer than 12 days. The column was regenerated by washing with approximately 4 bed volumes of e q u i l i b r a t i o n buffer containing 5 M-guanidinium chloride followed by at l e a s t 5 bed volumes of e q u i l i b r a t i o n buffer. 6. L a b e l l i n g With DIFP METHOD A: A modification of the method of Berman (1973) was used to l a b e l s p e c i f i c a l l y AchE p u r i f i e d by MAC-Sepharose 4B. Enzyme solutions (100-400 units ml 1 , 185-210 units mg 1 protein) i n a f i n a l volume of 0.810 ml i n 20 mM-potassium phosphate b u f f e r , pH 7.0, containing 0.0 or 0.2 M-NaCl were incubated f o r 15 min at 37°C with 15 y l of 52.8 mM-DIFP ( i n CaO dried isopropanol) or 50 01 of 324 mM-butylcholine c h l o r i d e , or both DIFP and butylcholine c h l o r i d e . The contents of the tubes were dialy.zed against 4 changes of the react i o n b u f f e r f o r 18 h at 4°C. Non-dialyzable mixtures were assayed f o r AchE a c t i v i t y and protein. METHOD B: Enzyme solutions (100-400 units ml" 1, 185-210 units mg _ 1 protein) i n 20 mM-potassium phosphate bu f f e r , pH 7.0, containing 1.0 M-NaCl ( f i n a l volume = 0.810 ml) were allowed to react f o r 15 min at 37°C 13 i n t h e p r e s e n c e o r ab sence o f 50 u l o f 1.35 m M - n e o s t i g m i n e b r o m i d e . 3 -1 -1 F i f t y - s i x u l o f H - D I F P (0.26 mg m l i n p r o p y l e n e g l y c o l , 3.4 C i mmol ) were added and t h e t u b e s were i n c u b a t e d f o r 20 m i n a t 37°C. R e a c t i o n m i x t u r e s w e r e d i a l y z e d a g a i n s t 4 changes o f t h e r e a c t i o n b u f f e r f o r 18 h a t 4 ° C ; t h e m i x t u r e s were a s s a y e d f o r A c h E a c t i v i t y and r a d i o a c t i v i t y . A l l p r o c e d u r e s w e r e p e r f o r m e d o n d u p l i c a t e p r e p a r a t i o n s . 7. D e t e r m i n a t i o n o f R a d i o a c t i v i t y Ten m l o f d i o x a n e b a s e d s c i n t i l l a t i o n f l u i d ( B r a y , 1960) we re added t o 20-600 u l s a m p l e s . Samples w e r e c o u n t e d a t 45% e f f i c i e n c y i n an I s o c a p 300 l i q u i d s c i n t i l l a t i o n s p e c t r o m e t e r ( N u c l e a r C h i c a g o ) f o r 2-20 m i n w i t h an 800 K cpm t e r m i n a t i o n . A l l a c t i v i t i e s w e r e b e l o w t h e c o i n c i d e n c e c o u n t i n g r a n g e o f t h e s p e c t r o m e t e r . B a c k g r o u n d was c o u n t e d i n d u p l i c a t e b e f o r e e a c h s e t o f s amp le s and s u b t r a c t e d f r o m cpm v a l u e s . A quench c u r v e was p r e p a r e d f o r a c t i v i t y d e t e r m i n a t i o n s by t h e c h a n n e l s r a t i o 3 method (Wang and W i l l i s , 1965) u s i n g H - t o l u e n e quench s t a n d a r d s . I n some e x p e r i m e n t s n e t cpm v a l u e s w e r e r e c o r d e d d i r e c t l y . A l l s amples w e r e c o u n t e d i n d u p l i c a t e . 8. P o l y a c r y l a m i d e D i s c G e l E l e c t r o p h o r e s i s M i x t u r e s c o n t a i n i n g 25-100 u l s a m p l e s o f enzyme p u r i f i e d by e i t h e r g e l f i l t r a t i o n o r c h r o m a t o g r a p h y on M A C - S e p h a r o s e 4B (30-150 ug o f p r o t e i n ) , 5 u l o f 0.05% ( w / v ) b r o m o p h e n o l b l u e i n w a t e r , and s o l i d s u c r o s e t o 5% ( w / v ) w e r e a p p l i e d t o 7% ( w / v ) a c r y l a m i d e g e l s . E l e c t r p h o r e s i s was p e r f o r m e d i n s m a l l p o r e g e l s a t pH 8.3 i n d u p l i c a t e on a t l e a s t d u p l i c a t e p r e p a r a t i o n s ( D a v i s , 1964). G e l s were s t a i n e d w i t h 1% ( w / v ) Amido S c h w a r t z i n 7% ( w / v ) a c e t i c a c i d and d e s t a i n e d i n 7% a c e t i c a c i d . The g e l s w e r e 14 sliced immediately after electrophoresis and individual 2 mm slices from duplicate gels were assayed for AchE activity i n the presence or absence of neostigmine. 9. SDS Gel Electrophoresis AchE purified by chromatography on MAC-Sepharose 4B and labeled with 3 H-DIFP by method B and standard proteins (BSA, 3-galactosidase, catalase, G-3-PDH, myoglobin, and ovalbumin) were dialyzed against 10 mM-sodium phosphate buffer, pH. 7.2, containing 1% (w/v) SDS for 18 h at room temperature. Samples (75 yl) were mixed with 75 y l of either 10 mM-sodium phosphate buffer, pH 7.2, containing 1% (w/v) SDS, 20% (w/v) sucrose, 0.002 % (w/v) Pyronin Y (a tracking dye), and 40 mM-dithioerythritol or the same solution without 40 mM-dithioerythritol. Mixtures were incubated at 100°C for 5 min, cooled, and 20-100 y l aliquots were applied to 5% (w/v) acrylamide gels (0.8X10 cm) containing 1% (w/v) SDS prepared as described by Weber and Osborne (1969). Electrophoresis was performed at 2 ma per gel for 1 hr and 5 ma per gel for an additional 5.5 h-. The center of the tracking dye was marked with India ink. Gels were stained with 0.25% (w/v) Coomassie B r i l l i a n t Blue R-250 in methanol:water:acetic acid (5:5:1, v/v) overnight at 60°C and destained with a 5% (v/v) methanol, 7.5% (v/v) acetic acid solution in water at 60°C for 24 h. Gels were scanned at 550 nm in a Gilford 240 spectrophotometer; absorbance was recorded by a Gilford 6050 chart recorder at 2 cm min ^. Gels were placed on dry ice for 15 min and sliced in 2-.'mm sections. Slices were placed in s c i n t i l l a t i o n vials containing 0.6 ml of NCS tissue solubilizer (Amersham/Searle):water (9:1,v/v) and kept for 20 h at room temperature followed by 2 h at 50°C. Samples were counted for 15 radioactivity as described above. Mobilities of a l l stained and radio-active peaks were determined relative to the mobility of the tracking dye; molecular weights were obtained by comparing the resulting values to a calibration curve prepared from R^  values of standard proteins. A l l gels were run in duplicate. 10. Sedimentation in Isokinetic Sucrose Gradients Isokinetic sucrose gradients (10-29.3% (w/v)) were prepared according to the theoretical formulations of Noll(1967) as applied by Morrod (1975). AchE (310 units ml \ 195 units mg ^ protein), or the same 3 preparation labelled with H-DIFP by method B, and a mixture of standard proteins (myoglobin, catalase and B-galactosidase) were dialyzed against 20 mM-phosphate buffer, pH 7.0, containing 1.0 M-NaCl. Mixtures containing 3 175 y l of the active enzyme.or 125 y l of the .3H-D IP-enzyme plus 50jul of the standard proteins mixture, and solid sucrose to 5% (w/v) were applied to the top of the gradient, overlaid with buffer, and centrifuged at 40 000 rpm i n a Beckman SW41 rotor.for 18 h at 5°C in a Beckman L3-50 ultracentrifuge. Gradients were eluted at a flow rate of approximately 12 ml h. A; 0.5 ml fractions were collected and assayed for AchE activity or radioactivity. Catalase and myoglobin were located by measuring the absorbance at 405 nm. 3-galactosidase was assayed as described by Massoulie and Rieger (1969). A calibration curve was prepared for each gradient using the standard proteins. A l l sedimentation experiments were run in duplicate. 11. Isoelectric Focusing Isoelectric focusing was performed by the thin layer method (Radola, 1973). Sephadex G-75 (7.0 g) was swollen in 100 ml of deionized water over b o i l i n g water for 1 h. Four ml of pHisolytes and 0.1 g each of l y s i n e and arginine were added to the cooled suspension. Glass plates (20X20 cm) were prepared as described by Radola (1973). Samples were dialyzed against 10 mM-potassium phosphate b u f f e r , pH 7.0, overnight at 4°C. These and standard proteins — BSA, cytochrome c and myoglobin — (10 mg ml ^) were applied i n d i v i d u a l l y i n 18 mm bands by d i f f u s i o n from a c o v e r s l i p . One or more applications of 20-50 y l samples t o t a l l i n g 100-200 yg of protein were made 7-10 cm from the cathode end of the p l a t e . Samples focussed i n 10 mm t h i c k dextran were applied by replacing a 1 cm band of dextran with s i m i l a r l y prepared dextran containing up to 1 ml of sample. Twenty y l of each standard were applied 1-3 cm from both the cathode and anode end of the plate. Plates were placed i n the TLE double chamber (Desaga). The platinum ribbon electrodes were put on t h i n s t r i p s of Whatman 7/1 paper moistened with 0.2 M-H^SO^ (anode) or 0.4 M-ethylenediamine (cathode). Focusing was c a r r i e d out at 4°C at 200 V f o r 8 h and 500 V f o r an a d d i t i o n a l 10 h at which time colored standard proteins were focused. Measurements of the pH were made d i r e c t l y with a f l a t membrane glass electrode (Desaga) or 0.3 cm bands were removed from the dextran layer, d i l u t e d with 200 y l of deionized water, and measured. Pr o t e i n was detected by the paper p r i n t method of Delincee and Radola (1972) using Coomassie B r i l l i a n t Blue R-250 or bromophenol blue. A c t i v i t y of AchE was detected by assaying samples (approximately 100 y l ) which were removed from the dextran layer. This method proved adequate for q u a l i t a t i v e purposes but quantitative estimation was not achieved. In two experiments, dextran layers removed from the plate were eluted from either a 25 ml syringe containing glas s wool or a 2.5 X 24 cm column of Sephadex G—25 with 10 mM-potassium phosphate b u f f e r , pH 7.0. A l l i s o e l e c t r i c focusing experiments were duplicated. I s o e l e c t r i c focusing was also performed i n a sucrose density gradient (0-64% (w/v)) containing 1% (w/v) ampholytes i n a 110 ml column (LKB) as described by Morrod (1975). The l i n e a r gradient was prepared by mixing two s o l u t i o n s , one s o l u t i o n containing 1.87 ml of Ampholines (LKB), 28 g of sucrose and 42 ml of water and another s o l u t i o n containing 0.63 ml of Ampholines, 1.5 ml of enzyme s o l u t i o n (251 units ml , 204 units mg ^ protein) i n 10 mM-potassium phosphate b u f f e r , pH 7.0 and 53.87 ml of water. Focusing was c a r r i e d out at 3°C f o r 44 h: at 300V f o r 5 h, at 700V f o r 18 h, at 1250V f o r 6 h, and at 1500V f o r 15 h. The column was eluted at 2.5 ml min and 1 ml f r a c t i o n s were assayed f o r AchE a c t i v i t y , p r o t e i n , and pH. 12. Determination of Stokes Radius and Molecular Weight A Sepharose 6B column £0.8 X 70 cm) was e q u i l i b r a t e d with 20 mM-potassium phosphate b u f f e r , pH 7.0, containing 1.0 M-NaCl. A s o l u t i o n containing Blue Dextran 2000 and K^Fe(CN)g (2 mg ml * each) i n e q u i l i b r a t i o n b u f f e r was applied at a flow rate of 5 ml hr'''. Fractions (0.8 ml) were c o l l e c t e d and absorbance at 650 nm and 410 nm was recorded to determine the void and t o t a l volumes, r e s p e c t i v e l y . In a second run, standard proteins (B-galactosidase (6.9 nm), catalase (5.2 nm), and myoglobin (1.9 nm) — 2 mg ml * each) were applied under the same conditions. Catalase and myoglobin were detected by absorbance at 405 nm and 6-galactosidase was assayed as described by Massoulie and Rieger (1969). The p a r t i t i o n c o e f f i c i e n t (K ) was determined as described by Pharmacia Fine Chemicals av J and a c a l i b r a t i o n curve was prepared by p l o t t i n g the Stokes r a d i i (R ) of 18 standard proteins vs /-log K av' The K of AchE was determined by the av 1 preparative gel f i l t r a t i o n described above. The Stokes radius of AchE was obtained from the c a l i b r a t i o n curve and molecular weight was determined by the combined Svedberg equation and Stokes-Einstein equation (Pang, 1975): M = S R 6N' TT n(l-v ) _ 1 e p where, 13 S = sedimentation c o e f f i c i e n t x 10 R = Stokes radius e n = 0.01002 poise (the v i s c o s i t y of water at 20 C) " 3 - 1 v = '0.75. cm g ( p a r t i a l specif i c volume :'of e e l AchE "-reported by P Bon, et a l . (19?3)) -3 p = 1.00 g cm The f r i c t i o n a l r a t i o (f/fo)was calculated from the equation; (Seigel and Monty, 1966): 13. Ion Exchange Chromatography on Deae-Sepharose CL-6B A 10 ml sample (45 units ml *) prepared by ex t r a c t i o n method B was applied to a column (2.8 X 57 cm) of DEAE-Sepharose CL-6B e q u i l i b r a t e d with 20 mM-pptassium phosphate buffer containing 0.03 M-NaCl and 0.01% (w/v) NaN^, pH 7.0, at a flow rate of 15 ml h ^. The column was eluted with 315 ml of buffer and a NaCl gradient (0.03-0.8 M). Fractions (7 ml) were co l l e c t e d and assayed for AchE a c t i v i t y and p r o t e i n . The method of a f f i n i t y e l u t i o n (Scopes, 1977) was performed i n 20 mM-potassium phosphate bu f f e r , pH 7.2. A 10 ml sample (45 units ml *) prepared by extraction method A was applied to a column (5.5 X 10 cm) of f T o (3v M/ 4TTN) Re 1/3 DEAE-Sepharose CL-6B equilibrated with buffer, at a flow rate of 45 ml h ^. The column was eluted with 170 ml of buffer containing 1 mM-guanidinium chloride, and 170 ml of buffer containing 1 mM-acetylcholine. Fractions (9 ml) were collected and assayed for AchE activity and protein. 14. Cell Wall Extraction Hypocotyl tissue (50 g) was ground to a fine powder in liquid nitrogen (8-10 min). The frozen powder was l e f t to melt at 0°C. The following extraction was carried out at 0-4°C. The homogenate was suspended in 500 ml of 10 mM-potassium phosphate buffer, pH 7.0, and allowed to settle u n t i l 2 layers appeared (10-20 min). The upper layer was transferred to centrifuge bottles. The lower layer was resuspended and the settling procedure was repeated twice. The c e l l wall fragments were collected from the pooled upper layers by centrifugation at 15 000 _g_ for 10 min. The pellet was resuspended in 200 ml of buffer. This suspension contained less than 2% of the c e l l fragments as intact cells (determined by light microscopy). The suspension was treated with a Blackstone Ultrasonic Probe for 2 min at 200 ± 50 W to release attached cytoplasmic contaminants. The fragments were collected by centrifugation at 10,000 g_ for 15 min, resuspended in buffer and treated again with the ultrasonic probe. This procedure was repeated unt i l the c e l l walls were free from cytoplasmic contaminants as determined by phase contrast microscopy. Four washings were usually sufficient to achieve the desired- purity. The c e l l wall suspension was assayed for AchE activity and protein. Assays were performed in duplicate or t r i p l i c a t e on two c e l l wall preparations. 20 15. Growth Regulator Experiments The following procedures were performed to determine whether a range of exogenous s t i m u l i a f f e c t AchE a c t i v i t y i n hypocotyl hooks of e t i o l a t e d seedlings. Surface s t e r i l i z e d beans were grown for 5 days i n darkness i n p l a s t i c trays (2 per treatment). The e t i o l a t e d seedlings -3 were sprayed with approximately 10 ml of one of the fol l o w i n g : 10 M- g i b b e r e l l i n , 10 4 M-kinetin, 100 ppm 2-chlorosulphonic acid (as E t h r e l , an ethylene source) or water. One tray of seedlings was exposed to 3 -2 -1 fluorescent l i g h t (cool white, 2 X 10 erg cm sec ). A sample (2-4 g) of hypocotyl hooks was harvested d a i l y from each f l a t and the spray treatment was repeated. These operations were performed i n dim green l i g h t (40 W f i l t e r e d by a Carolina,.Biological Supply (,GBJ3>) <GR 545 f i l t e r ) . Hoo.ks.uweire.. frozen -in ^ l i q u i d ..nitro'g.env andjcground to-..a •Mne p.owder i n a mortar. T^pav.blumes -.of ^ i l 0;-mM^ p.ho.sphat:.e"'- buffer," pH J,..0,' wjs&eidadjied .and :the_suspensions thawed i n an i c e bath and dialyzed f o r 18 h against buffer at 4°C. Samples (500 y l ) were assayed for AchE a c t i v i t y and p r o t e i n . Assays were performed i n duplicate or t r i p l i c a t e on three separate preparations. 16. Hypocotyl Hook Angle Measurements Nine-day-old e t i o l a t e d beans were harvested i n dim green l i g h t (25 W incandescent l i g h t f i l t e r e d by a CBS GR 545 f i l t e r ) and hooks were excised as described by K l e i n , et a l . (1956) and incubated i n -3 -5 -7 -9 p e t r i plates containing 10- ,10 ,10 and 10 M-acetylcholine chloride i n water f o r 24 h i n darkness. One group containing no acetylcholine chloride was exposed to red l i g h t from a 25 W incandescent bulb f i l t e r e d by a CBS red 650 f i l t e r . Hook angles were determined by the method of K l e i n , et a l . (1956). The experiments 21 were performed i n duplicate. 17. Elongation Measurements —6 Seven-day-old e t i o l a t e d bean plants were sprayed with 10 M-acetylcholine chloride i n water or water alone, measured i n dim green l i g h t (25 W incandescent l i g h t f i l t e r e d by a CBS GR 545 f i l t e r ) and measured again a f t e r 24 h. Measurements were made from the base of the hypocotyl along the length of the hypocotyl to the cotyledonary node using a f l e x i b l e r u l e r . C. RESULTS 1. The Assay Methods a) The e f f e c t of neostigmine on AchE a c t i v i t y The assumption that neostigmine-inhibitable hydrolysis of a c e t y l t h i o c h o l i n e i?aSxidenfHLc:al.j-with AchE.. actijVjity Twas tested, by determining the e f f e c t of neostigmine on the hydrolysis of acetylthioeb choline. The r e s u l t s are shown i n Figure 2. Concentrations of neostigmine greater than 10 uM i n h i b i t e d substrate h y d r o l y s i s by 90% i n preparations having a s p e c i f i c a c t i v i t y of 12 units mg 1 p r o t e i n . The curve was c h a r a c t e r i s t i c of neostigmine i n h i b i t i o n of AchEs (Karczmar; 1967; Riov and J a f f e , 1973) and v a l i d a t e d the assumption that neostigmine-inhibitable hydrolysis of a c e t y l t h i o c h o l i n e ; w a s i d e n t i c a l with AchE a c t i v i t y i n bean root preparations. Neostigmine-i n h i b i t e d preparations remained i n a c t i v e a f t e r d i a l y s i s against 3 changes of buffer f o r 36 hr at 4°C. A l l esterase a c t i v i t y i n preparations having a s p e c i f i c a c t i v i t y greater than 20 units mg 1 protein was neostigmine i n h i b i t a b l e . 22 F i g u r e 2 . The e f f e c t o f n e o s t i g m i n e on A c h E a c t i v i t y . R o o t e x t r a c t s w e r e p r e c i p i t a t e d w i t h ( N H ^ ^ S O , by method A . V a l u e s a r e means o f two a s s a y s f r o m a d u p l i c a t e d e x p e r i m e n t . fog Eneostigrnine] M 23 b) The effect of assay time on AchE activity The neostigmine-inhibitable hydrolysis of acetylthiocholine was time dependent (Figure 3). The absolute ^A^^ values and the slope varied depending on the preparation used, however linearity from 3-18 min was reproducible for soluble and particulate fractions of both root and hypocotyl preparations. Extrapolation of the line to t=0 yielded a positive ^-422 value, which reflected the time lag between termination of the assay incubation* and the measurement of absorbance. The assay was suitable for use when longer incubation times were needed to detect low enzyme ac t i v i t i e s . c) The effect of enzyme quantity on AchE activity The activity of AchE depended directly on the quantity of solution assayed (Figure 4). The relationship was linear for ^A^^ values ranging from 0.02 to approximately 0.85 (for the experiment presented in Figure 4 this corresponds to 5-100 y l ) . The regression coefficient was 0.996. 2. Extraction and (NH.^SO^ Precipitation of AchE From P_. vulgaris Roots and Hypocotyls The crude preparations which were obtained after homogenization of either root or hypocotyl tissues in 10 mM-potassium phosphate buffer, pH 7.0, had both low AchE activity and low specific activity (Table I ) . The activities were increased 7 fold in roots and 20 fold in hypocotyls after these preparations were dialyzed against 4 1 of deionized water and then 2 changes of 4 1 of 10 mM-potassium phosphate buffer, pH 7.0, for 36 h at 4°C. The activity of these preparations was reduced when the diffusate from either root or hypocotyl dialysis was Figure 4. 25 The e f f e c t of enzyme quantity on the AchE a c t i v i t y . Values are means^of two assays of a preparation containing 11.9 units mg protein. volume assayed (pi) Table I: The effect of dialysis and the reintroduction of the diffusate to the non-dialyzable homogenate on the AchE activity in _P. vulgaris root and hypocotyl. Each tissue was homogenized i n 10 mM-potassium phosphate buffer, pH 7.0, and immediately dialyzed against 4 1 of deionized water, and then buffer. The diffusate in water was reduced to the original sample volume and returned to the non-dialyzable fraction. N.D. denotes values not determined. % Decrease of Activity in Origin of Fraction Assayed Diffusate Activity ^ (units g )* Sp. Activity (units mg )** % of I n i t i a l Activity Non-Dialyzable Substances Tissue Origin: ROOT Homogenate Before Dialysis 3.15 0.25 100 Homogenate After Dialysis 20.4 2.83 648 0 Dialyzed Homo= >^ Root 15.2 N.D. 482 25 gem ate AAfiterAAddf-fddn yof f D:if f usat e Hypo co ty 1 8.5 N.D. 270 58 Tissue Origin: HYPOCOTYL Homogenate Before Dialysis 0.20 0.02 100 Homogenate After Dialysis 3.9 0.47 1950 0 Dialyzed Homo-g,z Root 2.2 N.D. 1100 43 genate.Mfit er. -Ad d i -Ciori ofitDiffusate Hypocotyl 1.3 N.D. 650 67 * weights are given as fresh weight ** weights are given as Lowry protein 27 reintroduced to e i t h e r root or hypocotyl preparations. However, t h i s a c t i v i t y l o s s d i d not f u l l y correspond to the increased a c t i v i t y produced by the d i a l y s i s . A r a pid e x t r a c t i o n method (method B) i n which the e x t r a c t i o n b u f f e r contained 5% (NH^^SO^ was tested on roots and hypocotyls. The r e s u l t s are shown i n Table I I . Although the 5% (NH.).SO. extraction 4 2 4 procedure was more e f f i c i e n t f o r hypocotyls, i n which 81% of the t o t a l a c t i v i t y was extracted, than for roots, i n which 43% of the t o t a l a c t i v i t y was extracted, subsequent (NH^^SO^ p r e c i p i t a t i o n r e s u l t e d i n higher a c t i v i t i e s on protein and fresh weight bases i n roots than i n hypocotyls. Thus, fur t h e r p u r i f i c a t i o n e f f o r t s were d i r e c t e d toward the root AchE. 3. L o c a l i z a t i o n of AchE i n P_. v u l g a r i s a) AchE a c t i v i t y i n excised pieces of root and hypocotyl The AchE a c t i v i t y of excised portions of e t i o l a t e d bean seedlings i s shown i n Table I I I . A c t i v i t y S.E.M. values were very large because small absorbance changes were recorded. Enzyme a c t i v i t y expressed on both a fresh weight and p r o t e i n basis was highest i n roots and lowest i n the basal regions of hypocotyls, but increased with distance up the hypocotyl. A c t i v i t y 4 o f Ventirechypocotyls; was estimated at 0. l-un-its>g: "vifreshhwdight" of _hyp.qco.t5yl &_ _,Thi-s Rvalue, wasipha'sed. on the-obser-vationOfhat the freshoweighteoft sub.ap.icalTarid basal segments -comprised greater.Tr,thany80%io£nthe gresh^ weight '.of.zhypoGoty^ls. Both the hypocotyl and root a c t i v i t i e s were lower than cprresp.o.nd;ing"V/alue.s obtained by assaying ti s s u e homogenates (Table I ) . Table II: Recovery of AchE from P. vulgaris roots and hypocotyls extracted and fractionated with (NH^^SO^. Values are averages of at least three preparations (method B). N.D. denotes values not determined. FRACTION 5% (w/v) (NH 4) 2S0 4 residue extract ROOT Activity ^ (units g )* 8.2 6.1 Sp. Activity (units mg )** N.D. 3.2 HYPOCOTYL Activity_^ (units g )* 0.06 0.25 Sp. Activity (units mg )** N.D. 0.2 10-40% Saturation precipitate 0.2 supernatant 5.8 N.D. 3.8 0.06 0.21 N.D. 1.1 40-70% Saturation precipitate 5.0 supernatant 0.9 7.1 N.D. 0.21 0.00 1.0 N.D. * weights are given as fresh weight ** weights are given as Lowry protein T a b l e I I I : A c t i v i t y o f A c h E i n e x c i s e d segments o f r o o t s and r e g i o n s o f t h e h y p o c o t y l s o f P_. v u l g a r i s shown i n F i g u r e 1. I n t a c t 12 cm segments o f t i s s u e were a s s a y e d as d e s c r i b e d i n M a t e r i a l s and M e t h o d s . A c h E a c t i v i t y v a l u e s a r e means o f f o u r d e t e r m i n a t i o n s ± S . E . M . P r o t e i n v a l u e s a r e means o f 3 a s s a y s on a t l e a s t d u p l i c a t e samples ± S . E . M . O r i g i n o f A c t i v i t y _^ T o t a l P r o t e i n S p e c i f i c ^ A c t i v i t y E x c i s e d Segments u n i t s mg f r e s h w t . ) (mg g - f r e s h w t . ) ( u n i t s mg p r o t e i n ) A p i c a l R e g i o n o f H y p o c o t y l 0.370 + 0.199 16.8 ± 0.8 0.022 Hook R e g i o n o f H y p o c o t y l 0.206 ± 0.066 15.3 ± 0.6 0.013 S u b a p i c a l R e g i o n o f H y p o c o t y l 0.087 ± 0.024 9.7 ± 0.7 0.009 B a s a l R e g i o n o f H y p o c o t y l 0.072 ± 0.038 8.7 ± 0.5 0.008 R o o t s 0.700 ± 0.140 7.3 ± 0.2 0.096 to 30 b) A c t i v i t y of AchE i n the c e l l w a l l The hypocotyl c e l l walls prepared by successive washes with buffer were assayed for AchE a c t i v i t y by the p a r t i c u l a t e assay procedure. The a c t i v i t y was 0.033 ± 0.005 units mg 1 dry c e l l w a l l , and the s p e c i f i c a c t i v i t y of these preparations was 0.25 ± 0.04 units mg 1 c e l l wall protein. 4. • P u r i f i c a t i o n of Root AchE Low a c t i v i t i e s were detected i n extracts of roots homogenized i n e i t h e r 10 or 20 mM-potassium phosphate b u f f e r , pH 7.0, a f t e r c e n t r i f u g a t i o n and f i l t r a t i o n (Table IV). Values f o r a c t i v i t y i n crude extracts were close to the detection l i m i t s of the assay procedure and consequently v a r i e d from one experiment to the next. These were consistently less than 5% of the t o t a l a c t i v i t y i n homogenates (Tables I and I I ) . The r e s u l t s presented i n Table IV were obtained when the r e s u l t i n g residue was extracted with buffer containing 5% (w/v) (NH^SO^ and p a r t i a l l y p u r i f i e d by (NH^SO^ f r a c t i o n a t i o n , gel f i l t r a t i o n on Sepharose 6B, and chromatography on MAC-Sepharose 4B. S p e c i f i c a c t i v i t y a f t e r (NH^^SO^ f r a c t i o n a t i o n was greater than that obtained a f t e r e x traction by method B (Table I I ) , although t o t a l recovery was 5.0 units g 1 fresh weight i n both cases. The e l u t i o n p r o f i l e shown i n Figure 5 was obtained when AchE was chromatographed on Sepharose 6B. AchE a c t i v i t y eluted as a s i n g l e peak having a K of 0.647. The e l u t i o n p r o f i l e shown i n Figure 6 was obtained when AchE prepared by gel f i l t r a t i o n on Sepharose 6B was chromatographed on the MAC-Sepharose 4B. S p e c i f i c a c t i v i t y of the concentrated 1 M-NaCl elution volume (ml) F i g u r e 5. E l u t i o n p r o f i l e o b t a i n e d a f t e r g e l f i l t r a t i o n o f A c h E on S e p h a r o s e 6B. Samples were p r e p a r e d by method A . A c h E a c t i v i t y ( • — • ) arid•'•A^.go' ( x x ) °^ e a c n 10 m l f r a c t i o n were m e a s u r e d . elution Volume (ml) F i g u r e 6. E l u t i o n p r o f i l e o b t a i n e d a f t e r ch roma tog raphy o f A c h E on M A C - S e p h a r o s e - A B c o n t a i n i n g 2.0 y m o l o f l i g a n d m l . The sample o b t a i n e d f r o m g e l f i l t r a t i o n was l o a d e d i n 20 m M - p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 0.2 M - N a C l . The co lumn was washed w i t h t h e b u f f e r t h e n w i t h t h e b u f f e r c o n t a i n i n g 1.0 M - N a C l . AchE a c t i v i t y (• e») and A^gQ ( x x ) o f e a c h f r a c t i o n were m e a s u r e d . The h o r i z o n t a l dashed w l i n e i n d i c a t e s t h e A 0 Q n o f t h e sample (0.365). Table IV: Purification of AchE from _P. vulgaris roots. Methods of extraction and purification are detailed in Materials and Methods. Values presented were obtained from one preparation and are representative of at least two other preparations. Purification values are based on the specific activity of dialyzed root homogenates from Table I. Fraction Volume - (ml) Protein (mg) Total Units (units) Specific Recovery Activity """'(%'of 5% (unitssmgv (NH^SO^ protein) Extract) Purification (-fold) Crude Extract (in buffer) 848 N.D. 508 5% (NH 4) 2S0 4 Extract of Residue 695 662.3 4239 80% (NH,)„S0, ppt. (resuspended) 30 149.2 1775 Sepharose 6B 87 74.2 1722 MAC-Sepharose 4B (after u l t r a f i l t r a t i o n ) 2.9 2.6 589 N.D. 6.4 11.9 23.2 222.9 100 42 41 14 2.3 4.2 8.2 78.8 LO Co 3 4 eluate varied from 1 9 0 - 2 3 0 units mg * prot e i n . 5. Characterization of P u r i f i e d AchE a) DIFP Labeling Figure 7 shows that AchE a c t i v i t y was completely i n h i b i t e d at DIFP concentrations greater than 10 M. The i n h i b i t i o n of enzyme a c t i v i t y by DIFP was reduced from 9 9 % to 2 9 % i n the presence of 2 0 mM-butylcholine i n 20 mM-phosphate b u f f e r , pH 7.0, and from 9 4 % to 6 3 % i n the presence of 2 0 0 mM-butylcholine i n 2 0 mM-phosphate bu f f e r , pH 7.0, containing 0.2 M-NaCl. The l a t t e r treatment completely protects animal AchEs from DIFP i n a c t i v a t i o n (Cohen, et a l . , 1 9 6 7 ) . The incomplete protection §gainst DIFP i n h i b i t i o n that butylcholine provided to the bean enzyme prevented the use of butylcholine i n experiments designed to s p e c i f i c a l l y l a b e l AchE with radioactive DIFP. Table V shows that there was greater incorporation of IH-DIFP into preparations 'labeled i n the absence rather than i n the presence of 125 uM-neostigmine. The c a t a l y t i c center a c t i v i t y , based on DIFP binding s i t e s , was 606 and 1 9 7 ± 5 mol.' of substrate min * mol ^ DIFP binding s i t e when corrected and uncorrected, r e s p e c t i v e l y , f o r binding i n the presence of neostigmine. The corrected value was based on the assumption that neostigmine completely i n h i b i t e d DIFP binding to AchE. b) Disc Gel Electrophoresis Results of polyacrylamide gel electrophoresis of AchE preparations from two stages of p u r i f i c a t i o n are shown i n Figure 8. There were two densely stained bands of protein. (R F=0.07±0.03 and R F = 0 . 6 9 ± 0 . 0 3 ) and a l i g h t l y stained region (from R^=0.39-0.49) a f t e r g e l electrophoresis of high s p e c i f i c a c t i v i t y ( 1 9 5 - 2 2 9 units mg * protein) preparations. A 35 Figure 7. The e f f e c t of DIFP on the AchE a c t i v i t y p u r i f i e d by chromatography on MAC-Sepharose 4B. Values are means of two assay| performed on a preparation containing 96 units ml and 229 units mg protein. log L DIFPH M 36 Figure 8. Polyacrylamide gel electrophoresis of AchE. Samples were prepared by e i t h e r a) gel f i l t r a t i o n on Sepharose 6B, or b) chromatography on MAC-Sepharose 4B and separated by electrophoresis i n 7% (w/v) polyacrylamide gels at pH 8.3. Gels were stained with 1% (w/v) Amido Schwartz ( a l and bl) or cut into 2 mm s l i c e s and assayed f o r AchE a c t i v i t y (b2). Protein zymograms are based on values averaged from duplicate gels of two (al) or three (bl) d i f f e r e n t preparations. The enzyme a c t i v i t y p r o f i l e shows values from one of two duplicate experiments. b 2 origin fnont origin front C P < t > o C • rT O r 0 . 2 A A 0 . 4 4 1 2 37 3. Table V: Incorporation of H-DIFP into AchE p u r i f i e d by chromatography mlg and 185-210 units mg protein were reacted with 10 M- H-DIFP i n the presence or absence of 125 uM-neostigmine and counted for r a d i o a c t i v i t y a f t e r exhaustive d i a l y s i s . Estimated c a t a l y t i c center a c t i v i t y was based on DIFP binding s i t e s . Values are means of three samples of duplicate preparations ± S.E.M. N.D. denotes value not determined. on iH-DIFP incorporated (pmol unit ) Estimated c a t a l y t i c center a c t i v i t y ^ (mol of substrate min mol DIFP) -1 No neostigmine 5.0710.12 197+5 Neostigmine 3.42±0.15 N.D. Net value 1.65 606 38 s i n g l e peak of AchE a c t i v i t y was located (R^=0.05) that corresponded with one of the bands of protein. This peak accounted f o r more than 90% of the applied a c t i v i t y . Preparations having lower s p e c i f i c a c t i v i t y (21530 units mg 1 protein) showed a d d i t i o n a l protein bands. The methods of Koelle (1951) as modified by Wright and Plummer (1973) and Karnovsky and Roots (1964) were applied without success to detect AchE a c t i v i t y i n polyacrylamide gels. c) SDS Gel Electrophoresis Figure 9 shows the c a l i b r a t i o n curve obtained a f t e r SDS polyacrylamide gel electrophoresis of standard proteins. AchE was p u r i f i e d by chromato-3 graphy:.onl-MAC7 .Sepharo'sep'ABr and labelediw"ithidH-MFP.; ••- The. scans .shown dnaEdguliresi. 10a ahduMa were* obtained^af t^cSDScgel ielec£r,pphpresis of this 1" preparationo.und'ersripnc-r.educing a©dc-re\4l^i»8d€SS^§'tigns..respectively. Figures-.10b andpllbixehpw thegiraddoacLtiyityj. i n s l i e e s h p f r=lfe§:; same;.-gels. Theimajor DiEFPr-labeledar'egipnsh of 61 .000 + 2 000i, 26 000 ± 1000 and 17 500 ± 500. A minor DIFP-labeled peak had a mol. wt. of 77 000 ± 2000. The 77 000, 61 000, and 26 000 DIFP-labeled components corresponded to d i s t i n c t p r o t e i n peaks i n the gel scans. A d d i t i o n a l low molecular weight (16 000) protein was observed i n the gel scans but no corresponding t r i t i a t e d peak was seen. The g e l scan of reduced AchE showed the same protein peaks. There was less r a d i o a c t i v i t y i n the t r i t i a t e d - 6 1 000 mol. wt. subunit and an increase i n a c t i v i t y i n a 30 000 ± 1000 mol. wt. component. Complete reduction of the 61 000 mol. wt. subunit was not observed. The same res u l t s were obtained f or AchE labeled with DIFP i n the presence of neostigmine. F i g u r e 9 . M o b i l i t y o f s t a n d a r d p r o t e i n s r e l a t i v e to the t r a c k i n g dye i n p o l y a c r y l a m i d e g e l s c o n t a i n i n g 1% SDS. V a l u e s o b t a i n e d f r o m d u p l i c a t e g e l s a r e p r e s e n t e d . Co 39a Figure 10. D i s t r i b u t i o n of (a) p r o t e i n and (b) t r i t i u m a f t e r SDS-acrylamide gel electrophoresis of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and labeled with H-DIFP. AchE was denatured i n the absence of any reducing agents. Gels were stained with Coomassie Blue, scanned for absorbance at 550 nm, s l i c e d into sections, and assayed for r a d i o a c t i v i t y . Molecular weights are averages from duplicate gels. 40a Figure 11. D i s t r i b u t i o n of a) protein and b) t r i t i u m a f t e r SDS-acrylamide gel electrophoresis of reduced AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and labeled with H-DIFP. AchE was denatured i n the presence of 40 mM d i t h i o e r y t h r i t o l . Gels were stained with Coomassie Blue, scanned f or absorbance at 550 nm, s l i c e d into sections and assayed for r a d i o a c t i v i t y . Molecular weights are averages from duplicate gels. 41 Figure11 a . 16K 42 d) Sedimentation in isokinetic gradients The results of isokinetic sedimentation of AchE purified by chromatography on MAC-Sepharose 4B are shown in Figure 12. AchE activity appeared as one major peak with a corresponding value of 4.2 S (4.1-4.3S). The preparations labeled with ^3H-DIFP in the presence and in the absence of 125 uM-neQstigmine each contained a single peak of radioactivity sedimenting at 5.6 S (5.3-5.9$) and 5.0 S (4.6-5.4S) respectively. These peaks a l l represent the same sedimenting particle within experimental error. It was concluded that neostigmine did not inhibit completely the binding of DIFP to AchE and that a l l of 3 the radioactivity recovered from sedimentation of H-preparations are located in AchE. e) - - Is oelectr ic'-f o cus ing A gradient from pH 3-9.5 was obtained consistently after thin layer isoelectric focusing. Standard proteins focused at pis reported previously (Radola, 1973). Figure 13 shows the result of a typical isoelectric focusing experiment. The isoelectric pH.. of AchE prepared by chromatography on MAC-Sepharose 4B was 5.3 ± 0.1. The single predominant protein band lacked AchE activity and focused at pH 9.2 ± 0.1; only a faint stain was detected at pH 5.3. An irreproducible peak of AchE activity focused at pH 6.7-7.7 (usually at pH 7.0). The peak appeared in most preparations but rarely focused at the same pH. Preparative scale experiments, in which 400-500 units of enzyme activity were applied to a 2-10 mm thick dextran layer, resulted in yields of less than 20 units after recovery of the enzyme by elution of the dextran 42a Figure 12. Elution profiles obtained after isokinetic sedimentation of standard proteins and AchE. Samples_contained: a) 54 units of AchE activity (195 units mg protein) purified by MAC-Sepharose 4B, b) the samg preparation as a) but quantitatively inhibited by 10 M- H-DIFP^ and c) the same preparation as a) but treated with 10 M- H-DIFP in the presence of 125 yM-neostigmine. Fractions (0.5 ml) were assayed for AchE activity (a) or radioactivity (b. and c) (A A ) , and g-galactosidase (S=15.9) activity (o o) ; absorbance at 405 nm was measured to localize catalase (S=11.3) (« •) and myoglobin (S=2.0) (a n). The profiles were duplicated t O v o b t a i n average S values. Figure 12 a 4 8 12 16 20 24 f r a c t i o n number Figure 12b fraction number fraction number -Or 4 8 12 16 distance from cathode (cm) Figure 13. AchE activity and pH. gradient after thin layer isoelectric focusing of enzyme purified by chromatography on MAC-Sepharose 4B. The 1 ml sample (480 units, 0 205 units mg protein) was applied as a band 8 cm from the cathode and focused for 8 h at 200 V and an additional 10 h at 500 V. The pH gradient (+ +) and AchE activity (• cm) were determined on approximately 100 yl samples removed from the dextran layer and diluted with 200 y l of water. ON 47 on Sephadex G-25 or f i l t r a t i o n through glass wool. Figure 14 shows the results of isoelectric fosucing in a sucrose gradient. The peak of AchE activity at pH 7.0 accounted for less than 8% of the applied activity. Protein precipitate was observed in the column. Neither method of isoelectric focusing was suitable for further purification of AchE activity because of low recovery and precipitation. f) • Stokes-e-Radius andsMoleculaf,,Weight The Stokes radius calibration curve obtained by gel f i l t r a t i o n of standard proteins is shown in Figure 15. Using the value of K & v =• 0.647 obtained for AchE prepared by method A, the Stokes radius of this enzyme was 4.00 nm. The calculated molecular weight of AchE was 76 000 ± 2000 using the value of S = 4.2 (Figure 12a) and assuming a V of 0.75 3-1 cm g (Bon, et a l . , 1973). The-frictional ratio, f/fo, resulting from these idata was. 1.37. .A mol.-wt^ of 78'- 000 ± 8-000 was obtained from a globular protein calibration curve of the same standard protein K data r av prepared by plotting log mol. wt. vs K (Figure 16). g) . Substrate ^Affinities and the^Effect of'Various Substances on • " AchE Activity. 1) The effect,of acetylthiocholine, propionylthiocholine, and butylthiocholihe on AchE*activity. The effects of acetylthiocholine, propionylthiocholine, and butylthiocholine on the AchE activity of MAC-Sepharose 4B-purified preparations are shown in Figure 17. The specific activity of AchE for hydrolysis of the three substrates (ImM) is shown in Table VI. Figure 18 shows a Lineweaver-Burke plot of the results shown in Figure 17 for acetylthiocholine. A regression line for points of the ascending Figure 14 Figure 14. E l u t i o n p r o f i l e a f t e r column i s o e l e c t r i c focusing of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B. The sample was focused as described i n Materials and Methods and the pH (• • ) , the &280 ^A A ^' anc* the AchE a c t i v i t y ( • — - — r • ) of each f r a c t i o n were measured. 49 Figure; 15. Stokes radius c a l i b r a t i o n curve. The p l o t was obtained a f t e r gel f i l t r a t i o n of standard proteins on Sepharose 6B. 50 Figure 16. Globular protein molecular weight c a l i b r a t i o n curve. The p l o t was obtained a f t e r g e l f i l t n a t i o n of the standard proteins shown i n Figure 15 on Sepharose 6B. 51 Figure 17. The e f f e c t of substrate concentration on AchE a c t i v i t y of MAC-Sepharose 4B-purified preparations. Values are averages of duplicate assays. Substrates tested included a c e t y l t h i o c h o l i n e (+ +)•, propionylthiocholine (• •) , and b u t y l t h i o c h o l i n e (x x ) . 52 Table VI: Substrate specificity of AchE prepared by chromatography on MAC-Sepharose AB for three choline esters. Values are means of duplicate assays. Specific activity Substrate : (units mg . ..protein) ACETYLTHIOCHOLINE 19 A.1 PROPIONYLTHIOCHOLINE 62.1 BUTYLTHIO CHOLINE 65.9 53 Figure 18. A Lineweaver-Burke pl o t f o r a c e t y l t h i o c h o l i n e . Values are averages of duplicate assays, based on r e s u l t s presented i n Figure 17. 54 limb of the acetylthiochdline curve in Figure 17 resulted i n a K of 56 yM. The K for acetylthiocholine, determined by the same method on low specific activity (11.5 units mg protein ^) preparations, was 58 yM. The enzyme did not obey Michaelis-Mente-n kinetics when either prQpionyl-thiocholine or butylthiocholine was used as the substrate. Substrate inhibition is characteristic of acetylcholinesterases from both plant and animal sources (Augustinsson and Nachmansohn, 1949; Wilson and Bergman, 1950; Riov and Jaffe, 1973; Kasturi and Vasantharajan, 1976) and both the high and low specific activity preparations from P_. vulgaris roots displayed this characteristic (Figure 17). 2) -The effect, of choline, decamethonium "(NH-^ S^O^ , and NaCl .. .Jon AchE activity. Figure 19 shows the effect of choline on AchE activity. Choline enhanced AchE activity at concentrations ranging from 0.5-10 mM. The enhancement was reduced at concentrations exceeding 10 mM. Decamethonium has been used, as ran eluant in affinity.chromatography studies involving the use of the N-methylacridinium ligand (Dudai, et a l . , 1972a). The inhibition of the AchE activity by decamethonium is shown in Figure 20. Activity was recovered following dialysis against three changes of buffer for 9 h at 4°C. Figure 21 shows the effect of NaCl on AchE activity of preparations partially purified by method B. Both root and hypocotyl AchE activity were inhibited by high concentrations of NaCl. Inhibition could be reversed by dilution of NaCl. There was no interference with the AchE assay when the NaCl concentration was reduced to less than 0.1 M in the assay solution. Figure 19. 55 The e f f e c t of choline on MAC-Sepharose,4B-purified AchE (195 units mg p r o t e i n ) . The values are means of duplicate assays. 56 Figure 20. The e f f e c t of decamethonium on AchE a c t i v i t y . The values are means of duplicate assays of samples prepared by method A. log [decamethonium] M 57 Figure 21. The e f f e c t of NaCl on AchE a c t i v i t y . Samples were prepared by 70% (NH^^SO^ p r e c i p i t a t i o n of root (a ^ ) and hypocotyl (o o) extracts. The values are means of duplicate assays. l o o t 58 High concentrations of (NH^^SO^ inhibited root AchE (Figure 22). This inhibition could be reversed by dilution of (NH^^SO^ but dialysis against buffer was preferred to regain activity. Sodium azide (0.01% (w/v)) had no effect on AchE activity of preparations partially purified by method. 6. Behavior of Low Specific Activity AchE on Chromatographic Media a) Chromatography on MAC-Sepharose 4B The i n i t i a l attempts to purify root AchE by aff i n i t y chromatography used conditions established for the purification of AchE from electroplaques of Electrophorus electricus (Dudai, et a l . , 1973; Webb and Clark, unpublished results). The results obtained are presented in Table VII. L i t t l e binding occurred when the enzyme was applied to the column in 10 mM-potassium phosphate buffer, pH 7.0, containing 1.0 M-NaCl. When the enzyme was applied in buffer without salt, 62% of the activity remained bound after a 20 ml buffer wash. A l l of that activity was recovered when the column was eluted with 1.0 M-NaCl in buffer. This step resulted in a 6.5 fold purification (Table VII). This purification factor suggested that under such conditions, the matrix was behaving as an ion exchanger. Conditions were modified in an attempt to obtain greater purification. Samples prepared by method B in 10 mM-potassium phosphate buffer, pH 7.0, containing 0.5 M-NaCl were applied to 4 columns (1.5 X 1.0 cm) of MAC-Sepharose 4B containing 0.4, 1.0, 1.6 and 2.0 Vmol of ligand ml The profiles shown in Figure 23 were obtained. Substantial activity was retarded only by the column containing 2.0 Umol of ligand ml 1. 59 Figure 22. The e f f e c t of (NH^SO^ on AchE a c t i v i t y . The values are means of duplicate assays of root extracts prepared by method B. 60 Table VII: Recovery of AchE activity from MAC-Sepharose 4B having a ligand concentration of 0.4 umol ml . Samples (8.8 units mg protein) of root extracts prepared by method B were applied to a 1.5 ml column i n 10 mM-potassium phosphate buffer, pH 7.0, either containing 1.0 M-NaCl or without NaCl. Values for recovery, specific activity and purification were obtained after elution of columns with buffer containing 1.0 M NaCl. Values are derived from averages of duplicate assays. N. D. denotes values not determined. Activity Bound •' - Specific to column Recovery Activity ^ Equilibration (% of applied (% of bound (units mg • Purification Buffer activity) activity) "protein) ,'. (-fold) 1 M-NaCl 6 N.D. N.D. N.D. no NaCl 62 100 56.8 6.5 60a F i g u r e 23. A c h E a c t i v i t y r e c o v e r e d f r o m f o u r M A C - S e p h a r o s e 4B co lumns o f d i f f e r i n g l i g a n d c o n c e n t r a t i o n s . L i g a n | c o n c e n t r a t i o n s _ | n t h e co lumns were^ A) 0.4 y m o l m l , B ) _ l . 0 y m o l m l , C) 1.6 y m o l m l , and D) 2.0 y m o l m l . Columns were l o a d e d w i t h 20 m l o f enzyme p r e p a r e d by method B i n 10 m M - p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 0.5 M - N a C l . A f t e r a l l enzyme was a p p l i e d , t h e co lumns were washed w i t h 15 m l o f e q u i l i b r a t i o n b u f f e r and e l u a t e f r a c t i o n s (1 m l ) were a s s a y e d f o r A c h E a c t i v i t y ( • • ) and A . . ( • • ) . 61 Figure 23 D. ~" 11.6 fraction number 62 Samples prepared by method B were dialyzed against 10 mM-potassium phosphate buffer, pH 7.0, containing either 0.05, 0.1, 0.2, or 0.4 M-NaCl. Each of these samples was applied to one of four columns containing 2.0 ymol of ligand ml ^. The elution profiles are shown in Figure 24. The amount of protein bound to the columns varied inversely with the ionic strength of the buffering system. The greatest amount of AchE activity was bound to the columns operated i n 0.2 M-NaCl. Elution of protein ceased abruptly after a l l 4 columns were washed with their equilibration buffers. Elution of AchE activity from the column operated i n 0.4 M-NaCl continued after ^280 v a - l u e s returned to baseline indicating that the enzyme was only retarded by the ligand. In. the other three columns, enzyme activity was retained by the columns. No activity was recovered after the four columns were eluted with 5 mM-neostigmine and the resulting fractions were dialyzed against buffer for 36 h at 4°C. Following elution with neostigmine, the columns previously operated in 0.05 and Oil M-NaCl were eluted with buffer containing 1.0 M NaCl. No activity was recovered. It was concluded that neostigmine released the bound enzyme but the decarbamylation reaction was too slow to yield active enzyme after dialysis. Enzyme prepared by method B was applied in 10 mM-potassium phosphate buffer, pH 7.0, containing 0.2 M-NaCl to columns containing 2.0 ymol of ligand ml The columns were eluted with either a NaCl gradient (0.2-1.0 M), 5 mM-acetylcholine, or a decamethonium gradient (0-50 mM). Fractions of the acetylcholine and decamethonium eluates were dialyzed against three changes of buffer for 10 h at 4°C prior to the AchE assay. The elution profiles obtained are shown in Figure 25a. Elution with the Figure 24 "D CD a o 100 50 A. t sample 10 f20 buffer 30 ^ 40 neostigmine 50f 1M NaCl < < I oo|-50 c. t sample F i g u r e 24. 10 buffer 30 . 40 . t neostigmine 50 B. P.4 > ro oo O neost igmine f r a c t i o n number R e c o v e r y o f A c h E f r o m MAC-Sepharo;-e-4B co lumns e q u i l i b r a t e d a t d i f f e r e n t i o n i c s t r e n g t h s . Twenty m l o f enzyme p r e p a r e d by method B was a p p l i e d t o e a c h o f f o u r c o l u m n s i n 10 mM-p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g a) 0.05, b ) 0.10, c ) 0^20, o r d) 0.40 M - N a C l . E a c h c o l u m n was washed w i t h i t s e q u i l i b r a t i o n b u f f e r and e l u t e d w i t h 5 mM-n e o s t i g m i n e i n e q u i l i b r a t i o n b u f f e r . Columns a and b were t h e n e l u t e d w i t h e q u i l i b r a t i o n b u f f e r c o n t a i n i n g 1.0 M - N a C l . F r a c t i o n s (1 m l ) were a s s a y e d f o r A c h E a c t i v i t y (± . ^ ) a n d *280 ( ' — * > s a m p l e s . The h o r i z o n t a l d a s h e d l i n e s i n d i c a t e t he A o n s o f t h e a p p l i e d zoU 6ja Figure 25. Recovery of AchE from MAC-Sepharose 4B column by elution with a) a NaCl gradient (0.2-1.0 M) , b) acetylcholine (5 mM0 or c) a decamethonium gradient (0-50 m M ) T h i r t y ml samples in 10 mM-potassium phosphate buffer, pH 7.0, containing 0.2 M-NaCl were applied to^columns having a ligand concentration of 2.0 umol ml . Columns were washed, eluted with the appropriate eluant, and columns b) and c) were eluted with a NaCl gradient. Fractions (2 ml) were assayed for AchE activity (• • ) and A2gQ (• - — • ) • Profiles presented were duplicated. Figure 2 5 a 64 sample 30 60 buffer acetylcholine N a L elution volume (ml) Figure 2 5 c 65 NaCl gradient produced a broad peak of relatively low specific activity (45 units mg * protein) accounting for 51% of the loaded activity. Similar results were obtained using enzyme prepared by method A. No activity was recovered by substrate elution (Figure 25b). When a 0.2-1.0 M-NaCl gradient was subsequently applied, 68% of the bound activity was recovered. It was concluded that acetylcholine did not affect AchE binding. Elution with a decamethonium gradient resulted in a slow emergence of protein and a peak of AchE activity at 25 mM-decamethonium (Figure 25c). This peak was asymmetric and broad suggesting non-specific elution. Twenty one percent of the bound activity was recovered in this peak and subsequent elution with the NaCl gradient yielded additional activity accounting for a total of 39% recovery. Specific activity was 60 units mg * protein. Comparable results were obtained from decamethonium eluates that had not been dialyzed prior to assaying. There was no additional recovery of AchE when 40 mM-decamethonium was applied after elution of a l l columns with 1.0 M-NaCl during routine purification (Figure 6). In the purification of eel AchE, an inverse relationship was found between the bed volume and recovery (G. Webb, personal communication). To examine this relationship in the plant enzyme, a comparison was made between two columns — 0.5 and 2.0 ml of MAC-Sepharose 4B (Table VIII). Greater recovery was obtained from the 2.0 ml column. In one experiment (Figure 26) a delay of one day was allowed before application of the NaCl gradient. Most of the protein and a minor amount of AchE activity was released in the i n i t i a l fraction. The major 66 Table VIII: Recovery of AchE activity in successive buffer (10 mM-potassium phosphate with 0.2 M-NaCl),decamethonium (0-50 mM<) ,and NaCl, (0.2-1.0 M) gradient eluates as a function of bed volume of MAC-Sepharose 4B. Eluate activities are expressed as percentages of the applied sample volume. Values represent results from one of two duplicate experiments. Bed volume (ml) Buffer (%) Decamethonium (%) NaCl (%) 0.5 24 7 9 40 2.0 27 21 12 60 67 Figure 26, Recovery of AchE activity following delayed NaCl elution on a MAC-Sepharose 4B column. The column was developed as described for Figure 25 except one day passed between the completion of the buffer wash and the application of the NaCl gradient. AchE activity (• •) and A 2„_ (• • ) were measured. 68 AchE peak contained a greater specific activity (67 units mg * protein) than the corresponding peak in Figure 25a. b) Gel f i l t r a t i o n on Sepharose 6B Four major peaks of AchE activity were eluted when samples from both roots and hypocotyls prepared by method B were applied to a 25 X 50 cm Sepharose 6B column (Figure 27). No increase i n specific activity was achieved. The f i r s t peak eluted in the void volume; the others had the following values for Kav.: 0.48, 0.66 and 0.87. These value corresponded to mol. wts. of 1,000,000, 350,000, 70,000, and 10 000 daltons, respectively, applying the calibration curve of Figure 16, assuming that a l l species were globular. c) Ion exchange chromatography on DEAE-Sepharose CL-GB. Four major peaks of AchE activity were eluted from a DEAE-Sepharose CL-6B column (Figure 28). No substantial increase in specific activity occurred in any of these peaks. The elution profile suggests that there may be either isoenzymes or aggregates of AchE. having different ion exchange properties. There was no activity recovered from DEAE-Sepharose CL-6B when the column was operated at pH 7.2 and eluted with 1 mM-guanidinium or 1 mM-acetylcholine. The column was operated at pH 7.2 because any AchE having a pi of 7.0 would be loosely bound to the ion exchange resin thereby f a c i l i t a t i n g elution with the substrate (Scopes, 1977). The technique was not used to attempt to purify AchE having a pi of 5.3 because enzyme prepared by both methods (A and B) precipitated at pH 5.6 resulting in a loss of more than 90% of the enzyme activity. 69 Figure 27. E l u t i o n of AchE prepared by method B from Sepharose 6B. Fractions (5 ml) were c o l l e c t e d and assayed f o r AchE a c t i v i t y ( A A ) and A (° ° ). The p r o f i l e presented was reproduced i n a separate preparation. elution volume (ml) 70 Figure 28. Elution profile of AchE elutedjfrom DEAE-Sepharose CL-6B. AchE (540 units, 8.8 units mg protein) was applied to the column in 20 mM-potassium phosphate buffer, pH 7.0, containing 0.03 M NaCl. After the addition of 315 ml of buffer, an NaCl gradient 0.03 to 1.0 M was applied. Five ml fractions were collected and assayed for AchE activity (A A ) and A 0 R n ( ) . —I T " « « • • * ^ 200 400 600 800 elution volume (ml) 7. Physiological Role of AchE in the Hypocotyl a) Effect of Growth Regulators on AchE Activity in the Hypocotyl Hooks Experiments were designed to screen regulatory substances and white light for their effects on AchE activity in the hypocotyl hook. 3 -2 -1 There was no effect of white light (1 X 10 erg cm sec ) or Ethrel (100 ppm; 2-chlorosulphonic acid) on AchE activity or specific activity in hypocotyl hooks of 5-day old etiolated seedlings over a 4 day period (ie. 9 days after germination) (Figure 29). The Ethrel treated plants displayed the characteristic short hypocotyl and thick subapical region after one day. Kinetin (10 ^  M) had no effect u n t i l the fourth day at which time a significant (t^ ^ = 44.5, a = 0.01) decrease in specific _3 activity of AchE was observed (Figure 30). Gibberellin (10 M) treated plants showed a significant (t^ ^ = 28.4, a = 0.01) increase in AchE specific activity by the third day. By the fourth day plumular hooks of these and Ethrel-treated plants showed effects of tissue damage. The possibility that effects were an artifact of early stages of hook injury in Ethrel- and gibberellin-treated plants cannot be dismissed. b) The Effect of Acetylcholine on the Hypocotyl The effect of acetylcholine on the hook opening response of excised hypocotyl hooks was tested. The hook angle of the control hooks was -46.5 ± 2.9° after 20 h in darkness (Figure 31). This value does not agree with the control value of 0° reported for the Black Valentine variety of bean typically used in hook angle experiments (Klein, 1956); however, no significant difference (a = 0.01) was -3 -5 -7 observed between the angle of hooks incubated in 10 ,10 ,10 , or -9 10 M-acetylcholine and the control hook angle. Hooks exposed to red 72 Figure 29. The effects of light and Ethrel on specific activity of AchE in hypocotyl hooks of 5-day old etiolated P_. vulgaris. Plants were sprayed daily in dim green light with either water ( A - — - A ) or Ethrel (100 ppm; 2-chlorosuphonic acid) (• •) and l e f t in darkness, or with^water^and exposed to continuous light (2 X 10 erg cm sec ) (• • ) . Hypocotyl hooks (15 to 20) were excised daily un t i l 9 days after germination, homogenized, and assayed for AchE activity by the particulate assay procedure. Values are means of 3 experiments and error bars represent + or -.S.E.M. 2 3 4 clays after initial "treatment 73 Figure 30. The effects of gibberellin and kinetin on specific activity of AchE in hypocotyl hooks of 5-day old etiolated J?. vulgaris. Plants were sprayed daily in dim green light with either water (A A ) , 10 M-gibberellin (••.—: , or 10~Ttf-kinetin —-„•). and;. l e f t ;in. darkness. Hypocotyl hooks were excised jdaily .-.until, 9"-days-after germination, . homogenized .and "assayed - for AchE activity; .^Values are means of 3 experiments and error bars represent + or - S.E.M. 74 Figure 31. The e f f e c t of ac e t y l c h o l i n e on the hook angle of 20 h excised hypocotyl hooks of P_. v u l g a r i s . Values are means of 30 i n d i v i d u a l hooks for two pooled experiments and error bars represent ± S.E.M. log facetylchol ine] M 75 light had a f i n a l angle of 72.4 ± 19.8°. Seven-day old etiolated P_. vulgaris hypocotyls elongated to 130.7 ±0.8% and 130 ± 1.3% of their i n i t i a l lengths after 24 h in —6 darkness when sprayed with water or 10 M-acetylcholine, respectively. There was no significant difference (a = 0.01) between these values. _ . ;, D. DISCUSSION 1. Identity of-AchE in.'P.;..vulgaris. . _ Criteria for the (identification ..ofj^AchE activity in- plants have been established (Fluck and Jaffe, 1974a). They include inhibition by neostigmine and maximal activity against acetyl esters of choline as fundamental features of the enzyme. AchE activity that satisfied these c r i t e r i a was identified in P_. vulgaris. Acetylcholine hydrolysis in the presence of partially purified extracts was inhibited by neostigmine (Figure 2) at concentrations effective against AchE activity in animals (Augustinsson, 1960 and 1963) and plants (Riov and Jaffe, 1973). This property was exploited i n activity assays to correct for spontaneous or non-specific hydrolysis of acetylcholine and a l l of the esterase activity in preparations purified beyond (NH^^SO^ precipitation was neostigmine inhibitable. The neostigmine inhibitable hydrolysis of acetylthiocholine was related linearly to the volume of the extract which was assayed (Figure 4) and the assay time (Figure 3). The observation that acetylthiocholine was hydrolyzed three times as fast as either butyl- or propionyl-thiocholine (Figure 17) further supported the argument that this plant cholinesterase was an acetylcholinesterase. 76 Both acetylcholine and i t s thiocholine analogue used as the substrate in this study, have been reported to occupy the same position in substrate a f f i n i t y and hydrolysis rate hierarchies when compared to other acylcholine esters. Both animal (Ellman, et a l . , 1961) and plant AchEs (Riov and Jaffe, 1973) hydrolyze the acetylthiocholine more slowly than acetylcholine and have slightly higher Kms for the analogue. The possibility of microbial contamination of extracts as a source of AchE activity was considered because a cholinesterase was identified in the bacterium Pseudomonas fluorescens (Fitch, 1963; Laing, et a l . , 1967). The present study did not address this problem directly but the following observations negate this possibility. AchE activity was located histochemically within the cells of Phaseolus aureus roots (Fluck and Jaffe, 1974b) and was located in purified c e l l walls in this study. It was extracted in similar amounts from one experiment to the next, a feature not to be expected from contaminating elements. Callus tissue grown axenically produced activities comparable to those from the parent tissue. Surface ste r i l i z e d seeds germinated in st e r i l e petri plates produced seedlings having the same activity as their vermiculite-grown counterparts (R. A. Fluck, personal communication). A group of cholinesterases exists in plants which has a low Km for acetylcholine or acetylthiocholine (56-200,UM) and is inhibited by the carbamates neostigmine (10 M) or eserine (10 *M) (Tzagaloff, 1963; Riov and Jaffe, 1973; Fluck and Jaffe, 195!4d; Kasturi and Vasantharajan, 1976). Variations exist in the response to choline and the hydrolysis rate hierarchy. The AchE in this study and the enzymes studied by Riov and Jaffe (1973) and Kasturi and Vasantharajan (1976) have been purified beyond (NH,)„S0, precipitation. Only the bean enzymes were 77 stimulated by choline (Figure 19 and Riov and Jaffe, 1973). A l l of the cholinesterases which have been partially purified by (NH^^SO^ precipitation are inhibited by choline (Tzagaloff, 1963; Schwartz, 1967; Fluck and Jaffe, 1975). Both Schwartz (1967) and Kasturi and Vasantharajan (1976) studied enzymes from Pisum sativum root but the latter workers did not examine the effect of choline on their preparations. Cholinesterase activity was identified in 23 species from 5 families (Fluck and Jaffe, 1974d). Which of these cholinesterases are acetylcholinesterase remains uncertain because of low level of purity of these preparations and the undetermined substrate hydrolysis rate hierarchy of each enzyme. AchE was located in the roots of P_. vulgaris and tentatively identified in the hypocotyl. The hypocotyl enzyme was not purified beyond (NH^^SO^ precipitation and must be considered as a cholinesterase even though i t resembled the root enzyme in several respects (Figures 4, 21, and 27, and Table II). AchE has been identified and partially purified from roots of two other members of the Fabaceae (Riov and Jaffe, 1973; Kasturi and Vasantharajan, 1976). 2. Extraction and Localization .of AchE - • The highest :extractable . AchE. a c t i v i t i e s : have been reported x in roots. Leaves contained nearly as much as roots; and buds, hypocotyls, and stems contained the least (Fluck and Jaffe, 1974b and d). Activity measured in vulgaris tissue under different conditions was always greater in the roots but quantitation of activity proved d i f f i c u l t . Enzyme activities were measured in situ by assaying tissue slices (Table III) but values obtained by this method were slightly (in 78 hypocotyls) or substantially (in roots) lower than activities i n homogenates. This suggested that not a l l of the enzyme was accessible to the substrate and that the assay was not suitable for cholinesterase determinations in. situ. Fresh homogenates contained substantially lower activities than the same homogenates after dialysis against water (Table I ) . The increase of activity during dialysis is attributed in part to the presence of an endogenous dialyzable inhibitor (Table I ) . However, other factors removed during dialysis contributed to the low activity since return of concentrated diffusate did not reduce activity to the level detected before dialysis. The inhibitory agent in the hypocotyl was greater than in the root. Insufficient information is available to determine the nature or the significance of i t s effect. Kasturi and Vasantharajan (1976) found increased total AchE activity after (NH^^SO^ precipitation. Fluck and Jaffe (1974d) identified a 2-nitro-5-thiobenzoate decolorizing activity in buffer extracts of P_. aureus roots which was partially dialyzable and heat-inactivated. A similar decolorizing agent has been reported in sea urchin extracts (Wolfson, 1972) which was heat-inactivated and completely dialyzable. Regardless of the nature of the inhibitor values for AchE activity in fresh homogenate appear to be minimal. The presence of inhibitors of presumably variable quantity made assay of crude extracts d i f f i c u l t . Values were highly dependent on the extraction and assay procedures (see Tables I, II, and III; Figures 29 and 30) and no quantitative estimate was obtained for activities in situ. Enzyme activity was detected in c e l l walls extracted from P_. vulgaris hypocotyls. The result was expected since Fluck and Jaffe 79 (1974b) found that 95% of the AchE activity in P_. aureus roots was located in the c e l l walls. Jansen, et a l . , (1960) showed that c e l l walls were capable of binding soluble proteins which were not native components of the wall. The AchE activity was considered a real component of the c e l l wall because the lOmM-buffer insoluble activity was greater than 5% of the total activity. The 5% (NH^SO^ extracted 43% of the root acetylcholinesterase (Fluck and Jaffe's (1974b) value was 37%) and 81% of the hypocotyl enzyme. The different efficacies of this extractant i n roots and hypocotyls suggests that the localization or binding properties of AchE differed between these two organs. Ammonium sulphate also extracted a greater portion of AchE from aerial organs than from roots of light grown mung bean plants (Fluck and Jaffe, 1974b). The observation that only 57% of the light grown mung bean hypocotyl AchE (Table 6 in Fluck and Jaffe, 1974b), but 81% of the etiolated P. vulgaris hypocotyl enzyme was extraced further suggests that the developmental status of these plants affects the properties of AchE. These extraction data indicate that two populations of AchE (or cholinesterase) activity exist in the c e l l wall and they differ in their binding properties to the wall (Fluck and Jaffe, 1974b). At least five other c e l l wall enzymes exhibit this property and in the case of peroxidases, covalently and noncovalently bound populations of the enzymes have been proposed (Hall and Butt, 1968; Nevins, 1970; Ridge and Osborne, 1970; K l i s , 1971; Copping and Street, 1972). The selection of 5% (NH^SO^ to extract AchE from P. vulgaris was based on work of Riov and Jaffe (1973), and Fluck and Jaffe (1974b) 80 who found 4% (NH^^SO^ to be the most effective of 10 extraction media. This was also the most effective extractant used in the purification of autolyzed or trypsin-digested eel AchE (Leuzinger and Baker, 1967). 3. Purification and Characterization The use of 5% (NH,)^,S0. favored extraction of one - form ~of AchE. 4 2 4 -A total 79-fold' > purification of AchE was achieved by the use of (NH^^SO^ precipitation, gel f i l t r a t i o n and chromatography on MAC-Sepharose 4B. The purification shown in Table III yielded enzyme of 283 fold greater purity than previously reported (Riov and Jaffe, 1973). Greater yields (2.15 compared to 1.71 units g 1 fresh weight) were obtained by Riov and Jaffe (1973) but they achieved a lower specific activity (96 compared to 223 units mg 1 protein). The specific activity of the pea AchE was only 37.8 units mg 1 protein (Kasturi and Vasantharajan, 1976). Precipitation with (NH^^SO^ has been used in the purification of cholinesterases (Tzagoloff, 1963; Schwartz, 1967; Fluck and Jaffe, 1974d) and acetylcholinesterases (Kremzner and Wilson, 1963; Leuzinger and Baker, 1967; Dudai,*'-et^.al. , 1972b; Riov and Jaffe, 1973; Kasturi and Vasantharajan, 1976). By the use of gel f i l t r a t i o n in the purification protocol, a 5-fold increase in specific activity was achieved (220 units mg 1 protein (Figure 6) vs 45 units mg - 1 protein (Figure 25a)). For this reason i t provided a substantial contribution to the purification. The elution profile for gel f i l t r a t i o n on Sepharose 6B in the presence of 0.2M-NaCl contained one peak of AchE activity. This had a migration corresponding to a globular protein mol. wt. of 78 000 (Figure 16). However, the elution profile from gel f i l t r a t i o n of 81 preparations extracted directly with 5% (NH^^SO^ (method B) contained multiple peaks of the activity i n the absence of 0.2 M-NaCl (Figure 27). Gel f i l t r a t i o n of the mung bean root AchE prepared by a method similar to method A of this study (Riov and Jaffe, 1973) produced one peak of activity which was eluted in the void volume of a column of Sephadex G-200. This corresponded to a globular protein mol. wt. of 200 000. When that enzyme was prepared by the direct extraction of roots with 4% (NH^^SO^ the void volume peak occurred as well as one corresponding to a globular protein mol. wt. of 80 000. In both cases, gel f i l t r a t i o n was performed in 20mM-phosphate buffer (pH 7.0) in the absence of NaCl. This apparent inconsistency in gel f i l t r a t i o n results could be explained, in part, by analogy with animal AchE. Asymmetric forms of the eel AchE, which contain an elongated structure referred to in the literature as a t a i l (Dudai, et a l . , 1973; Reiger, ej: a l . , 1973), and the bovine erythrocyte AchE form large aggregates at low (<0.3) ionic strength (Rothenberg and Nachmansohn, 1947; Lawler, 1963; Changeux, 1966; Massoulie and Reiger, 1969; Dudai, et a l . , 1972a; Crone, 1973). The two peaks in Figure 27 correspond to globular protein mol. wts. of 350 000 and 1 000 000. These may represent 4x and 16x aggregates of the 78 000 mol. wt. species. These high mol. wt. forms would correspond to the peak of AchE activity excluded from Sephadex G-200 (Riov and Jaffe, 1973). An ionic strength dependent equilibrium may exist between these forms such that at a high (0.22) ionic strength a l l activity would exist in the 78 000 mol. wt. .(IX) form. At a lower (0.02) ionic strength some molecules would exist in higher (4X, 16X) aggregation states. Riov and Jaffe (1973) suggested that protease activity in (NH^^SO, extracts of 82 roots would account for the presence of the 80 000 mol. wt. form of AchE. They reasoned that most of the protease activity would be removed by the preliminary buffer extract and no degradation of higher mol. wt. forms would occur in the time between (NH^^SO^ extraction of the buffer insoluble material and gel f i l t r a t i o n . This proposal is supported by the observation that s t i l l lower mol. wt. forms appeared in the elution profile of material extracted directly with 5% (NH^^SO^ (Figure 27) but were absent in the profile of material extracted from the buffer insoluble residue (Figure 5). Proteolytic treatment of eel or torpedo AchE with trypsin or storage of electric tissue in toluene for months (Rothenberg and Nachmansohn, 1947) results in the formation of an active globular form of the enzyme with a sedimentation coefficient of 11 S (Dudai, e_t. a l . , 1972a; Rosenberry, et a l . , 1972; Taylor, et al., 1974) and a mol. wt. of 310 000-350 000 (Dudai, et a l . , 1973; Taylor, et a l . , 1974; Morrod, 1975) which is incapable of forming aggregates at low ionic strength (Massoulie and Rieger, 1969). Electron microscopy revealed the absence of the " t a i l " structure in this form (Rieger, et a l . , 1973). A thorough investigation of the behavior of bean root AchE on columns containing MAC-Sepharose 4B led to i t s use in the purification protocol. Of a l l of the conditions tested, those presented in Figure 6 produced the greatest recovery and specific activity. Even when the greatest binding of AchE occurred, the most effective eluant was. NaCl. Elution with a NaCl gradient resulted in a single broad peak of activity (Figure 25a). This suggested that the interaction between the ligand and the enzyme was of a non-specific nature. Had the interaction been 83 specific, a precise ionic strength would have been effective i n eluting a l l of the enzyme (Rosenberry, et a l . , 1972) and specific elution by decamethonium would have been observed. Use of chromatography with MAC-Sepharose 4B gave a 10-fold purification and this procedure was less effective than in the purification of eel AchE (Dudai, ejt a_l. , 1972a). The MAC-Sepharose 4B behaved as a true a f f i n i t y chromatography column against the eel enzyme by the c r i t e r i a of Cuatrecasas:and Anfinsen'(1971). The -inefficacy of acetylcholine to elute the enzyme (Figure 25b) suggested that the binding was not exclusive to the active center. Furthermore, when there was a delay before the application of NaCl, a portion of the AchE was eluted in the i n i t i a l volume suggesting a very weak association with the ligand. The observation that decamethonium eluted a smaller quantity of AchE in an equally broad and asymmetric peak (Figure 25c) suggests that the effect of this substance was also nonspecific and related to i t s contribution to ionic strength rather than i t s chemical properties. This is supported by the observation that A^QQ values increased gradually in proportion to the ionic strength of decamethonium (Figure 25c). It is known (Adams and Whittaker, 1950; Wilson and Bergman, 1950; Wilson, 1954; Koelle, 1970; Rosenberry, 1975) that the active center of the AchE from a variety of animal sources contains two primary binding sites — an esteratic site which contains a nucleophilic group specific for the acyl carbonyl and an anionic site which is attracted to the quaternary nitrogen of the choline and also contains a hydrophobic region. Studies with quaternary ammonium, carbamate, and organophosphate inhibitors suggested that the mung bean AchE active center resembles that 84 of the animal AchE (Riov and Jaffe, 1973). The results of my experiments add further support to this view. The enzyme from P_. vulgaris was inhibited by neostigmine (Figure 3) and the inhibition product remained inactive after dialysis suggesting the formation of a carbamy1-enzyme similar to that reported for carbamate inhibition of eel AchE (Wilson, 1954). The bean enzyme was inactivated by DIFP and remained inactive after exhaustive dialysis (Figure 7). Complete characterization of the active center of the enzyme must await further studies using strategies which have proven successful in eel AchE active center investigations. A major difference between the plant and animal enzymes is in their responses to eserine. Whereas the animal AchE is inhibited by 10 ^M-eserine (Augustinsson, 1963; Jackson and Aprison, 1966), the enzyme from roots is inhibited at 10 (Riov and Jaffe, 1973; Kasturi and Vasantharaja, 1976). The inhibition of eel AchE by eserine depends on the hydrophobic a f f i n i t y of: CHir CH~ because, unlike neostigmine, this substance lacks quaternary nitrogen. If the enzyme from roots lacks a substantial hydrophobic component in the anionic binding site, then eserine would not be as effective an inhibitor as of the animal enzyme. This may explain why greater N.-methylcacridinium concentrations were required to bind the AchE from bean roots than from eel tissue (Figure 23) but would not necessarily answer the question of the ligands apparent nonspecificity. On the other hand, i f N-methylacridinium binding was specific for peripheral anionic sites of the eel enzyme, such as those considered to be important CH '3 85 in the binding of decamethonium (Froede and Wilson, 1971; Rosenberry, 1975), then the absence of a corresponding site in the bean root AchE would explain both the non-specific binding of N-methylacridinium and the non-specific elution with decamethonium in both 0.2 and 1.0-M-NaCl. Massoulie and Bon (1976) have pointed out the importance of knowing the binding properties of the enzyme in the application of affinity chromatography to the purification of AchEs. Insufficient information is available pertaining to such properties of both _P. vulgaris and Electrophorus AchEs to resolve satisfactorily the inapplicability of this ligand to a f f i n i t y chromatography of P_. vulgaris AchE. It may be possible to purify the bean AchE by other a f f i n i t y chromatography ligands. The use of N-acyl-p-aminophenyltrimethylammonium (Dudai, est al_. , 1972b; Rosenberry, et a l . , 1972) appears to be especially promising because of the similarity of i t s structure to that of neostigmine. The results of electrophoresis in 7% acrylamide gels showed that the AchE was not purified to homogeneity (Figure 8). Other acetyl-cholinesterases migrate slowly in 7-7.5% acrylamide gels (Dudai, et a l . , 1972b; Chen, et a l . , 1974; Steele and Smallman, 1976; Das, et a l . , 1977). The presence of two bands after gel f i l t r a t i o n (Figure 8a) in the v i c i n i t y of the enzyme activity suggests that more than one active form of the enzyme may exist in the (NH^^SO^ extracts. The gels were not sliced to detect enzyme activity following gel f i l t r a t i o n because the bands were too close together to be resolved. Two bands of AchE activity were observed in acrylamide electrophoregrams of pea root extracts after gel f i l t r a t i o n on Sephadex G-200 by Kasturi and Vasantharajan (1976). They did not report the location of these bands. Closely migrating 86 multiple bands have been observed in the eel (Chen, e_t a_l. , 1974), house fl y head (Steele and Smallman,.1976), and human erythrocyte (Das, et a l . , 1977) AchEs. Electrophoresis of the globular 11 S form of the eel enzyme appeared as two bands of enzyme activity only one of which was detectable using protein stain (Chen, et al.., 1974). An elegant study involving gel f i l t r a t i o n , sedimentation, and electrophoresis revealed that the different forms of the f l y head AchE were not simply oligomers of the smallest active species (Steele and Smallman, 1976) . In the case of the erythrocyte enzyme, a single peak of activity was eluted from Sephadex G-200 in the void volume. This peak was resolved into four unique bands by gel electrophoresis (Das, et a l . , 1977). The protein contaminant, R^=0.69, (Figure 8b), could be a non-active component of the AchE. Das, et a l , , (1977) observed that the single active peak from gel f i l t r a t i o n was separated as one peak of activity on DEAE-cellulose. When this was rechromatographed with smaller mol. wt. protein peaks lacking activity, at least two active AchEs appeared i n the DEAE-cellulose elution profile. This supports the existence of either degradation products or a non-AchE matrix which could reassemble with the active enzyme into an AchE having different charge properties. Support of the existence of root.AchEs differing in charge properties is apparent from Figure 28. The nature or significance of the major contaminating protein remains undetermined but i t could represent a structure similar to the one reported by Das, et a l . (1977) because the conditions of gel electrophoresis differed from the conditions of purification. The contaminant may represent a protein not associated with the AchE. The isoelectric point of AchE was at pH 5.3. The enzyme precipitated with a loss of greater than 90% of the activity at this pH and pH 5.6. 87 These observations account for low recoveries from thin dextran layers (Figure 13) and the isoelectric focusing column (Figure 14)}and explains why I did not use isoelectric focusing in the purification protocol. This property also prevented the successful application of affinity elution chromatography (Scopes, 1977). The peak at pH 7.0 (Figure 14) is d i f f i c u l t to explain. The values in the peak resulted from lower values in the absence of neostigmine which typify AchE activity. The autolyzed 11 S globular form, of the eel enzyme has a p i of 5.3 (Leuzinger, et a l . , 1968) or 4.5 (Chen, et a l . , 1974). Morrod (1975) has reported variable values (pI=3.6-4.9) for the asymmetric forms of that enzyme. Precise values for the forms of the erythrocyte AchE are unavailable but pis range from 3-r5.3 (Das, et a l . , 1977). The var i a b i l i t y of these values probably reflects the instability of a l l AchEs.at their isoelectric points as well as variation i n the isoelectric points of the enzyme from different sources. The low p i found in both this and other AchEs suggests the presence of many negatively charged groups. It is lik e l y that both the amino acid and carbohydrate composition of the purified bean AchE w i l l reveal the source of negatively charged groups and explain the anion exchange behavior with the affinity ligand. The results of the isokinetic sedimentation indicate the presence of a single major sedimenting particle having S=4.2 (Figure 12a). This value used in conjunction with the Stokes radius (4.00 nm) yielded a mol. wt. of 76 000. This approximated the mol. wt. of 78 000 obtained (Figure 16) by assuming that the AchE had a globular structure. An additional calculation yielded the f r i c t i o n a l ratio (f/f~=1.37).. This 88 value is only slightly greater than an average globular protein value of 1.2 (Lehninger, 1970) and provides further evidence as to the globular nature of this enzyme. The accuracy of these values i s limited by the assumption that the partial specific volume (v) of this enzyme is equivalent to that of the eel AchE as reported by Bon, et a l , , (1973). 3 -1 Their value (v=0.75cm g ) corresponds with an average protein value but 3-1 3-1 differs from other values of 0.714 cm g and 0.793 cm g for the 11 S form of the eel AchE (Bon, et a l . , 1976) and Lubrol extracted erythrocyte AchE (Beauregard and Roufogalis, 1977), respectively. The high value obtained i n the latter case was attributed to the integral association of phosphoiipidiwithi the..enzyme. The .usefof /these avalues..resulted; in mol. wts. of 67 000 and 92 000, respectively. The use of the value of the AchE obtained during i t s purification rather than the use of another K by rechromatography of the purified enzyme may have led to clV additional errors. However, the accuracy of the calculated mol. wt. is further supported by the similarity of the mol. wt. obtained by SDS gel electrophoresis (77 000, Figure 10) and the glubular protein mol. wt. obtained by gel f i l t r a t i o n (78 000, Figure 16), and the observation that K & v was reproducible to within 0.01. An important observation of the sedimentation studies was the presence of a single t r i t i a t e d particle (Figures 12b and 12c). This 3 particle was established as H-DIP-AchE since AchE was stoichiometrically 3 inactivated by H-DIFP. The greater sedimentation coefficient of this particle than of the active AchE may have been a function of an altered conformation of the enzyme because a more globular structure would have shown a greater sedimentation coefficient. 89 Radioactive DIFP labelling strategies have been used to detect catalytic subunits i n either AchE purified to homogeneity (Dudai and Silman, 1974; Rosenberry, et a l . , 1974) or contaminated preparations in which the AchE was protected with butylcholine against activation by non-radioactive DIFP followed, after dialysis, by exclusive AchE labelling with radioactive DIFP (Cohen et a l . , 1967; Bellhorn, et a l . , 1970; Berman, 1973). The latter strategy was attempted in this study. Although butylcholine did offer protection against inactivation, the protection was incomplete under the two conditions tested. An alternative method was examined by labeling in the presence or absence of 3 neostigmine. Neostigmine was ineffective in preventing H-DIFP labeling (Table V, Figure 12). This may have resulted from incomplete carbamylation of AchE by 125 uM-neostigmine, phosphorylation of some of the carbamylated enzyme, or a more rapid decarbamylation of the enzyme in the presence of DIFP. In any case, neostigmine did not seem to act as an appropriate control for DIFP labeling of AchE. The observations that DIP-AchE sedimented as a single particle corresponding to a slightly more globular form of AchE, that no other t r i t i a t e d particles were observed in the sedimentation gradients, and that the preparations were free from contaminating esterase activity led to the conclusion that DIP-AchE was a suitable form of the enzyme in which to detect catalytic subunits. The similarity of the value of 77 000 obtained from SDS gel electrophoresis (Figure 10) and the mol. wt. obtained by gel f i l t r a t i o n (Figure 16) suggested that this was a trace of the active form of the AchE undenatured by SDS. However, denaturation yielded a major catalytic 90 subunit having a mol. wt.- of 61 000. Much of the proteinaceous material which centered at 16 000 probably corresponded to the major contaminating protein observed in polyacrylamide gel electrophoresis and isoelectric focusing. The observation that no t r i t i a t e d peak corresponded to the 16 000 mol. wt. protein (Figures 10a and 11a) supports this statement. The 26 000 mol. wt. shoulder (Figures 10a and 11a) corresponding to a radioactive peak suggested that this represents a second subunit of the AchE. whether this represented a catalytic subunit is d i f f i c u l t to t e l l . The observation that, unlike the 61 000 mol. wt. subunit, the radioactivity was slight compared to the protein content suggests that i t is not catalytic. From the results of preparations containing protein contaminants i t is d i f f i c u l t to assess the significance of the 17 500 mol. wt. radioactive peak, however, i t may represent a degradation product of the .61 000 or 26 000 mol. wt. subunit. With the appearance in reduced samples of radioactivity corresponding to a 30 000 mol. wt. component and a concomitant decrease of activity in the .61 000 dalton component (Figures 10b and.lib) i t is tempting to suggest that two 30 000 mol; wt. catalytic subunits are linked by a. disulphide bond to form the .61 000 mol. wt. subunit of the active AchE. This observation is tentative because complete reduction was not observed. Conditions must be found in which the 61 000 mol. wt. peak is completely eliminated and replaced by a 30 000 mol. wt. peak. Such conditions were not established i n this study. A tentative subunit structure of the bean AchE can be postulated based on the foregoing information (Figure 32). 90a F i g u r e 3 2 . P o s t u l a t e d s t r u c t u r e o f P . v u l g a r i s A c h E . E O > 4—« O CO "O CD CO c c CO Q CT) + c 1 o o ' -4—' o 1_ __ "O > CD k_ CD O .CD _r a) Q. 73 + E o CO o c Q CO + active centers ~80 000 s 80 000 -20 000 f t - J -60 000 - 8 0 000 HSH •20 000 -60 000 HS-I ! -30 000 91 A remarkable similarity exists with the subunit composition of animal AchE. Upon denaturation of the globular (11 S) form of the enzyme, four forms were observed representing tetramer, trimer, and dimer of a monomer having a mol. wt. of 70 000-82 000 in eel (Millar and Grafius, 1970; Dudai and Silman, 1971; Chen, et a l . , 1974; Dudai and Silman, 1974; . Morrod, \1975; Rosenberry, 1977) and 78 000-82 000 in torpedo (Taylor, e_t al. , 1974). When the eel enzyme was reduced in the presence of 2-mercaptoethanol or dithiothreitol, bands appeared having mol. wts. of 75 000-82 000, 50 000-59 000, 25 000-28 000, and 23 000 (Chen, et a l . , 1974; Dudai and Silman, 1974; Morrod, 1975; Rosenberry, ejt a l . , 1974; Rosenberry, 197^.)..Molecular weightrvalues are •" c suspected to vary because of the glycoprotein nature of the AchE (Rosenberry, 1977). A model has been proposed for the subunit structure of the eel AchE on the basis of these and other observations (Figure 33). The model accounts for the presence of a l l fragments except the smallest one which is suspected to be a degradation product of the 25 000 mol. wt. form (Morrod, 1975). In the erythrocyte ghost AchE, the smallest active isolable component has a MW of 200 000 which appears to consist of a dimer with subunits having mol. wts. of 126'000 and 75 000 (Berman, 1973) and containing an integral phospholipid component (Beauregard and Roufogalis, 1977). In the case of the house f l y head, the smallest active component had a mol. wt. of approximately 80 000 but no subunits studies have been undertaken (Steele and Smallman, 1976). Because of the inefficacy of neostigmine i n the prevention of DIFP labeling discussed above, the best estimate of catalytic center activity Figure 33. Schematic model f o r the 11 S form of e e l AchE from Dudai and Silman (1974). £ o o <n T3 0 .-.-. .>» 3 B s ? 3 I COOM c _ _ A A - s - s -MW= 3 30H " >^ 1 1 2 0 - 3 5 0 , 0 COOM COOM A A | y - s - s -NK, NH, 00 H \ MW = r*1 w Y c -1? - s - s --S—3-OOH c< A V T hi 3 30M • 20-350.C Y C )0 1 »2 COOM A - s - s -VI - s - s -_.0H NM2 0 m o 05 U"! 1 l/l COO" c A A - s - s -v n ~I65,00C v» i i-»2> > MW = N i Y c H ? . - s - s -- s - s --I63.C c A T-rj )0 OOM H, 0 N CCCi-' 8 0 , 0 0 0 i o © c> o . o u « E i C Q . n o CCOH CO'JM HS -A A 5H * v V w t v V H S 1 -SM - SH MS • IT- W MW= ~ 8 0 , 0 O O ; ~ 6 O P O O , ~ 23,000 COUH zoo* wf yvy Uv MW= . ~ 8 0 , 0 0 0 ; ~ 6 0 , 0 0 0 , ~ 25 ,000 93 ( F l o r k i n and S t o l t z , 1973) was o b t a i n e d by DIFP l a b e l i n g i n t h e absence o f n e o s t i g m i n e ( T a b l e V ) . T h i s v a l u e (197 m o l o f s u b s t r a t e m i n ^mol 1 D I F P ) was n e a r l y 5000 t i m e s l e s s t h a n t h e v a l u e f o r t h e e e l A c h E (960 000 m o l s u b s t r a t e m i n ^mol 1 a c t i v e c e n t e r , R o s e n b e r r y , 1975). The a c c u r a c y o f t h i s e s t i m a t e was l i m i t e d by t h e p u r i t y o f t h e p r e p a r a t i o n 3 and t h e a c c u r a c y o f t h e s p e c i f i c a c t i v i t y o f H - D I F P . A r i g o r o u s t e s t o f t h i s v a l u e a w a i t s t h e d e v e l o p m e n t o f a s u i t a b l e c o n t r o l f o r n o n A c h E l a b e l i n g by D I F P . ' The maximum t h e o r e t i c a l s p e c i f i c a c t i v i t y was e s t i m a t e d f r o m t h i s c a t a l y t i c c e n t e r a c t i v i t y by d i v i d i n g t h e c a l c u l a t e d DIFP b i n d i n g (mol DIFP mg 1 p r o t e i n b y t h e p o s t u l a t e d maximum b i n d i n g o f DIFP a s s u m i n g t h e 61 000 m o l . w t . s u b u n i t c o n t a i n e d o n l y one a c t i v e c e n t e r p e r m o l e c u l e (mol DIFP m o l ' ' "AchE). B e c a u s e t h e p r e p a r a t i o n s c o n t a i n e d 210 u n i t s mg 1 p r o t e i n and t h e b i n d i n g c o r r e s p o n d e d t o an " e q u i v a l e n t w e i g h t " o f 950 000, a maximum t h e o r e t i c a l s p e c i f i c a c t i v i t y f o r t h i s enzyme w o u l d be 3270 u n i t s mg 1 Lowry p r o t e i n . B o t h a p u r e r p r e p a r a t i o n and a more a c c u r a t e o r more s p e c i f i c t i t r a t i o n o f a c t i v e c e n t e r s w o u l d be r e q u i r e d t o o b t a i n c a t a l y t i c s u b u n i t e q u i v a l e n t w e i g h t s and t h e r e b y e s t a b l i s h w h e t h e r t h e 61 000 d a l t o n s u b u n i t was t h e o n l y c a t a l y t i c s u b u n i t . The l o w c a t a l y t i c c e n t e r a c t i v i t y o f t h i s enzyme compared t o t h a t o f t h e e e l s u g g e s t s t h a t t h e r e q u i r e m e n t s f o r r a p i d h y d r o l y s i s o f a c e t y l c h o l i n e i n t h e s e o r g a n i s m s d i f f e r t r e m e n d o u s l y . T h i s i s n o t s u r p r i s i n g c o n s i d e r i n g t h e r e l a t i v e l y h i g h q u a n t i t i e s and e s t a b l i s h e d r o l e o f a c e t y l c h o l i n e i n t h e e l e c t r i c o r g a n ( F l o r e y , 1966) and t h e l o w q u a n t i t i e s o b s e r v e d i n p l a n t s (Har tmann and K i l b i n g e r , 1974; W h i t e and P i k e , 1974). 94 The Km of P_. vulgaris root AchE was 56 yM (Figure 18) for acetylthiocholine. Other Kms have been reported: P. aureus root AchE i 1 had a Km of 84 yM (Riov and Jaffe, 1973); Pisum sativum root AchE had a Km of 200 yM (Kasturi and Vasantharajan, 1976). The Kms of animal AchEs for acetylcholine vary depending on the ionic strength and range from 90-130 yM in Torpedo and 230-340 yM in Electrophorus , (Massoulie and Bon, 1976). No consideration has been given to ionic strength during Km determinations in this or other plant AchEs. Such studies may reveal interesting properties of the enzyme which would f a c i l i t a t e i t s purification. Furthermore,no rigorous kinetic studies have been performed on any plant cholinesterases. The response of this enzyme to butyl- and propionylthiocholine (Table VI) was similar to that observed by Riov and Jaffe (1973) although they did not determine the effect of variable concentrations of these substrates on the enzyme activity. In this study, Michaelis-Mentin kinetics were not observed for either of these two substrates -5 -2 between 10 and 10 M (Figure 17). Unfortunately, a control containing no substrate was not included in this experiment, although extrapolation of the curves to zero substrate suggests a peculiar phenomenon. The response to propionyl- or butylthiocholine suggests that the use of neostigmine in the assay with propionyl- or butylthiocholine was inappropriate. The true zero for these substrates may correspond to AA4I2 of approximately 0.1,in which case the actual specific activity for these two substrates in Table VI approaches zero. Riov and Jaffe (1973) have demonstrated that at a substrate concentration of 0.5 mM, neostigmine inhibited the hydrolysis of a l l three substrates equally. This may not have applied to a l l substrate concentrations however. 95 Alternatively,the enzyme may become saturated at a substrate concentration below 10 M^. This >seems unlikely i n view of the substrate inhibition demonstrated by acetylcholine. Kinetic studies using a different assay method would be required to resolve the problem of substrate a f f i n i t y . A reevaluation of relative hydrolysis rates and specificity for other esters (Riov and Jaffe, 1973) would be necessary considering the tentative status of hydrolysis rate data for acetyl-, butyl-, and propionyl-thiocholine. 4. Physiological Role of the Acetylcholine/Acetylcholinesterase System. The possibility that, developmental -changes are correlated with changes in the status of AchE was suggested earlier i n this discussion. Fluck and Jaffe (1974b) observed that light grown mung bean stems have a 10-fold greater extractable AchE activity than etiolated hypocotyls. This suggested the possibility that light may mediate AchE levels as well as acetylcholine levels. The AchE activity in hypocotyls of 5-day old etiolated seedlings exposed to continuous, relatively low intensity white light increased by the same amount over a four day period as the etiolated controls (Figure 29). The increase of activity in etiolated controls was comparable to that observed by Fluck and Jaffe (1974b) for total activity in mung bean roots and by Kasturi and Vasantharajan (1976) for extractable activity in pea roots. Such values show that as the hypocotyl develops,it too increases i t s AchE activity. The inefficacy of 2-chlorosulphonic acid, an ethylene generating growth regulator applied i n concentrations which were effective i n inducing the " t r i p l e response" symptoms (Neljubow, 1901), in altering AchE levels (Figure 29) suggests that any role that AchE may have in the 96 hook opening response is not affected by ethylene. This could mean that the inhibitory effect of acetylcholine on ethylene generation and subsequent hook closure (Parups, 1976) is independent of the AchE level in the tissues, although no conclusive evidence has been obtained. -4 -3 Kinetin at 10 M inhibits hook opening and GA at 10 M enhances opening (Kang and Ray, 1969). It is interesting that these regulators have opposite and s t a t i s t i c a l l y significant effects on AchE of etiolated P_. vulgaris hypocotyls (Figure 30) . The time course involved in this effect is substantially longer than the 8-12 hours required for the i n i t i a t i o n of hook opening in excised hooks. Based on the time course of the response, any regulation of acetylcholine in the hook opening response would be attributed to constitutive enzyme activity and the variation in AchE activity more lik e l y would be linked with a longer term developmental change. Considering the observation that AchE activity increased with the age of the tissue, i t is conceivable that AchE may be involved in the actual process of aging i t s e l f . The observation that kinetin, a growth regulator active in counteracting many senescent processes (Sacher, 1973) reduced AchE activity in hypocotyls (Figure 30) supports this idea. Even though a l l growth regulators studied in this project were tested at concentrations in which they are effective in inducing hook opening responses, the possibility that other concentrations may exert different effects cannot be discounted, nor can the possibility that the concentrations effective in the excised hook opening assay differ from optimal concentrations of sprayed applications to intact plants. 97 Parups (1976) observed that acetylcholine partially prevented the IAA-promoted delay of hook opening and inhibited IAA-induced ethylene production. By these actions, acetylcholine appears to act as an antagonist of IAA thereby preventing ethylene from becoming available to induce hook closure. Parups (1976) postulated further that the action of acetylcholine was in mimicking the effect of red light on IAA concentration and ethylene production. On this basis i t would be likely that at some concentration, acetylcholine alone would enhance hook -9 opening. This was not observed between the concentrations of 10 and -3 —6 10 M (Figure .31) nor was any elongation response induced by 10 M acetylcholine. In view of the increasing number of observations of acetylcholine effects on assorted plant processes (Dekhuijzen, 1973; Grerrin, et a l . , 1973; Penel, et a l . , 1976; Sharna et a l . , 1977) and i t s endogenous existence (Jaffe, 1970; Hartmann and Kilbinger, 1974), i t would not be surprising to find that more plant responses or developmental changes than secondary root formation were mediated by acetylcholine. The results of this portion of the study are inconclusive but point the way to an obvious area of study in developmental biology. E. SUMMARY Acetylcholinesterase activity was studied to identify, purify and characterize the enzyme which may be responsible for regulating acetylcholine levels in plant tissue and to assess the role of the enzyme activity in plant tissues. A unique AchE was identified i n etiolated P_. vulgaris roots and tentatively identified i n the hypocotyl by use of a colorimetric assay 98 which included the cholinesterase i n h i b i t o r neostigmine as a c o n t r o l . A form of the AchE was extracted with 5% (NH^^SO^ from a b u f f e r i n soluble residue of root t i s s u e and p u r i f i e d to a s p e c i f i c a c t i v i t y of 210±20 units mg 1 protein-greater than twice the previously reported maximum. This preparation contained one major contaminating protein which may or may not have been associated with the a c t i v e AchE but did not contain any other esterase a c t i v i t y . The requirement of high p u r i t y has been established as a necessity f o r the d i s t i n c t i o n between cholinesterase and acetylcholinesterase a c t i v i t i e s i n plant t i s s u e s . An u n i d e n t i f i e d d i a l y z a b l e i n h i b i t o r of AchE a c t i v i t y was located i n buffer homogenates of both root and hypocotyl tissues and the p o s s i b i l i t y of a non-dialyzable i n h i b i t o r was proposed. The presence of i n h i b i t o r s i l l u s t r a t e d the need f o r a d i f f e r e n t assay to e s t a b l i s h quantitative l e v e l s of enzyme a c t i v i t y i n s i t u . The existence of other forms of AchE including a 4x and possibly a 16x aggregate..-of the p u r i f i e d AchE was demonstrated and i t was suggested that both aggregates and iso enzymes might e x i s t . The major f r a c t i o n of the hypocotyl enzyme was located i n the c e l l w a l l . P h y s i c a l and c a t a l y t i c properties of the root AchE were studied and are summarized i n Table IX. Many of these properties are s i m i l a r to those of the AchE from various animal sources. A subunit structure has been t e n t a t i v e l y proposed on the basis of r e s u l t s from SDS gel electrophoresis and K measurements. Of the information obtained i n t h i s and two av previous studies of plant AchEs, i t was concluded that although c a t a l y t i c properties of t h i s enzyme d i f f e r between plants and animals, s t r u c t u r a l T a b l e I X . Summary o f P h y s i c a l P r o p e r t i e s o f P_. v u l g a r i s A c h E P h y s i c a l p a r a m e t e r (Method) V a l u e S „ _ ( s e d i m e n t a t i o n v e l o c i t y ) 4.2 ± 0.1 S 20 w J M o l . w t . ( g e l f i l t r a t i o n and s e d i m e n t a t i o n v e l o c i t y ) * 76 000 ± 2 000 M o l . w t . (SDS g e l e l e c t r o p h o r e s i s ) 77 000 ± 2 000 S u b u n i t m o l . w t s . (SDS g e l e l e c t r o p h o r e s i s ) 61 000 ± 2 000 (2 X 30 000 ± 1 000) 26 000 ± 2 000 S t o k e s r a d i u s ( g e l f i l t r a t i o n ) 4.00 nm f / f Q * 1.37 C a t a l y t i c c e n t e r a c t i v i t y (DIFP t i t r a t i o n ) 197 + 5 mol . . s u b s t r a t e m i n m o l a c t i v e c e n t e r I s o e l e c t r i c p o i n t 5.3 ± 0.1 3-1 * v = 0.75 cm g (assumed) properties are remarkably s i m i l a r . The conclusion that t h i s enzyme i s involved i n the hydrolysis of acetylcholine to the exclusion of other esters i n s i t u cannot be drawn from e x i s t i n g data. I t was suggested that AchE may not be important i n the short.term r e g u l a t i o n of acetylcholine i n such responses as hook opening but may be involved i n regulation of acetyl c h o l i n e l e v e l s over longer time periods and may thus be important f o r normal plant development. BIBLIOGRAPHY Chapter I Adams, D. H. and V. P. Whittaker. 1950. The cholinesterases of human blood. II. 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Rapid r e s p i r a t o r y changes due to red l i g h t or acetylcholine during the early events of phytochrome-mediated photomorphogenesis. Plant P h y s i o l . 49:1-7. CHAPTER II THE ABSENCE OF NUCLEOPHILIC SITES IN THE CELL WALLS OF ETIOLATED PHASEOLUS VULGARIS L. HYPOCOTYLS AND ITS RELATION TO CELL ELONGATION A. INTRODUCTION Plant c e l l elongation has been a topic of interest for decades and has been extensively reviewed (Heyn, 1940; Setterfield and Bayley, 1961; Frey-Wyssling, 1962; Wilson, 1964; Lockhart, 1965; Morreuand Eisinger, 1-968; Cleland, 1971; Preston, 1974). It is generally agreed that 1) turgor pressure is a driving force for c e l l extension, 2) c e l l extension involves changes in the viscoelastic properties of the primary c e l l wall resulting i n reduced c e l l wall resistance to stress, and 3) deposition of c e l l wall polymers is required for continuation of c e l l extension. However, the arrangement of c e l l wall polymers before, during, and after c e l l extension, the cause and the molecular location of the viscoelastic changes, and the controls which operate upon c e l l extension remain uncharacterized. An understanding of the arrangement and linkages of c e l l wall r 0 polymers is necessary for characterization of the structural changes which occur during c e l l wall extension. The primary c e l l wall of higher plants consists of cellulosic microfibrils surrounded by an apparently amorphous matrix of hemicelluloses, pectic acids,.and both enzymatic and structural proteins. Our present understanding of the primary c e l l wall structure is based upon observations using such techniques as polar and 110 interference microscopy (Ruge, 1937; Diehl, et a l . , 1939), x-ray diffraction (Meyer and Misch, 1936; Berkeley and Kerr, 1946), electron microscopy (Frey-Wyssling, ejt a l . , 1948; Heyn, 1966), histochemistry (Reis and Roland, 1974), chemical (Selvendran, et a l . , 1975; Monro, et a l . , 1976) and enzymatic (Karr and Albersheim, 1970) degradation, and chemical analysis (Bauer, et al., 1973; Talmadge, est al_., 1973). In lateral primary c e l l walls of many c e l l types (Roelofsen, 1951; Wardrop, 1956; Setterfield and Bayley, 1958; Preston, 1974) the orientation of microfibrils on the inner wall surface is perpendicular to the major c e l l axis becoming parallel as the c e l l extends and more material is added to the existing c e l l wall. Thus, according to the multinet growth hypothesis (Roelofsen and Houwink, 1951) , movement of the microfibrils with respect to the matrix comprises a major event during c e l l extension. But microfibrillar movement depends upon a recorganization of the matrix accompanying reduced c e l l wall resistance to stress (Barnicki-Garcia, 1973; Preston, 1974). It is postulated that bonds either are broken, newly formed, or both broken and reformed during c e l l wall extension. Recently, specific linkages have been examined with respect to their susceptibility to cleavage or formation under conditions which increase, decrease, or terminate c e l l wall extension. These linkages include hydrogen bonding involving the xyloglucan component of hemicellulose (Labavitch and Ray, 1974), arabinosyl-hydroxyproline (Lamport, 1965; Lamport, 1970) and galactosyl-serine (Lamport, et a l . , 1973; Cho and Chrispeels, 1976) of the c e l l wall glycoprotein, g-1, 4-glucosyl linkages in the cellulose (Wong, et a l . , 1977), and ferulic 112 acid-polysaccharide esters (Hartley, e_t a l . , 1976). Linkages involved in the glycosylation of c e l l wall protein are formed during biosynthesis of c e l l wall components before export and incorporation into the c e l l wall (Chrispeels, 1976). However, i f the cross-linking of c e l l wall glycoprotein that is believed to accompany the cessation of elongation (Lamport, 1965; Sadava, et d . , 1973) occurs between pre-existing c e l l wall protein and newly exported c e l l wall polysaccharides, then glycosylation would also occur in the c e l l wall. The probability of such a modification occurring in the c e l l wall would be supported i f there were some sites in the glycoprotein which were not glycosylated in young actively extending c e l l walls but were found glycosylated in older, f u l l y extended c e l l walls. Positive evidence for such sites would be obtained i f the sites were reactive toward an a r t i f i c i a l modification reagent and i f the reagent could be incorporated into preparations of actively elongating c e l l walls. A failure to detect binding would suggest the absence of suitably reactive sites.- Diisopropylfluorophosphate (DIFP) could act as such a reagent. DIFP has been widely used in protein modification and enzyme inhibition studies (Cohen, et a l . , 1967). In the reaction: ( C H J 2 C H - 0 ^ // ( C H 3 ) 2 C H - 0 ^ P + H-PROTEIN / PROTEIN <» (CH 3) 2CH - 0 F ( C H3 }2 C H ~ ° + lP + F © the electrophilic phosphorus reacts with a nucleophilic group of the protein leading to a departure of F© and formation of a diisopropyl-phosphoryl-(DIP-)protein. A major product of partial acid hydrolysis of most DIP-proteins is phosphoserine (Schaffer, et a l . , 1953; Schaffer, 113 et a l . , 1954; Cohen, et a l . , 1967) i n d i c a t i n g that the serine hydroxyl group i s the nucleophile i n r e a c t i o n (1). As hydrolysis procedes, the phosphate ester i s also cleaved y i e l d i n g orthophosphoric a c i d . In the case of DIFP i n h i b i t i o n of acid phosphomonoesterase, no phosphoserine was recovered, i n d i c a t i n g that other n u c l e o p h i l i c s i t e s could react with DIFP (Greenberg and Nachmansohn, 1965). The purpose of t h i s study was to examine the c e l l walls of hypocotyls of the bush bean (Phaseolus v u l g a r i s L.) f o r the presence of serine residues which were reactive by n u c l e o p h i l i c s u b s t i t u t i o n . The bean hypocotyl o f f e r s a developmental continuum of elongation stages from the a p i c a l region consisting of non-elongated c e l l s to the basal region consisting of elongated c e l l s (Bailey and Kauss, 1974). By sampling regions along t h i s continuum, the a p p l i c a t i o n of exogenous s t i m u l i to induce or terminate elongation i s avoided. The growth patterns of the c e l l types present conform to the multinet growth hypothesis with the exception of the epidermal c e l l s (Bayley, et a l , , 1957). Though the g l y c o s y l linkages to c e l l w a l l protein have not been examined i n P_. v u l g a r i s , compositional studies of the primary c e l l walls derived from t h i s plant (Wilder and Albersheim, 1973) suggest that i t i s s i m i l a r to other members of the Dicotyledonae whose g l y c o s y l linkages have been studied. In t h i s study, phosphoserine was analyzed following the reaction of DIFP with a) serine, b) a-chymotrypsin, and c) c e l l walls of elongating and f u l l y elongated c e l l s from e t i o l a t e d bush bean hypocotyls. A comparison was made between phosphoserine recovered from c e l l walls labeled with 3 2P-DIFP and c e l l walls labeled with 3 2P-DIFP following a pretreatment with non-radioactive DIFP to correct for non-specific labeling or adsorption of the radioisotope. This comparison led to the identification of serine labeling in c e l l wall proteins under.conditions which yielded a nearly complete modification of serine in the a-chymotrypsin active center. Reactivity of a-chymotrypsin and the c e l l walls toward a spin-labeled analogue of DIFP (2,2,6,6-hydroxytetram-ethylpiperidinooxyl monoethylfluorophosphate (HTMFP)) was also examined. B. MATERIALS AND METHODS 1. Chemicals Supplies were obtained from sources as indicated: DIFP: Aldrich 32 32 Chemical Co., Milwaukee, Wi.; P-orthophosphoric acid and P-DIFP: Amersham/Searle, Arlington Heights, II.; HTMFP: Syva Assoc. Inc., Palo Alto, Ca.; cellulose powder: W. & R. Balston Ltd., England; ethylene glycol monomethyl ether (methylcellosolve) and ninhydrin: Pierce Chemical Co., Rockford, II.; Folin-Ciocalteu phenol reagent: Harleco, Philadelphia, Pa.; trichloroacetic acid (TCA), p-terphenyl, l,4-bis-2-(5-phenyloxazolyl)-benzene and National Bureau of Standards calibrated HC1: Fisher Scientific Co., Fairlawn, N.J.; isoprbpanol, dioxane, and naphthalene: Mallinckrodt Chemical Works, St. Louis, Mo.; Beckman Amino Acid Calibration Mixture Type I: Beckman Instruments Inc., Spinco Div., Palo Alto, Ca.; phosphoserine: California Biochemical Corp., Los Angeles, Ca.; N-acetyl tyrosine ethyl ester: Sigma Chemical Co., St. Louis, Mo.; a-chymotrypsin: Worthington Biochemical Corp.; Freehold, N.J. A l l other chemicals were obtained locally. "Baker Analyzed" grade (J. T. Baker Chemical Co., Phillipsburg, N.J.) was used when available. 115 2. Plant M a t e r i a l Bush bean (Phaseolus v u l g a r i s L. var. Top Crop Green Pod) seeds were obtained from Buckerfields Ltd., Vancouver, B.C. They were grown i n v e r m i c u l i t e f o r 7 days i n a dark wooden cabinet at room temperature. Hypocotyls were separated from the cotyledons and roots. Segments (1-5 cm) of eit h e r e n t i r e hypocotyls, hook regions, or basal regions (Figure 1) were prepared f o r c e l l w a l l extractions. 3. C e l l Wall Extraction Hypocotyl t i s s u e (50g) was ground toaa f i n e powder i n l i q u i d nitrogen (8-10 min). The frozen powder was l e f t to melt at 0°C. The following extraction was.carried out at 0-4°C. The homogenate was suspended i n 500 ml of deionized water and allowed to s e t t l e u n t i l 2 layers appeared (10-20 min). The upper layer was transferred to centrifuge b o t t l e s . The lower layer was resuspended and the s e t t l i n g procedure was repeated twice. The c e l l w all fragments were c o l l e c t e d from the pooled upper layers by c e n t r i f u g a t i o n at 15 000 £ for 10 min. The p e l l e t was resuspended i n 200 ml of deionized water. This suspension contained less than 2% of i n t a c t c e l l s as determined by l i g h t microscopy. The suspension was treated with a Blackstone U l t r a s o n i c Probe f o r 2 min at 200±50 W to release attached cytoplasmic contaminants. The fragments were c o l l e c t e d by cen t r i f u g a t i o n at 10 000 j» for 15 min, resuspended i n water and treated again with the u l t r a s o n i c probe. This procedure was repeated u n t i l the c e l l walls were free from cytoplasmic contaminants as determined by phase contrast microscopy. Four washings were usually enough to achieve the desired p u r i t y . C e l l walls were l y o p h i l i z e d and stored at 0°C. Yie l d s varied from 1-4 mg dry c e l l w all per g of hypocotyl. 116 Figure 1. Diagram of an etiolated bean hypocotyl ill u s t r a t i n g the regions from which c e l l walls were extracted. H - hook region; B - basal region. H 117 4. Reaction of Serine with DIFP The reaction of serine with DIFP was established by addition of 1 ml of 2% (w/v) D I F P i n CaO-dried isopropanol to 1 ymol.of serine i n 350 y l of 0.1 M-sodium phsphate b u f f e r , pH 7.3. The mixture was incubated for 45 min at room temperature, and 0.27, 0.45, or 1.35 ml of 12 M-HC1 was added to i n i t i a t e h y d r o l y s i s . Tubes were hydrolyzed i n vacuo f o r periods ranging from 2 to 30 h. Hydrolyzates were dried i n a dessicator over KOH p e l l e t s and resuspended i n 500 y l of deionized water. Each tube was prepared i n duplicate. Two hundred y l of each sample and 200 y l of Beckman Amino Acid C a l i b r a t i o n Mixture Type I , containing 20 nmol of the common protein amino acids, were applied to a 0.9 x 57 cm column on a Beckman Amino Acid Analyzer Model 120 C. Amino acids were separated and phosphoserine was analyzed according to the method of Spackman, e_t a l . , (1958) using asparatic acid as an i n t e r n a l standard. 5. • Reaction of Alpha-chymotrypsin with DIFP The reaction of a-chymotrypsin with r a d i o a c t i v e and non-radioactive DIFP was performed by a modification of the method of Schaffer, e_t a l . 32 (1954). A t y p i c a l reaction mixture contained 50 y l of a P - D I F P (470 Ci/mg) s o l u t i o n (0.96 mg/ml of CaO-dried isopropanol), 1 ml of an a-chymotrypsin so l u t i o n (5 mg/ml of 0.05M-sodium phosphate bu f f e r , pH, 7.3), and 10 y l of a 2% (v/x) s o l u t i o n of DIFP i n CaO-dried isopropanol. A reaction control contained 1 ml of a-chymotrypsin s o l u t i o n (5 mg/ml of Q.05M-sodium phosphate b u f f e r , pH 7.3) and 10 y l of a 10% (w/v) s o l u t i o n of DIFP i n CaO-dried isopropanol and was incubated for 118 32 90 min at room temperature before 50 y l of a P-DIFP (470 yCi/mg) so l u t i o n (0.96 mg/ml of CaO-dried isopropanol) was added. 32 Tubes were incubated for 45 min at room temperature; excess P-DIFP was removed ei t h e r by d i a l y s i s against deionized water f o r 36 h at 4°C, or by p r e c i p i t a t i o n of the protein with 20% TCA, c e n t r i f u g a t i o n at 600 g_ for 1 min, and seven washes with water. Labeled protein was l y o p h i l i z e d and stored at 0°C. In one experiment, the temperature ana reaction time were v a r i e d to test f or conditions y i e l d i n g optimal i n h i b i t i o n of enzymatic a c t i v i t y by DIFP. Alpha-chymotrypsin was allowed to react with DIFP ei t h e r by the method of Cohen, et a l . , (1967) or Schaffer, et a l . (1954). Tubes were placed on i c e u n t i l a-chymotrypsin assays began. 6. Reaction of C e l l Walls with DIFP One ml of 0.05 M-sodium phosphate b u f f e r , pH 7.5, was added to 30 mg of c e l l w a l l s ; radioactive and non-radioactive DIFP were added to t h i s suspension i n amounts described i n the previous section. Reactions were c a r r i e d out as described above. Excess reagent was removed e i t h e r by d i a l y s i s against deionized water f o r 36 h at 4°C or by c e n t r i f u g a t i o n at 600 g_ f o r 1 min and resuspension i n deionized water repeated u n t i l no r a d i o a c t i v i t y was detected i n the supernatant (10 times). C e l l walls were l y o p h i l i z e d and stored at 0°C, 7. Recovery of Phosphoserine To confirm and quantitate the extent of diisopropylphosphorylation of serine by DIFP, samples of the d e r i v a t i z e d a-chymotrypsin were subjected to s e r i a l h y d r o l y s i s . The l y o p h i l i z e d p rotein (3.00 mg) 119 was dissolved i n 0.4 ml of 2 M-HC1 and hydrolyzed f o r 6, 12, 18, 21, and 24 h at 100°C i n vacuo. Derivatized c e l l walls were hydrolyzed i n 2 M-HC1 f o r 21 hr at 100°C i n vacuo. The acid was removed i n vacuo over KOH p e l l e t s and s i l i c a g e l . Each hydrolyzate was dissolved i n 0.5 ml deionized water, 2 p i of dansyl hydroxide was added as a fluorescent i n t e r n a l standard, and the sample was applied as a spot to a sheet of Whatman 3MM chromatography paper. Phosphoserine, orthophosphoric acid and a colored marker (DNP-aspartic acid) were applied beside the sample. The mixture was resolved by electrophoresis for 25-30 min at pH 3.5 at 50 V/cm (Ambler, 1963). Phosphoserine and orthophosphoric acid standards were detected by eadmium-ninhydrin reagent (Heilmann, et a l . , 1957) and ammonium molybdate-ascorbic acid reagent (Ames, 1966), r e s p e c t i v e l y . M o b i l i t i e s of standard phosphoserine and orthophosphate were determined with reference to DNP-aspartate and dansyl hydroxide. Electrophoregrams were cut into s t r i p s and scanned 32 for P a c t i v i t y using an Actigraph I I Model 1025 s t r i p scanner (Nuclear Chicago) coupled to a Nuclear Chicago Model CR8416 chart recorder. A l l radioactive compounds had m o b i l i t i e s corresponding to e i t h e r phosphoserine or orthophosphate standards. Samples having m o b i l i t i e s corresponding to these standards were cut from electrophoregrams and f i n e l y minced with s c i s s o r s f o r l i q u i d s c i n t i l l a t i o n counting. Two hydrolyzates were separated by ion exchange chromatography as described f o r the reaction of serine with DIFP. On both occasions, the t o t a l a c t i v i t y recovered i n phsophoserine and orthophosphate was within 4% of the corresponding a c t i v i t y recovered following high voltage paper electrophoresis. 120 8. Determination of Ra d i o a c t i v i t y 10 ml of diozane-based s c i n t i l l a t i o n f l u i d (Bray, 1960) were added to aqueous s o l u t i o n , suspensions, l y o p h i l i z e d s o l i d s or f i n e l y cut pieces of Whatman 3MM paper. Samples were counted i n an Isocap 300 l i q u i d s c i n t i l l a t i o n spectrometer (Nuclear Chicago) f o r 20 min with an 32 800 K cpm termination at a window of 25 to 1700 Kev. A ^ P quench curve was prepared using the external standard r a t i o method (Wang and W i l l i s , 1965) by the addition, i n t r i p l i c a t e , of 0.00, 0.15, 0.30, 0.50, 1.00, and 3.00 ml of deionized water to 10 y l of undiluted 32 P?phosphate s o l u t i o n i n 10 ml of Bray's s o l u t i o n . A l l a c t i v i t i e s were below the coincidence counting range of the spectrometer. A c t i v i t i e s of ^^P-DIFP stock solutions were determined to correct f or incomplete 32 transf e r of P-DIFP to isopropanol. Thus, the quench curve ordinate 32 was cpm/dpm calculated f o r P-DIFP and sample a c t i v i t i e s were expressed 32 as a mole f r a c t i o n of o r i g i n a l P-DIFP. Background was counted i n t r i p l i c a t e before each seri e s of samples and subtracted from sample counts p r i o r to a c t i v i t y determinations. A l l determinations were performed i n t r i p l i c a t e on at l e a s t two preparations. 9. Calculations 32 A correction was made f o r non-specific P-DIFP l a b e l i n g by subtracting mean values of DIFP-pretreated groups from mean values of treatments lacking DIFP pretreatment. These differences were tested f o r s t a t i s t i c a l s i g n i f i c a n c e using the Student t - d i s t r i b u t i o n and weighted 32 variance f o r unequal sample s i z e s . A l l corrected values f o r P recovery were s i g n i f i c a n t at 3=0.01 unless indicated otherwise. 121 10. Protein Determination Cell walls were extracted in 1 M-KOH for 30 min in a boiling water bath. The mixture was diluted ten-fold, shaken, and allowed to settle. The protein in the supernatant was determined by the method of Lowry, e_t a l . , (1951) as modified by Eggstein and Kreutz (1955). The residue of extracted c e l l walls washed twice with deionized water showed,no protein by the qualitative microbiuret assay (Goa, 1953). A l l determinations were performed in trip l i c a t e on at least two c e l l wall preparations. 11. Assay of Alpha-chymotrypsin Activity Activity of a-chymotrypsin was determined by a modification of the method of Cohen, et a l . , (1967) using a Radiometer pH-stat. Twenty ml of 0.05 M-KC1 containing 22.5 mg of N-acetyl tyrosine ethyl ester were equilibrated at 30°C. One hundred y l of the appropriate dilution of native and DIP-a-chymotrypsin in 0.05 M-Tris HC1, pH 7.5, were added. The volume of 0.01 M-NaOH required to titrate liberated acetic acid to pH 7.5 was automatically recorded for 1-2 min. Activities were determined from i n i t i a l slopes and recorded as mol of substrate hydrolyzed min m^g ^ (units) of a-chymotrypsin. Titrant molarity was confirmed by t i t r a t i o n against Nations Bureau of Standards-calibrated 1 M-HC1. 12. Spin Labeling The spin labeling method was based on procedures described by Schaffer, e_t a l . , (1954) for eel cholinesterase diisopropylphosphorylation with modifications relevant to the use of the spin label l-oxyl-2,2,6,6-tetramethyl-4-piperidinylmethylphosphonofluoridate (Morrisett, et a l . , 1969). Fifty yl of 5% (w/v) HTMFP in benezene were added to 3 mg of c e l l walls or Whatman cellulose powder in 1 ml of 0.1 M-sodium phosphate buffer, pH 7.3. Other tubes contained 50 yl of HTMFP alone in buffer and 50 yl of HTMFP with 3 mg of c e l l walls that had been pre-treated with 100 yl of 2% (w/v) DIFP in CaO-dried isopropanol for 45 min then washed with approximately 1 ml of buffer and centrifuged at 600 for 1 min at room temperature. This washing procedure was repeated 12 times. The mixtures were shaken and incubated at room temperature for 1 h. Excess spin label was removed by centrigugation at 600 £ for 1 min and resuspension in deionized water 12 times u n t i l no spin label was detected in the wash or diffusate, respectively. Spectra of suspensions (approximately 6 mg/ml) were recorded at room temperature in a low temperature aqueous solution quartz c e l l (James F. Scalon, Solvang, Ca.) using a Varian E-3 electron spin resonance spectrometer (Department of Chemistry, University of British Columbia) with an H f i e l d range of 3435 - 3535 Gauss and detector power of 5.00 mW at 9.523 GHz. Alpha-chymotrypsin was spin labeled by the same method. Excess reagent was removed by the method described for the removal of excess 32 P-DIFP from labeled a-chymotrypsin. C. RESULTS 1. Modification of Serine with DIFP Table I shows the recoveries of phosphoserine from hydrolyzates of mixtures following the reaction of serine with DIFP. Recovery depended upon hydrolytic conditions. A maximum of 1.1% of the original 1-23 Table I. Phosphoserine recovered following the reaction between serine and DIFP and s e r i a l hydrolysis with 2, 3, or 6 M-HCl. Values are means of duplicate reactions and are expressed as a percentage of serine i n the i n i t i a l r e a c t i o n mixture. N.D. • denotes values not determined. HC1 Duration of hydrolysis (hr) concentration 2 .4 „• 8 15 24 30 2 M N.D. N.D. N.D. 1.0 0.0 1.1 3 M N.D. Trace 0.1 0.3 N.D. N.D. 6 M 0.7 0.8 0.1 N.D. N.D. N.D. 124 serine was recovered as phosphoserine. No phosphoserine was detected in the serine solution used for the reaction. Only phosphoserine and serine were observed in any analysis of ninhydrin positive substances. The presence of isopropanol in the reaction mixture did not effect chromatography of products. Unreacted serine was not measured after hydrolysis because, based on phsophoserine recovery, the quantity of DIP-serine and thus the difference between serine quantities before and after diisopropylphosphprylation and hydrolysis was smaller than the average error i n an analysis. 2. Modification of Alpha-chymotrypsin with DIFP Alpha-chymotrypsin was treated with DIFP and assayed for i t s ab i l i t y to hydrolyze N-acetyl tyrosine ethyl ester. The activity was inhibited by DIFP under both reaction conditions tested and inhibition was essentially complete after 20 min at 30°C (Table I I ) . The inhibited enzyme was used as a test system to measure recovery of 32 phosphoserine following diisopropylphosphyorylation with P - D I F P . There were 0.583 mol< of phosphorus-32 recovered per mol of 32 a-chymotrypsin, regardless of the method used to remove excess P - D I F P 32 from P-DIP-a-chymotrypsin (Table I I I ) . Under reaction conditions which result in 68.2% inhibition of a-chymotrypsin (Table II), 58.3% of 32 the a-chymotrypsin was labeled with P . Thus, the molar ratio of 32 bound P - D I F P to active centers inhibited by DIFP is 0.855. This is in close agreement with the value of 1.00 established by Jansen, e_t a l . (1950. 125 Table I I . A c t i v i t y of native and DIFP-inhibited a-chymotrypsin measured by hydrolysis of the synthetic substrate, N-acetyl tyrosine e t h y l ester. Values represent means of duplicate assays. I n h i b i t i o n S p e c i f i c a c t i v i t y Eeaction conditions (units mg a-chymotrypsin) % i n h i b i t i o n Native enzyme 216.0 0.0 (no i n h i b i t i o n ) Enzyme a f t e r reaction with 1.2 99.3 DIFP f o r 20 min at 30°C Enzyme a f t e r r e a c t i o n with 64.2 68.2 DIFP f o r 45 min at 24°C 126 Table I I I . Recovery of P ^ a c t i v i t y from DIFP pretreated ai^cj non-pretreated P^DIP-a-chymotrypsin. Excess P-DIFP was. removed from P-DIP-a-chymotrypsin by eit h e r d i a l y s i s or 20% TCA p r e c i p i t a t i o n of the protein. Phosphorus-32 recoveries are expressed as a mole f r a c t i o n of a-chymotrypsin ± S.E.M. Method of P-DIFP DIFP pretrea^ment _jNo DIFP pretreatment Corrected removal (mol., P mol a-chymotrypsin) value D i a l y s i s 0.116 ± 0.001 0.699 ± 0.028 0.583 Protein 0.003 ± 0.002 0.586 ± 0.056 0.583 p r e c i p i t a t i o n 127 32 3. Phosphoserine Recovery from P-DIP-a-chymotrypsin The products of the p a r t i a l hydrolysis of diisopropylphosphoryl enzymes — phosphorserine and orthophosphoric a c i d - (Schaffer, et a l . , 1953; Schaffer, et a l a . , 1954) — w e r e assayed for r a d i o a c t i v i t y a f t e r 32 s e r i a l hydrolysis of P-DIP-a-chymotrypsin to e s t a b l i s h conditions f o r 32 quantitative or optimal recovery of P from c e l l walls treated with 32 P-DIFP. When hydrolysis products were separated by high voltage paper electrophoresis, the t o t a l a c t i v i t y recovered from electrophoregrams varied from 14 to 38% (Table IV). Optimal but incomplete recovery of phosphoserine occurred a f t e r 12 h of h y d r o l y s i s . Phosphate recovery increased with hydrolysis time and t o t a l phosphorus-32 recovery increased a f t e r 18 h. The molar r a t i o of phosphoserine to a-chymotrypsin was at l e a s t one order of magnitude less than the corresponding r a t i o f o r DIP-a-chymotrypsin from Table I I I . I f I assumed that unrecovered a c t i v i t y was d i s t r i b u t e d proportionately i n phosphoserine and phosphate, then the corrected values presented i n Table IV were obtained. Each sample was corrected independently. These values are consistently less than h a l f of the corresponding molar r a t i o f o r DIP-a-chymotrypsin from Table I I I . This r e s u l t i s i n agreement with those of others (Schaffer, et a l . , 1953; Schaffer, et a l . , 1954). 4. Modification of C e l l Walls with DIFP C e l l walls i s o l a t e d from e n t i r e hypocotyls of e t i o l a t e d P. v u l g a r i s 32 were not labeled by P-DIFP a f t e r correcting f o r non-specific binding 32 32 of P-DIFP, when excess P-DIFP was.removed by d i a l y s i s (Table V). C e l l walls i s o l a t e d from hook and basal regions of hypocotyls of 32 -1 e t i o l a t e d P_. v u l g a r i s contained 5.4 and 3.4 pmol of P mg of dry Table IV. Recovery of P from electrophoregrams a f t e r s e r i a l hydrolysis of DIP-a-chymotrypsin. Phosphoserine and orthophosphate recoveries are expressed as molar r a t i o s ' q j ct-chymo t r y p s i n . Total P recoveries are expressed as percentages of the P a c t i v i t i e s i n DIP-a-chymotrypsin before hydrolysis. Phosphoserine recoveries are corrected f o r loss during hydrolysis and electrophoresis r e f l e c t e d i n i n d i v i d u a l t o t a l recoveries. Values are means of duplicate analyses. Duration of hydrolysis (h) 6 12 - 18 - 21 24 0.0001 0.0382 0.0381 0.0006 0.0553 0.0547 0.0002 0.0432 0.0430 0.0001 0.0387 0.0386 0.0001 0.0450 0.0449 0.0004 0.0281 0.0277 0.0035 0.0680 0.06.45 0.0009 0.0641 0.0632 0.0027 0.0770 0.0743 0.0001 0.0930 0.0929 14 32 20 33 38 0.072 0.171 0.215 .0.116 0.118 Phosphoserine Recovery (mol phosphoserine/ mol a-chymotrypsin) DIFP pcetreatment No DIFP pretreatment Corrected value Orthophosphate Recovery DIFP pretreatment (mol: orthophosphate/ No DIFP pretreatment mol a-chymotrypsin) Corrected value T o t a l Recovery Corrected Phosphoserine Recovery (mol. phosphoserine/mol a-chymotrypsin) Table V. Phosphorus-32 recovery from "'''P-DIFP treated c e l l walls i s o l a t e d from entire hypocotyls and regions of hypocotyls of e t i o l a t e d P_. vulgaris shown i n Figure 1. Excess P-DIFP was removed from hypocotyl c e l l walls by d i a l y s i s and from hook and basal c e l l walls ' by repeated washings. Values are means of at le a s t t r i p l i c a t e analyses ± S.E.M. Corrected values are the difference between groups pretreated and not pretreated with non-radioactive DIFP. N.S. denotes no s i g n i f i c a n t difference at a=0.01. N.D. denotes value not determined. O r i g i n of C e l l Walls DIFP No DIFP Corrected pretreatment pretreatment value 32 -1 (pmol' P mg c e l l wall) Protein content (yg protein/ mg c e l l wall) S p e c i f i c a c t i v i t y (pmol. 3 2 P / mg c e l l w a l l protein) Entire hypocotyl Hook region Basal region 7.7 ±0.1 7.7 ± 0.7 9.1 ± .1.7 14.5 ± 1.8 8.9 ± 0.4 12.2 ± 1.9 0.0(N.S.) 5.4 3.4 N.D. 128 9 110 6 0.0 42.2 30.9 t o VO 130 32 c e l l w a l l , r e s p e c t i v e l y , a f t e r removal of excess P-DIFP by repeated washing. Recoveries calculated on a c e l l wall protein basis were 42.2 32 -1 and 30.9 pmols of P mg of c e l l w a l l protein for hook and basal c e l l walls r e s p e c t i v e l y . 5. Phosphoserine Recovery from C e l l Walls 32 -1 There were 0.56 pmol. of P-phosphoserine mg of dry c e l l w a l l 32 -1 and 4.38 pmol of P-phosphoserine mg of c e l l w a l l p r o t e i n recovered from high voltage, paper electrophoresis of p a r t i a l hydrolyzates of 32 P-DIFP labeled c e l l walls i s o l a t e d from hook regions of hypocotyls of e t i o l a t e d P. v u l g a r i s (Table VI). No s i g n i f i c a n t ( t . =1.49, a=0.01) — 4dr 32 phosphoserine recovery occurred i n P-DIFP treated c e l l walls i s o l a t e d from basal regions of s i m i l a r hypocotyls when compared to DIFP-pretreated controls. T o t a l r a d i o a c t i v e product recovery from electrophoregrams ranged from 12 to 25%. A mean corrected value of 2.71 pmol;: of phosphoserine mg of c e l l w a l l (21.2 pmol of phosphoserine mg ^ of c e l l w a l l protein) was obtained when phosphoserine recoveries from labeled hypocotyl hook c e l l walls were cor-rected f o r loss during hydrolysis and electrophoresis. 6. Spin Labeling The spectrum shown i n Figure 2a was obtained f o r HTMFP-labeled a-chymotrypsin i n deionized water a f t e r removal of excess spin l a b e l by e i t h e r d i a l y s i s or TCA p r e c i p i t a t i o n of the protein. The spectrum obtained f o r HTMFP i n phosphate buffer i s shown i n Figure 2b. No resonance was observed between 3435 and 3535 G at highest gain i n i n t a c t e t i o l a t e d P_. v u l g a r i s hypocotyls, 1-5 cm segments of the Table VI. Phosphorus-32-phosphoserine recovered from P-DIFP treated c e l l walls i s o l a t e d from regions of hypocotyls of e t i o l a t e d P. v u l g a r i s shown i n Figure 1. Values are expressed as means of t r i p l i c a t e analyses S.E.M. Corrected values are the difference between groups pretreated and not pretreated with DIFP. Phosphoserine recoveries were corrected i n d i v i d u a l l y f o r loss during hydrolysis and electrophoresis. N.S. denotes no s i g n i f i c a n t d i f f e r e n c e at a=0.01. N.D. denotes value not determined. O r i g i n of C e l l Walls Phosphoserine recovery 32T Corrected phosphoserine recovery (pmol . P-phosphoserine/ mg c e l l wall) S p e c i f i c Corrected phosphoserine s p e c i f i c recovery phosphoserine 22 recovery (pmoL P-phosphoserine/ mg c e l l w a l l protein) Hook region DIFP pretreatment No DIFP pretreatment Corrected value Basal region DIFP pretreatment No DIFP pretreatment Corrected value 0.07 ± 0.00 0.63 ± 0.14 0.56 0.27 ± 0.03 0.34 ± 0.07 0.07 (N.S.) 0.34 ± 0.02 3.05 ± 0.67 2.71 1.91 ± 0.16 1.79 ± 0.30 -0.12 (N.S.) 4.38 21.2 N.D. N.D. 131a Figure 2. Electron spin resonance spectra of a) a-chymotrypsin labeled with HTMFP, b) HTMFP alone in phosphate buffer, and c) entire hypocotyl c e l l walls labeled with HTMFP. Spectra were recorded at room temperature at a receiver gain of a) 8 x 10 , b) 2.4 x 10 , and c) 2 x 10 . f-lgurc Figure 20 Figure 2c 133 hypocotyls, c e l l w a l l preparations, or any of the following HTMFP-treated preparations a f t e r removal of excess HTMFP: c e l l u l o s e powder, DIFP-,pretreated c e l l walls i s o l a t e d from en t i r e hypocotyls or hook regions of the hypocotyl, and non-pretreated c e l l walls i s o l a t e d from the hook region. On one occasion, c e l l walls i s o l a t e d from e n t i r e hypocotyls were labeled a f t e r removal of excess HTMFP by the washing method. This r e s u l t was not reproducible. The spectrum i s shown i n Figure 2c. A f t e r four days at room temperature, the l a b e l was released q u a n t i t a t i v e l y by one wash with deionized water. D. DISCUSSION Phosphoserine and orthophosphate were the major radioactive hydrolysis products following the reaction of DIFP with both a-chymotrypsin and P_. v u l g a r i s hypocotyl hook c e l l w a l l s . This observation indicated that serine was the labeled nucleophile i n these preparations. Cohen et a l . , (1967) stated that "serine i t s e l f and i t s peptides do not react (with DIFP)." The r e s u l t s shown i n Table 1 do not support t h e i r observation. Phosphoserine was detected i n p a r t i a l hydrolyzates a f t e r the. reaction of DIFP and serine (Table I ) . Ah estimate of the serine which was phosphorylated was d i f f i c u l t to obtain from these values fo r the following reason. During acid hydrolysis of a DIP ester, a l l of the bonds indicated i n Scheme 1 would be hydrolyzed. The R-phosphate ester would be recovered q u a n t i t a t i v e l y only i f the d i i s o p r o p y l esters 1 were cleaved q u a n t i t a t i v e l y f i r s t . Under conditions which y i e l d complete hydrolysis of diisopropylphosphate esters, some hydrolysis of a serine phosphate ester would be expected. The best estimate (a minimal value) 134 Scheme 1. 1 (CH3)2CH R 1 2 would be the maximal value for phosphoserine recovery in Table I (1.1%). 32 The nearest integer of the mole ratio of P-DIP-a-chymotrypsin to a-chymotrypsin active centers was 1.0. This result agreed with observations of Jansen, et a l . , (1950)and Koshland (1960) and suggested that of a l l of the serine residues in a-chymotrypsin, only the one i n the active center of the enzyme was reactive toward DIFP. Although the phosphoserine recovered after hydrolysis accounted for less than half of the radioactivity in DIP-a-chymotrypsin, the DIFP labeling strategy was applied to c e l l walls to establish maximum and minimum values for DIFP binding to serine. Any c e l l wall serine reactive toward DIFP would most likely be a component of an active center of an enzyme but could represent any serine which was sterically available to DIFP and sufficiently nucleophilic. 32 The method of removal of excess P-DIFP had no effect on the mole 32 fraction of a-chymotrypsin labeled with P-DIFP (Table I I ) . This was expected since the spontaneous hydrolysis of the DIP-protein ester procedes very slowly (Cohen, et a l . , 1967). Greenberg and Nachmansohn 32 (1965) showed that when P-DIFP inhibited phosphomonoesterase and no serine was phosphorylated, dialysis resulted i n complete release, of 32 P-DIFP from the enzyme and recovery of enzymatic activity. This was not observed, however, when the protein was precipitated and washed. 135 There was no s i g n i f i c a n t l a b e l i n g of e n t i r e hypocotyl c e l l walls when excess reagent was removed by d i a l y s i s (Table V). The s i n g l e c e l l w a l l preparation that was spin labeled released the spin l a b e l a f t e r 4 days. These observations suggested that a c e l l w a l l preparation treated with the phosphofluoridate may have been labeled but the l a b e l was released with the passage of time, e i t h e r by spontaneous or phosphatase catalyzed h y d r o l y s i s . Although spontaneous hydrolysis occurs, i t procedes at a very slow rate. On the other hand, acid phosphatase i s a known constituent of c e l l walls (Lamport and Northcote, 1960; K i v i l a a n , et a l . , 1961; Suzuki, 1972) and may have existed i n these preparations. The release of the spin l a b e l may have resulted from enzymatic reduction of the n i t r o x i d e (Smith, 1972). Although c e l l walls i s o l a t e d from both hook and basal regions of the hypocotyl contained r a d i o a c t i v i t y a f t e r removal of excess reagent by repeated washing (Table V), only the hook c e l l walls contained detectable phosphoserine i n the hydrolysis products (Table VI). This 32 p e c u l i a r i t y may be a t t r i b u t e d to P-DIFP l a b e l i n g of c e l l w a l l components other than serine that were not adequately prelabeled by DIFP, however no other evidence supports the i d e n t i t y of such a s i t e . The only c e l l w a l l preparations which contained any s i g n i f i c a n t r eactive serine were those extracted from the hypocotyl hook. The corrected phosphoserine recovery from hypocotyl hook c e l l walls (2.71 pmol;. mg 1 c e l l w a l l , Table VI) represented a minimum value based on the fac t that recovery from a-chymotrypsin was incomplete under a l l conditions tested; the value i n Table V (5.4 pmol.- mg 1 c e l l wall) represents a maximum value of r e a c t i v e serine residues. 136 To detect spin labeled c e l l w all suspensions (6 mg ml ^) by electron spin resonance spectrometry, the spin l a b e l concentration would have to be approximately 2nnmol mg * c e l l w a l l based on s e n s i t i v i t y c a l c u l a t i o n s described by Bolton, et a l . , (1972). This i s s u b s t a n t i a l l y greater than the maximum value for DIFP binding to hook c e l l w a l l serine. The r e a c t i v e serine i n hypocotyl hook c e l l walls which was absent i n basal hypocotyl walls could have been part of the active center of a c e l l w a l l enzyme or an enzyme adsorbed to the c e l l w a l l , or a part of a s t r u c t u r a l p r o t e i n , such as extensin (Lamport, 1965) which was s t i l l .capable of being glycosylated. Numerous enzymes have been i d e n t i f i e d i n the c e l l walls of various members of the Dicotyledonae. These include phosphatase (Lamport and Northcote, 1960; K i v i l a a n , et a l . , 1961; Suzuki, 1972), pec t i n methyl esterase (Glasziou, '1959), ascorbic acid oxidase (Newcomb, 1951; Mertz, 1964), invertase ( K l i s and Akster, 1974; Edelman and H a l l , 1964), a- (Murray and Bandurski, 1975) and 3-glycosidases (Ashford and McCully, 1970; K l i s , et a l . , 1974; Murray and Bandurski, 1975), ATPase ( K i v i l a a n , et a l . , 1961), phosphorylase (Kivilaan,.et a l . , 1961) and a c e t y l cholinesterase (Fluck and J a f f e , 1974; Chapter I of t h i s t h e s i s ) . A l k a l i n e phosphatase and phosphorylase from animal sources are i n a c t i v a t e d by DIFP ( M i l s t e i n , 1964 and Fisher, et a l . , 1959, respectively) and i t i s probable that the plant enzymes are s i m i l a r l y i n a c t i v a t e d . Acetylcholinesterase was ina c t i v a t e d by DIFP (Chapter I, Figure 7) and many other esterases are i n h i b i t e d by DIFP so pectin-methylesterase may be i n a c t i v a t e d by DIFP although no d i r e c t evidence exists i n support of t h i s argument. I t 137 would be possible to account f o r the l a b e l i n g of hook c e l l walls i f any of these (or other) DIFP s e n s i t i v e enzymes had a greater s p e c i f i c a c t i v i t y i n the hypocotyl hook than i n the basal region of the hypocotyl. This i s the case f o r acetylcholinesterase (Chapter I, Table I I I ) . Murray and Bandurski (1975) have i d e n t i f i e d B-galactosidase i n higher s p e c i f i c a c t i v i t y i n the hook of pea stems than i n the ba s a l stem segments, but DIFP s e n s i t i v e enzymes have not been studied from t h i s perspective. Using the value of estimated c a t a l y t i c center a c t i v i t y of acetylcholinesterase (0.197 units pmol 1 DIFP; Chapter 1^ Table V) and the s p e c i f i c a c t i v i t y of acetylcholinesterase i n whole hypocotyls (0.25 units mg 1 c e l l w a l l p r o t e i n ) , the predicted amount of DIFP l a b e l i n g i n the whole hypocotyl c e l l walls a t t r i b u t a b l e to acetylcholinesterase i s 1.25 pmol of DIFP. This quantity represents only 6% of the minimum value f o r rea c t i v e serine (Table VI). However, t h i s value i s maximal because the s p e c i f i c a c t i v i t y i n hook c e l l walls may be greater than i n e n t i r e hypocotyl c e l l walls (see Chapter I, Table I I I ) . This DIFP s e n s i t i v e enzyme cannot account f o r a l l DIFP binding i n hook c e l l w a lls. I t i s also possible that cytoplasmic enzymes adsorbed to the c e l l w a l l by i o n i c i nteractions (Jansen, e t ' a l . , I960) would bind DIFP. Hook c e l l walls may bind more cytoplasmic enzymes or a greater proportion of DIFP s e n s i t i v e enzymes may e x i s t i n the cytoplasm of hook c e l l s than i n that of basal c e l l s . Before resolving these p o s s i b i l i t i e s , the p u r i t y of the c e l l w a l l preparations must be established. 138 During the c e l l wall extraction procedure, the high purity of the preparations was established by the observation that there were fewer than 2% of intact cells and also by failure to find contaminants during examination by phase contrast microscopy. The settling procedure chosen for the separation of intact cells from c e l l wall fragments, produced good yields of c e l l wall fragments but some intact cells were present. These were eliminated during the washing procedure, but soluble c e l l constituents not v i s i b l e by phase contract microscopy may have been released during the extraction and settling procedures. Adsorbed proteins, for example, may have had a measurable impact on reactive serine since soluble proteases and esterases abound in plant c e l l extracts. In any c e l l wall purification method, one is faced with the problem of retaining native c e l l wall enzymes yet removing adventitious ones. My procedure was chosen to avoid the use of reagents which would remove ionically bound c e l l wall enzymes but the risk of contamination by soluble cytoplasmic enzymes was increased. It is conceivable that hook c e l l walls would bind more contaminating enzymes than would basal c e l l walls. Ordin, et a l . , (1957) observed that methylation of the pectin component of c e l l walls accompanies auxin-induced c e l l extension in Avena coleoptiles. If more methyl esters of uronic acids existed i n the f u l l y extended c e l l walls of the basal region, the hook would contain more negatively charged uronic acid carbonyls and provide for ionic interactions with contaminating proteins. No evidence supports the alternative that there are a greater proportion of DIFP sensitive enzymes in the hook than in 139 the basal region of the hypocotyl. If the reactive serine observed in the hook c e l l walls absent from the basal c e l l walls was glycosylated during c e l l extension, then the quantity of serine labeled by DIFP should equal the quantity of non-glycosylated serine present in young c e l l walls which would be glycosylated i n situ and exist as glycosyl serine in the fully extended c e l l wall. Klis (1976) examined the quantity of glycosylated (hydrazine labile) and non-glycosylated (hydrazine stable) serine in segments excised from etiolated pea epicotyl during elongation and after elongation had ceased. He found that glycosylated serine increased from 3.4 to 20.0 nmol mg ^ c e l l wall while non-glycosylated serine increased from 14.6 to 20.9 nmol mg ^ c e l l wall. From these data, i f a l l of the non-glycosylated serine existing in the c e l l wall became glycosylated during c e l l extension (which would s t i l l not account for a l l of the glycosylated serine) and i f the bean hypocotyl behaved similarly, then at least 14.6 nmol reactive serine would have been expected per mg of hook c e l l wall. The maximum value (Table V) was actually 3 orders of magnitude less than this. Klis concluded that the glycosylation site is not l i k e l y to be in the c e l l wall because the hydroxyproline: glycosyl-serine ratio remained constant during elongation. The results of this study similarly refute the possibility that the serine glycosylation site is in the c e l l wall. It may be possible to use the radioactive DIFP labeling strategy as a rapid method to detect ionically bound proteins, whether native or adventitious, and thereby establish a criterion of purity, i f c e l l walls which have been purified by washing with 1 M-NaCl, 8 M-urea, 1 M-NH.OH 140 or 0.5 M-formic acid (Mitchell and Taylor, 1969) can f i r s t be demonstrated to contain no reactive serine. Such a strategy would use the labeling procedure described in Methods and the washing procedure to remove 32 excess P - D I F P (or any other radioactive isotope of D I F P ) . The difference between the counts per minute of DIFP pretreated and non-pretreated c e l l walls would be a direct measure of purity. BIBLIOGRAPHY CHAPTER II Ambler, R. P. 1963. The amino acid sequence of Pseudomonas cytochrome C-551. Biochem. J . 89:349-378. Ames, B. N. 1966. Assay of inorganic phosphate, t o t a l phosphate, and phosphatases, p. 115-118. In E. F. Neufeld and V. 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