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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 C o l l e g e , 1973  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES (Botany Department)  We a c c e p t t h i s t h e s i s as conforming to t h e r e q u i r e d s t a n d a r d  THE UNIVERSITY OF BRITISH COLUMBIA September, 1977  ©  Donald Holmes M a n s f i e l d , 1977  In p r e s e n t i n g  this  an a d v a n c e d d e g r e e the L i b r a r y I  further  for  thesis  in p a r t i a l  fulfilment  o f the  at the U n i v e r s i t y  of B r i t i s h  C o l u m b i a , I agree  s h a l l make i t  agree  scholarly  that permission  this  written  thesis  It  gain  of  The U n i v e r s i t y  T > 0 1 of B r i t i s h  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  September  15, 1977  A^/ Columbia  r e f e r e n c e and copying  of this  shall  that  copying or  for  that  study. thesis  by t h e Head o f my Department  i s understood  for financial  for  for extensive  permission.  Department  Date  available  p u r p o s e s may be g r a n t e d  by h i s r e p r e s e n t a t i v e s . of  freely  requirements  or  publication  n o t be a l l o w e d w i t h o u t my  ABSTRACT  Acetylcholinesterase (AchE) a c t i v i t y r was and hypocotyls  i d e n t i f i e d i n roots  of e t i o l a t e d Phaseolus vulgaris L. by means of a  colorimetric assay which included the cholinesterase i n h i b i t o r neostigmine as a control.  An i n h i b i t o r of this a c t i v i t y was  i n tissue homogenates but was  removed by d i a l y s i s .  of the a c t i v i t y i n the hypocotyl was enzyme was (NHp2 4 S0  observed  Greater than 95%  l o c a l i z e d i n the c e l l walls.  The  extracted from the buffer-insoluble residue of roots with 5% a n d  p u r i f i e d by (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 on  Sepharose 6B and chromatography on N-methylacridinium-Sepharose 4B. P u r i f i e d preparations had a s p e c i f i c a c t i v i t y of 210 ± 20 units -mg—L_protein and contained one active protein and one major inactive protein as determined by polyaerylamide gel electrophoresis and t h i n layer i s o e l e c t r i c focusing.  The AchE had at least 3-fold greater  a c t i v i t y against acetylthiocholine than,butyl- or propionylthiocholine. The  of AchE f o r acetylthiocholine was  stimulated by choline (0.5-50 mMO  56 uM.  The enzyme was  and t o t a l l y i n h i b i t e d by -4  diisopropylf luorophosphate (DIFP., 10  M) and decamethonium (60 mM.) .  c a t a l y t i c center a c t i v i t y determined by DIFP t i t r a t i o n was substrate min was  5.3  ± 0.1,  ^ mol  ^ active center.  4.00  nm.  197 ± 5 mol  The i s o e l e c t r i c point of AchE  the sedimentation c o e f f i c i e n t (S„_ ) was zu ,w  and the Stokes radius was  The  The mol. wt.  4.2  ± 0.1  S,  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 r a t ii oi (f/fo) of 1.37.  A t h e o r e t i c a l model  of t h e quaternary s t r u c t u r e 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 f o l l o w i n g g e l f i l t r a t i o n i n low i o n i c s t r e n g t h and i o n exchange chromatography o f preparations  having  low s p e c i f i c a c t i v i t y .  I t was suggested t h a t an  i o n i c s t r e n g t h dependent e q u i l i b r i u m e x i s t e d between aggregates of the 77 000 mol. with  wt. s p e c i e s .  P r o p e r t i e s of the bean r o o t AchE were compared  t h e AchEs from e e l and o t h e r animal t i s s u e s .  d i f f e r e n c e s e x i s t e d , i n ' c a t a l y t i c " cent er z'a'bt r a t e s , and b e h a v i o r the d i f f e r e n t The  i v i t i e s V substrate 'hydrolysis  on N-methylacridinium-Sepharose 4B, the AchE from  sources  wereosimilariin^manyrespects.  s p e c i f i c a c t i v i t y of h y p o c o t y l  exposure o f e t i o l a t e d s e e d l i n g s The  Though l a r g e  hook AchE was u n a f f e c t e d by  to l i g h t o r an e t h y l e n e  s o u r c e f o r 3 days.  _3 s p e c i f i c a c t i v i t y o f hook AchE i n c r e a s e d 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 p l a n t s and decreased a f t e r 4 days i n 10 treated plants.  M-kinetin-  These r e s u l t s were i n t e r p r e t e d i n terms of a p o s s i b l e  r o l e of AchE i n h y p o c o t y l  aging.  In a second s t u d y , w a l l s of e t i o l a t e d P_. v u l g a r i s h y p o c o t y l s  were  32 treated with  P-DIFP under c o n d i t i o n s which c o r r e c t e d f o r  adsorption  of t h e r a d i o i s o t o p e , to determine t h e number o f n u c l e o p h i l i c s i t e s i n c e l l w a l l s from r e g i o n s h a v i n g  different elongation rates.  Alpha-  chymotrypsin and s e r i n e were t r e a t e d s i m i l a r l y t o e s t a b l i s h optimum 32 conditions for diisopropylphosphorylation. The P-phosphoserine content of p a r t i a l l y h y d r o l y z e d c e l l w a l l s was determined. C e l l w a l l s from 32 regions  of a c t i v e c e l l e l o n g a t i o n  contained  mg * c e l l w a l l and those from r e g i o n s iii  2.71 pmol  P-phosphoserine  i n which c e l l e l o n g a t i o n had  terminated contained results  no s i g n i f i c a n t  P-phosphoserine.  From these  I concluded t h a t the few n u c l e o p h i l i c s i t e s present' were, not  g l 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 r a t h e r were s i t e s centers  of enzymes which were bound to c e l l w a l l  iv  i n active  preparations.  V  TABLE OF CONTENTS  Page ABSTRACT  i i  LIST OF TABLES - CHAPTER I . . . . . . . . . . . . . . . . . . . LIST OF FIGURES - CHAPTER I LIST OF TABLES - CHAPTER I I  .  . . . . . . . .  x  •  xiii  LIST OF FIGURES - CHAPTER I I  xiv  ACKNOWLEDGEMENTS CHAPTER I .  ix  xv  STUDIES ON THE ACETYLCHOLINESTERASE OF 'RHASEOLUS VULGARIS L  . .  1 •  A.  INTRODUCTION . . .  1  B.  MATERIALS AND METHODS  6  1.  Chemicals . .  6  2.  Plant Material  7  3.  Assay of A c e t y l c h o l i n e s t e r a s e Activity  (AchE)  7  •  4.  Protein Determination  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 . . . .  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)  G e l F i l t r a t i o n on Sepharose 6B  11  c)  Chromatography  11  on MAC-Sepharose 4B . . . .  6.  L a b e l l i n g With DIFP  12  7.  D e t e r m i n a t i o n of R a d i o a c t i v i t y . . . . . . . .  13  8.  Polyacrylamide Disc Gel Electrophoresis  13  9.  SDS G e l E l e c t r o p h o r e s i s  10.  S e d i m e n t a t i o n i n I s o k i n e t i c Sucrose Gradients . . . . . . . . . . . . .  . . .  14  . . . .  15  Chapter I .  Page 11.  I s o e l e c t r i c Focusing  12.  D e t e r m i n a t i o n of Stokes Radius and M o l e c u l a r Weight . . . .  13.  . . . . . .  15  •  Ion Exchange Chromatography on DEAESepharose CL-6B  C.  17  18  14.  C e l l Wall Extraction  19  15.  Growth R e g u l a t o r Experiments  20  16.  H y p o c o t y l Hook Angle Measurements  20  17.  E l o n g a t i o n Measurements  21  RESULTS . . . 1.  . . .  The Assay Methods a)  b)  c)  2.  . .  The e f f e c t activity  21  of neostigmine on AchE , . .  21  The e f f e c t o f assay time on AchE activity . .  23  The e f f e c t of enzyme q u a n t i t y on AchE a c t i v i t y . . . . . . .  23  E x t r a c t i o n and (NH,) S0, 2  Precipitation  of AchE . . . . . . . . . . . 3.-  Localization a)  .  o f AchE i n P_. v u l g a r i s  23 27  AchE a c t i v i t y i n e x c i s e d p i e c e s of r o o t and h y p o c o t y l -  b)  21  A c t i v i t y of AchE i n the c e l l w a l l  27 . . . .  30  4.  P u r i f i c a t i o n o f Root AchE  30  5.  C h a r a c t e r i z a t i o n o f P u r i f i e d AchE  34  a)  34  DIFP L a b e l i n g  b) D i s c G e l E l e c t r o p h o r e s i s . . . . . . . c) . SDS G e l E l e c t r o p h o r e s i s ,. vi  . .  34 38  Chapter I .  Page  6.  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  f)  Stokes r a d i u s and m o l e c u l a r weight  47  g)  S u b s t r a t e a f f i n i t i e s and the e f f e c t o f v a r i o u s substances on AchE a c t i v i t y . . . .  47  .  B e h a v i o r of Low S p e c i f i c A c t i v i t y AchE on Chromatographic Media  7.  Chromatography on MAC-Sepharose  b)  G e l f i l t r a t i o n on Sepharose 6B  68  c)  Ion exchange chromatography  68  4B  58  P h y s i o l o g i c a l Role of AchE i n the H y p o c o t y l E f f e c t o f growth r e g u l a t o r s i n the h y p o c o t y l hooks  b)  on AchE  . . .  71 71  DISCUSSION  75  1.  I d e n t i t y of AchE i n P_. v u l g a r i s  75  2.  E x t r a c t i o n 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 C h a r a c t e r i z a t i o n  80  4.  P h y s i o l o g i c a l r o l e of the A c e t y l c h o l i n e / System  . . .  SUMMARY  101  THE ABSENCE OF NUCLEOPHILIC SITES IN THE CELL WALLS OF ETIOLATED PHASEOLUS VULGARIS L. HYP0C0TYLS AND ITS RELATION TO CELL ELONGATION  A.  INTRODUCTION  B.  MATERIALS AND METHODS 1.  95 97  BIBLIOGRAPHY - CHAPTER I . . . • CHAPTER I I .  71  activity  The e f f e c t o f a c e t y l c h o l i n e on the hypocotyl . . . . . . . .  Acetylcholinesterase E.  58  a)  a)  D.  42  '  Chemicals  110 .110  •  114 114  vii  Chapter I I .  C.  Page 2.  Plant Material  115  3.  C e l l Wall Extraction  4.  R e a c t i o n of S e r i n e with DIFP  5.  R e a c t i o n of Alpha-chymotrypsin  6.  R e a c t i o n of C e l l W a l l s w i t h DIFP  7.  Recovery  8.  D e t e r m i n a t i o n of R a d i o a c t i v i t y  9.  Calculations  .  of Phosphoserine  U5 117  w i t h DIFP  . . . . .  1  . . . . . . . . . . . .  P r o t e i n Determination  11.  Assay  12.  Spin Labeling  .  . . . .  1  8  H8 120  . .  10.  117  . . .  120  . . . . . . . . 1 2 1  of Alpha-chymotrypsin  Activity  . . . . . . .  121 121  RESULTS  122  1.  M o d i f i c a t i o n of S e r i n e w i t h DIFP  122  2.  M o d i f i c a t i o n of Alpha-chymotrypsin  3. • Phosphoserine  Recovery  chymotrypsin  from  32  w i t h DIFP  . . .  P-DIP-S. . . . . . . .  4.  M o d i f i c a t i o n of C e l l W a l l s w i t h DIFP  5.  Phosphoserine  6.  Spin Labeling  Recovery  124  from C e l l W a l l s  127 127  . . . . . .  130 130  D. DISCUSSION BIBLIOGRAPHY - CHAPTER I I  .133 141  viii  ix  LIST OF TABLES  CHAPTER I  Table I.  II;  Page The e f f e c t of d i a l y s i s and t h e r e i n t r o d u c t i o n o f t h e d i f f u s a t e to the n o n - d i a l y z a b l e homogenate on the AchE a c t i v i t y i n ]?. v u l g a r i s r o o t and h y p o c o t y l  26  Recovery of AchE from P_. v u l g a r i s r o o t s and h y p o c o t y l s e x t r a c t e d and f r a c t i o n a t e d w i t h (NH ) S0 , .  28  A c t i v i t y of AchE i n e x c i s e d segments of 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 of P_. v u l g a r i s shown i n Figure 1 . . . . . . . . . . .  29  P u r i f i c a t i o n of AchE from P. v u l g a r i s r o o t s  33  4  III.  IV. V. VI.  VII.  VIII.  2  4  . . . . . . . .  3 I n c o r p o r a t i o n of H-DIFP i n t o AchE p u r i f i e d by chromatography on MAC-Sepharose 4B S u b s t r a t e s p e c i f i c i t y o f AchE prepared by chromatography on MAC-Sepharose 4B f o r t h r e e choline esters .  37  . .  Recovery o f AchE a c t i v i t y from MAC-Sepharose 4B h a v i n g a l i g a n d c o n c e n t r a t i o n of 0.4 pmol ml . . . . . . . . .  60  Recovery o f AchE a c t i v i t y i n s u c c e s s i v e b u f f e r , decamethonium , and.NaCl .gradient e l u a t e s as a; f u n c t i o n o f bed volume of , MAC-Sepharose 4B  66  :  IX.  52  Summary o f p h y s i c a l p r o p e r t i e s of P_. v u l g a r i s AchE  . . . .  99  X  LIST OF FIGURES  CHAPTER I  Figure 1.  Page Diagram of an e t i o l a t e d bean s e e d l i n g showing the r e g i o n s used i n d e t a i l e d s t u d i e s of enzyme . distribution  2.  The e f f e c t of neostigmine on AchE a c t i v i t y  3.  Neostigmine-inhibitable  .... 8 22  h y d r o l y s i s of AchE as a  f u n c t i o n of time .  '  24  4.  The e f f e c t of enzyme q u a n t i t y  5.  E l u t i o n p r o f i l e obtained on Sepharose 6B  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 chromatography of AchE on MAC-Sepharose 4B . . . . . . .  7.  on the AchE a c t i v i t y  . . . .  a f t e r g e l f i l t r a t i o n of AchE • ••  . .  25  31 32  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.  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of AchE . .  36  9.  M o b i l i t y o f standard 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 . . . . . .  39  D i s t r i b u t i o n of p r o t e i n and t r i t i u m a f t e r SDSa c r y l a m i d e g e l e l e c t r o p h o r e s i s of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and l a b e l e d w i t h H-DIFP  40  D i s t r i b u t i o n of p r o t e i n and t r i t i u m a f t e r SDSa c r y l a m i d e g e l e l e c t r o p h o r e s i s of reduced AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and labeled with H-DIFP •  41  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 of s t a n d a r d p r o t e i n s and AchE  43  10.  11.  12.  13.  sedimentation •  AchE a c t i v i t y and pH g r a d i e n t a f t e r t h i n l a y e r i s o e l e c t r i c f o c u s i n g of enzyme p u r i f i e d by chromatography on MAC-Sepharose 4B  -  .  J >. . .  46  Figure 14.  Page 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 f o c u s i n g  of  AchE p u r i f i e d by chromatography on MAC-Sepharose  4B . . .  48  15.  Stokes r a d i u s c a l i b r a t i o n curve . . . . . . . . . . . . . .  49  16.  G l o b u l a r p r o t e i n m o l e c u l a r weight c a l i b r a t i o n curve . . . .  50  17.  The e f f e c t of s u b s t r a t e c o n c e n t r a t i o n on AchE a c t i v i t y o f MACjSepharose 4 B - p u r i f i e d  preparations  .  51  18.  A Lineweaver-B'urke p l o t for- a c e t y l t h i o c h o l i n e  19.  The e f f e c t o f c h o l i n e  20.  The e f f e c t of decamethonium on. AchE a c t i v i t y . . . . . .  21.  The e f f e c t of NaCl on AchE a c t i v i t y  22.  The e f f e c t of ( N H ^ S O ^ on AchE a c t i v i t y  23.  AchE a c t i v i t y r e c o v e r e d from f o u r MAC-Sepharose 4B columns of 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 . Recovery of AchE from MAC-Sepharose 4B columns e q 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. . . . .  24. 25.  26.  27.  28.  29.  30.  31.  on MAC-Sepharose  4B-purified  53 AchE . .  .56 57  . .  59  61 . . .  Recovery of AchE from MAC-Sepharose 4B columns by e l u t i o n w i t h a NaCl g r a d i e n t , a c e t y l c h o l i n e , or a decamethonium g r a d i e n t . . . . . . . . . . . . . . . Recovery of AchE a c t i v i t y f o l l o w i n g on MAC-Sepharose 4B  . .  64  67  69  from DEAE-Sepharose ••.  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 h y p o c o t y l hooks of 5-day o l d e l i o l a t e d P_. v u l g a r i s . 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 h y p o c o t y l hooks of 5-day o l d e t i o l a t e d P. v u l g a r i s The e f f e c t of a c e t y l c h o l i n e on the hook angle o f 20 h e x c i s e d h y p o c o t y l hooks of P_. v u l g a r i s xi  63  d e l a y e d NaCl e l u t i o n  E l u t i o n of AchE, prepared by method B, from Sepharose CL-6B E l u t i o n p r o f i l e o f AchE e l u t e d CL-6B  55  70  72  . .  73  74  Figure  Page  32.  Postulated  s t r u c t u r e o f P_. v u l g a r i s AchE'. . . . . . . . .  33.  Schematic model f o r the 11 S form of e e l AchE from Dudai and Silman (1974) . .  xii  90a  92  xiii  LIST OF TABLES  CHAPTER I I  Table  Page i  I.  II.  III.  IV.  V.  VI.  P h o s p h o s e r i n e r e c o v e r e d f o l l o w i n g the r e a c t i o n between s e r i n e and DIFP and s e r i a l h y d r o l y s i s w i t h 2, 3, or 6 M-HCl A c t i v i t y of n a t i v e and D I F P - i n h i b i t e d a-chymotrypsin measured by h y d r o l y s i s of t h e s y n t h e t i c s u b s t r a t e , N-acetyl tyrosine e t h y l ester 32 Recovery of P a c t i v i t y from DIFP p r e t r e a t e d non-pretreated P-DIP-a-chymotrypsin Recovery of hydrolysis  32  P from electrophoregrams a f t e r of DIP-a-chymotrypsin. . .  and  serial  32 Phosphorus-32 r e c o v e r y from P-DIFP t r e a t e d c e l l w a l l s i s o l a t e d from e n t i r e h y p o c o t y l s and r e g i o n s of h y p o c o t y l s of e t i o l a t e d _P. v u l g a r i s shown i n Figure 1 . . 32 •• Phosphorus-32-phosphoserine r e c o v e r e d from P-DIFP t r e a t e d c e l l w a l l s i s o l a t e d from r e g i o n s of h y p o c o t y l s of e l i o l a t e d P_. v u l g a r i s shown i n Figure 1 . . . .  123  . . .  125  126  128  129  . 131  xiv  LIST OF FIGURES  CHAPTER.II  Figure 1.  2.  Page Diagram of an e t i o l a t e d bean h y p o c o t y l i l l u s t r a t i n g the r e g i o n s from which c e l l w a l l s were e x t r a c t e d . . . .  116  E l e c t r o n s p i n resonance s p e c t r a of a-chymotrypsin l a b e l e d w i t h HTMEP, e n t i r e h y p o c o t y l c e l l w a l l s l a b e l e d w i t h HTMFP, and HTMFP alone  132  \  XV  ACKNOWLEDGEMENT S  Dr.  I a i n E . P . T a y l o r has provided r e s p o n s i v e ,  encouraging have  supervision.  t a u g h t me a v a r i e t y  suggestions. scientist. supplies  D r . D o n a l d G . C l a r k a n d M r . G e o f f r e y Webb of techniques  A l l have been  instrumental  are extended  am g r a t e f u l  I have  grateful  a l s o extended  to friends  and A d r i a n n e Ross.  times  typing this  excellence.  who h e l p e d me m a i n t a i n s a n i t y notably  o f need.  and Anne W a l t e r . Thanks  are also  Thanks a r e during the  Gary Court has h e l p e d  extended  to Carrol  Hudson  thesis. -Research C o u n c i l o f  Ganad_a ; ^ 4 d f e i y e £ S j f c t 3 6 t ° £ . J ^ i f i§frj Qj&ifflbia and a U n i v e r s i t y l o f  understanding  I am  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  iTjaeJcnowledg.e:^  Finally,  the thesis.  Illimaar Altosaar,  t o M r . Denny f o r h i s d r a f t i n g  Dianne Roe, D r . M i c h a e l S w i f t countless  T o w e r s , B . A . Bohm,  and c r i t i c i z i n g  discussions with  Mark Denny, Sue K r e p p ,  second and t h i r d y e a r s ,  for  as a  equipment.  to Drs. Taylor, Clark,  appreciated  Guy B e a u r e g a r d ,  in  i n my d e v e l o p m e n t  helpful  to D r . B . D. R o u f o g a l i s , D r . G. H . N . Towers,  Camm a n d A . D . M . G l a s s f o r r e a d i n g  also  numerous  D r s . T a y l o r and C l a r k a r e acknowledged f o r p r o v i d i n g  a n d D r . P . W, H o c h a c h k a f o r l o a n i n g  E.  and o f f e r e d  and equipment.  Thanks  I  o p t i m i s t i c ' , and  British  C o l u m b i a Summer  I w i s h t o t h a n k my f a m i l y and l o v e .  grants t o D r s . T a y l o r a n d C l a r k (1974)  Research  and. N a n c y S c h e r e r  Scholarship.  for  encouragement,  CHAPTER I .  STUDIES ON THE ACETYLCHOLINESTERASE OF PHASEOLUS VULGARIS L.  A.  INTRODUCTION  A c e t y l c h o l i n e mediates b i o e l e c t r i c responses i n a number of animal tissues  (Hebb and K r n j v e i c , 1962; Ruch and P a t t o n , 1965).  Regulation  of a c e t y l c h o l i n e l e v e l s i s o f paramount importance i n the c o n t r o l o f the i n f o r m a t i o n  conveyed.by  t h i s substance.  Biosynthesis  i s completed  by t h e c h o l i n e a c e t y l t r a n s t e r a s e r e a c t i o n (Nachmansohn and Machado, 1943).  D e g r a d a t i o n i n v o l v e s e i t h e r an a c e t y l c h o l i n e s t e r a s e  ( a c e t y l c h o l i n e h y d r o l a s e EC 3.1.1.7) o r a p s e u d o c h o l i n e s t e r a s e , (acylcholine acylhydrolase  EC 3.1.1.8).  Acetylcholinesterases  (AchEs) a r e membrane bound enzymes l o c a t e d i n nervous t i s s u e , e f f e c t o r organs i n n e r v a t e d  by c h o l i n e r g i c neurons, and e r y t h r o c y t e s .  s p e c i f i c i t y i s f o r the a c y l c a r b o x y l i c a c i d of the e s t e r Oosterbahn, 1963). i n serum.  Their  (Cohen and  P s e u d o c h o l i n e s t e r a s e s a r e s o l u b l e enzymes l o c a t e d  T h e i r s p e c i f i c i t y i s f o r the c h o l i n e o f the e s t e r  ( A u g u s t i n s s o n , 1963). i n some r e s p e c t s ,  A l t h o u g h i t appears t h a t b o t h enzymes are s i m i l a r  their tissue l o c a l i z a t i o n , substrate  specificities,  and responses to i n h i b i t o r s , modulators and excess s u b s t r a t e (Rosenberry, 1977).  The p r o p e r t i e s o f t h e s e enzymes have been reviewed  (Froede and W i l s o n , 1971; O ' B r i e n , 1971; Rosenberry, 1975; 1977). vivo  differ  Rosenberry,  AchE i s i n v o l v e d i n r e g u l a t i o n o f a c e t y l c h o l i n e l e v e l s i n  (Nachmansohn, 1959).  diisopropylfluorophosphate  I t i s i n h i b i t e d by organophosphates, such as (DIFP), and carbamates, such as n e o s t i g m i n e 1  2 or e s e r i n e .  I t i s f i v e times more a c t i v e a g a i n s t a c e t y l c h o l i n e than  a g a i n s t p r o p i o n y l c h o l i n e and a t l e a s t a c e t y l c h o l i n e than b u t y l c h o l i n e .  100 times more a c t i v e a g a i n s t  Phenylacetate  rate s i m i l a r to that f o r a c e t y l c h o l i n e . c h o l i n e and by excess  The enzyme i s i n h i b i t e d by  i n a v a r i e t y of plant tissues  secondary r o o t f o r m a t i o n  and s t i m u l a t e s  promotes t h e a d h e s i o n o f r o o t t i p s  surfaces ATP  ( F l u c k and  1974a). • E x o g e n o u s a p p l i c a t i o n s of a c e t y l c h o l i n e t o mung bean  r o o t s have e f f e c t s s i m i l a r to t h o s e o f r e d l i g h t .  it  at a  substrate.  A c e t y l c h o l i n e i s present Jaffe,  i s hydrolyzed  It inhibits  e f f l u x i n roots  to n e g a t i v e l y  (Jaffe,  charged  1970);  glass  (Tanada, 1972); and i t i n c r e a s e s oxygen uptake and d e c r e a s e s  levels  i n roots  (Junghans and J a f f e ,  1972).  A p p l i c a t i o n s of  a c e t y l c h o l i n e promote f l o w e r i n g o f .Lemna p e r p u s i l l a ( a s h o r t day p l a n t ) under continuous i l l u m i n a t i o n b u t p r e v e n t f l o w e r i n g o f Lemna (a l o n g day p l a n t ) under t h e same c o n d i t i o n s the same e f f e c t as l i g h t four species  (Kandler,  i n i n c r e a s i n g seed g e r m i n a t i o n  (Holm and M i l l e r ,  1972).  gibba  1972), and have of at l e a s t  Both a c e t y l c h o l i n e and i n d u c t i v e  p h o t o p e r i o d s a l t e r t h e a c t i o n spectrum o f b i o e l e c t r i c responses i n spinach  ( G r e r r i n , 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 i n c r e a s e  i n endogenous a c e t y l c h o l i n e l e v e l s i n mung bean r o o t s and  other plants  ( J a f f e , 1970)  (Hartmann and K i l b i n g e r , 1974).  A c e t y l c h o l i n e a f f e c t s growth responses of wheat s e e d l i n g s 1973) It  (Dekhuijzen,  and Avena c o l e o p t i l e s (Evans, 1972), as w e l l as mung bean r o o t s .  inhibits  i n d o l e a c e t i c a c i d ( I A A T ) - i n d u c e d e t h y l e n e r p r o d u c t i o n .and  p a r t i a l l y p r e v e n t s t h e lAA-promoted d e l a y vulgaris peroxidase  o f hook opening i n Phaseolus  (Parups, 1976) y e t mimics t h e e f f e c t o f IAA on e l o n g a t i o n and a c t i v i t y patterns  i n l e n t i l roots  ( P e n e l , e t a l . , 1976).  3 On the basis of these observations,  i t may be that acetylcholine  mediates the e f f e c t of l i g h t on b i o e l e c t r i c responses i n 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 i n plants have been undertaken.  The presence of a choline  acetyltransferase has been demonstrated only i n one plant species  —  U r t i c a d i o i c a (Barlow and Dixon, 1973). In plant tissues, acetylcholine i s hydrolyzed  by a v a r i e t y of  esterases including c i t r u s 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 J a f f e , 1973; Kasturi and Vasantharajan, 1976). The citrus acetylesterase and wheat germ esterase have K^s of 1.6 and 1.0 M, respectively, f o r acetylcholine but have not been tested for DIFP or carbamate i n h i b i t i o n .  The cucurbitacin esterase a c t i v i t y i s  unaffected by 10 "*M- DIFP and i s s l i g h t l y stimulated by 10 ^M-eserine. The sinapine esterase has a lower (660yM) Km for acetylcholine but -3 i s only marginally  i n h i b i t e d by 10  M-eserine.  However, the cholinesterase, p a r t i a l l y p u r i f i e d from mung bean roots and subsequently from a v a r i e t y of other plant tissues (Riov and J a f f e , 1973; Fluck and J a f f e , 1974d; Kasturi and Vasantharajan, 1976) , resembles animal AchE i n having i n h i b i t i o n by neostigmine and eserine, maximal a c t i v i t y against acetylesters, a K of less than m 200uM for acetylcholine or acetylthiocholine, and substrate i n h i b i t i o n .  4 I n h i b i t i o n of t h e pea r o o t enzyme by an organophosphate, F e n s u l f o t h i o n , r e s u l t s i n an i n c r e a s e  i n the endogenous  a c e t y l c h o l i n e l e v e l s over  c o n t r o l p l a n t s w i t h concomitant i n h i b i t i o n o f l a t e r a l r o o t ( K a s t u r i and V a s a n t h a r a j a n , 1976).  A rigorous  formation  p u r i f i c a t i o n of t h i s  enzyme has not been a c h i e v e d , though a v a r i e t y of chromatographic procedures have been a p p l i e d  (R. A. F l u c k , p e r s o n a l  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 p l a n t t i s s u e s or e x t r a c t s because p l a n t s c o n t a i n 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  ( F l u c k 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 s u b s t r a t e acetylthiocholine  (Ellman, e t a l . , 1961) r e q u i r e s an i n c u b a t i o n  of s e v e r a l minutes t o d e t e c t  enzyme a c t i v i t y  i n plant extracts  than the seconds r e q u i r e d i n the a n i m a l enzyme a s s a y . n o n - s p e c i f i c h y d r o l y s i s of a c e t y l c h o l i n e d u r i n g period  analogue period rather  Spontaneous o r  the l o n g  incubation  i s c o n t r o l l e d by the a d d i t i o n o f 10 ^M-neostigmine t o assay  mixtures  ( F l u c k and J a f f e , 1974b).  P u r i f i c a t i o n o f AchE to homogeneity from v a r i o u s  animal t i s s u e s  has i n v o l v e d the use o f e i t h e r the c o m b i n a t i o n of (NH^^SO^ p r e c i p i t a t i o n , g e l f i l t r a t i o n and i o n exchange chromatography i n many media and W i l s o n , 1963; L e u z i n g e r and Baker, 1967) or a f f i n i t y  (Kremzner  chromatography  ( K a l d e r o n , et a l . , 1970; Berman and Young, 1971; Dudai, e t a l . , 1972a; Rosenberry, e t a l . , 1972).  A v a r i e t y of a f f i n i t y  m a t r i c i e s have been used to p u r i f y e e l AchE.  chromatography  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 o f the n a t i v e m o l e c u l a r form o f t h e enzyme c o n s i s t s of the N-methyl a c r i d i n i u m d e r i v a t i v e : l - m e t h y l - 9 - ^ N ^ - ( e - a m i n o c a p r o y l ) - Y aminopropylaminoj a c r i d i n i u m bromide hydrobromide; c o v a l e n t l y l i n k e d to  CNBr-activated Sepharose 4B (MAC-Sepharose 4B) (Dudai, et a l , , 1972a). MAC-Sepharose 4B: Sepharose 4B —  N H ( C H ) CONH 2  2  5  (CH ) 2  CH T h i s s t u d y was u n d e r t a k e n t o exists which regulates objectives (1)  of the  study  (Ellman,  a c t s as of  BP  3  examine the p o s s i b i l i t y  t h a t an AchE  l e v e l of acetylcholine i n plant  tissues.  feasibility  o f u s i n g a c o l o r i m e t r i c enzyme  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 o n t r o l f o r spontaneous  or n o n - s p e c i f i c h y d r o l y s i s  substrate.  To i d e n t i f y segments  AchE a c t i v i t y  and  i n Phaseolus v u l g a r i s L .  tissue  extracts.  (3)  To p u r i f y  t h i s AchE 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  (4)  To d e t e r m i n e some p h y s i c a l enzyme a n d t o  and c h e m i c a l p r o p e r t i e s  compare t h e s e p r o p e r t i e s w i t h  those  of of  4B. this  other  AchEs. (5)  The  were:  To d e t e r m i n e t h e assay  (2)  the  3  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 o f P_. v u l g a r i s .  i n h y p o c o t y l hooks  6 B.  1.  MATERIALS AND  METHODS  Chemicals S u p p l i e s were o b t a i n e d from s o u r c e s as i n d i c a t e d :  a c r y l a m i d e , N,  N,  N', N ' - t e t r a m e t h y l e t h l y l e n e diamine, and N, N'-methylene b i s a c r y l a m i d e : Eastman Kodak Co., R o c h e s t e r , N.Y.;  t r i c h l o r a c e t i c acid, etheylene  diamine, p - t e r p h e n y l , and 1 , 4 - b i s - 2 - ( 5 - p h e n y l o x a z o l y l ) - b e n z e n e : S c i e n t i f i c Co., P i t t s b u r g h , Pa.;  i s o p r o p a n o l , dioxane and  M a l l i n c k r o d t Chemical Works, S t . L o u i s , Mo.;  Fisher  naphthalene:  guanidinium c h l o r i d e  and  sodium  l a u r y l s u l p h a t e (SDS): B r i t i s h Drug House Chemicals, T o r o n t o ,  Ont.;  H-DIFP:  Amersham/Searle, A r l i n g t o n H e i g h t s , 111.:  A l d r i c h Chemical Co., Milwaukee, Wn.; Wesbury, N.Y.;  p H i s o l y t e s : Brinkman Instruments,  acetylcholine chloride, acetylthiocholine  a r g i n i n e , b o v i n e serum albumin  iodide,  (BSA), b u t y l c h o l i n e c h l o r i d e , b u t y l t h i o -  c h o l i n e i o d i d e , c a t a l a s e , c h o l i n e c h l o r i d e , cytochrome (trimethyl-ammonium  DIFP:  c, d e c a m e t h y l e n e b i s -  bromide) decamethonium b r o m i d e ) , d i e t h y l a m i n o e t h y l -  (DEAE-) Sepharose CL-6B, 5 , 5 ' - d i t h i o b i s - ( 2 - n i t r o b e n z o i c a c i d ) (DTNB), • e s e r i n e , 6 - g a l a c t o s i d a s e , ;,gibberellic'.acid<j' glycera'ldehyde-3-phosphate dehydrogenase  (G-3-PDH) (m-hydroxy p h e n y l ) - t r i m e t h y l ammonium bromide  dimethylcarbamate  ( n e o s t i g m i n e ) , k i n e t i n , l y s i n e , myoglobin,  and p r o p i o n y l t h i o c h o l i n e i o d i d e : Sigma Chemical Co., Sepharose Sweden;  6B, and Sephadex G-75: Ethrel:  Pharmacia  St. Louis  Mo.;  F i n e Chemicals A . B i , U p p s a l a ,  Amehem P r o d u c t s I n c . , Ambler, Pa.  were o b t a i n e d l o c a l l y .  "Baker A n a l y z e d " grade  P h i l l i p s b u r g , N.J.)  used when a v a i l a b l e .  was  ovalbumin  A l l o t h e r chemicals  ( J . T. Baker  Chemical  Electrophorus e l e c t r i c u s  AchE and MAC-Sepharose 4B were g e n e r o u s l y donated by Dr. D. G. Department o f Chemistry, U n i v e r s i t y o f B r i t i s h  Co.,  Columbia.  Clark,  7 2.  Plant Material Bush bean (Phaseolus v u l g a r i s L . v a r . Top Crop Green Pod)  seeds were  s u r f a c e s t e r i l i z e d w i t h 0.5% (v/v) sodium h y p o c h l o r i t e o r 10% (v/v) commercial  b l e a c h f o r 15 min,  vermiculite i n p l a s t i c trays  r i n s e d f o u r times w i t h w a t e r , and grown i n (McConkey and Co.,  i n a dark c a b i n e t a t room temperature  Sumner, Wn.) f o r 9 days  (24 ± 2 ° C ) .  Roots were s e p a r a t e d  and washed w i t h c o l d tap water t o remove v e r m i c u l i t e .  H y p o c o t y l s were  s e p a r a t e d from c o t y l e d o n s and r i n s e d w i t h c o l d tap water.  I n one experiment,  the h y p o c o t y l was f u r t h e r d i v i d e d i n t o t h e r e g i o n s d e p i c t e d i n F i g u r e 1.  3.  Assay o f A c e t y l c h o l i n e s t e r a s e (AchE) A c t i v i t y The a c t i v i t y o f AchE was•determined by the p h o t o m e t r i c method o f  E l l m a n , e t a l . , (1961) as m o d i f i e d by R i o v and J a f f e mixtures  (1973).  Assay  i n f i n a l volume o f 1.62 m l c o n t a i n e d 1.00-1.48 ml o f 0.5 M-  potassium phosphate b u f f e r , pH 8.0, 60 V l o f 2.6 mM-DTNB prepared i n t h e same b u f f e r c o n t a i n i n g 4.5 mM-NaHCO^, and 0.02-0.5 m l o f the sample t o be assayed.  F o r each sample, a second  tube was p r e p a r e d i n which 30 V l  of 1.35 mM-neostigmine bromide r e p l a c e d 30 u l o f b u f f e r . The assay m i x t u r e s were i n c u b a t e d f o r 15 min a t 37°C; then 60 u l o f 12.5  m M - a c e t y l t h i o c h o l i n e i o d i d e was added.  proceed f o r 10-20 bath.  The r e a c t i o n was a l l o w e d t o  min and was t e r m i n a t e d by c h i l l i n g  t o 0°C i n an i c e  Absorbance o f c l e a r assay mixtures was determined  d i r e c t l y a t 4.12 nm.  P a r t i c u l a t e assay m i x t u r e s were a l l o w e d t o s e t t l e f o r 10 min and t h e upper l a y e r was removed w i t h a P a s t e u r p i p e t t e .  T h i s s u s p e n s i o n was  c e n t r i f u g e d a t 15,000 g_ f o r 15 min and the absorbance was  recorded.  o f the supernatant  8 Figure  1.  Diagram o f an e t i o l a t e d bean s e e d l i n g showing the 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 — plumular hook, S A — s u b a p i c a l , B basal.  regions  9 Enzyme a c t i v i t i e s were determined by the f o l l o w i n g One  calculation:  u n i t = AA.,„ v t * e * 412  where, AA412 = - •  of. the m i x t u r e l a c k i n g neostigmine minus  °f the  m i x t u r e c o n t a i n i n g neostigmine  v  = volume o f the assay m i x t u r e  (ul)  t  = assay time  E  = e x t i n c t i o n c o e f f i c i e n t of 2 - n i t r o - 5 - t h i o b e n z o a t e  (min)  (1.36 x 1 0 n l n m o l " ) 4  1  With the e x c e p t i o n of column o r g r a d i e n t e f f l u e n t enzyme assays were performed I n the experiment  i n duplicate or  designed to determine  enzyme a c t i v i t y , enzyme s o l u t i o n was  triplicate. the e f f e c t of n e o s t i g m i n e  on  b o i l e d f o r 30 min and c l e a r e d by  c e n t r i f u g a t i o n a t 15 000 g_ f o r 10 min. d i f f e r e n c e between absorbances  fractions, a l l  The  AA^.^  v a l u e was  the  of c o r r e s p o n d i n g assay m i x t u r e s c o n t a i n i n g  b o i l e d and u n b o i l e d enzyme s o l u t i o n s . In experiments  designed t o examine the e f f e c t of a g i v e n substance  on AchE a c t i v i t y , 30-60 ul o f s t o c k s o l u t i o n of the substance was  prepared  i n water (or i s o p r o p a n o l i n the case o f DIFP) and t h i s r e p l a c e d the same volume of b u f f e r i n the assay m i x t u r e s . The method d e s c r i b e d by F l u c k and J a f f e assay of e x c i s e d t i s s u e .  (1974b) was  used f o r the  Assay m i x t u r e s h a v i n g a f i n a l volume 20.25 ml  c o n t a i n e d 18.75  ml of 0.5 M-potassium phosphate  DTNB s o l u t i o n .  These were i n c u b a t e d f o r 15 min a t 37°C w i t h 1-3  u n i f o r m l y e x c i s e d t i s s u e ; 0.75 8 min, was  ml of s u b s t r a t e was  0.375 ml of n e o s t i g m i n e s o l u t i o n  added.  Aliquots  b u f f e r , pH 8.0,  and 0.75 g of  added and a f t e r  ( f i n a l c o n c e n t r a t i o n o f 25  (1 ml) were taken at 2 min i n t e r v a l s , the  ml  exactly UM)  absorbance  10 was  r e c o r d e d , and  the a l i q u o t was  m a i n t a i n a c o n s t a n t volume.  The  poured back i n t o the r e a c t i o n medium to absorbances v e r s u s time were p l o t t e d  the d i f f e r e n c e between the s l o p e s and  of the r e g r e s s i o n  a f t e r the a d d i t i o n of n e o s t i g m i n e was  and  l i n e s obtained before  used as ^ A ^ ^  t ^ in  the  c a l c u l a t i o n of enzyme a c t i v i t y .  4.  Protein The  one  Determination  p r o t e i n i n p a r t i c u l a t e f r a c t i o n s was  volume of 2 M-  shaken, and  Eggstein  f o r 30 min.  allowed to s e t t l e .  f r a c t i o n s was by  KOH  determined by  and  f r a c t i o n s was  eluates-  5.  The  washed w i t h water and  diluted ten-fold,  p r o t e i n i n these and  assay (Goa,  residue  of e x t r a c t e d  contained  no  was  e s t i m a t e d by  E x t r a c t i o n and  phosphate b u f f e r , pH f o r 3 min.  centrifuge  The  absorbance a t 280  The  Roots (344 7.0,  the  density  gradient  nm.  g) were added to 788  i n a Waring b l e n d o r and centrifuged  of the  ml  of 20 mM-potassium  homogenized at 20  at 4800 £  f o r 20 min  through Whatman #1  s l u r r y was The  by  Vulgaris  homogenate was  filtered  4800 j» f o r 20 min.  particulate  (NH^^SO^ P r e c i p i t a t i o n  resuspended i n 788 ml  (NH^^SO^.  modified  1953).  ( I n t e r n a t i o n a l Equipment Co.)  s u p e r n a t a n t was  a l l soluble  protein detectable  p r o t e i n content of chromatography column and  METHOD A:  was  s o l u t i o n was  the method of Lowry, et a l . , (1951) as  P u r i f i c a t i o n of AchE From P_. a)  rpm  The  K r e u t z (1955).  q u a l i t a t i v e microbiuret The  The  s o l u b i l i z e d by b o i l i n g w i t h  i n a BR  at -4°C.  f i l t e r paper and  extraction buffer containing  stirred  f o r 30 min  s u p e r n a t a n t was  at 4°C  and  6000  The the 5%  residue (w/v)  centrifuged  f i l t e r e d through two  000  layers  at of  Whatman #1 f i l t e r paper, brought to 80% saturation with s o l i d at 4°C and centrifuged at 4800 g_ for 20 min.  (NH^J^SO^  The p e l l e t was resuspended  in 30-100 ml of 20 mM-potassium phosphate buffer, pH 7.0, containing M-NaCl and dialyzed overnight  against the same buffer at 4°C.  0.2  The non-  d i f f u s i b l e material was c l a r i f i e d by centrifugation at 15 000 £ f o r 10 min. The supernatant could be used immediately or stored at 0°C f o r up to one month without loss of a c t i v i t y . 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 i n two steps with s o l i d (NH^^SO^ to 40% and 70% saturation. The f i n a l p e l l e t was resuspended with 10 mM-potassium phosphate b u f f e r , pH 7.0, and dialyzed overnight  against the same buffer, at 4°C.  non-diffusible material was treated the same as i n method A.  The  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. flow rate of 50-60 ml h.  Gel f i l t r a t i o n was performed at a  , Fractions were collected and assayed f o r  AchE a c t i v i t y and protein; the active fractions were pooled. c)  Chromatography on MAC-Sepharose 4B The AchE f r a c t i o n (87 ml) from g e l 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  ml in ; the ligand concentration was 2.0 umol ml  -1  . A f t e r the e n t i r e  12 sample had e n t e r e d , t h e column was washed w i t h 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 e l u t e d w i t h a p p r o x i m a t e l y 5 column volumes of 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 M-NaCl.  F r a c t i o n s were  c o l l e c t e d throughout t h e l o a d i n g , washing and e l u t i n g p r o c e d u r e s , and assayed f o r AchE a c t i v i t y and p r o t e i n .  The t o t a l e l u t e d a c t i v i t y was  compared w i t h t h a t o f t h e loaded sample t o determine t h e r e c o v e r y of AchE. The 1 M-NaCl e l u a t e  ( a p p r o x i m a t e l y 30 ml) was c o n c e n t r a t e d t o 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 s t o r e d a t OC  f o r no l o n g e r  than 12 days.  The column was r e g e n e r a t e d by washing w i t h  a p p r o x i m a t e l y 4 bed volumes of 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 5 M-guanidinium c h l o r i d e f o l l o w e d  by a t 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  o f t h e method o f Berman (1973) was used t o  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 (100-400 u n i t s ml , 185-210 u n i t s mg 1  0.810 ml i n 20 mM-potassium  1  protein)  4B.  Enzyme s o l u t i o n s  i n a f i n a l volume of  phosphate b u f f e r , pH 7.0, c o n t a i n i n g  0.0 o r  0.2 M-NaCl were i n c u b a t e d f o r 15 min a t 37°C w i t h 15 y l of 52.8 mM-DIFP ( i n CaO d r i e d i s o p r o p a n o l ) o r 50 01 o f 324 mM-butylcholine c h l o r i d e , o r b o t h DIFP and b u t y l c h o l i n e  chloride.  The c o n t e n t s of t h e tubes were dialy.zed a g a i n s t r e a c t i o n b u f f e r f o r 18 h  a t 4°C.  4 changes of t h e  N o n - d i a l y z a b l e m i x t u r e s were assayed  f o r AchE a c t i v i t y and p r o t e i n . METHOD B: protein)  Enzyme s o l u t i o n s  i n 20 mM-potassium  (100-400 u n i t s m l " , 185-210 u n i t s 1  phosphate b u f f e r , pH 7.0, c o n t a i n i n g  mg  _ 1  1.0  M-NaCl ( f i n a l volume = 0.810 ml) were a l l o w e d t o r e a c t f o r 15 min a t 37°C  13 in  the presence  Fifty-six  o r a b s e n c e o f 50 u l o f 3 H-DIFP  u l of  were added and t h e  (0.26 mg m l  All  7.  -1  i n propylene g l y c o l ,  tubes were i n c u b a t e d f o r  m i x t u r e s were d i a l y z e d a g a i n s t at  1.35 m M - n e o s t i g m i n e b r o m i d e .  4 changes  of the  procedures were performed on d u p l i c a t e  Determination of  20-600 u l s a m p l e s .  I s o c a p 300 l i q u i d  -1  )  Reaction for  18 h  and r a d i o a c t i v i t y .  preparations.  Radioactivity fluid  Samples were counted a t  scintillation  spectrometer  m i n w i t h a n 800 K cpm t e r m i n a t i o n .  each s e t  q u e n c h c u r v e was p r e p a r e d  (Bray,  1960) w e r e  45% e f f i c i e n c y  A l l a c t i v i t i e s were below  2-20  the  B a c k g r o u n d was c o u n t e d  o f samples and s u b t r a c t e d for activity  added  i n an  (Nuclear Chicago) for  coincidence counting range o f the spectrometer. duplicate before  37°C.  reaction buffer  4°C; the m i x t u r e s were assayed f o r AchE 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 to  20 m i n a t  3.4 C i mmol  f r o m cpm v a l u e s .  determinations  by the  in  A  channels  ratio  3 method  (Wang a n d W i l l i s ,  some e x p e r i m e n t s n e t  1965) u s i n g  H - t o l u e n e quench s t a n d a r d s .  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 .  In  A l l samples  were  counted i n d u p l i c a t e . 8.  Polyacrylamide Disc Gel Electrophoresis 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  Mixtures gel f i l t r a t i o n  (w/v) were a p p l i e d to  in  7% ( w / v ) a c r y l a m i d e g e l s .  i n small pore gels  preparations  either  o r c h r o m a t o g r a p h y o n M A C - S e p h a r o s e 4B (30-150 ug o f  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 ,  performed  by  (Davis,  1964).  and s o l i d  t o 5%  E l e c t r p h o r e s i s was  a t p H 8.3 i n d u p l i c a t e o n a t G e l s were s t a i n e d w i t h  7% ( w / v ) a c e t i c a c i d a n d d e s t a i n e d  sucrose  protein),  least  duplicate  1% ( w / v ) A m i d o S c h w a r t z  i n 7% a c e t i c a c i d .  The g e l s  were  14  s l i c e d immediately after electrophoresis and i n d i v i d u a l 2 mm s l i c e s from duplicate gels were assayed f o r AchE a c t i v i t y i n the presence or absence of neostigmine. 9.  SDS Gel Electrophoresis AchE p u r i f i e d 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 f o r 18 h at room temperature.  Samples (75 y l ) were mixed with 75 y l of either 10  sodium phosphate buffer, pH 7.2, containing 1% (w/v) SDS, 20% sucrose, 0.002 % (w/v) Pyronin Y (a tracking dye), and 40  mM-  (w/v)  mM-  d i t h i o e r y t h r i t o l 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 g e l for 1 hr and 5 ma per g e l f o r an additional 5.5 h-. India ink.  The center of the tracking dye was marked with  Gels were stained with 0.25%  (w/v) Coomassie B r i l l i a n t Blue  R-250 i n methanol:water:acetic acid (5:5:1, v/v) overnight at 60°C and destained with a 5% (v/v) methanol, 7.5% water at 60°C f o r 24 h.  (v/v) a c e t i c acid solution i n  Gels were scanned at 550 nm i n a G i l f o r d  240  spectrophotometer; absorbance was recorded by a G i l f o r d 6050 chart recorder at 2 cm min ^. sections.  Gels were placed on dry i c e f o r 15 min and s l i c e d i n 2-.'mm  Slices were placed i n s c i n t i l l a t i o n v i a l s containing 0.6 ml  of NCS tissue s o l u b i l i z e r (Amersham/Searle):water 20 h at room temperature followed by 2 h at 50°C.  (9:1,v/v) and kept f o r Samples were counted f o r  15  r a d i o a c t i v i t y as described above.  M o b i l i t i e s of a l l stained and radio-  active peaks were determined r e l a t i v e to the mobility of the tracking dye; molecular weights were obtained by comparing the r e s u l t i n g  values  to a c a l i b r a t i o n curve prepared from R^ values of standard proteins. A l l gels were run i n duplicate. 10.  Sedimentation i n Isokinetic Sucrose Gradients Isokinetic sucrose gradients (10-29.3% (w/v)) were prepared according  to the t h e o r e t i c a l formulations of Noll(1967) as applied by Morrod (1975).  AchE (310 units ml \  195 units mg ^ protein), or the same  3 preparation l a b e l l e d 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. 3  Mixtures containing  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 s o l i d 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 i n a Beckman L3-50 ultracentrifuge. 12 ml h. A;  Gradients were eluted at a flow rate of approximately  0.5 ml fractions were collected and assayed f o r AchE a c t i v i t y  or r a d i o a c t i v i t y .  Catalase and myoglobin were located by measuring the  absorbance at 405 nm.  3-galactosidase was assayed as described by  Massoulie and Rieger (1969).  A c a l i b r a t i o n curve was prepared f o r each  gradient using the standard proteins. A l l sedimentation experiments were run i n duplicate. 11.  I s o e l e c t r i c Focusing I s o e l e c t r i c focusing was performed by the t h i n layer method (Radola,  1973).  Sephadex G-75 (7.0 g) was swollen i n 100 ml of deionized water  over b o i l i n g water f o r 1 h.  Four ml of p H i s o l y t e s and 0.1  l y s i n e and a r g i n i n e were added t o the c o o l e d s u s p e n s i o n . (20X20 cm) were prepared as d e s c r i b e d by Radola  g each of Glass p l a t e s  (1973).  Samples were d i a l y z e d a g a i n s t 10 mM-potassium phosphate b u f f e r , 7.0,  o v e r n i g h t a t 4°C.  and myoglobin —  (10 mg  These and s t a n d a r d p r o t e i n s — ml  One  bands by  o r more a p p l i c a t i o n s of 20-50 y l samples  100-200 yg of p r o t e i n were made 7-10  of the p l a t e .  cytochrome c  ^) were a p p l i e d i n d i v i d u a l l y i n 18 mm  d i f f u s i o n from a c o v e r s l i p . totalling  BSA,  pH  Samples f o c u s s e d i n 10 mm  cm from the cathode  end  t h i c k d e x t r a n were a p p l i e d by  r e p l a c i n g a 1 cm band of d e x t r a n w i t h s i m i l a r l y prepared d e x t r a n c o n t a i n i n g up to 1 ml of sample. both the cathode TLE double  Twenty y l of each s t a n d a r d were a p p l i e d 1-3  and anode  end of the p l a t e .  chamber (Desaga).  M-ethylenediamine ( c a t h o d e ) .  f o r 8 h and  from  P l a t e s were p l a c e d i n the  The p l a t i n u m r i b b o n e l e c t r o d e s were put  t h i n s t r i p s of Whatman 7/1 paper moistened 0.4  cm  w i t h 0.2  F o c u s i n g was  on  M-H^SO^ (anode) or  c a r r i e d out a t 4°C a t 200  V  500 V f o r an a d d i t i o n a l 10 h at which time c o l o r e d s t a n d a r d  p r o t e i n s were f o c u s e d . Measurements of the pH were made d i r e c t l y w i t h a f l a t membrane g l a s s electrode  (Desaga) or 0.3  d i l u t e d w i t h 200  cm bands were removed from the d e x t r a n  y l of d e i o n i z e d water, and measured.  by the paper p r i n t method of D e l i n c e e and Radola B r i l l i a n t B l u e R-250 or bromophenol b l u e . by a s s a y i n g samples dextran l a y e r .  (approximately  T h i s method proved  q u a n t i t a t i v e e s t i m a t i o n was  layer,  P r o t e i n was  detected  (1972) u s i n g Coomassie  A c t i v i t y of AchE was  detected  100 y l ) which were removed from  the  adequate f o r q u a l i t a t i v e purposes  not a c h i e v e d .  I n two  experiments,  but  dextran  l a y e r s removed from the p l a t e were e l u t e d from e i t h e r a 25 ml s y r i n g e  containing  g l a s s wool or a 2.5  X 24 cm  mM-potassium phosphate b u f f e r , pH  column of Sephadex G—25 w i t h  7.0.  A l l isoelectric  10  focusing  experiments were d u p l i c a t e d . I s o e l e c t r i c f o c u s i n g was (0-64% (w/v)) c o n t a i n i n g as d e s c r i b e d two  by Morrod  s o l u t i o n s , one  of s u c r o s e and Ampholines,  (w/v)  (1975).  ampholytes i n a 110 ml  The  l i n e a r gradient  s o l u t i o n containing  42 ml  1.5  1%  a l s o performed i n a s u c r o s e d e n s i t y  of water and  1.87  u n i t s ml  i n 10 mM-potassium phosphate b u f f e r , pH  Focusing  was  at  1250V f o r 6 h, and 1 ml  a t 1500V f o r 15 h.  The  column was  and  12.  D e t e r m i n a t i o n of Stokes Radius and M o l e c u l a r  potassium phosphate b u f f e r , pH  proteins  was  absorbance at 650  containing  (B-galactosidase  nm  (6.9 nm),  —  and  myoglobin were d e t e c t e d  was  assayed as d e s c r i b e d  coefficient  (K  ml  u n i t s mg  53.87 ml  g of  ^  of water.  e l u t e d at 2.5  and  410  M-NaCl.  ml  ml  (5.2  (0.8 ml)  recorded  nm),  av  ) was  absorbance at 405  by M a s s o u l i e and  Rieger  nm  standard  and myoglobin  and  (1969).  a c a l i b r a t i o n curve was  p r e p a r e d by p l o t t i n g the  (1.9  Catalase  6-galactosidase The  partition  determined as d e s c r i b e d by Pharmacia F i n e J  were  to determine  * each) were a p p l i e d under the same c o n d i t i o n s . by  mM-  A solution  I n a second run,  catalase  pH.  * each) i n e q u i l i b r a t i o n  Fractions  nm was  h,  Weight  1.0  K^Fe(CN)g (2 mg  t o t a l volumes, r e s p e c t i v e l y .  2 mg ml  0.63  e q u i l i b r a t e d w i t h 20  a p p l i e d at a f l o w r a t e of 5 ml hr'''.  nm)  and  X 70 cm)  7.0,  c o n t a i n i n g Blue D e x t r a n 2000 and  the v o i d and  (LKB), 28  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  A Sepharose 6B column £0.8  c o l l e c t e d and  and  mixing  f o r 44 h: at 300V f o r 5 h, at 700V f o r 18  min  b u f f e r was  , 204  7.0  (LKB)  p r e p a r e d by  ml of Ampholines  protein)  at 3°C  column  another s o l u t i o n c o n t a i n i n g  ml of enzyme s o l u t i o n (251  c a r r i e d out  was  gradient  Chemicals  Stokes r a d i i  (R ) of  18 standard proteins vs  /-log K  av'  The K  av  p r e p a r a t i v e g e l f i l t r a t i o n d e s c r i b e d above. was  of AchE was  determined by the 1  The Stokes r a d i u s of AchE  o b t a i n e d from t h e c a l i b r a t i o n curve and m o l e c u l a r w e i g h t was  by the combined Svedberg e q u a t i o n and S t o k e s - E i n s t e i n e q u a t i o n M  =  S R  =  s e d i m e n t a t i o n c o e f f i c i e n t x 10  =  Stokes r a d i u s  n  =  0.01002 p o i s e  v  =  e  6N' TT n ( l - v )  _  determined  (Pang, 1975):  1  p  where, 13 S R  e  P  " 3 - 1 '0.75. cm g  ( t h e v i s c o s i t y o f water a t 20 C) ( p a r t i a l s p e c i f i c volume :'of e e l AchE "-reported by  Bon, e t 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 / f o ) w a s c a l c u l a t e d from t h e equation; ( S e i g e l and  Monty, 1966): f  Re  To  13.  (3v M/  4TTN)  1/3  Ion Exchange Chromatography on Deae-Sepharose CL-6B A 10 ml sample  (45 u n i t s ml *) p r e p a r e d by e x t r a c t i o n method  B was  a p p l i e d t o a column (2.8 X 57 cm) of DEAE-Sepharose CL-6B e q u i l i b r a t e d 20 mM-pptassium NaN^,  phosphate b u f f e r c o n t a i n i n g 0.03 M-NaCl and 0.01%  pH 7.0, a t a f l o w r a t e of 15 ml h ^.  ml o f b u f f e r and a NaCl g r a d i e n t  (0.03-0.8 M).  c o l l e c t e d and assayed f o r AchE a c t i v i t y The method mM-potassium  of a f f i n i t y  The column was  elution  (w/v)  eluted with  315  (7 ml) were  and p r o t e i n .  (Scopes, 1977) was  phosphate b u f f e r , pH 7.2.  p r e p a r e d by e x t r a c t i o n method A was  Fractions  with  performed i n 20  A 10 ml sample (45 u n i t s ml  *)  a p p l i e d to a column (5.5 X 10 cm) o f  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  guanidinium chloride, and 170 ml of buffer containing Fractions 14.  1 mM-  1 mM-acetylcholine.  (9 ml) were collected and assayed f o r AchE a c t i v i t y and protein.  C e l l Wall Extraction Hypocotyl tissue (50 g) was ground to a 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 10 mM-potassium phosphate buffer, pH 7.0, and allowed to s e t t l e u n t i l 2 layers appeared (10-20 min). transferred to centrifuge b o t t l e s .  The upper layer was  The lower layer was resuspended and  the s e t t l i n g 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 p e l l e t was resuspended i n 200 ml of buffer.  This suspension  contained less than 2% of the c e l l fragments as intact c e l l s (determined by l i g h t microscopy).  The suspension was treated with a Blackstone Ultrasonic  Probe f o r 2 min at 200 ± 50 W to release attached cytoplasmic contaminants. The fragments were collected by centrifugation at 10,000 g_ f o r 15 min, resuspended i n buffer 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. were usually s u f f i c i e n t to achieve the desired- purity.  Four washings The c e l l wall  suspension was assayed f o r AchE a c t i v i t y and protein.  Assays were  performed i n duplicate or t r i p l i c a t e on two c e l l wall  preparations.  20 15.  Growth R e g u l a t o r  Experiments  The f o l l o w i n g procedures were performed range  t o determine whether a  of exogenous s t i m u l i a f f e c t AchE a c t i v i t y  etiolated seedlings. darkness  in plastic  i n h y p o c o t y l hooks of  S u r f a c e s t e r i l i z e d beans were grown f o r 5 days i n  trays  (2 per t r e a t m e n t ) .  The e t i o l a t e d s e e d l i n g s -3  were sprayed w i t h a p p r o x i m a t e l y M - g i b b e r e l l i n , 10  4  M - k i n e t i n , 100 ppm  an e t h y l e n e source) or water.  fluorescent l i g h t  light  One  repeated.  of the f o l l o w i n g :  2-chlorosulphonic acid  t r a y of s e e d l i n g s was 3 -2 -1  ( c o o l w h i t e , 2 X 10  of h y p o c o t y l hooks was treatment was  10 ml of one  erg cm  sec  ).  h a r v e s t e d d a i l y from each f l a t  Hoo.ks.uweire.. f r o z e n -in ^ l i q u i d ..nitro'g.env andjcground T^pav.blumes -.of ^ i l 0;-mM^p.ho.sphat:.e"'- buffer," pH  J,..0,'  exposed to  A sample (2-4  i n dim  (,GBJ3>) <GR  performed 16.  green  545  filter).  wjs&eidadjied .and :the_suspensions  and p r o t e i n .  4°C. Assays were  i n d u p l i c a t e o r t r i p l i c a t e on t h r e e s e p a r a t e p r e p a r a t i o n s .  H y p o c o t y l Hook Angle Measurements N i n e - d a y - o l d e t i o l a t e d beans were h a r v e s t e d i n dim green  (25 W i n c a n d e s c e n t l i g h t f i l t e r e d by a CBS  GR 545  light  f i l t e r ) and hooks  were e x c i s e d as d e s c r i b e d by K l e i n , e t a l . (1956) and i n c u b a t e d i n -3 -5 -7 -9 p e t r i p l a t e s c o n t a i n i n g 10- , 1 0 ,10 and 10 M-acetylcholine c h l o r i d e i n water f o r 24 h i n darkness. a c e t y l c h o l i n e c h l o r i d e was  group c o n t a i n i n g no  exposed to r e d l i g h t  i n c a n d e s c e n t b u l b f i l t e r e d by a CBS determined  One  r e d 650  by the method of K l e i n , e t a l .  g)  to-..a •Mne p.owder i n a mortar.  thawed i n an i c e bath and d i a l y z e d f o r 18 h a g a i n s t b u f f e r at Samples (500 y l ) were assayed f o r AchE a c t i v i t y  (as E t h r e l ,  and the spray  These o p e r a t i o n s were performed  (40 W f i l t e r e d by a C a r o l i n a , . B i o l o g i c a l Supply  10  from a 25 W  filter. (1956).  Hook a n g l e s were The  experiments  21  were performed i n d u p l i c a t e .  17.  E l o n g a t i o n Measurements Seven-day-old e t i o l a t e d bean p l a n t s were sprayed  M-acetylcholine light  measured a g a i n a f t e r  l i g h t f i l t e r e d by  24 h.  a CBS  —6  GR  545  filter)  green  and  Measurements were made from the base of  a l o n g the l e n g t h of the h y p o c o t y l  node u s i n g a f l e x i b l e  to the  cotyledonary  ruler.  C.  1.  10  c h l o r i d e i n water o r water a l o n e , measured i n dim  (25 W i n c a n d e s c e n t  the h y p o c o t y l  with  RESULTS  The  Assay Methods  a)  The  e f f e c t of neostigmine  The  assumption t h a t n e o s t i g m i n e - i n h i b i t a b l e h y d r o l y s i s of  on AchE a c t i v i t y  a c e t y l t h i o c h o l i n e i?aSxidenfHLc:al.j-with AchE.. a c t i j V j i t y w a s t e s t e d , by T  determining choline.  the e f f e c t  The  neostigmine  g r e a t e r than  curve was  on the h y d r o l y s i s of a c e t y l t h i o e b  r e s u l t s are shown i n F i g u r e 2.  i n preparations having The  of neostigmine  10 uM  of  i n h i b i t e d s u b s t r a t e h y d r o l y s i s by  a s p e c i f i c a c t i v i t y of 12 u n i t s mg  c h a r a c t e r i s t i c of neostigmine  (Karczmar; 1967;  Concentrations  R i o v and J a f f e , 1973)  1  protein.  i n h i b i t i o n of AchEs  and v a l i d a t e d the  assumption  t h a t n e o s t i g m i n e - i n h i b i t a b l e h y d r o l y s i s of a c e t y l t h i o c h o l i n e ; i d e n t i c a l w i t h AchE a c t i v i t y  i n bean r o o t p r e p a r a t i o n s .  i n h i b i t e d p r e p a r a t i o n s remained i n a c t i v e a f t e r d i a l y s i s changes of b u f f e r f o r 36 h r a t 4°C. having  neostigmine  inhibitable.  20 u n i t s mg  1  w a s  Neostigmineagainst  A l l esterase a c t i v i t y  a s p e c i f i c a c t i v i t y g r e a t e r than  90%  3  i n preparations  protein  was  22 Figure  2.  The e f f e c t o f n e o s t i g m i n e on AchE a c t i v i t y . Root e x t r a c t s were 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 are 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 a c t i v i t y 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 l i n e a r i t y from 3-18 min was  reproducible for soluble and p a r t i c u l a t e f r a c t i o n s of  both root and hypocotyl preparations.  Extrapolation of the l i n e to  t=0 yielded a p o s i t i v e ^-422 value, which r e f l e c t e d 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 a c t i v i t i e s . c)  The effect of enzyme quantity on AchE a c t i v i t y The a c t i v i t y of AchE depended d i r e c t l y on the quantity of  solution assayed (Figure 4). values ranging from 0.02 presented  The relationship was  to approximately  0.85  i n Figure 4 this corresponds to 5-100  l i n e a r for  ^A^^  (for the experiment y l ) . The regression  c o e f f i c i e n t was 0.996.  2.  Extraction and (NH.^SO^ P r e c i p i t a t i o n of AchE From P_. v u l g a r i s Roots and Hypocotyls The crude preparations which were obtained a f t e r homogenization  of either root or hypocotyl tissues i n 10 mM-potassium phosphate buffer, pH 7.0, had both low AchE a c t i v i t y and low s p e c i f i c a c t i v i t y (Table I ) . The a c t i v i t i e s were increased 7 f o l d i n roots and 20 f o l d i n 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 a c t i v i t y of these preparations was  when the d i f f u s a t e from either root or hypocotyl d i a l y s i s  was  reduced  25 F i g u r e 4.  The e f f e c t o f enzyme q u a n t i t y on the AchE a c t i v i t y . Values are means^of two assays of a p r e p a r a t i o n c o n t a i n i n g 11.9 u n i t s mg protein.  volume assayed (pi)  Table I:  The e f f e c t of d i a l y s i s and the reintroduction of the diffusate to the non-dialyzable homogenate on the AchE a c t i v i t y i n _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 i n water was reduced to the o r i g i n a l sample volume and returned to the non-dialyzable f r a c t i o n . N.D. denotes values not determined.  Fraction Assayed Tissue Origin:  Origin of Diffusate  Activity ^ (units g )*  Sp. A c t i v i t y (units mg ) * *  % of I n i t i a l Activity  % Decrease of Activity in Non-Dialyzable Substances  ROOT  Homogenate Before Dialysis  0.25  100  20.4  2.83  648  0  15.2  N.D.  482  25  8.5  N.D.  270  58  Homogenate Before Dialysis  0.20  0.02  100  Homogenate After Dialysis  3.9  0.47  1950  0  2.2  N.D.  1100  43  1.3  N.D.  650  67  Homogenate After Dialysis Dialyzed Homo= >^ Root gem ate AAfiterAAddffddn yof f D:if f usat e Hypo co ty 1 Tissue Origin:  3.15  HYPOCOTYL  Dialyzed Homo-g,z Root genate.Mfit er. -Ad d i Ciori ofitDiffusate Hypocotyl  * weights are given as fresh weight ** weights are given as Lowry protein  27 r e i n t r o d u c e d to e i t h e r r o o t o r h y p o c o t y l p r e p a r a t i o n s .  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 c o r r e s p o n d  activity  produced by  the  to t h e i n c r e a s e d  dialysis.  A r a p i d e x t r a c t i o n method (method B) b u f f e r contained The  5%  (NH^^SO^ was  t e s t e d on r o o t s and  r e s u l t s are shown i n T a b l e I I .  p r o c e d u r e was  i n which the e x t r a c t i o n  Although  the 5%  hypocotyls.  (NH.).SO. e x t r a c t i o n 4 2 4  more e f f i c i e n t f o r h y p o c o t y l s , i n which 81% of the  a c t i v i t y was  e x t r a c t e d , than f o r r o o t s , i n which 43%  a c t i v i t y was  e x t r a c t e d , subsequent  total  (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 h i g h e r a c t i v i t i e s on p r o t e i n and i n hypocotyls.  of the  total  f r e s h weight bases i n r o o t s  than  Thus, f u r 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 r o o t 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 e x c i s e d p i e c e s of r o o t and  hypocotyl  The AchE a c t i v i t y of e x c i s e d p o r t i o n s of e t i o l a t e d bean s e e d l i n g s i s shown i n T a b l e I I I .  A c t i v i t y S.E.M. v a l u e s were v e r y  l a r g e because s m a l l absorbance changes were r e c o r d e d .  Enzyme a c t i v i t y  expressed  on both a f r e s h weight and p r o t e i n b a s i s was  highest i n  r o o t s and  lowest  d i s t a n c e up  i n the b a s a l r e g i o n s of h y p o c o t y l s , but  the h y p o c o t y l .  increased with  A c t i v i t y o f V e n t i r e c h y p o c o t y l s ; was 4  estimated  0. l-un-its>g: "vifreshhwdight" of _hyp.qco.t5yl &_ _,Thi-s Rvalue, wasipha'sed. on vationOfhat  and  the-obser-  the freshoweighteoft sub.ap.icalTarid b a s a l segments -comprised  greater.Tr,thany80%io£nthe gresh^ weight '.of.zhypoGoty^ls.  Both the  r o o t a c t i v i t i e s were lower than cprresp.o.nd;ing"V/alue.s  a s s a y i n g t i s s u e homogenates (Table I ) .  hypocotyl  obtained  at  by  Table I I :  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.  Activity ^ (units g )* 5% (w/v) (NH ) S0 4  10-40% Saturation  40-70% Saturation  * **  HYPOCOTYL  ROOT  FRACTION  2  4  Sp. A c t i v i t y (units mg )**  Activity_^ (units g )*  Sp. A c t i v i t y (units mg )**  residue  8.2  N.D.  0.06  N.D.  extract  6.1  3.2  0.25  0.2  precipitate  0.2  N.D.  0.06  N.D.  supernatant  5.8  3.8  0.21  1.1  precipitate  5.0  7.1  0.21  1.0  supernatant  0.9  N.D.  0.00  N.D.  weights are given as fresh weight weights are given as Lowry protein  Table 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 s e g m e n t s o f r o o t s a n d 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 s h o w n i n F i g u r e 1. I n t a c t 12 cm s e g m e n t s o f t i s s u e w e r e 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 a n d 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 o n a t l e a s t d u p l i c a t e s a m p l e s ± S . E . M .  O r i g i n of E x c i s e d Segments  Activity _^ u n i t s mg fresh wt. )  A p i c a l Region of Hypocotyl  0.370 + 0.199  16.8 ± 0.8  0.022  Hook R e g i o n o f Hypocotyl  0.206 ± 0.066  15.3 ± 0.6  0.013  0.087 ± 0.024  9.7 ± 0.7  0.009  Hypocotyl  0.072 ± 0.038  8.7 ± 0.5  0.008  Roots  0.700 ± 0.140  7.3 ± 0.2  0.096  Subapical  Total Protein (mg g fresh wt.)  Specific^Activity ( u n i t s mg protein)  Region  of H y p o c o t y l Basal Region of  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 h y p o c o t y l c e l l w a l l s p r e p a r e d by s u c c e s s i v e washes w i t h  b u f f e r were assayed The  a c t i v i t y was  f o r AchE a c t i v i t y by the p a r t i c u l a t e assay  0.033 ± 0.005 u n i t s mg  1  dry c e l l w a l l , and  s p e c i f i c a c t i v i t y of these p r e p a r a t i o n s was  0.25  ± 0.04  procedure.  the  u n i t s 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 d e t e c t e d i n e x t r a c t s of r o o t s homogenized i n  e i t h e r 10 or 20 mM-potassium phosphate b u f f e r , pH 7.0, c e n t r i f u g a t i o n and  filtration  (Table I V ) .  Values  after  for activity in  crude e x t r a c t s were c l o s e to the d e t e c t i o n l i m i t s of the assay and  consequently  v a r i e d from one  experiment to the next.  c o n s i s t e n t l y l e s s than 5% of the t o t a l a c t i v i t y (Tables I and  II).  The  r e s u l t s presented  when the r e s u l t i n g r e s i d u e was (w/v)  Specific activity after  These were  i n homogenates  i n T a b l e IV were o b t a i n e d  e x t r a c t e d w i t h b u f f e r c o n t a i n i n g 5%  ( N H ^ S O ^ and p a r t i a l l y p u r i f i e d by  f i l t r a t i o n on Sepharose 6B,  procedure  and  (NH^SO^ fractionation, gel  chromatography on MAC-Sepharose  (NH^^SO^ f r a c t i o n a t i o n was  4B.  g r e a t e r than t h a t  o b t a i n e d a f t e r e x t r a c t i o n by method B (Table I I ) , a l t h o u g h  total  r e c o v e r y was  elution  5.0  units g  1  f r e s h weight i n both  p r o f i l e shown i n F i g u r e 5 was on Sepharose 6B.  cases.  o b t a i n e d when AchE was  The  chromatographed  AchE a c t i v i t y e l u t e d as a s i n g l e peak h a v i n g a K  of  0.647. The  e l u t i o n p r o f i l e shown i n F i g u r e 6 was  o b t a i n e d when AchE  prepared by g e l f i l t r a t i o n on Sepharose 6B was  chromatographed on  MAC-Sepharose 4B.  S p e c i f i c a c t i v i t y of the c o n c e n t r a t e d 1 M-NaCl  the  elution volume (ml)  Figure  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 of AchE o n S e p h a r o s e 6B. S a m p l e s w e r e p r e p a r e d b y m e t h o d A . A c h E a c t i v i t y ( • — • ) arid•'•A^.go' f r a c t i o n were measured.  (x  x  ) °^  e a c n  10  m  l  elution Volume (ml) Figure  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 c h r o m a t o g r a p h y 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 s a m p l e 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 . T h e c o l u m n was w a s h e d 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 . A c h E a c t i v i t y (• e») and A^gQ ( x x) of each f r a c t i o n were measured. The h o r i z o n t a l d a s h e d l i n e i n d i c a t e s the A o f t h e s a m p l e (0.365). 0  Q  n  w  Table IV:  P u r i f i c a t i o n of AchE from _P. vulgaris roots. Methods of extraction and p u r i f i c a t i o n are detailed i n Materials and Methods. Values presented were obtained from one preparation and are representative of at least two other preparations. P u r i f i c a t i o n values are based on the s p e c i f i c a c t i v i t y of dialyzed root homogenates from Table I.  Volume - (ml)  Fraction  Protein (mg)  Total Units (units)  Crude Extract (in buffer) 5% ( N H ) S 0 4  2  848  508  4  Extract of Residue 80%  N.D.  695  662.3  (resuspended)  30  149.2  1775  Sepharose 6B  87  74.2  1722  2.6  589  2.9  Recovery """'(%'of 5% (NH^SO^ Extract)  Purification (-fold)  N.D.  6.4  100  2.3  11.9  42  4.2  23.2  41  8.2  222.9  14  78.8  4239  (NH,)„S0, ppt.  MAC-Sepharose 4B (after u l t r a f i l t r a t i o n )  Specific Activity (unitssmgv protein)  LO Co  34  e l u a t e v a r i e d from 1 9 0 - 2 3 0 u n i t s mg  5.  C h a r a c t e r i z a t i o n of P u r i f i e d a)  DIFP  * protein.  AchE  Labeling  Figure  7 shows t h a t AchE a c t i v i t y was  DIFP c o n c e n t r a t i o n s  greater  a c t i v i t y by DIFP was  than 1 0  M.  completely i n h i b i t e d at  The i n h i b i t i o n o f enzyme  reduced from 9 9 % t o 2 9 % i n t h e p r e s e n c e of 2 0  mM-butylcholine i n 2 0 mM-phosphate b u f f e r , pH 7 . 0 , and from 9 4 % t o 6 3 % i n t h e presence o f 2 0 0 mM-butylcholine i n 2 0 mM-phosphate b u f f e r , pH 7 . 0 , c o n t a i n i n g protects  0.2 M-NaCl.  The l a t t e r treatment  animal AchEs from DIFP i n a c t i v a t i o n  completely  (Cohen, e t a l . , 1 9 6 7 ) .  incomplete p r o t e c t i o n §gainst DIFP i n h i b i t i o n t h a t b u t y l c h o l i n e  The  provided  to the bean enzyme p r e v e n t e d t h e use o f b u t y l c h o l i n e i n experiments designed to s p e c i f i c a l l y  l a b e l AchE w i t h r a d i o a c t i v e DIFP.  T a b l e V shows t h a t t h e r e was g r e a t e r preparations  'labeled i n the absence r a t h e r than i n t h e p r e s e n c e of  1 2 5 uM-neostigmine.  The c a t a l y t i c c e n t e r  binding  s i t e s , was  binding  s i t e when c o r r e c t e d  a c t i v i t y , based on DIFP  6 0 6 and 1 9 7 ± 5 mol.' of s u b s t r a t e and u n c o r r e c t e d ,  i n the presence of neostigmine.  min * mol ^ DIFP  respectively, f o r binding  The c o r r e c t e d v a l u e was based on the  assumption t h a t n e o s t i g m i n e completely b)  i n c o r p o r a t i o n of I H - D I F P i n t o  i n h i b i t e d DIFP b i n d i n g  Disc Gel Electrophoresis Results  of polyacrylamide  gel electrophoresis  from two s t a g e s o f p u r i f i c a t i o n a r e shown i n F i g u r e  8.  d e n s e l y s t a i n e d bands o f p r o t e i n . ( R = 0 . 0 7 ± 0 . 0 3  F  F  a l i g h t l y stained region of h i g h  t o AchE.  specific activity  (from R ^ = 0 . 3 9 - 0 . 4 9 ) ( 1 9 5 - 2 2 9 u n i t s mg  of AchE  preparations  There were two  and R = 0 . 6 9 ± 0 . 0 3 )  and  after gel electrophoresis * 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 a r e means of two a s s a y | performed on a p r e p a r a t i o n containing 96 u n i t s ml and 229 u n i t s mg protein.  log  L DIFPH M  36 F i g u r e 8.  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of AchE. Samples were prepared by e i t h e r a) g e l f i l t r a t i o n on Sepharose 6B, o r b) chromatography on MAC-Sepharose 4B and s e p a r a t e d by e l e c t r o p h o r e s i s i n 7% (w/v) p o l y a c r y l a m i d e g e l s at pH 8.3. Gels were s t a i n e d w i t h 1% (w/v) Amido Schwartz ( a l and b l ) or cut i n t o 2 mm s l i c e s and assayed f o r AchE a c t i v i t y (b2). P r o t e i n zymograms are based on values averaged from d u p l i c a t e g e l s of two ( a l ) or t h r e e ( b l ) d i f f e r e n t p r e p a r a t i o n s . The enzyme a c t i v i t y p r o f i l e shows v a l u e s from one of two d u p l i c a t e experiments.  b  origin  2  origin CP  <t>  C  o  r  •  rT O  fnont  front 0 . 2  A A  0 . 4  412  37 T a b l e V:  3. I n c o r p o r a t i o n o f H-DIFP i n t o AchE p u r i f i e d by chromatography on mlg and 185-210 u n i t s mg p r o t e i n were r e a c t e d w i t h 10 M- H-DIFP i n the presence o r absence of 125 uM-neostigmine and counted f o r r a d i o a c t i v i t y a f t e r e x h a u s t i v e d i a l y s i s . E s t i m a t e d c a t a l y t i c c e n t e r a c t i v i t y was based on DIFP binding s i t e s . Values are means of t h r e e samples of d u p l i c a t e p r e p a r a t i o n s ± S.E.M. N.D. denotes v a l u e not determined.  Estimated iH-DIFP incorporated (pmol u n i t )  No  Net  (mol  c a t a l y t i c center activity ^ -1 of s u b s t r a t e min mol DIFP)  neostigmine  5.0710.12  197+5  Neostigmine  3.42±0.15  N.D.  value  1.65  606  38 s i n g l e peak o f AchE a c t i v i t y was l o c a t e d w i t h one o f t h e bands o f p r o t e i n . 90% o f t h e a p p l i e d a c t i v i t y . activity  (21530 u n i t s mg  1  (R^=0.05) t h a t  T h i s peak accounted  P r e p a r a t i o n s having lower  corresponded f o r more than specific  p r o t e i n ) showed a d d i t i o n a l p r o t e i n bands.  The methods o f K o e l l e (1951) as m o d i f i e d by Wright  and Plummer  (1973) and Karnovsky  and Roots (1964) were a p p l i e d without success t o  d e t e c t AchE a c t i v i t y  i n polyacrylamide gels.  c)  SDS G e l E l e c t r o p h o r e s i s  F i g u r e 9 shows t h e c a l i b r a t i o n curve o b t a i n e d a f t e r SDS p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of standard p r o t e i n s .  AchE was p u r i f i e d by chromato3  graphy:.onl-MAC7 .Sepharo'sep'ABr and labelediw"ithidH-MFP.; ••- The. scans .shown dnaEdguliresi. 10a a h d u M a were* o b t a i n e d ^ a f t^cSDScgel elec£r,pphpresis o f i  t h i s " preparationo.und'ersripnc-r.educing a©dc-re\4l^i»8d€SS^§'tigns..respectively. 1  Figures-.10b andpllbixehpw Theimajor  thegiraddoacLtiyityj. i n s l i e e s h p f r=lfe§:; same .-gels. ;  DiEFPr-labeledar'egipnsh  of  61 .000 + 2 000i, 26 000 ± 1000 and 17 500 ± 500. had a mol. wt. o f 77 000 ± 2000.  The 77 000, 61 000, and 26 000  D I F P - l a b e l e d components corresponded g e l scans. observed  A minor D I F P - l a b e l e d peak  t o d i s t i n c t p r o t e i n peaks i n t h e  A d d i t i o n a l low m o l e c u l a r weight  (16 000) p r o t e i n was  i n t h e g e l scans b u t no c o r r e s p o n d i n g t r i t i a t e d peak was seen.  The g e l s c a n o f reduced AchE showed t h e same p r o t e i n peaks.  There was  l e s s r a d i o a c t i v i t y i n t h e t r i t i a t e d - 6 1 000 mol. wt. s u b u n i t and an i n c r e a s e i n a c t i v i t y i n a 30 000 ± 1000 mol. wt. component. r e d u c t i o n o f t h e 61 000 mol. wt. s u b u n i t was not observed.  Complete The same  r e s u l t s were o b t a i n e d f o r AchE l a b e l e d w i t h DIFP i n t h e presence o f 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% S D S . 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  F i g u r e 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 SDSa c r y l a m i d e g e l e l e c t r o p h o r e s i s of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and l a b e l e d w i t h H-DIFP. AchE was denatured i n the absence of any r e d u c i n g agents. G e l s were s t a i n e d w i t h Coomassie B l u e , scanned f o r absorbance a t 550 nm, s l i c e d i n t o s e c t i o n s , and assayed f o r r a d i o a c t i v i t y . M o l e c u l a r weights are averages from d u p l i c a t e g e l s .  40a  Figure  11.  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 SDSa c r y l a m i d e g e l e l e c t r o p h o r e s i s of reduced AchE p u r i f i e d by chromatography on MAC-Sepharose 4B and l a b e l e d 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 s t a i n e d w i t h Coomassie B l u e , scanned f o r absorbance a t 550 nm, s l i c e d i n t o s e c t i o n s and assayed f o r r a d i o a c t i v i t y . M o l e c u l a r weights are averages from d u p l i c a t e g e l s .  41  Figure11 a  .  16K  42 d)  Sedimentation i n i s o k i n e t i c gradients  The results of i s o k i n e t i c sedimentation of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B are shown i n Figure 12.  AchE  a c t i v i t y appeared as one major peak with a corresponding value of 4.2 S (4.1-4.3S).  The preparations labeled with  ^ H-DIFP i n the 3  presence and i n the absence of 125 uM-neQstigmine each contained a single peak of r a d i o a c t i v i t y 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  p a r t i c l e within experimental error.  It was concluded that neostigmine  did not i n h i b i t completely the binding of DIFP to AchE and that a l l of 3 the r a d i o a c t i v i t y recovered from sedimentation of  H-preparations are  located i n AchE. e) - - Is oelectr ic'-f o cus ing A gradient from pH 3-9.5 i s o e l e c t r i c focusing.  was  obtained consistently a f t e r thin layer  Standard proteins focused at pis reported  previously (Radola, 1973).  Figure 13 shows the result of a t y p i c a l  i s o e l e c t r i c 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 a c t i v i t y and focused at pH 9.2 ± 0.1; only a f a i n t s t a i n was detected at pH 5.3.  An irreproducible  peak of AchE a c t i v i t y focused at pH 6.7-7.7 (usually at pH 7.0). peak appeared  The  i n most preparations but r a r e l y focused at the same pH.  Preparative scale experiments, i n which 400-500 units of enzyme a c t i v i t y were applied to a 2-10 mm thick dextran layer, resulted i n y i e l d s of less than 20 units a f t e r recovery of the enzyme by elution of the dextran  42a  Figure 12.  Elution 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. Samples_contained: a) 54 units of AchE a c t i v i t y (195 units mg protein) p u r i f i e d by MAC-Sepharose 4B, b) the samg preparation as a) but quantitatively i n h i b i t e d by 10 M- H-DIFP^ and c) the same preparation as a) but treated with 10 M- H-DIFP i n the presence of 125 yM-neostigmine. Fractions (0.5 ml) were assayed for AchE a c t i v i t y (a) or r a d i o a c t i v i t y (b. and c) (A A ) , and g-galactosidase (S=15.9) a c t i v i t y (o o) ; absorbance at 405 nm was measured to l o c a l i z e catalase (S=11.3) (« •) and myoglobin (S=2.0) (a n). The p r o f i l e s were duplicated t O v o b t a i n average S values.  F i g u r e 12 a  4  8  12  16 20 f r a c t i o n number  24  Figure  12b  fraction number  fraction number -Or  4 8 distance from cathode Figure 13.  12 (cm)  16  AchE a c t i v i t y and pH. gradient after thin layer 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. 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 a c t i v i t y (• cm) were determined on approximately 100 y l 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 i s o e l e c t r i c fosucing i n a sucrose gradient.  The peak of AchE a c t i v i t y at pH 7.0 accounted f o r less than  8% of the applied a c t i v i t y . column.  Protein p r e c i p i t a t e was observed i n the  Neither method of i s o e l e c t r i c focusing was suitable f o r further  p u r i f i c a t i o n of AchE a c t i v i t y because of low recovery and p r e c i p i t a t i o n . f)  • Stokes-e-Radius andsMoleculaf,,Weight  The Stokes radius c a l i b r a t i o n curve obtained by g e l f i l t r a t i o n of standard proteins i s shown i n Figure 15.  Using the value of K  =• 0.647  & v  obtained f o r AchE prepared by method A, the Stokes radius of t h i s 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).  T h e - f r i c t i o n a l r a t i o , f / f o , r e s u l t i n g from  these idata was. 1.37. .A mol.-wt^ of 78'- 000 ± 8-000 was obtained globular protein c a l i b r a t i o n curve of the same standard  from a  protein K data av r  prepared by p l o t t i n g l o g mol. wt. vs K  (Figure 16).  g) . Substrate ^ A f f i n i t i e s and the^Effect of'Various • " AchE A c t i v i t y . 1)  Substances on  The effect,of acetylthiocholine, propionylthiocholine, and butylthiocholihe on AchE*activity.  The effects of acetylthiocholine, propionylthiocholine, and butylthiocholine on the AchE a c t i v i t y of MAC-Sepharose 4B-purified preparations  are shown i n Figure 17.  for hydrolysis of the three substrates  The s p e c i f i c a c t i v i t y of AchE (ImM) i s shown i n Table VI.  Figure 18 shows a Lineweaver-Burke plot of the results shown i n Figure 17 for acetylthiocholine.  A regression l i n e f o r 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 f o c u s i n g of AchE p u r i f i e d by chromatography on MAC-Sepharose 4B. The sample was focused as d e s c r i b e d i n M a t e r i a l s and Methods and the pH (• • ) , the &280 ^ ^' * the AchE a c t i v i t y ( • — - — r • ) of each f r a c t i o n were measured. A  A  anc  49 Figure; 15.  Stokes r a d i u s c a l i b r a t i o n curve. a f t e r g e l f i l t r a t i o n of standard  The p l o t was obtained p r o t e i n s on Sepharose 6B.  50 F i g u r e 16.  G l o b u l a r p r o t e i n m o l e c u l a r weight c a l i b r a t i o n c u r v e . The p l o t was o b t a i n e d a f t e r g e l f i l t n a t i o n of the s t a n d a r d p r o t e i n s shown i n F i g u r e 15 on Sepharose 6B.  51 Figure  17.  The e f f e c t of s u b s t r a t e c o n c e n t r a t i o n on AchE a c t i v i t y of MAC-Sepharose 4 B - p u r i f i e d p r e p a r a t i o n s . V a l u e s a r e averages of d u p l i c a t e assays. Substrates tested included a c e t y l t h i o c h o l i n e (+ +)•, p r o p i o n y l t h i o c h o l i n e (• •) , and b u t y l t h i o c h o l i n e (x x).  52 Table VI: Substrate s p e c i f i c i t y of AchE prepared by chromatography on MAC-Sepharose AB f o r three choline esters. Values are means of duplicate assays.  Substrate ACETYLTHIOCHOLINE  :  S p e c i f i c activity (units mg . ..protein) 19 A.1  PROPIONYLTHIOCHOLINE  62.1  BUTYLTHIO CHOLINE  65.9  53 F i g u r e 18.  A Lineweaver-Burke p l 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 o f d u p l i c a t e a s s a y s , based on r e s u l t s p r e s e n t e d i n F i g u r e 17.  54  limb of the acetylthiochdline curve i n Figure 17 resulted i n a K 56 yM.  The K  of  f o r acetylthiocholine, determined by the same method on  low s p e c i f i c a c t i v i t y (11.5 units mg protein ^) preparations, was 58 yM. The enzyme did not obey Michaelis-Mente-n  k i n e t i c s when either prQpionyl-  thiocholine or butylthiocholine was used as the substrate.  Substrate  i n h i b i t i o n i s c h a r a c t e r i s t i c of acetylcholinesterases from both plant and animal sources  (Augustinsson and Nachmansohn, 1949; Wilson and  Bergman, 1950; Riov and J a f f e , 1973; Kasturi and Vasantharajan,  1976) and  both the high and low s p e c i f i c a c t i v i t y preparations from P_. v u l g a r i s roots displayed this c h a r a c t e r i s t i c (Figure 17). 2) -The effect, of choline, decamethonium "(NH-^^SO^, and NaCl .. .Jon AchE a c t i v i t y . Figure 19 shows the effect of choline on AchE a c t i v i t y . Choline enhanced AchE a c t i v i t y at concentrations ranging from 0.5-10 mM. The enhancement was reduced at concentrations exceeding  10 mM.  Decamethonium has been used, as ran eluant i n affinity.chromatography studies involving the use of the N-methylacridinium 1972a).  ligand (Dudai, et a l . ,  The i n h i b i t i o n of the AchE a c t i v i t y by decamethonium i s shown  i n Figure 20.  A c t i v i t y was recovered following d i a l y s i s against three  changes of buffer f o r 9 h at 4°C. Figure 21 shows the effect of NaCl on AchE a c t i v i t y of preparations p a r t i a l l y p u r i f i e d by method B.  Both root and hypocotyl AchE a c t i v i t y  were inhibited by high concentrations of NaCl. reversed by d i l u t i o n of NaCl.  I n h i b i t i o n could be  There was no interference with the AchE  assay when the NaCl concentration was reduced to less than 0.1 M i n the assay solution.  55 Figure  19.  The e f f e c t of c h o l i n e on MAC-Sepharose,4B-purified AchE (195 u n i t s mg protein). The v a l u e s are means of duplicate assays.  56 F i g u r e 20.  The e f f e c t of decamethonium on AchE a c t i v i t y . The v a l u e s a r e means of d u p l i c a t e assays of samples p r e p a r e d by method A.  log [decamethonium] M  57 Figure  loot  21.  The e f f e c t o f NaCl on AchE a c t i v i t y . Samples were p r e p a r e d by 70% (NH^^SO^ p r e c i p i t a t i o n of r o o t ( a ^ ) and h y p o c o t y l (o o) e x t r a c t s . The v a l u e s a r e means of d u p l i c a t e a s s a y s .  58 High concentrations  of (NH^^SO^ inhibited root AchE (Figure 22).  This i n h i b i t i o n could be reversed by d i l u t i o n of (NH^^SO^ but d i a l y s i s against buffer was preferred to regain a c t i v i t y . Sodium azide (0.01% (w/v)) had no effect on AchE a c t i v i t y of preparations p a r t i a l l y p u r i f i e d by method. 6.  Behavior of Low S p e c i f i c A c t i v i t y AchE on Chromatographic Media a)  Chromatography on MAC-Sepharose 4B The i n i t i a l attempts to purify root AchE by a f f i n i t y chromatography  used conditions established f o r the p u r i f i c a t i o n of AchE from electroplaques of Electrophorus and Clark, unpublished Table VII.  results).  electricus  (Dudai, et a l . , 1973; Webb  The results obtained are presented i n  L i t t l e binding occurred when the enzyme was applied to the  column i n 10 mM-potassium phosphate buffer, pH 7.0, containing 1.0 M-NaCl.  When the enzyme was applied i n buffer without s a l t , 62% of the  a c t i v i t y remained bound a f t e r a 20 ml buffer wash. was  A l l of that a c t i v i t y  recovered when the column was eluted with 1.0 M-NaCl i n buffer.  step resulted i n a 6.5 f o l d p u r i f i c a t i o n (Table V I I ) .  This  This p u r i f i c a t i o n  factor suggested that under such conditions, the matrix was behaving as an ion exchanger.  Conditions were modified i n an attempt to obtain  greater p u r i f i c a t i o n . Samples prepared by method B i n 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 p r o f i l e s shown i n Figure 23 were obtained.  Substantial a c t i v i t y was  retarded only by the column containing 2.0 Umol of ligand ml . 1  59  F i g u r e 22.  The e f f e c t o f ( N H ^ S O ^ on AchE a c t i v i t y . The v a l u e s a r e means of d u p l i c a t e assays of r o o t e x t r a c t s p r e p a r e d by method B.  60 Table VII:  Recovery of AchE a c t i v i t y 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 f o r recovery, s p e c i f i c a c t i v i t y and p u r i f i c a t i o n were obtained after e l u t i o n of columns with buffer containing 1.0 M NaCl. Values are derived from averages of duplicate assays. N. D. denotes values not determined.  Equilibration Buffer 1 M-NaCl no NaCl  A c t i v i t y Bound to column (% of applied activity)  Recovery (% of bound activity)  6  N.D.  62  •'  -  100  Specific Activity ^ (units mg • Purification "protein) ,'. (-fold) N.D. 56.8  N.D. 6.5  60a  Figure  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 columns of 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 . Ligan| c o n c e n t r a t i o n s _ | n t h e c o l u m n s were^ A ) 0.4 y m o l m l , B ) _ l . 0 ymol ml , C) 1.6 y m o l m l , a n d D) 2.0 y m o l ml . C o l u m n s w e r e l o a d e d w i t h 20 m l o f enzyme p r e p a r e d b y m e t h o d 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 c o l u m n s w e r e w a s h e d 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 a n d e l u a t e f r a c t i o n s (1 m l ) w e r e 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.  fraction  number  ~"  11.6  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 ^. Figure 24.  The e l u t i o n p r o f i l e s are shown i n  The amount of protein bound to the columns varied inversely  with the i o n i c strength of the buffering system.  The greatest amount  of AchE a c t i v i t y was bound to the columns operated i n 0.2 M-NaCl. E l u t i o n of protein ceased abruptly a f t e r a l l 4 columns were washed with t h e i r e q u i l i b r a t i o n buffers.  Elution of AchE a c t i v i t y from the column  operated i n 0.4 M-NaCl continued a f t e r ^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 a c t i v i t y was retained by the columns. No a c t i v i t y was recovered a f t e r the four columns were eluted with 5 mMneostigmine and the r e s u l t i n g fractions were dialyzed against for 36 h at 4°C.  Following  buffer  e l u t i o n with neostigmine, the columns  previously operated i n 0.05 and O i l M-NaCl were eluted with buffer containing 1.0 M NaCl.  No a c t i v i t y was recovered.  I t was concluded  that neostigmine released the bound enzyme but the decarbamylation reaction was too slow to y i e l d active enzyme a f t e r d i a l y s i s . Enzyme prepared by method B was applied i n 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 p r i o r to the AchE assay. The elution p r o f i l e s obtained are shown i n Figure 25a.  E l u t i o n with the  Figure  24 A.  B.  100  P.4  50 "D CD  10  t  a  f20  30  sample  ^ 40 neostigmine  buffer  >  50f  ro oo O  1M NaCl  c.  o  <  <  I oo|50  t  30  10 buffer  sample  Figure  24.  . t  40 .  50  neostigmine  neostigmine fraction  number  R e c o v e r y o f A c h E f r o m MAC-Sepharo;-e-4B c o l u m n s 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 . T w e n t y m l o f enzyme p r e p a r e d b y 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 mMp 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 w a s h e d 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 mMneostigmine i n e q u i l i b r a t i o n buffer. 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 ) w e r e a s s a y e d f o r A c h E a c t i v i t y ( ± . ^ ) 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 the A s o f t h e a p p l i e d *280 ' — *> zoU samples. a n d  (  o n  6ja  Figure 25.  Recovery of AchE from MAC-Sepharose 4B column by e l u t i o n 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 i n 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 f o r AchE a c t i v i t y (• • ) and A gQ (• - — • ) • P r o f i l e s presented were duplicated. 2  Figure 25 a 64  30  sample  buffer  60 acetylcholine  NaL  elution volume (ml) Figure 2 5 c  65  NaCl gradient produced a broad peak of r e l a t i v e l y low s p e c i f i c a c t i v i t y (45 units mg * protein) accounting for 51% of the loaded a c t i v i t y . Similar results were obtained using enzyme prepared by method A. No a c t i v i t y was recovered by substrate e l u t i o n (Figure 25b). When a 0.2-1.0 M-NaCl gradient was subsequently applied, 68% of the bound a c t i v i t y was recovered.  I t was concluded that acetylcholine  did not a f f e c t AchE binding. E l u t i o n with a decamethonium gradient resulted i n a slow emergence of protein and a peak of AchE a c t i v i t y at 25 mM-decamethonium (Figure 25c).  This peak was asymmetric and broad suggesting non-specific  elution.  Twenty one percent of the bound a c t i v i t y was recovered i n  this peak and subsequent e l u t i o n with the NaCl gradient yielded additional a c t i v i t y accounting f o r a t o t a l of 39% recovery. a c t i v i t y was 60 units mg * protein.  Specific  Comparable r e s u l t s were obtained  from decamethonium eluates that had not been dialyzed p r i o r to assaying. There was no additional recovery of AchE when 40 mM-decamethonium was applied a f t e r e l u t i o n of a l l columns with 1.0 M-NaCl during routine p u r i f i c a t i o n (Figure 6). In the p u r i f i c a t i o n of e e l AchE, an inverse relationship was found between the bed volume and recovery (G. Webb, personal communication). To examine this relationship i n 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 a c t i v i t y was released i n the i n i t i a l f r a c t i o n .  The major  66  Table VIII:  Bed volume (ml)  Recovery of AchE a c t i v i t y i n successive buffer (10 mMpotassium 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 a c t i v i t i e s are expressed as percentages of the applied sample volume. Values represent r e s u l t s from one of two duplicate experiments.  Buffer (%)  Decamethonium  (%)  NaCl (%)  0.5  24  7  9  40  2.0  27  21  12  60  67 Figure 26,  Recovery of AchE a c t i v i t y following delayed NaCl e l u t i o n on a MAC-Sepharose 4B column. The column was developed as described f o r Figure 25 except one day passed between the completion of the buffer wash and the application of the NaCl gradient. AchE a c t i v i t y (• • ) and A „ _ (• • ) were measured. 2  68  AchE peak contained a greater s p e c i f i c a c t i v i t y (67 units mg * protein) than the corresponding peak i n Figure 25a. b)  Gel f i l t r a t i o n on Sepharose 6B Four major peaks of AchE a c t i v i t y 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). a c t i v i t y was achieved.  No increase i n s p e c i f i c  The f i r s t peak eluted i n the void volume; the  others had the following values f o r K .: 0.48, 0.66 and 0.87. av  value corresponded to mol. wts. of  These  1,000,000, 350,000, 70,000, and  10 000 daltons, respectively, applying the c a l i b r a t i o n 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 a c t i v i t y were eluted from a DEAE-  Sepharose CL-6B column (Figure 28).  No substantial increase i n s p e c i f i c  a c t i v i t y occurred i n any of these peaks.  The e l u t i o n p r o f i l e suggests  that there may be either isoenzymes or aggregates of AchE. having d i f f e r e n t ion exchange properties. There was no a c t i v i t y 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 p i of 7.0 would be loosely bound to the ion exchange r e s i n 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 p i of 5.3 because enzyme prepared by both methods  (A and B) precipitated at pH  5.6 r e s u l t i n g i n a loss of more than 90% of the enzyme a c t i v i t y .  69 F i g u r e 27.  E l u t i o n of AchE prepared by method B from Sepharose 6B. F r a c t i o n s (5 ml) were c o l l e c t e d and assayed f o r AchE activity ( A A ) and A (° ° ) . The p r o f i l e p r e s e n t e d was reproduced i n a s e p a r a t e p r e p a r a t i o n .  elution v o l u m e (ml)  70 Figure 28.  E l u t i o n p r o f i l e of AchE elutedjfrom DEAE-Sepharose CL-6B. AchE (540 units, 8.8 units mg protein) was applied to the column i n 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 f o r AchE a c t i v i t y (A  A ) and A  —I  200  0  R  T  n  (  ) .  "  400  elution  ««  •  •  600  v o l u m e (ml)  *  ^  800  7.  Physiological Role of AchE i n the Hypocotyl a)  Effect of Growth Regulators on AchE A c t i v i t y i n the Hypocotyl Hooks Experiments were designed to screen regulatory substances and  white l i g h t f o r t h e i r effects on AchE a c t i v i t y i n the hypocotyl hook. 3 There was no e f f e c t of white l i g h t (1 X 10  -2 erg cm  -1  sec  ) or Ethrel  (100 ppm; 2-chlorosulphonic acid) on AchE a c t i v i t y or s p e c i f i c a c t i v i t y i n hypocotyl hooks of 5-day old etiolated seedlings over a 4 day period ( i e . 9 days a f t e r germination) (Figure 29). The Ethrel treated plants displayed the c h a r a c t e r i s t i c 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 s i g n i f i c a n t ( t ^ ^ =  44.5, a = 0.01) decrease i n s p e c i f i c _3  a c t i v i t y of AchE was observed (Figure 30). G i b b e r e l l i n (10 treated plants showed a s i g n i f i c a n t ( t ^ ^ = AchE s p e c i f i c a c t i v i t y by the t h i r d day.  M)  28.4, a = 0.01) increase i n  By the fourth day plumular  hooks of these and Ethrel-treated plants showed effects of tissue damage. The p o s s i b i l i t y that effects were an a r t i f a c t of early stages of hook injury i n E t h r e l - and gibberellin-treated plants cannot be dismissed. b)  The E f f e c t of Acetylcholine on the Hypocotyl The e f f e c t 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° a f t e r 20 h i n darkness (Figure 31). This value does not agree with the control value of 0° reported for the Black Valentine v a r i e t y of bean t y p i c a l l y used i n hook angle experiments (Klein, 1956); however, no s i g n i f i c a n t difference (a = 0.01) was -3 -5 -7 observed between the angle of hooks incubated i n 10 -9 10 M-acetylcholine and the control hook angle.  ,10  ,10  , or  Hooks exposed to red  72 Figure 29.  The effects of l i g h t and Ethrel 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 o l d e t i o l a t e d P_. v u l g a r i s . Plants were sprayed d a i l y i n dim green l i g h t with either water ( A - — - A ) or Ethrel (100 ppm; 2-chlorosuphonic acid) (• •) and l e f t i n darkness, or with^water^and exposed to continuous l i g h t (2 X 10 erg cm sec ) (• • ) . Hypocotyl hooks (15 to 20) were excised d a i l y u n t i l 9 days a f t e r germination, homogenized, and 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. Values are means of 3 experiments and error bars represent + or -.S.E.M.  2  3  clays after initial "treatment  4  73 Figure 30.  The effects 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 J?. v u l g a r i s . Plants were sprayed d a i l y i n dim green l i g h t 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 a c t i v i t y ; .^Values are means of 3 experiments and error bars represent + or - S.E.M.  74 F i g u r e 31.  The e f f e c t of a c e t y l c h o l i n e on the hook angle of 20 h e x c i s e d h y p o c o t y l hooks of P_. v u l g a r i s . V a l u e s a r e means o f 30 i n d i v i d u a l hooks f o r two pooled experiments and e r r o r b a r s r e p r e s e n t ± S.E.M.  log facetylcholine] M  75 l i g h t had a f i n a l angle of 72.4  ± 19.8°.  Seven-day old e t i o l a t e d P_. vulgaris hypocotyls 130.7  ±0.8%  and  130 ± 1.3%  elongated to  of their i n i t i a l lengths a f t e r 24 h i n —6  darkness when sprayed with water or 10 There was  no s i g n i f i c a n t difference (a = 0.01) _ . ;,  1.  M-acetylcholine, r e s p e c t i v e l y .  D.  DISCUSSION  Identity of-AchE in.'P.;..vulgaris. Criteria  for  the  between these values.  .  _  ( i d e n t i f i c a t i o n ..ofj^AchE  have been established (Fluck and J a f f e , 1974a).  activity  in- plants  They include i n h i b i t i o n  by neostigmine and maximal a c t i v i t y against acetyl esters of choline as fundamental features of the enzyme. c r i t e r i a was  AchE a c t i v i t y that s a t i s f i e d these  i d e n t i f i e d i n P_. v u l g a r i s .  Acetylcholine hydrolysis i n the presence of p a r t i a l l y p u r i f i e d extracts was  i n h i b i t e d by neostigmine (Figure 2) at  concentrations  e f f e c t i v e against AchE a c t i v i t y i n animals (Augustinsson, 1960 and plants (Riov and J a f f e , 1973).  This property was  and  1963)  exploited i n  a c t i v i t y assays to correct f o r spontaneous or non-specific hydrolysis of acetylcholine and a l l of the esterase a c t i v i t y i n preparations p u r i f i e d beyond (NH^^SO^ p r e c i p i t a t i o n was  neostigmine i n h i b i t a b l e .  The  neostigmine i n h i b i t a b l e hydrolysis of acetylthiocholine was related l i n e a r l y to the volume of the extract which was the assay time (Figure 3). hydrolyzed  assayed (Figure 4) and  The observation that acetylthiocholine was  three times as fast as either b u t y l - or propionyl-thiocholine  (Figure 17) further supported the argument that this plant was  an acetylcholinesterase.  cholinesterase  76 Both acetylcholine and i t s thiocholine analogue used as the  substrate  i n t h i s study, have been reported to occupy the same p o s i t i o n i n 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  AchEs (Riov and J a f f e , 1973)  plant  hydrolyze the acetylthiocholine more slowly  than acetylcholine and have s l i g h t l y higher Kms  for the analogue.  The p o s s i b i l i t y of microbial contamination of extracts as a source of AchE a c t i v i t y was  considered  because a cholinesterase was  i n the bacterium Pseudomonas fluorescens 1967).  (Fitch, 1963;  identified  Laing, et a l . ,  The present study did not address t h i s problem d i r e c t l y but  following observations  negate t h i s p o s s i b i l i t y .  AchE a c t i v i t y  the  was  located histochemically within the c e l l s of Phaseolus aureus roots (Fluck and J a f f e , 1974b) and was study.  It was  located i n p u r i f i e d c e l l walls i n t h i s  extracted i n s i m i l a r amounts from one experiment to the  next, a feature not to be expected from contaminating elements.  Callus  tissue grown axenically produced a c t i v i t i e s comparable to those from the parent tissue.  Surface s t e r i l i z e d seeds germinated i n s t e r i l e p e t r i  plates produced seedlings having the same a c t i v i t y as t h e i r v e r m i c u l i t e grown counterparts  (R. A. Fluck, personal  communication).  A group of cholinesterases exists i n plants which has a low Km for acetylcholine or acetylthiocholine (56-200,UM) and i s i n h i b i t e d by carbamates neostigmine (10 and J a f f e , 1973; 1976).  M) or eserine  (10 *M) (Tzagaloff, 1963;  the Riov  Fluck and J a f f e , 195!4d; Kasturi and Vasantharajan,  Variations exist i n the response to choline and the hydrolysis  rate hierarchy.  The AchE i n t h i s study and the enzymes studied by Riov  and J a f f e (1973) and Kasturi and Vasantharajan (1976) have been p u r i f i e d beyond (NH,)„S0, p r e c i p i t a t i o n . Only the bean enzymes were  77 stimulated by choline (Figure 19 and Riov and J a f f e , 1973).  A l l of the  cholinesterases which have been p a r t i a l l y p u r i f i e d by (NH^^SO^ p r e c i p i t a t i o n are i n h i b i t e d by choline (Tzagaloff, 1963; Schwartz, 1967; Fluck and J a f f e , 1975).  Both Schwartz (1967) and Kasturi and  Vasantharajan (1976) studied enzymes from Pisum sativum root but the l a t t e r workers did not examine the effect of choline on t h e i r preparations. Cholinesterase a c t i v i t y was i d e n t i f i e d i n 23 species from 5 families (Fluck and J a f f e , 1974d).  Which of these cholinesterases are  acetylcholinesterase remains uncertain because of low l e v e l of purity of these preparations and the undetermined substrate hydrolysis rate hierarchy of each enzyme. AchE was located i n the roots of P_. v u l g a r i s and t e n t a t i v e l y i d e n t i f i e d i n the hypocotyl.  The hypocotyl enzyme was not p u r i f i e d beyond  (NH^^SO^ p r e c i p i t a t i o n and must be considered as a cholinesterase even though i t resembled the root enzyme i n several respects  (Figures 4, 21,  and 27, and Table I I ) . AchE has been i d e n t i f i e d and p a r t i a l l y p u r i f i e d from roots of two other members of the Fabaceae (Riov and J a f f e , 1973; Kasturi and Vasantharajan, 1976). 2.  Extraction and L o c a l i z a t i o n .of AchE The  i n roots.  - •  highest :extractable . AchE. a c t i v i t i e s : have  been  reported  x  Leaves contained nearly as much as roots; and buds, hypocotyls,  and stems contained the least (Fluck and J a f f e , 1974b and d). measured i n  Activity  vulgaris tissue under d i f f e r e n t conditions was always  greater i n the roots but quantitation of a c t i v i t y proved d i f f i c u l t . Enzyme a c t i v i t i e s were measured i n s i t u by assaying tissue s l i c e s (Table III) but values obtained by this method were s l i g h t l y ( i n  78  hypocotyls) or s u b s t a n t i a l l y ( i n roots) lower than a c t i v i t i e s i n homogenates.  This suggested that not a l l of the enzyme was accessible  to the substrate and that the assay was determinations  in. s i t u .  not suitable for cholinesterase  Fresh homogenates contained s u b s t a n t i a l l y lower  a c t i v i t i e s than the same homogenates after d i a l y s i s against water (Table I ) .  The increase of a c t i v i t y during d i a l y s i s i s attributed i n  part to the presence of an endogenous dialyzable i n h i b i t o r (Table I ) . However, other factors removed during d i a l y s i s contributed to the  low  a c t i v i t y since return of concentrated d i f f u s a t e did not reduce a c t i v i t y to the l e v e l detected before d i a l y s i s . hypocotyl was  greater than i n the root.  The i n h i b i t o r y agent i n the I n s u f f i c i e n t information i s  available to determine the nature or the s i g n i f i c a n c e of i t s e f f e c t . K a s t u r i and Vasantharajan  (1976) found increased t o t a l AchE a c t i v i t y  a f t e r (NH^^SO^ p r e c i p i t a t i o n . 2-nitro-5-thiobenzoate  Fluck and J a f f e (1974d) i d e n t i f i e d a  decolorizing a c t i v i t y i n buffer extracts of P_.  aureus roots which was p a r t i a l l y dialyzable and heat-inactivated.  A  similar decolorizing agent has been reported i n sea urchin extracts (Wolfson, 1972) which was heat-inactivated and completely d i a l y z a b l e . Regardless  of the nature of the i n h i b i t o r values for AchE a c t i v i t y i n  fresh homogenate appear to be minimal.  The presence of i n h i b i t o r s of  presumably v a r i a b l e 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, I I , and I I I ; Figures 29 and 30) and no quantitative estimate was  obtained for a c t i v i t i e s i n s i t u . Enzyme a c t i v i t y was  vulgaris hypocotyls.  detected i n c e l l walls extracted from P_.  The result was  expected since Fluck and Jaffe  79 (1974b) found that 95% of the AchE a c t i v i t y i n P_. aureus roots  was  located i n the c e l l walls.  cell  Jansen, et a l . , (1960) showed that  walls were capable of binding soluble proteins which were not native components of the w a l l .  The AchE a c t i v i t y was  considered a r e a l  component of the c e l l wall because the lOmM-buffer insoluble a c t i v i t y was  greater than 5% of the t o t a l a c t i v i t y . The 5% (NH^SO^ extracted 43% of the root acetylcholinesterase  (Fluck and Jaffe's (1974b) value was enzyme.  37%)  and 81% of the  hypocotyl  The d i f f e r e n t e f f i c a c i e s of this extractant i n roots  hypocotyls  and  suggests that the l o c a l i z a t i o n or binding properties of  AchE d i f f e r e d between these two organs.  Ammonium sulphate  also  extracted a greater portion of AchE from a e r i a l organs than from roots of l i g h t grown mung bean plants (Fluck and J a f f e , 1974b). observation  The  that only 57% of the l i g h t grown mung bean hypocotyl  AchE (Table 6 i n Fluck and J a f f e , 1974b), but 81% of the e t i o l a t e d 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) a c t i v i t y exist i n the c e l l wall and they d i f f e r i n t h e i r binding properties to the wall (Fluck and J a f f e , 1974b).  At  least f i v e other c e l l wall enzymes exhibit this property and i n the case of peroxidases,  covalently and noncovalently  bound populations  enzymes have been proposed (Hall and Butt, 1968; Osborne, 1970;  K l i s , 1971;  Nevins, 1970;  of the Ridge and  Copping and Street, 1972).  The s e l e c t i o n of 5% (NH^SO^ to extract AchE from P. vulgaris was  based on work of Riov and J a f f e (1973), and Fluck and J a f f e (1974b)  80 who  found 4% (NH^^SO^ to be the most e f f e c t i v e of 10 extraction media.  This was  also the most e f f e c t i v e extractant used i n the p u r i f i c a t i o n of  autolyzed or trypsin-digested eel AchE (Leuzinger and Baker, 1967). 3.  P u r i f i c a t i o n and The  use  of  Characterization  5%  (NH,)^,S0. 4 2  A  total  favored extraction  of one - form ~of AchE.  4-  79-fold' > p u r i f i c a t i o n of AchE was  achieved by the use 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 chromatography on Sepharose 4B.  MAC-  The p u r i f i c a t i o n shown i n Table I I I yielded enzyme of  f o l d greater purity than previously reported Greater y i e l d s (2.15  compared to 1.71  units g  283  (Riov and J a f f e , 1973). 1  fresh weight) were  obtained by Riov and J a f f e (1973) but they achieved a lower s p e c i f i c a c t i v i t y (96 compared to 223 units mg of the pea AchE was  only 37.8  protein).  1  units mg  1  The s p e c i f i c a c t i v i t y  protein (Kasturi and  Vasantharajan, 1976). P r e c i p i t a t i o n with (NH^^SO^ has been used i n the p u r i f i c a t i o n of cholinesterases  (Tzagoloff, 1963;  1974d) and acetylcholinesterases Baker, 1967;  Schwartz, 1967;  Fluck and J a f f e ,  (Kremzner and Wilson, 1963;  Dudai,*'-et^.al. , 1972b; Riov and J a f f e , 1973;  Vasantharajan, 1976).  1  protein (Figure 6) vs 45 units mg  this reason i t provided  Kasturi and  By the use of gel f i l t r a t i o n i n the p u r i f i c a t i o n  protocol, a 5-fold increase i n s p e c i f i c a c t i v i t y was mg  Leuzinger and  -1  achieved (220 units  protein (Figure 25a)).  For  a substantial contribution to the p u r i f i c a t i o n .  The elution p r o f i l e for gel f i l t r a t i o n on Sepharose 6B i n the presence of 0.2M-NaCl contained migration  one peak of AchE a c t i v i t y .  corresponding to a globular protein mol. wt. of 78  (Figure 16).  This had a 000  However, the e l u t i o n p r o f i l e from gel f i l t r a t i o n of  81 preparations extracted d i r e c t l y with 5% (NH^^SO^ (method B)  contained  multiple peaks of the a c t i v i t y 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 s i m i l a r to method A of this study (Riov and J a f f e , 1973) a c t i v i t y which was G-200.  produced one peak of  eluted i n the void volume of a column of Sephadex  This corresponded to a globular protein mol. wt. of  When that enzyme was  000.  prepared by the direct extraction of roots with  4% (NH^^SO^ the void volume peak occurred as w e l l as one to a globular protein mol. wt. of 80 000. was  200  corresponding  In both cases, gel f i l t r a t i o n  performed i n 20mM-phosphate buffer (pH 7.0)  i n the absence of NaCl.  This apparent inconsistency i n gel f i l t r a t i o n results could be i n part, by analogy with animal AchE. AchE, which contain an elongated as a t a i l (Dudai, et a l . , 1973;  explained,  Asymmetric forms of the e e l  structure referred to i n the l i t e r a t u r e Reiger, ej: a l . , 1973), and the bovine  erythrocyte AchE form large aggregates at low (<0.3) ionic strength (Rothenberg and Nachmansohn, 1947; Massoulie and Reiger, 1969;  Lawler, 1963;  Changeux,  1966;  Dudai, et a l . , 1972a; Crone, 1973).  The  peaks i n Figure 27 correspond to globular protein mol. wts. of 350 and  1 000 000.  mol. wt. species.  These may  two  000  represent 4x and 16x aggregates of the 78  000  These high mol. wt. forms would correspond to the  peak of AchE a c t i v i t y excluded from Sephadex G-200 (Riov and J a f f e , 1973). An i o n i c strength dependent equilibrium may  exist between these forms  such that at a high (0.22) i o n i c strength a l l a c t i v i t y would exist i n the 78 000 mol. wt. .(IX) form.  At a lower (0.02) i o n i c strength some  molecules would exist i n higher (4X, 16X) aggregation  states.  Riov  and  J a f f e (1973) suggested that protease a c t i v i t y i n (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 a c t i v i t y would be  removed by the preliminary buffer extract and no degradation of higher mol. wt. forms would occur i n the time between (NH^^SO^ extraction of the buffer insoluble material and gel f i l t r a t i o n .  T h i s proposal i s  supported by the observation that s t i l l lower mol. wt. forms appeared i n the e l u t i o n p r o f i l e of material extracted d i r e c t l y with 5% (NH^^SO^ (Figure 27) but were absent i n the p r o f i l e of material extracted from the buffer insoluble residue  (Figure 5).  P r o t e o l y t i c treatment of e e l  or torpedo AchE with trypsin or storage of e l e c t r i c tissue i n toluene for months (Rothenberg and Nachmansohn, 1947)  results i n the  formation  of an active globular form of the enzyme with a sedimentation c o e f f i c i e n t of 11 S (Dudai, e_t. a l . , 1972a; Rosenberry, et a l . , 1972; Taylor, et al., 1974) 1973;  and a mol. wt. of 310 000-350 000  Taylor, et a l . , 1974;  Morrod, 1975)  aggregates at low i o n i c strength  (Dudai, et a l . ,  which i s incapable of forming  (Massoulie  and Rieger, 1969).  Electron  microscopy revealed the absence of the " t a i l " structure i n this form (Rieger, et a l . , 1973). A thorough i n v e s t i g a t i o n of the behavior of bean root AchE on columns containing MAC-Sepharose 4B led to i t s use i n the p u r i f i c a t i o n protocol.  Of a l l of the conditions tested, those presented i n Figure 6  produced the greatest recovery and s p e c i f i c a c t i v i t y .  Even when the  greatest binding of AchE occurred, the most e f f e c t i v e eluant was. NaCl. E l u t i o n with a NaCl gradient resulted i n a single broad peak of a c t i v i t y (Figure 25a).  This suggested that the i n t e r a c t i o n between the ligand  and the enzyme was  of a non-specific nature.  Had  the i n t e r a c t i o n been  83  s p e c i f i c , a precise ionic strength would have been e f f e c t i v e i n e l u t i n g a l l of the enzyme (Rosenberry, et a l . , 1972) decamethonium would have been observed.  and s p e c i f i c e l u t i o n by  Use of chromatography with  MAC-Sepharose 4B gave a 10-fold p u r i f i c a t i o n and this procedure was  less  e f f e c t i v e than i n the p u r i f i c a t i o n of e e l 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 suggested that the binding was Furthermore, when there was portion of the AchE was  25b)  not exclusive to the active center.  a delay before the application of NaCl, a  eluted i n 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 i n 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 i o n i c strength rather than i t s chemical properties.  This i s supported by the observation that A^QQ  values increased gradually i n proportion to the i o n i c strength of decamethonium (Figure 25c). It i s known (Adams and Whittaker, 1950; Wilson, 1954;  Koelle, 1970;  Rosenberry, 1975)  Wilson and Bergman,  1950;  that the active center of  the AchE from a v a r i e t y of animal sources contains two primary binding sites —  an e s t e r a t i c s i t e which contains a n u c l e o p h i l i c group s p e c i f i c  for the a c y l carbonyl and an anionic s i t e which i s attracted to the quaternary nitrogen of the choline and also contains a hydrophobic region.  Studies with quaternary ammonium, carbamate, and organophosphate  i n h i b i t o r s suggested that the mung bean AchE active center resembles that  84 of the animal AchE (Riov and J a f f e , 1973). add further support to this view.  The results of my experiments  The enzyme from P_. vulgaris was  i n h i b i t e d by neostigmine (Figure 3) and the i n h i b i t i o n product remained inactive a f t e r d i a l y s i s suggesting  the formation of a carbamy1-enzyme  similar to that reported for carbamate i n h i b i t i o n of eel AchE (Wilson, 1954).  The bean enzyme was  inactivated by DIFP and remained i n a c t i v e  after exhaustive d i a l y s i s (Figure 7).  Complete characterization of the  active center of the enzyme must await further studies using strategies which have proven successful i n eel AchE active center investigations. A major difference between the plant and animal enzymes i s i n t h e i r responses to eserine.  Whereas the animal AchE i s i n h i b i t e d by  10 ^M-eserine (Augustinsson, 1963; from roots i s i n h i b i t e d at 10 Vasantharaja, 1976).  Jackson and Aprison, (Riov and J a f f e , 1973;  1966), the enzyme Kasturi and  The i n h i b i t i o n of eel AchE by eserine depends on  the hydrophobic a f f i n i t y of:  CHir  CH  CH~  '3  because, unlike neostigmine, this substance lacks quaternary nitrogen. If the enzyme from roots lacks a substantial hydrophobic component i n the anionic binding s i t e , then eserine would not be as e f f e c t i v e an i n h i b i t o r as of the animal enzyme.  This may  explain why  greater  N.-methylcacridinium concentrations were required to bind the AchE from bean roots than from e e l tissue (Figure 23) but would not necessarily answer the question of the ligands apparent n o n s p e c i f i c i t y . On other hand, i f N-methylacridinium binding was  the  s p e c i f i c for peripheral  anionic s i t e s of the eel enzyme, such as those considered  to be important  85 i n the binding of decamethonium (Froede and Wilson, 1971;  Rosenberry,  1975), then the absence of a corresponding s i t e i n the bean root AchE would explain both the non-specific binding of N-methylacridinium and the non-specific e l u t i o n with decamethonium i n both 0.2 Massoulie and Bon  and 1.0-M-NaCl.  (1976) have pointed out the importance of knowing the  binding properties of the enzyme i n the application of a f f i n i t y chromatography to the p u r i f i c a t i o n of AchEs.  I n s u f f i c i e n t information  i s available pertaining to such properties of both _P. vulgaris and Electrophorus  AchEs to resolve s a t i s f a c t o r i l y the i n a p p l i c a b i l i t y of  this ligand to a f f i n i t y chromatography of P_. vulgaris AchE. I t may  be possible to p u r i f y the bean AchE by other  chromatography ligands.  affinity  The use of N-acyl-p-aminophenyltrimethylammonium  (Dudai, est al_. , 1972b; Rosenberry, et a l . , 1972)  appears to be especially  promising because of the s i m i l a r i t y of i t s structure to that of neostigmine. The results of electrophoresis i n 7% acrylamide gels showed that the AchE was  not p u r i f i e d to homogeneity (Figure 8).  Other a c e t y l -  cholinesterases migrate slowly i n 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 a f t e r gel f i l t r a t i o n (Figure 8a) i n the v i c i n i t y of the enzyme a c t i v i t y suggests that more than one active form of the enzyme may  e x i s t i n the (NH^^SO^ extracts.  The gels were not  s l i c e d to detect enzyme a c t i v i t y 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 a c t i v i t y  were observed i n acrylamide electrophoregrams of pea root extracts a f t e r 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 i n the eel (Chen, e_t a_l. , 1974), house f l y head (Steele and Smallman,.1976), and human erythrocyte 1977)  AchEs.  (Das, et a l . ,  Electrophoresis of the globular 11 S form of the e e l  enzyme appeared as two bands of enzyme a c t i v i t y only one of which was detectable using protein s t a i n (Chen, et al.., 1974).  An elegant study  involving g e l f i l t r a t i o n , sedimentation, and electrophoresis revealed that the d i f f e r e n t forms of the f l y head AchE were not simply of the smallest active species (Steele and Smallman, 1976) .  oligomers In the case  of the erythrocyte enzyme, a single peak of a c t i v i t y was eluted from Sephadex G-200 i n 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 DEAE-cellulose.  as one peak of a c t i v i t y on  When this was rechromatographed with smaller mol. wt.  protein peaks lacking a c t i v i t y , 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 d i f f e r e n t charge properties.  Support  of the existence of root.AchEs d i f f e r i n g i n charge properties i s apparent from Figure 28. The nature or s i g n i f i c a n c e of the major contaminating protein remains undetermined but i t could represent  a structure s i m i l a r  to the one reported by Das, et a l . (1977) because the conditions of g e l electrophoresis d i f f e r e d from the conditions of p u r i f i c a t i o n . contaminant may represent  The  a p r o t e i n not associated with the AchE.  The i s o e l e c t r i c point of AchE was at pH 5.3.  The enzyme p r e c i p i t a t e d  with a loss of greater than 90% of the a c t i v i t y at this pH and pH 5.6.  87 These observations  account f o r low recoveries from thin dextran layers  (Figure 13) and the i s o e l e c t r i c focusing column (Figure 14) and explains }  why I did not use i s o e l e c t r i c focusing i n the p u r i f i c a t i o n protocol. This property also prevented the successful a p p l i c a t i o n of a f f i n i t y e l u t i o n chromatography (Scopes, 1977). i s d i f f i c u l t to explain. lower  The  The peak at pH 7.0 (Figure 14)  values i n the peak resulted from  values i n the absence of neostigmine which t y p i f y 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). has reported v a r i a b l e values that enzyme.  Morrod (1975)  (pI=3.6-4.9) f o r the asymmetric forms of  Precise values f o r the forms of the erythrocyte AchE  are unavailable but pis range from 3-r5.3 (Das, et a l . , 1977).  The  v a r i a b i l i t y of these values probably r e f l e c t s the i n s t a b i l i t y of a l l AchEs.at t h e i r i s o e l e c t r i c points as well as v a r i a t i o n i n the i s o e l e c t r i c points of the enzyme from d i f f e r e n t sources.  The low p i found i n both  t h i s and other AchEs suggests the presence of many negatively charged groups.  I t i s l i k e l y that both the amino acid and carbohydrate  composition  of the p u r i f i e d bean AchE w i l l reveal the source of negatively charged groups and explain the anion exchange behavior with the a f f i n i t y ligand. The r e s u l t s of the i s o k i n e t i c sedimentation  indicate the presence  of a single major sedimenting p a r t i c l e having S=4.2 (Figure 12a).  This  value used i n 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. additional c a l c u l a t i o n yielded the f r i c t i o n a l r a t i o (f/f~=1.37)..  An This  88  value i s only s l i g h t l y 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 l i m i t e d  by the assumption that the p a r t i a l s p e c i f i c volume (v) of this enzyme i s equivalent to that of the e e l 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 d i f f e r s from other values of 0.714 cm g and 0.793 cm g f o r the 11 S form of the e e l AchE (Bon, et a l . , 1976) and Lubrol extracted erythrocyte AchE (Beauregard and Roufogalis, 1977), respectively. The high value obtained i n the l a t t e r case was attributed to the i n t e g r a l association of phosphoiipidiwithi the..enzyme.  The .usefof /these avalues..resulted; i n  mol. wts. of 67 000 and 92 000, respectively. The use of the value of the AchE obtained during i t s p u r i f i c a t i o n rather than the use of another K  by rechromatography of the p u r i f i e d enzyme may have l e d to clV  additional errors.  However, the accuracy of the calculated mol. wt.  i s further supported by the s i m i l a r i t y 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 p a r t i c l e (Figures 12b and 12c). This 3 p a r t i c l e was established as  H-DIP-AchE since  AchE was s t o i c h i o m e t r i c a l l y  3 inactivated by  H-DIFP.  The greater sedimentation  c o e f f i c i e n t of this  p a r t i c l e 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 l a b e l l i n g strategies have been used to detect c a t a l y t i c subunits i n e i t h e r AchE p u r i f i e d to homogeneity (Dudai and Silman, 1974;  Rosenberry, et a l . , 1974)  which the AchE was  or contaminated preparations i n  protected with butylcholine against a c t i v a t i o n by  non-radioactive DIFP followed, after d i a l y s i s , by exclusive AchE l a b e l l i n g with radioactive DIFP (Cohen et a l . , 1967; Bellhorn, et a l . , 1970; Berman, 1973).  The l a t t e r strategy was  attempted i n this study.  Although butylcholine did o f f e r protection against i n a c t i v a t i o n , the protection was method was  incomplete  under the two conditions tested.  An a l t e r n a t i v e  examined by labeling i n the presence or absence of 3  neostigmine.  Neostigmine was  (Table V, Figure 12).  i n e f f e c t i v e i n preventing  This may  have resulted from  H-DIFP labeling  incomplete  carbamylation of AchE by 125 uM-neostigmine, phosphorylation of some of the carbamylated enzyme, or a more rapid decarbamylation i n the presence of DIFP.  of the enzyme  In any case, neostigmine did not seem to act  as an appropriate control for DIFP labeling of AchE.  The  that DIP-AchE sedimented as a single p a r t i c l e corresponding  observations to a s l i g h t l y  more globular form of AchE, that no other t r i t i a t e d p a r t i c l e s were observed i n the sedimentation free from contaminating DIP-AchE was  gradients, and that the preparations were  esterase a c t i v i t y led to the conclusion that  a suitable form of the enzyme i n which to detect  c a t a l y t i c subunits. The s i m i l a r i t y 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 AchE undenatured by SDS.  a trace of the active form of the  However, denaturation yielded a major c a t a l y t i c  90 subunit having a mol. wt.- of 61 000.  Much of the proteinaceous material  which centered at 16 000 probably corresponded  to the major  protein observed i n polyacrylamide gel electrophoresis and focusing.  The observation that no t r i t i a t e d  contaminating isoelectric  peak corresponded  to the  16 000 mol. wt. protein (Figures 10a and 11a) supports this statement. The 26 000 mol. wt. shoulder radioactive peak suggested AchE.  (Figures 10a and 11a) corresponding to a  that this represents a second subunit of the  whether this represented a c a t a l y t i c subunit i s 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 r a d i o a c t i v i t y was  s l i g h t compared to the protein content suggests that  i t i s not c a t a l y t i c .  From the results of preparations containing protein  contaminants i t i s d i f f i c u l t to assess the significance of the 17 mol. wt. radioactive peak, however, i t may of the .61 000 or 26 000 mol. wt.  500  represent a degradation  product  subunit.  With the appearance i n reduced samples of r a d i o a c t i v i t y  corresponding  to a 30 000 mol. wt. component and a concomitant decrease of a c t i v i t y i n the .61 000 dalton component (Figures 10b and.lib) i t i s tempting  to  suggest that two 30 000 mol; wt. c a t a l y t i c subunits are linked by a. disulphide bond to form the .61 000 mol. wt. subunit of the active AchE. This observation i s tentative because complete reduction was observed.  not  Conditions must be found i n which the 61 000 mol. wt. peak  i s 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  Figure 32.  Postulated structure  of P . v u l g a r i s  AchE.  E  O > 4—«  O CO  "O CD  active  centers  CO  c  ~80 000  c  CO Q CT) +  f t - J  s  80 000  1  o  -4—'  1_  - 2 0 000  -60 000  c o  '  o  HSH  __ "O > CD k_  CD O .CD _ r a) Q. 73 + E  o o CO c Q CO +  HS-I  - 8 0 000  •20 000  - 6 0 000  !  - 3 0 000  91 A remarkable s i m i l a r i t y 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 i n e e l ( M i l l a r 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 i n torpedo (Taylor, e_t al. , 1974).  When the e e l enzyme was reduced i n the  presence of 2-mercaptoethanol or d i t h i o t h r e i t o l , 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; ejt a l . ,  Rosenberry,  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 e e l AchE on the basis of these and other observations (Figure 33). The model accounts f o r the presence of a l l fragments except the smallest one which i s suspected to be a degradation product of the 25 000 mol. wt. form (Morrod,  1975).  In the erythrocyte ghost AchE, the smallest active i s o l a b l e 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 i n t e g r a l 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 i n e f f i c a c y of neostigmine i n the prevention of DIFP labeling discussed above, the best estimate of c a t a l y t i c center a c t i v i t y  F i g u r e 33.  I  Schematic model f o r the 11 S form o f e e l AchE from Dudai and Silman (1974).  c 30H  COOM  £ o o  COOM A  " ^> 1 _ _ A  1  H  -? 1  r*1- s - s -  |  y  A  <n  -s-s-  T3 0 .-.-..>» 3  NK,  U"!  A  cOOH  COOM A  -s-s-  V •  -s-sY C _.0H  T hi  MW =  VI  NM  2  3 2 0 - 3 5 0 . C )0 0  N? .  i  v»  -s-s-  N  c OOM  H  CCCi-  A  i  1  o 05  Y  NH,  c A  COO"  m  »2  \w  32 0 - 3 5 0 , 0 0 0  MW=  1  c<30M A  -S—3-  -s-s-  B  s ? 3  COOM A  l/l -s-s-  v  n  -s-sY c  i-»2>  ~I65,00C >  i  CCOH  o  HS  o u «  HS  -A *v  © c> o.  MW=  V w  -I63.C )0 0  ' 80,000  COUH  zoo*  A 5H  tvV  wf  yvy  Uv  -SM  IT-  n o  CO'JM  1 - SH  E i CQ.  MW =  Trj H,  MS •  W  ~ 8 0 , 0 O O ; ~ 6 O P O O , ~ 23,000  MW=  .  ~ 8 0 , 0 0 0 ; ~ 6 0 , 0 0 0 , ~ 25,000  93 ( F l o r k i n a n d S t o l t z , 1973) was o b t a i n e d b y D I F P l a b e l i n g i n t h e of  neostigmine  (Table V ) .  D I F P ) was n e a r l y (960 000 m o l  This value  5000 t i m e s  less  s u b s t r a t e m i n ^mol  The a c c u r a c y o f t h i s  estimate  (197 m o l  absence  o f s u b s t r a t e m i n ^mol  1  t h a n t h e v a l u e f o r the e e l AchE 1  active center,  Rosenberry,  1975).  was l i m i t e d b y t h e p u r i t y o f t h e  preparation  3 and the of  accuracy of the  this value awaits  s p e c i f i c a c t i v i t y of  the  development  l a b e l i n g by DIFP.  H-DIFP.  of a s u i t a b l e  A rigorous  c o n t r o l f o r non AchE  '  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 catalytic  center  D I F P mg  p r o t e i n by the  1  61 000 m o l . w t .  a c t i v i t y by d i v i d i n g the  subunit  ( m o l D I F P m o l ''"AchE). mg of  1  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 Because the  1  preparations  Lowry p r o t e i n .  required to o b t a i n c a t a l y t i c subunit the  the  e e l suggests  surprising  that  the  a c t i v i t y of t h i s requirements  considering the  weight"  Pike,  observed i n plants  1974).  enzyme  preparation  centers would and  only  and a be  thereby  catalytic  enzyme c o m p a r e d t o  subunit. that  f o r r a p i d h y d r o l y s i s of  tremendously.  r e l a t i v e l y high quantities  a c e t y l c h o l i n e i n the e l e c t r i c organ  quantities  Both a purer  equivalent weights  a c e t y l c h o l i n e i n these organisms d i f f e r  of  per molecule  to an " e q u i v a l e n t  61 000 d a l t o n s u b u n i t was t h e  The l o w c a t a l y t i c c e n t e r  the  c o n t a i n e d 210 u n i t s  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  e s t a b l i s h whether  this  c a l c u l a t e d DIFP b i n d i n g (mol  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  w o u l d b e 3270 u n i t s mg  of  from  p o s t u l a t e d maximum b i n d i n g o f D I F P a s s u m i n g  p r o t e i n and the b i n d i n g corresponded  more a c c u r a t e  test  This  is  and e s t a b l i s h e d  ( F l o r e y , 1966) a n d t h e  (Hartmann and K i l b i n g e r ,  not  low  1974; W h i t e a n d  role  94 The Km of P_. vulgaris root AchE was acetylthiocholine.  Other Kms  56 yM  (Figure 18) f o r  have been reported:  P. aureus root AchE  i  had a Km of 84 yM  1  (Riov and J a f f e , 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 i n Torpedo and 230-340 yM i n Electrophorus , (Massoulie and Bon,  1976).  No consideration has been given to i o n i c strength  Km determinations i n this or other plant AchEs.  Such studies may  during 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 k i n e t i c studies have been performed  on any plant cholinesterases. The response of this enzyme to b u t y l - and (Table VI) was  propionylthiocholine  s i m i l a r to that observed by Riov and J a f f e (1973)  although they did not determine the effect of v a r i a b l e concentrations these substrates on the enzyme a c t i v i t y .  In this study, Michaelis-  Mentin k i n e t i c s were not observed f o r either of these two -5 between 10  substrates  -2 and  no substrate was  10  M (Figure 17).  Unfortunately,  a control containing  not included i n 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 i n the assay with propionyl- or butylthiocholine was inappropriate.  The true zero f o r these substrates may  correspond to  AA4I2 of approximately 0.1,in which case the actual s p e c i f i c a c t i v i t y for these two substrates i n Table VI approaches zero.  Riov and  Jaffe  (1973) have demonstrated that at a substrate concentration of 0.5 neostigmine i n h i b i t e d the hydrolysis of a l l three substrates This may  of  not have applied to a l l substrate concentrations  mM,  equally.  however.  95 Alternatively,the enzyme may below 10 ^M.  become saturated at a substrate  concentration  This >seems u n l i k e l y i n view of the substrate i n h i b i t i o n  demonstrated by acetylcholine.  K i n e t i c studies using a d i f f e r e n t assay  method would be required to resolve the problem of substrate  affinity.  A reevaluation of r e l a t i v e hydrolysis rates and s p e c i f i c i t y f o r other esters (Riov and J a f f e , 1973) would be necessary considering  the  tentative status of hydrolysis rate data for a c e t y l - , b u t y l - , and propionyl-thiocholine. 4.  Physiological Role of the Acetylcholine/Acetylcholinesterase System. The  possibility  that, developmental -changes  with changes i n the status of AchE was discussion.  are  correlated  suggested e a r l i e r i n this  Fluck and J a f f e (1974b) observed that l i g h t grown mung bean  stems have a 10-fold greater extractable AchE a c t i v i t y than e t i o l a t e d hypocotyls.  This suggested the p o s s i b i l i t y that l i g h t may  levels as well as acetylcholine l e v e l s .  mediate AchE  The AchE a c t i v i t y i n hypocotyls  of 5-day old e t i o l a t e d seedlings exposed to continuous, r e l a t i v e l y i n t e n s i t y white l i g h t increased by the same amount over a four period as the e t i o l a t e d controls (Figure 29). i n e t i o l a t e d controls was  low  day  The increase of a c t i v i t y  comparable to that observed by Fluck and J a f f e  (1974b) for t o t a l a c t i v i t y i n mung bean roots and by Kasturi and Vasantharajan (1976) f o r extractable a c t i v i t y i n pea roots.  Such values  show that as the hypocotyl develops,it too increases i t s AchE a c t i v i t y . The i n e f f i c a c y of 2-chlorosulphonic  acid, an ethylene  generating  growth regulator applied i n concentrations which were e f f e c t i v e i n inducing the " t r i p l e response" symptoms (Neljubow, 1901), i n a l t e r i n g AchE levels (Figure 29) suggests that any role that AchE may  have i n the  96 hook opening response i s not affected by ethylene.  This could mean that  the inhibitory effect of acetylcholine on ethylene generation subsequent hook closure (Parups, 1976)  and  i s independent of the AchE l e v e l  i n the t i s s u e s , although no conclusive evidence has been obtained. Kinetin at 10  -4  M i n h i b i t s hook opening and GA at 10  opening (Kang and Ray,  1969).  -3 M enhances  I t i s i n t e r e s t i n g that these regulators  have opposite and s t a t i s t i c a l l y s i g n i f i c a n t e f f e c t s on AchE of etiolated P_. vulgaris hypocotyls  (Figure 30) .  The time course involved i n this  effect i s s u b s t a n t i a l l y longer than the 8-12 i n i t i a t i o n of hook opening i n excised hooks.  hours required for the Based on the time course  of the response, any regulation of acetylcholine i n the hook opening response would be attributed to constitutive enzyme a c t i v i t y and  the  v a r i a t i o n i n AchE a c t i v i t y more l i k e l y would be linked with a longer term developmental change.  Considering the observation that AchE a c t i v i t y  increased with the age of the t i s s u e , i t i s conceivable that AchE may be involved i n the actual process of aging i t s e l f .  The observation that  k i n e t i n , a growth regulator active i n counteracting many senescent processes  (Sacher, 1973)  reduced AchE a c t i v i t y i n hypocotyls  (Figure  30)  supports t h i s idea. Even though a l l growth regulators studied i n this project were tested at concentrations  i n which they are e f f e c t i v e i n inducing hook  opening responses, the p o s s i b i l i t y that other concentrations may  exert  d i f f e r e n t e f f e c t s cannot be discounted, nor can the p o s s i b i l i t y that the concentrations e f f e c t i v e i n the excised hook opening assay d i f f e r from optimal concentrations  of sprayed applications to intact plants.  97 Parups (1976) observed that acetylcholine p a r t i a l l y prevented the IAA-promoted delay of hook opening and i n h i b i t e d IAA-induced ethylene production. antagonist  By these actions, acetylcholine appears to act as an of IAA thereby preventing  induce hook closure. of acetylcholine was concentration  ethylene from becoming available to  Parups (1976) postulated further that the action i n mimicking the e f f e c t of red l i g h t on  and ethylene production.  that at some concentration,  IAA  On this basis i t would be  likely  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 effects on assorted plant processes (Dekhuijzen, 1973; 1973;  Penel, et a l . , 1976;  existence  (Jaffe, 1970;  Sharna et a l . , 1977)  of acetylcholine G r e r r i n , et a l . ,  and i t s endogenous  Hartmann and K i l b i n g e r , 1974), i t would not  be  surprising to f i n d 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 i n developmental biology. E.  SUMMARY  Acetylcholinesterase a c t i v i t y was characterize the enzyme which may  studied to i d e n t i f y , purify and  be responsible for regulating  acetylcholine l e v e l s i n plant tissue and to assess the r o l e of the enzyme a c t i v i t y i n plant tissues. A unique AchE was  i d e n t i f i e d i n e t i o l a t e d P_. vulgaris roots  and  tentatively i d e n t i f i e d i n the hypocotyl by use of a colorimetric assay  98 which i n c l u d e d the  cholinesterase  form of the AchE was  e x t r a c t e d w i t h 5%  s o l u b l e r e s i d u e of r o o t t i s s u e and 210±20 u n i t s mg  protein-greater  1  maximum.  This preparation  which may  or may  c o n t a i n any has  i n h i b i t o r n e o s t i g m i n e as a c o n t r o l . (NH^^SO^ from a b u f f e r i n  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 than t w i c e the p r e v i o u s l y  contained  one  esterase  activity.  major c o n t a m i n a t i n g p r o t e i n  The  requirement of h i g h  did  not  purity  been e s t a b l i s h e d as a n e c e s s i t y f o r the d i s t i n c t i o n between  c h o l i n e s t e r a s e and  acetylcholinesterase activities  i n plant tissues.  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 i n b u f f e r homogenates of b o t h r o o t and h y p o c o t y l p o s s i b i l i t y of a n o n - d i a l y z a b l e of i n h i b i t o r s i l l u s t r a t e d  i n h i b i t o r was  The  existence  of o t h e r  The  proposed.  the  The  presence  in situ.  forms of AchE i n c l u d i n g a 4x and  aggregate..-of the p u r i f i e d AchE was  t h a t b o t h aggregates and  t i s s u e s and  located  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  q u a n t i t a t i v e l e v e l s of enzyme a c t i v i t y  16x  of  reported  not have been a s s o c i a t e d w i t h the a c t i v e AchE but  other  A  demonstrated and  possibly a  i t was  suggested  i s o enzymes might e x i s t .  major f r a c t i o n of the h y p o c o t y l  enzyme was  l o c a t e d i n the  cell  wall. P h y s i c a l and  c a t a l y t i c p r o p e r t i e s of the r o o t AchE were s t u d i e d  are summarized i n T a b l e IX. of the AchE from v a r i o u s  Many of these p r o p e r t i e s are s i m i l a r to those  animal s o u r c e s .  A subunit  s t r u c t u r e has  t e n t a t i v e l y proposed on the b a s i s of r e s u l t s from SDS and K  av  previous  measurements.  and  Of the i n f o r m a t i o n  s t u d i e s of p l a n t AchEs, i t was  obtained  gel  electrophoresis  i n t h i s and  concluded t h a t a l t h o u g h  p r o p e r t i e s of t h i s enzyme d i f f e r between p l a n t s  and  been  two catalytic  animals, s t r u c t u r a l  Table IX.  Summary o f P h y s i c a l P r o p e r t i e s  P h y s i c a l parameter  (Method)  Value  S„_ (sedimentation v e l o c i t y ) 20 w J  Mol. wt.  (gel f i l t r a t i o n  4.2 ± 0.1 S  and  sedimentation v e l o c i t y ) * Mol. wt.  o f P_. v u l g a r i s A c h E  (SDS g e l e l e c t r o p h o r e s i s )  S u b u n i t m o l . w t s . (SDS g e l electrophoresis)  76 000 ± 2 000 77 000 ± 2 000  61 000 ± 2 000 (2 X 30 000 ± 1 000) 26 000 ± 2 000  Stokes radius f/f  Q  (gel f i l t r a t i o n )  *  Catalytic  4.00 nm 1.37  center  a c t i v i t y (DIFP t i t r a t i o n )  Isoelectric point  3-1 * v = 0.75 cm g  (assumed)  197 + 5 m o l . . s u b s t r a t e min mol active center 5.3 ± 0.1  p r o p e r t i e s a r e remarkably The  conclusion  similar.  t h a t t h i s enzyme i s i n v o l v e d i n the h y d r o l y s i s of  a c e t y l c h o l i n e to the e x c l u s i o n o f o t h e r e s t e r s i n s i t u from e x i s t i n g d a t a .  cannot be drawn  I t was suggested t h a t AchE may not be important i n  the s h o r t . t e r m r e g u l a t i o n of a c e t y l c h o l i n e i n such responses as hook opening b u t may be i n v o l v e d i n r e g u l a t i o n o f a c e t y l c h o l i n e l e v e l s l o n g e r time p e r i o d s and may thus be important f o r normal development.  plant  over  BIBLIOGRAPHY  Chapter I Adams, D. 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L. and D. T. Plummer. 1973. M u l t i p l e forms of a c e t y l c h o l i n e s t e r a s e from human e r y t h r o c y t e s . Biochem.J. 133:521-527. Yunghans, H. and M. J . J a f f e . 1972. Rapid r e s p i r a t o r y changes due t o red l i g h t o r a c e t y l c h o l i n e d u r i n g t h e e a r l y events of phytochromemediated photomorphogenesis. P l a n t 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 i n t e r e s t for decades and has been extensively reviewed (Heyn, 1940; Frey-Wyssling, 1962;  Wilson, 1964;  1-968; Cleland, 1971;  Preston,  turgor pressure  S e t t e r f i e l d and Bayley,  Lockhart, 1965;  1974).  1961;  Morreuand E i s i n g e r ,  It i s generally agreed that  i s a driving force for c e l l extension,  2) c e l l  1)  extension  involves changes i n the v i s c o e l a s t i c properties of the primary c e l l wall r e s u l t i n g i n reduced c e l l wall resistance to s t r e s s , and  3)  deposition of c e l l wall polymers i s 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 l o c a t i o n of the v i s c o e l a s t i c 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 i s necessary for characterization of the s t r u c t u r a l changes which occur during c e l l wall extension.  The primary c e l l wall of higher  plants consists of c e l l u l o s i c m i c r o f i b r i l s surrounded by an  apparently  amorphous matrix of hemicelluloses, pectic acids,.and both enzymatic and s t r u c t u r a l proteins.  Our present understanding of the primary c e l l  wall structure i s based upon observations 110  using such techniques as polar  and  interference microscopy (Ruge, 1937; D i e h l , et a l . , 1939), x-ray d i f f r a c t i o n (Meyer and Misch, 1936; microscopy (Frey-Wyssling,  ejt a l . , 1948; Heyn, 1966), histochemistry  (Reis and Roland, 1974), chemical et a l . , 1976)  Berkeley and Kerr, 1946), electron  (Selvendran, et a l . , 1975; Monro,  and enzymatic (Karr and Albersheim,  chemical analysis (Bauer, et al.,  1973;  1970)  S e t t e r f i e l d and Bayley,  and  Talmadge, est al_., 1973).  In l a t e r a l primary c e l l walls of many c e l l types Wardrop, 1956;  degradation,  (Roelofsen,  1958; Preston, 1974)  1951;  the  orientation of m i c r o f i b r i l s on the inner wall surface i s perpendicular to the major c e l l axis becoming p a r a l l e l as the c e l l extends and more material i s added to the existing multinet growth hypothesis  c e l l wall.  Thus, according to the  (Roelofsen and Houwink, 1951) , movement of  the m i c r o f i b r i l s with respect to the matrix comprises a major event during c e l l extension.  But m i c r o f i b r i l l a r movement depends upon a  recorganization of the matrix accompanying reduced c e l l w a l l resistance to stress (Barnicki-Garcia, 1973; Preston, 1974).  It i s postulated  that bonds either are broken, newly formed, or both broken and reformed during c e l l w a l l extension. Recently, s p e c i f i c linkages have been examined with respect to t h e i r s u s c e p t i b i l i t y to cleavage or formation under conditions which increase, decrease, or terminate c e l l w a l l extension.  These linkages  include hydrogen bonding involving the xyloglucan component of hemicellulose (Labavitch and Ray, (Lamport, 1965; 1973;  Lamport, 1970)  Cho and Chrispeels, 1976)  1974), arabinosyl-hydroxyproline  and galactosyl-serine (Lamport, et a l . , of the c e l l wall glycoprotein, g-1,  4-glucosyl linkages i n the c e l l u l o s e  (Wong, et a l . , 1977), and f e r u l i c  112 acid-polysaccharide  esters (Hartley, e_t a l . , 1976).  Linkages involved i n 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 i s 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, wall.  then glycosylation would also occur i n the  The p r o b a b i l i t y of such a modification occurring i n the  cell  cell  wall would be supported i f there were some s i t e s i n the glycoprotein which were not glycosylated i n young a c t i v e l y extending c e l l walls but were found glycosylated i n older, f u l l y extended c e l l walls. evidence for such s i t e s would be obtained  Positive  i f the s i t e s 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 a c t i v e l y elongating c e l l walls.  A  f a i l u r e to detect binding would suggest the absence of suitably reactive sites.-  Diisopropylfluorophosphate  (DIFP) could act as such a reagent.  DIFP has been widely used i n protein modification and enzyme i n h i b i t i o n studies (Cohen, et a l . , 1967). (CHJ CH- 0 ^  //  2  (CH ) CH-0^ 3  P (CH ) CH 3  the  2  0  In the reaction:  + F  H-PROTEIN  2  + lP  ( C H  3 2 }  C H  /  PROTEIN  <»  ~° +  F ©  e l e c t r o p h i l i c phosphorus reacts with a n u c l e o p h i l i c group of the  protein leading to a departure of F © and formation of a d i i s o p r o p y l phosphoryl-(DIP-)protein.  A major product of p a r t i a l acid hydrolysis  of most DIP-proteins i s phosphoserine (Schaffer, et a l . , 1953;  Schaffer,  113 et  a l . , 1954;  Cohen, e t a l . ,  1967)  i n d i c a t i n g t h a t the s e r i n e h y d r o x y l  group i s the n u c l e o p h i l e i n r e a c t i o n  (1).  As h y d r o l y s i s p r o c e d e s ,  phosphate e s t e r i s a l s o c l e a v e d y i e l d i n g o r t h o p h o s p h o r i c a c i d . case of DIFP i n h i b i t i o n o f a c i d phosphomonoesterase, no was  In the  phosphoserine  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 The purpose of  the  and Nachmansohn, 1965). of t h i s study was  to examine the c e l l w a l l s of h y p o c o t y l s  the bush bean (Phaseolus v u l g a r i s L.) f o r the presence of s e r i n e  r e s i d u e s which were r e a c t i v e 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 . h y p o c o t y l o f f e r s a developmental  continuum of e l o n g a t i o n s t a g e s  the a p i c a l r e g i o n c o n s i s t i n g of non-elongated c o n s i s t i n g of e l o n g a t e d c e l l s  The bean  cells  from  to the b a s a l r e g i o n  ( B a i l e y and Kauss, 1974).  By  sampling  r e g i o n s a l o n g 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 o r t e r m i n a t e e l o n g a t i o n i s a v o i d e d . cell  types p r e s e n t conform  The growth p a t t e r n s of the  t o the m u l t i n e t growth h y p o t h e s i s w i t h  e x c e p t i o n of the e p i d e r m a l c e l l s  (Bayley, et a l , ,  1957).  the  Though the  g l y c o s y l l i n k a g e s to c e l l w a l l p r o t e i n have not been examined i n P_. v u l g a r i s , c o m p o s i t i o n a l s t u d i e s of the primary c e l l w a l l s d e r i v e d from this plant to  ( W i l d e r and A l b e r s h e i m , 1973)  suggest t h a t i t i s s i m i l a r  o t h e r members of the D i c o t y l e d o n a e whose g l y c o s y l l i n k a g e s have been  studied. In of  t h i s study, phosphoserine was  a n a l y z e d f o l l o w i n g the  DIFP w i t h a) s e r i n e , b) a-chymotrypsin,  reaction  and c) c e l l w a l l s of  e l o n g a t i n g and f u l l y e l o n g a t e d c e l l s from e t i o l a t e d bush bean h y p o c o t y l s . A comparison labeled with  was 32  made between phosphoserine  r e c o v e r e d from c e l l w a l l s  P - D I F P and c e l l w a l l s l a b e l e d w i t h  32  P-DIFP following a  pretreatment with non-radioactive DIFP to correct f o r non-specific labeling or adsorption of the radioisotope.  This comparison led to the  i d e n t i f i c a t i o n of serine labeling i n c e l l wall proteins under.conditions which yielded a nearly complete modification of serine i n 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. 1.  MATERIALS AND METHODS  Chemicals Supplies were obtained from sources as indicated:  Chemical Co., Milwaukee, Wi.;  32  DIFP: A l d r i c h  P-orthophosphoric acid and  32 P-DIFP:  Amersham/Searle, Arlington Heights, I I . ; HTMFP: Syva Assoc. Inc., Palo A l t o , Ca.; c e l l u l o s e powder:  W. & R. Balston Ltd., England; ethylene  g l y c o l monomethyl ether (methylcellosolve) and ninhydrin:  Pierce  Chemical Co., Rockford, I I . ; Folin-Ciocalteu phenol reagent:  Harleco,  Philadelphia, Pa.; t r i c h l o r o a c e t i c acid (TCA), p-terphenyl, l,4-bis-2(5-phenyloxazolyl)-benzene and National Bureau of Standards calibrated HC1: Fisher S c i e n t i f i c Co., Fairlawn, N.J.; isoprbpanol, dioxane, and naphthalene: Mallinckrodt Chemical Works, St. Louis, Mo.;  Beckman Amino  Acid C a l i b r a t i o n Mixture Type I: Beckman Instruments Inc., Spinco Div., Palo Alto, Ca.; phosphoserine: C a l i f o r n i a 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 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 available.  115 2.  Plant Material Bush bean (Phaseolus v u l g a r i s L. v a r . Top Crop Green Pod) seeds  were o b t a i n e d from B u c k e r f i e l d s L t d . , 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 c a b i n e t a t room temperature. H y p o c o t y l s were s e p a r a t e d from t h e c o t y l e d o n s and r o o t s .  Segments  (1-5 cm) of e i t h e r e n t i r e h y p o c o t y l s , hook r e g i o n s , o r b a s a l r e g i o n s ( F i g u r e 1) were p r e p a r e d f o r c e l l w a l l 3.  extractions.  C e l l Wall Extraction Hypocotyl t i s s u e  (50g) was ground t o a a f i n e powder i n l i q u i d  n i t r o g e n (8-10 m i n ) .  The f r o z e n powder was l e f t  f o l l o w i n g e x t r a c t i o n w a s . c a r r i e d out a t 0-4°C. suspended  t o melt a t 0°C.  The  The homogenate was  i n 500 ml o f d e i o n i z e d water and a l l o w e d to s e t t l e u n t i l 2  l a y e r s appeared  (10-20 m i n ) .  centrifuge bottles.  The upper l a y e r was t r a n s f e r r e d to  The lower l a y e r was resuspended and the s e t t l i n g  procedure was r e p e a t e d t w i c e .  The c e l l w a l l fragments were c o l l e c t e d  from the p o o l e d upper l a y e r s by c e n t r i f u g a t i o n a t 15 000 £ f o r 10 min. The p e l l e t was resuspended i n 200 ml of d e i o n i z e d water.  This suspension  c o n t a i n e d l e s s than 2% of i n t a c t c e l l s as determined by l i g h t  microscopy.  The s u s p e n s i o n was t r e a t e d w i t h a B l a c k s t o n e U l t r a s o n i c Probe f o r 2 min at  200±50 W to r e l e a s e a t t a c h e d c y t o p l a s m i c contaminants.  The fragments  were c o l l e c t e d by c e n t r i f u g a t i o n a t 10 000 j» f o r 15 min, resuspended i n water and t r e a t e d a g a i n w i t h the u l t r a s o n i c probe.  T h i s procedure was  r e p e a t e d u n t i l the c e l l w a l l s were f r e e from c y t o p l a s m i c contaminants as determined by phase  contrast microscopy.  enough to a c h i e v e the d e s i r e d p u r i t y . s t o r e d a t 0°C.  Four washings were u s u a l l y  C e l l w a l l s were l y o p h i l i z e d and  Y i e l d s v a r i e d from 1-4 mg dry c e l l w a l l p e r g of h y p o c o t y l .  116  Figure 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. H - hook region; B - basal region.  H  117 Reaction of Serine with DIFP  4.  The  r e a c t i o n of s e r i n e w i t h D I F P was e s t a b l i s h e d by a d d i t i o n of  1 ml o f 2% (w/v) D I F P i n CaO-dried i s o p r o p a n o l 350  to 1 ymol.of s e r i n e i n  y l of 0.1 M-sodium phsphate b u f f e r , pH 7.3.  The m i x t u r e was  i n c u b a t e d f o r 45 min a t room temperature, and 0.27, 0.45, or 1.35 ml of  12 M-HC1 was added t o i n i t i a t e h y d r o l y s i s .  i n vacuo f o r p e r i o d s  r a n g i n g from 2 to 30 h .  Tubes were h y d r o l y z e d H y d r o l y z a t e s were d r i e d  i n a d e s s i c a t o r over KOH p e l l e t s and resuspended i n 500 y l o f d e i o n i z e d water.  Each tube was prepared i n d u p l i c a t e .  Two hundred y l o f each sample and 200 y l of Beckman Amino A c i d C a l i b r a t i o n M i x t u r e Type I ,  containing  20 nmol of t h e common p r o t e i n  amino a c i d s , were a p p l i e d to a 0.9 x 57 cm column on a Beckman Amino A c i d A n a l y z e r Model 120 C. was  analyzed according  using  5. •  Amino a c i d s were s e p a r a t e d and phosphoserine  to t h e method of Spackman, e_t a l . , (1958)  a s p a r a t i c a c i d as an i n t e r n a l  standard.  Reaction of Alpha-chymotrypsin with DIFP The  r e a c t i o n o f a-chymotrypsin w i t h r a d i o a c t i v e and  D I F P was performed by a m o d i f i c a t i o n  non-radioactive  o f t h e method of S c h a f f e r ,  e_t a l .  32 (1954). (470  A t y p i c a l r e a c t i o n mixture contained  50 y l of a  P-DIFP  Ci/mg) s o l u t i o n (0.96 mg/ml o f CaO-dried i s o p r o p a n o l ) ,  1 ml of  an a-chymotrypsin s o l u t i o n (5 mg/ml o f 0.05M-sodium phosphate b u f f e r , pH,  7.3), and 10 y l o f a 2% (v/x)  isopropanol.  s o l u t i o n of D I F P i n CaO-dried  A r e a c t i o n c o n t r o l contained  1 ml of a-chymotrypsin s o l u t i o n  (5 mg/ml o f 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 o f D I F P i n CaO-dried i s o p r o p a n o l  and was i n c u b a t e d f o r  118 90 min  at room temperature b e f o r e  solution  (0.96  at 4°C, at 600  y l of a  32  mg/ml of CaO-dried i s o p r o p a n o l )  Tubes were i n c u b a t e d DIFP was  50  removed e i t h e r by  P-DIFP (470 was  l y o p h i l i z e d and I n one  and  dialysis  a g a i n s t d e i o n i z e d water f o r 36 20%  TCA,  Ph  centrifugation  Labeled  protein  was  0°C.  experiment, the temperature ana  to t e s t f o r c o n d i t i o n s y i e l d i n g o p t i m a l by DIFP.  32  at room temperature; excess  seven washes w i t h water.  s t o r e d at  added.  f o r 45 min  or by p r e c i p i t a t i o n of the p r o t e i n w i t h g_ f o r 1 min,  yCi/mg)  A l p h a - c h y m o t r y p s i n was  r e a c t i o n time were v a r i e d  i n h i b i t i o n of enzymatic  allowed  activity  to r e a c t w i t h DIFP e i t h e r by  the method of Cohen, e t a l . , (1967) o r S c h a f f e r , et a l . (1954).  Tubes  were p l a c e d on i c e u n t i l a-chymotrypsin assays began.  6.  Reaction One  30 mg  ml  of C e l l W a l l s w i t h DIFP  of 0.05  M-sodium phosphate b u f f e r , pH  7.5,  of c e l l w a l l s ; r a d i o a c t i v e and n o n - r a d i o a c t i v e  was  DIFP were added  to t h i s s u s p e n s i o n i n amounts d e s c r i b e d  i n the p r e v i o u s  Reactions  above.  were c a r r i e d out as d e s c r i b e d  added to  section.  Excess reagent  was  removed e i t h e r by d i a l y s i s a g a i n s t d e i o n i z e d water f o r 36 h at 4°C by  c e n t r i f u g a t i o n at 600  water repeated (10  7.  times).  u n t i l no  g_ f o r 1 min  and  r a d i o a c t i v i t y was  resuspension detected  C e l l w a l l s were l y o p h i l i z e d and  i n deionized  i n the  s t o r e d at  or  supernatant  0°C,  Recovery of Phosphoserine To  c o n f i r m and  q u a n t i t a t e the extent  of  diisopropylphosphorylation  of s e r i n e 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 r o t e i n (3.00  mg)  119 was d i s s o l v e d i n 0.4 ml o f 2 M-HC1 and h y d r o l y z e d f o r 6, 12, 18, 21, and in  24 h a t 100°C i n vacuo.  D e r i v a t i z e d c e l l w a l l s were h y d r o l y z e d  2 M-HC1 f o r 21 h r a t 100°C i n vacuo.  over KOH p e l l e t s and s i l i c a  gel.  The a c i d was removed i n vacuo  Each h y d r o l y z a t e was d i s s o l v e d i n  0.5 ml d e i o n i z e d water, 2 p i o f d a n s y l h y d r o x i d e was added as a f l u o r e s c e n t i n t e r n a l s t a n d a r d , and the sample was a p p l i e d as a s p o t t o a sheet o f Whatman 3MM chromatography paper.  Phosphoserine,  o r t h o p h o s p h o r i c a c i d and a c o l o r e d marker (DNP-aspartic a c i d ) were a p p l i e d b e s i d e the sample. for  The m i x t u r e was r e s o l v e d by e l e c t r o p h o r e s i s  25-30 min a t pH 3.5 a t 50 V/cm (Ambler, 1963).  Phosphoserine and  o r t h o p h o s p h o r i c a c i d standards were d e t e c t e d by eadmium-ninhydrin reagent  (Heilmann,  et a l . ,  1957) and ammonium m o l y b d a t e - a s c o r b i c  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 s t a n d a r d  and orthophosphate  were determined  dansyl hydroxide.  Electrophoregrams  acid  phosphoserine  w i t h r e f e r e n c e t o DNP-aspartate and were c u t i n t o s t r i p s and scanned  32 for  P a c t i v i t y u s i n g an A c t i g r a p h I I Model 1025 s t r i p scanner  Chicago)  (Nuclear  coupled t o a N u c l e a r Chicago Model CR8416 c h a r t r e c o r d e r . A l l  r a d i o a c t i v e compounds had m o b i l i t i e s c o r r e s p o n d i n g t o e i t h e r or  orthophosphate  standards.  Samples h a v i n g m o b i l i t i e s  to  these standards were c u t from electrophoregrams  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  phosphoserine  corresponding  and f i n e l y minced  counting.  Two h y d r o l y z a t e s were s e p a r a t e d by i o n exchange chromatography as d e s c r i b e d f o r the r e a c t i o n o f s e r i n e w i t h DIFP. t o t a l a c t i v i t y r e c o v e r e d i n phsophoserine 4% o f the c o r r e s p o n d i n g a c t i v i t y electrophoresis.  On b o t h o c c a s i o n s , the  and orthophosphate  was w i t h i n  r e c o v e r e d f o l l o w i n g h i g h v o l t a g e paper  120 8.  D e t e r m i n a t i o n of R a d i o a c t i v i t y 10 ml of diozane-based  scintillation fluid  (Bray, 1960)  were added  to aqueous s o l u t i o n , s u s p e n s i o n s , l y o p h i l i z e d s o l i d s or f i n e l y p i e c e s of Whatman 3MM  paper.  Samples were counted  l i q u i d s c i n t i l l a t i o n spectrometer  cut  i n an Isocap  ( N u c l e a r Chicago)  300  f o r 20 min w i t h  an  32 800 K cpm  t e r m i n a t i o n at a window o f 25 to 1700  curve was  p r e p a r e d u s i n g the e x t e r n a l s t a n d a r d r a t i o method (Wang and  Willis, 0.50,  1965)  1.00,  by  Kev.  A ^ P quench  the a d d i t i o n , i n t r i p l i c a t e , of 0.00,  and 3.00  0.15,  0.30,  ml of d e i o n i z e d water to 10 y l of u n d i l u t e d  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 c o i n c i d e n c e c o u n t i n g range o f the s p e c t r o m e t e r . A c t i v i t i e s of ^^P-DIFP s t o c k s o l u t i o n s were determined  to c o r r e c t  f o r incomplete  32 t r a n s f e r of  P-DIFP to i s o p r o p a n o l .  Thus, the quench curve o r d i n a t e  32 was  cpm/dpm c a l c u l a t e d f o r  P-DIFP and sample a c t i v i t i e s were e x p r e s s e d 32  as a mole f r a c t i o n o f 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 b e f o r e each s e r i e s of samples and s u b t r a c t e d from counts p r i o r to a c t i v i t y performed 9.  determinations.  sample  A l l d e t e r m i n a t i o n s were  i n t r i p l i c a t e on at l e a s t two p r e p a r a t i o n s .  Calculations 32 A c o r r e c t i o n was  made f o r n o n - s p e c i f i c  P-DIFP l a b e l i n g  by  s u b t r a c t i n g mean v a l u e s of D I F P - p r e t r e a t e d groups from mean v a l u e s of treatments  l a c k i n g DIFP p r e t r e a t m e n t .  These d i f f e r e n c e s were t e s t e d 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 u s i n g the Student  v a r i a n c e f o r unequal  sample s i z e s .  r e c o v e r y were s i g n i f i c a n t  t - d i s t r i b u t i o n and  weighted 32  A l l corrected values f o r  at 3=0.01 u n l e s s i n d i c a t e d  otherwise.  P  121 10.  Protein Determination C e l l walls were extracted i n 1 M-KOH for 30 min i n a b o i l i n g water  bath.  The mixture was diluted ten-fold, shaken, and allowed to s e t t l e .  The protein i n 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 q u a l i t a t i v e microbiuret assay (Goa, determinations  1953). A l l  were performed i n t r i p l i c a t e on at least two c e l l wall  preparations.  11.  Assay of Alpha-chymotrypsin A c t i v i t y A c t i v i t y 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 d i l u t i o n of  native and DIP-a-chymotrypsin i n 0.05 M-Tris HC1, pH 7.5, were added. The volume of 0.01 M-NaOH required to t i t r a t e l i b e r a t e d a c e t i c acid to pH 7.5 was automatically recorded for 1-2 min.  A c t i v i t i e s were determined  from i n i t i a l slopes and recorded as mol of substrate min  ^mg ^ (units) of a-chymotrypsin.  hydrolyzed  Titrant molarity was confirmed  by t i t r a t i o n against Nations Bureau of Standards-calibrated  12.  1 M-HC1.  Spin Labeling The spin l a b e l i n g method was based on procedures described by  Schaffer, e_t a l . , (1954) for e e l cholinesterase diisopropylphosphorylation with modifications relevant to the use of the spin l a b e l tetramethyl-4-piperidinylmethylphosphonofluoridate  l-oxyl-2,2,6,6-  (Morrisett, et a l . , 1969).  F i f t y y l of 5% (w/v) HTMFP i n benezene were added to 3 mg of c e l l walls or Whatman c e l l u l o s e powder i n 1 ml of 0.1 M-sodium phosphate buffer, pH 7.3. Other tubes contained 50 y l of HTMFP alone i n buffer and 50 y l of HTMFP with 3 mg of c e l l walls that had been pre-treated with 100 y l of  2% (w/v) DIFP i n CaO-dried isopropanol for 45 min then washed with  approximately 1 ml of buffer and centrifuged at 600 temperature.  for 1 min at room  This washing procedure was repeated 12 times.  The mixtures were shaken and incubated at room temperature f o r 1 h. Excess spin l a b e l was removed by centrigugation at 600 £ for 1 min and resuspension i n deionized water 12 times u n t i l no spin l a b e l was detected i n the wash or d i f f u s a t e , respectively. Spectra of suspensions (approximately 6 mg/ml) were recorded at room temperature i n 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 B r i t i s h 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 f o r the removal of excess 32 P-DIFP from labeled a-chymotrypsin. C. 1.  RESULTS  Modification of Serine with DIFP Table I shows the recoveries of phosphoserine from hydrolyzates  of mixtures following the reaction of serine with DIFP. depended upon hydrolytic conditions.  Recovery  A maximum of 1.1% of the o r i g i n a l  1-23 Table I .  Phosphoserine r e c o v e r e d f o l l o w i n g the r e a c t i o n between s e r i n e and DIFP and s e r i a l h y d r o l y s i s w i t h 2, 3, o r 6 M-HCl. Values are means of d u p l i c a t e r e a c t i o n s and a r e expressed as a percentage of s e r i n e i n the i n i t i a l r e a c t i o n m i x t u r e . N.D. • denotes v a l u e s not determined.  HC1 concentration  2  .4  D u r a t i o n of h y d r o l y s i s (hr) „• 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  i n the serine solution used for the reaction.  Only phosphoserine and  serine were observed i n any analysis of ninhydrin p o s i t i v e substances. The presence of isopropanol i n 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 a f t e r diisopropylphosphprylation and hydrolysis was smaller than the average error i n an analysis.  2.  Modification of Alpha-chymotrypsin with D I F P Alpha-chymotrypsin was  treated with D I F P and assayed f o r i t s  a b i l i t y to hydrolyze N-acetyl tyrosine ethyl ester.  The a c t i v i t y  was  i n h i b i t e d by D I F P under both reaction conditions tested and i n h i b i t i o n was e s s e n t i a l l y complete after 20 min at 30°C (Table I I ) .  The  i n h i b i t e d enzyme was used as a test system to measure recovery of 32 phosphoserine following diisopropylphosphyorylation with  P-DIFP.  There were 0.583 mol< of phosphorus-32 recovered per mol of 32 a-chymotrypsin, regardless of the method used to remove excess  P-DIFP  32 from P-DIP-a-chymotrypsin (Table I I I ) . Under reaction conditions which r e s u l t i n 68.2% i n h i b i t i o n of a-chymotrypsin (Table I I ) , 58.3% of 32 the a-chymotrypsin was labeled with  P.  Thus, the molar r a t i o of  32 bound  P - D I F P to active centers i n h i b i t e d by D I F P i s 0.855.  This i s i n  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 n a t i v e and D I F P - i n h i b i t e d a-chymotrypsin measured by h y d r o l y s i s of the s y n t h e t i c s u b s t r a t e , N - a c e t y l t y r o s i n e ethyl ester. Values r e p r e s e n t means of d u p l i c a t e a s s a y s .  Inhibition Eeaction conditions  Specific activity ( u n i t s mg a-chymotrypsin)  N a t i v e enzyme (no i n h i b i t i o n )  % inhibition  216.0  0.0  Enzyme a f t e r r e a c t i o n w i t h DIFP f o r 20 min a t 30°C  1.2  99.3  Enzyme a f t e r r e a c t i o n w i t h DIFP f o r 45 min a t 24°C  64.2  68.2  126 Table I I I .  Recovery of P ^ a c t i v i t y from DIFP p r e t r e a t e d ai^cj non-pretreated P^DIP-a-chymotrypsin. Excess P-DIFP was. removed from P-DIP-a-chymotrypsin by e i t h e r d i a l y s i s o r 20% TCA p r e c i p i t a t i o n o f the p r o t e i n . Phosphorus-32 r e c o v e r i e s a r e e x p r e s s e d as a mole f r a c t i o n of a-chymotrypsin ± S.E.M.  Method o f P-DIFP removal  DIFP pretrea^ment _jNo DIFP p r e t r e a t m e n t (mol., P mol a-chymotrypsin)  Corrected value  Dialysis  0.116 ± 0.001  0.699 ± 0.028  0.583  Protein precipitation  0.003 ± 0.002  0.586 ± 0.056  0.583  127 3.  Phosphoserine  Recovery from  32  P-DIP-a-chymotrypsin  The products o f the p a r t i a l h y d r o l y s i s o f d i i s o p r o p y l p h o s p h o r y l enzymes — 1953;  phosphorserine  and o r t h o p h o s p h o r i c a c i d  S c h a f f e r , e t a l a . , 1954) — w e r e  s e r i a l h y d r o l y s i s of  assayed  (Schaffer, et a l . ,  -  for radioactivity  after  32 P-DIP-a-chymotrypsin to e s t a b l i s h c o n d i t i o n s f o r 32  q u a n t i t a t i v e or o p t i m a l r e c o v e r y of  P from c e l l w a l l s t r e a t e d w i t h  32 P-DIFP.  When h y d r o l y s i s products were s e p a r a t e d by h i g h v o l t a g e paper  e l e c t r o p h o r e s i s , the t o t a l a c t i v i t y r e c o v e r e d from v a r i e d from  14 to 38% (Table I V ) .  phosphoserine  occurred a f t e r  electrophoregrams  Optimal but incomplete r e c o v e r y of  12 h of h y d r o l y s i s .  Phosphate r e c o v e r y  i n c r e a s e d w i t h h y d r o l y s i s time and t o t a l phosphorus-32 r e c o v e r y i n c r e a s e d a f t e r 18 h.  The molar r a t i o o f phosphoserine  to a-chymotrypsin  was  a t l e a s t one o r d e r o f magnitude l e s s than the c o r r e s p o n d i n g r a t i o f o r DIP-a-chymotrypsin from T a b l e I I I .  I f I assumed t h a t  unrecovered  a c t i v i t y was d i s t r i b u t e d p r o p o r t i o n a t e l y i n phosphoserine  and phosphate,  then the c o r r e c t e d v a l u e s p r e s e n t e d i n T a b l e IV were o b t a i n e d . sample was c o r r e c t e d i n d e p e n d e n t l y .  Each  These v a l u e s a r e c o n s i s t e n t l y  less  than h a l f o f the c o r r e s p o n d i n g molar r a t i o f o r DIP-a-chymotrypsin from T a b l e I I I .  T h i s r e s u l t i s i n agreement w i t h those of o t h e r s  ( S c h a f f e r , e t a l . , 1953; S c h a f f e r , e t a l . , 1954). 4. M o d i f i c a t i o n of C e l l W a l l s w i t h DIFP C e l l walls isolated  from e n t i r e h y p o c o t y l s of e t i o l a t e d P. v u l g a r i s  32 were not l a b e l e d by P-DIFP a f t e r c o r r e c t i n g f o r n o n - s p e c i f i c b i n d i n g 32 32 of P-DIFP, when excess P-DIFP was.removed by d i a l y s i s ( T a b l e V ) . C e l l w a l l s i s o l a t e d from hook and b a s a l r e g i o n s of h y p o c o t y l s of e t i o l a t e d P_. v u l g a r i s c o n t a i n e d 5.4 and 3.4 pmol  of  32  P mg  -1  of dry  T a b l e IV.  Recovery of P from electrophoregrams a f t e r s e r i a l h y d r o l y s i s of P h o s p h o s e r i n e and orthophosphate r e c o v e r i e s a r e expressed as molar ct-chymo t r y p s i n . Total P r e c o v e r i e s are expressed as percentages a c t i v i t i e s i n DIP-a-chymotrypsin b e f o r e h y d r o l y s i s . Phosphoserine c o r r e c t e d f o r l o s s d u r i n g h y d r o l y s i s and e l e c t r o p h o r e s i s r e f l e c t e d r e c o v e r i e s . V a l u e s are means o f d u p l i c a t e a n a l y s e s .  DIP-a-chymotrypsin. ratios'qj of the P recoveries are i n individual total  D u r a t i o n o f h y d r o l y s i s (h) 6  Phosphoserine (mol mol  Recovery  phosphoserine/ a-chymotrypsin)  Orthophosphate (mol:  Recovery  orthophosphate/  mol a-chymotrypsin)  Total  18  - 21  24  DIFP pcetreatment  0.0001  0.0006  0.0002  0.0001  0.0001  No DIFP p r e t r e a t m e n t  0.0382  0.0553  0.0432  0.0387  0.0450  Corrected value  0.0381  0.0547  0.0430  0.0386  0.0449  DIFP pretreatment  0.0004  0.0035  0.0009  0.0027  0.0001  No DIFP pretreatment  0.0281  0.0680  0.0641  0.0770  0.0930  Corrected value  0.0277  0.06.45  0.0632  0.0743  0.0929  14  32  20  33  38  0.072  0.171  0.215  Recovery  C o r r e c t e d Phosphoserine  Recovery  (mol.  a-chymotrypsin)  phosphoserine/mol  12 -  .0.116  0.118  T a b l e V.  Phosphorus-32 r e c o v e r y from "'''P-DIFP t r e a t e d c e l l w a l l s i s o l a t e d from e n t i r e h y p o c o t y l s and r e g i o n s o f h y p o c o t y l s of e t i o l a t e d P_. v u l g a r i s shown i n F i g u r e 1. Excess P-DIFP was removed from h y p o c o t y l c e l l w a l l s by d i a l y s i s and from hook and b a s a l c e l l w a l l s ' b y repeated washings. Values a r e means o f a t l e a s t t r i p l i c a t e analyses ± S.E.M. C o r r e c t e d v a l u e s a r e the d i f f e r e n c e between groups p r e t r e a t e d and n o t p r e t r e a t e d w i t h n o n - r a d i o a c t i v e DIFP. 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 a t a=0.01. N.D. denotes v a l u e n o t determined.  DIFP pretreatment O r i g i n of C e l l W a l l s  (pmol'  No DIFP pretreatment 32  -1  P mg  Corrected value  c e l l wall)  Protein  content  (yg p r o t e i n / mg c e l l w a l l )  Specific  activity  (pmol. P/ mg c e l l w a l l p r o t e i n ) 3 2  0.0  Entire hypocotyl  7.7 ± 0 . 1  7.7 ± 0.7  Hook region  9.1 ± .1.7  14.5 ± 1.8  5.4  128  9  42.2  Basal region  8.9 ± 0.4  12.2 ± 1.9  3.4  110  6  30.9  0.0(N.S.)  N.D.  to  VO  130 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 washing.  30.9  P-DIFP by  repeated  R e c o v e r i e s c a l c u l a t e d on a c e l l w a l l p r o t e i n b a s i s were 32  and  32  pmols of  42.2  -1 P mg  of c e l l w a l l p r o t e i n f o r hook and b a s a l  cell  walls respectively. 5.  Phosphoserine Recovery from C e l l W a l l s There were 0.56  and  4.38  pmol  pmol. of  32  of  32  -1  P-phosphoserine mg  P-phosphoserine mg  -1  of dry  cell  of c e l l w a l l p r o t e i n  wall  recovered  from h i g h voltage, paper e l e c t r o p h o r e s i s of p a r t i a l h y d r o l y z a t e s  of  32 P-DIFP l a b e l e d c e l l w a l l s  i s o l a t e d from hook r e g i o n s  e t i o l a t e d P. v u l g a r i s (Table V I ) . —  No  significant  of h y p o c o t y l s  of  ( t . =1.49, a=0.01) 4dr  32 phosphoserine r e c o v e r y from b a s a l r e g i o n s pretreated  occurred  in  P-DIFP t r e a t e d c e l l w a l l s i s o l a t e d  of s i m i l a r h y p o c o t y l s  controls.  T o t a l r a d i o a c t i v e product r e c o v e r y  electrophoregrams ranged from 12 to 25%. 2.71  pmol;  :  of phosphoserine mg  phosphoserine mg recoveries  from l a b e l e d h y p o c o t y l  Spin The  from  A mean c o r r e c t e d v a l u e  of c e l l w a l l  ^ of c e l l w a l l p r o t e i n ) was  l o s s d u r i n g h y d r o l y s i s and 6.  when compared to DIFP-  (21.2  pmol  obtained  of  of  when phosphoserine  hook c e l l w a l l s were cor-rected f o r  electrophoresis.  Labeling spectrum shown i n F i g u r e  2a was  obtained  f o r HTMFP-labeled  a-chymotrypsin i n d e i o n i z e d water a f t e r removal of excess s p i n l a b e l e i t h e r d i a l y s i s or TCA obtained  p r e c i p i t a t i o n of the p r o t e i n .  The  f o r HTMFP i n phosphate b u f f e r i s shown i n F i g u r e  resonance was  observed between 3435 and  i n t a c t e t i o l a t e d P_. v u l g a r i s h y p o c o t y l s ,  3535 G at h i g h e s t 1-5  spectrum 2b.  No  gain i n  cm segments of  the  by  Table VI.  Phosphorus-32-phosphoserine r e c o v e r e d from P-DIFP t r e a t e d c e l l w a l l s i s o l a t e d from r e g i o n s of h y p o c o t y l s of e t i o l a t e d P. v u l g a r i s shown i n F i g u r e 1. Values a r e expressed as means of t r i p l i c a t e a n a l y s e s S.E.M. C o r r e c t e d v a l u e s a r e the d i f f e r e n c e between groups p r e t r e a t e d and not p r e t r e a t e d w i t h DIFP. Phosphoserine r e c o v e r i e s were c o r r e c t e d i n d i v i d u a l l y f o r l o s s d u r i n g h y d r o l y s i s and e l e c t r o p h o r e s i s . 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 a t a=0.01. N.D. denotes v a l u e not determined. Phosphoserine recovery  Corrected phosphoserine recovery  32 (pmol . P-phosphoserine/ mg c e l l w a l l ) T  O r i g i n of C e l l  Walls  Specific Corrected phosphoserine specific recovery phosphoserine 22 recovery (pmoL P-phosphoserine/ mg c e l l w a l l p r o t e i n )  Hook r e g i o n  Basal  DIFP pretreatment  0.07 ± 0.00  0.34 ± 0.02  No DIFP pretreatment  0.63 ± 0.14  3.05 ± 0.67  Corrected value  0.56  2.71  DIFP pretreatment  0.27 ± 0.03  1.91 ± 0.16  No DIFP pretreatment  0.34 ± 0.07  1.79 ± 0.30  Corrected value  0.07  4.38  21.2  N.D.  N.D.  region  (N.S.)  -0.12  (N.S.)  131a  Figure 2.  Electron spin resonance spectra of a) a-chymotrypsin labeled with HTMFP, b) HTMFP alone i n 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 wall preparations,  treated preparations  of the h y p o c o t y l ,  the hook r e g i o n .  On  of the f o l l o w i n g HTMFP-  a f t e r removal of excess HTMFP:  DIFP-,pretreated c e l l w a l l s regions  or any  one  c e l l u l o s e powder,  i s o l a t e d from e n t i r e h y p o c o t y l s or hook and  non-pretreated c e l l walls  occasion,  c e l l walls  i s o l a t e d from  i s o l a t e d from e n t i r e  h y p o c o t y l s were l a b e l e d a f t e r removal of excess HTMFP by method. Figure  T h i s r e s u l t was 2c.  not  reproducible.  one wash w i t h d e i o n i z e d  D.  Phosphoserine and  orthophosphate were the major r a d i o a c t i v e r e a c t i o n of DIFP w i t h b o t h a-chymotrypsin  the l a b e l e d n u c l e o p h i l e  Cohen e t a l . , (1967) s t a t e d t h a t react  (with DIFP)."  t h e i r observation.  The  for  serine  p h o s p h o r y l a t e d was  the f o l l o w i n g reason.  This observation  i n these  preparations.  "serine i t s e l f  and  detected  were c l e a v e d  (Table  difficult  I).  Ah  e s t i m a t e of  to o b t a i n  quantitatively f i r s t .  h y d r o l y s i s of d i i s o p r o p y l p h o s p h a t e expected.  from these v a l u e s ester, a l l  The  R-phosphate  which y i e l d  e s t e r s , some h y d r o l y s i s best  the  i f the d i i s o p r o p y l e s t e r s  Under c o n d i t i o n s  The  support  hydrolyzates  D u r i n g a c i d h y d r o l y s i s of a DIP  r e c o v e r e d q u a n t i t a t i v e l y only  phosphate e s t e r would be  i t s peptides  in partial  of the bonds i n d i c a t e d i n Scheme 1 would be h y d r o l y z e d . e s t e r would be  indicated  r e s u l t s shown i n T a b l e 1 do not  Phosphoserine was  a f t e r the. r e a c t i o n of DIFP and s e r i n e which was  released  water.  P_. v u l g a r i s h y p o c o t y l hook c e l l w a l l s .  t h a t s e r i n e was  l a b e l was  DISCUSSION  h y d r o l y s i s p r o d u c t s f o l l o w i n g the  do not  spectrum i s shown i n  A f t e r f o u r days at room temperature, the  q u a n t i t a t i v e l y by  and  The  the washing  of a  1  complete serine  e s t i m a t e (a minimal v a l u e )  134 1  Scheme 1.  (CH ) CH 3  R  2  1  2  would be the maximal value f o r phosphoserine recovery i n Table I (1.1%). 32 The nearest integer of the mole r a t i o of to a-chymotrypsin active centers was  1.0.  P-DIP-a-chymotrypsin  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 i n a-chymotrypsin, only the one i n the  active center of the enzyme was reactive toward DIFP. Although the phosphoserine recovered a f t e r hydrolysis accounted  for less than h a l f of the r a d i o a c t i v i t y i n DIP-a-chymotrypsin, the DIFP l a b e l i n g strategy was applied to c e l l walls to establish maximum and minimum values f o r DIFP binding to serine.  Any c e l l w a l l serine  reactive toward DIFP would most l i k e l y be a component of an active center of an enzyme but could represent any serine which was  sterically  available to DIFP and s u f f i c i e n t l y n u c l e o p h i l i c . 32 The method of removal of excess P-DIFP had no effect on the mole 32 f r a c t i o n 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 i n h i b i t e d phosphomonoesterase and no serine was phosphorylated, d i a l y s i s resulted i n complete release, of 32 P-DIFP from the enzyme and recovery of enzymatic a c t i v i t y . not  This was  observed, however, when the protein was precipitated and washed.  135 There was when excess  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 h y p o c o t y l c e l l w a l l s  reagent was  removed by d i a l y s i s  w a l l p r e p a r a t i o n t h a t was days.  The  single  cell  s p i n l a b e l e d r e l e a s e d the s p i n l a b e l a f t e r 4  These o b s e r v a t i o n s suggested  that a c e l l w a l l preparation  t r e a t e d w i t h the p h o s p h o f l u o r i d a t e may was  (Table V ) .  have been l a b e l e d but the  label  r e l e a s e d w i t h the passage of time, e i t h e r by spontaneous or phosphatase  catalyzed hydrolysis.  Although  procedes at a v e r y slow r a t e .  spontaneous h y d r o l y s i s o c c u r s , i t On the o t h e r hand, a c i d phosphatase i s  a known c o n s t i t u e n t of c e l l w a l l s K i v i l a a n , e t a l . , 1961; preparations. enzymatic  The  Suzuki,  (Lamport and N o r t h c o t e ,  1972)  have e x i s t e d i n t h e s e  r e l e a s e of the s p i n l a b e l may  r e d u c t i o n of the n i t r o x i d e  Although  and may  (Smith,  1960;  have r e s u l t e d  from  1972).  c e l l w a l l s i s o l a t e d from b o t h hook and b a s a l r e g i o n s of  the h y p o c o t y l c o n t a i n e d r a d i o a c t i v i t y a f t e r removal of excess by r e p e a t e d washing  (Table V ) , o n l y the hook c e l l w a l l s  d e t e c t a b l e phosphoserine  i n the h y d r o l y s i s products  reagent  contained  (Table V I ) .  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 s e r i n e t h a t were not adequately no o t h e r e v i d e n c e  supports  p r e l a b e l e d by DIFP, however  the i d e n t i t y of such a s i t e .  The o n l y c e l l w a l l p r e p a r a t i o n s which c o n t a i n e d any  significant  r e a c t i v e s e r i n e were those e x t r a c t e d from the h y p o c o t y l hook. c o r r e c t e d phosphoserine pmol;. mg  1  r e c o v e r y from h y p o c o t y l hook c e l l w a l l s  The (2.71  c e l l w a l l , T a b l e VI) r e p r e s e n t e d a minimum v a l u e based on  f a c t t h a t r e c o v e r y from a-chymotrypsin t e s t e d ; the v a l u e i n Table V  was  (5.4 pmol.- mg  maximum v a l u e of r e a c t i v e s e r i n e r e s i d u e s .  incomplete 1  the  under a l l c o n d i t i o n s  c e l l wall) represents a  136 To d e t e c t  s p i n l a b e l e d c e l l w a l l suspensions  (6 mg  e l e c t r o n s p i n resonance s p e c t r o m e t r y , the s p i n l a b e l would have to be sensitivity  approximately 2nnmol mg  c a l c u l a t i o n s described  s u b s t a n t i a l l y greater  ml  ^)  by  concentration  * c e l l w a l l based  on  by B o l t o n , e t a l . , (1972).  than the maximum v a l u e  f o r DIFP b i n d i n g  This i s to hook  c e l l wall serine. The  reactive serine i n hypocotyl  i n basal hypocotyl  walls  hook c e l l w a l l s which was  absent  c o u l d have been p a r t of the a c t i v e c e n t e r  of  a  c e l l w a l l enzyme o r an enzyme adsorbed to the c e l l w a l l , or a p a r t of  a  s t r u c t u r a l p r o t e i n , such as e x t e n s i n .capable of b e i n g i n the  c e l l walls  glycosylated.  Suzuki,  of v a r i o u s members of the D i c o t y l e d o n a e .  Edelman and H a l l ,  3-glycosidases  N o r t h c o t e , 1960;  1972), p e c t i n methyl e s t e r a s e  a c i d o x i d a s e (Newcomb, 1951; 1974;  which was  Numerous enzymes have been  i n c l u d e phosphatase (Lamport and 1961;  (Lamport, 1965)  (Ashford  These  ( G l a s z i o u , '1959), a s c o r b i c  (Murray and  and M c C u l l y , 1970;  identified  K i v i l a a n , et a l . ,  M e r t z , 1964), i n v e r t a s e  1964), a-  still  ( K l i s and  B a n d u r s k i , 1975)  K l i s , et a l . , 1974;  Akster, and Murray  and B a n d u r s k i , 1975), ATPase ( K i v i l a a n , et a l . , 1961), p h o s p h o r y l a s e ( K i v i l a a n , . e t a l . , 1961) 1974;  and  acetyl cholinesterase  Chapter I of t h i s t h e s i s ) .  ( F l u c k and J a f f e ,  A l k a l i n e phosphatase  and  p h o s p h o r y l a s e 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  F i s h e r , et a l . , 1959,  p l a n t enzymes are s i m i l a r l y  r e s p e c t i v e l y ) and  inactivated.  i n a c t i v a t e d by DIFP (Chapter I , F i g u r e  although no  Acetylcholinesterase  7) and  i n h i b i t e d by DIFP so p e c t i n - m e t h y l e s t e r a s e  i t i s probable that  may  many o t h e r be  was  esterases  i n a c t i v a t e d by  d i r e c t e v i d e n c e e x i s t s i n s u p p o r t of t h i s argument.  the  are  DIFP It  137 would be p o s s i b l e to account f o r the l a b e l i n g of hook c e l l w a l l s i f any  of these  activity  (or o t h e r ) DIFP s e n s i t i v e enzymes had a g r e a t e r  specific  i n the h y p o c o t y l hook than i n the b a s a l r e g i o n of the h y p o c o t y l .  T h i s i s the case f o r a c e t y l c h o l i n e s t e r a s e (Chapter Murray and B a n d u r s k i specific activity  (1975) have i d e n t i f i e d  I , Table I I I ) .  B-galactosidase  i n higher  i n the hook of pea stems than i n the b a s a l stem  segments, but DIFP s e n s i t i v e enzymes have not been s t u d i e d from t h i s perspective. Using  the v a l u e of e s t i m a t e d  acetylcholinesterase  c a t a l y t i c c e n t e r a c t i v i t y of  (0.197 u n i t s pmol  1  DIFP; Chapter 1^ T a b l e V) and  the s p e c i f i c a c t i v i t y of a c e t y l c h o l i n e s t e r a s e i n whole (0.25 u n i t s mg  1  hypocotyls  c e l l w a l l p r o t e i n ) , the p r e d i c t e d amount of DIFP  l a b e l i n g i n the whole h y p o c o t y l a c e t y l c h o l i n e s t e r a s e i s 1.25  c e l l w a l l s a t t r i b u t a b l e to  pmol  of DIFP.  This quantity  represents  only 6% of the minimum v a l u e f o r r e a c t i v e s e r i n e ( T a b l e V I ) . this value  i s maximal because the s p e c i f i c a c t i v i t y  may be g r e a t e r than i n e n t i r e h y p o c o t y l Table I I I ) .  c e l l walls  However,  i n hook c e l l  walls  (see Chapter I ,  T h i s DIFP s e n s i t i v e enzyme cannot account f o r a l l DIFP  b i n d i n g i n hook c e l l w a l l s . It  i s also p o s s i b l e that cytoplasmic  w a l l by i o n i c i n t e r a c t i o n s (Jansen, c e l l w a l l s may b i n d more c y t o p l a s m i c DIFP s e n s i t i v e enzymes may t h a t of b a s a l c e l l s .  exist  Before  enzymes  et'al.,  adsorbed to the c e l l  I960) would b i n d DIFP.  Hook  enzymes o r a g r e a t e r p r o p o r t i o n of  i n the cytoplasm  of hook c e l l s  than i n  r e s o l v i n g 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 p r e p a r a t i o n s must be e s t a b l i s h e d .  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 c e l l s and also by f a i l u r e to find contaminants during examination by phase contrast microscopy. for  The s e t t l i n g procedure chosen  the separation of intact c e l l s from c e l l wall fragments, produced  good y i e l d s of c e l l wall fragments but some intact c e l l s 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 released during the extraction and s e t t l i n g procedures. proteins, f o r example, may  have been Adsorbed  have had a measurable impact on reactive  serine since soluble proteases  and esterases abound i n plant c e l l  extracts. In any c e l l wall p u r i f i c a t i o n method, one i s 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  i o n i c a l l y bound c e l l wall enzymes but the r i s k of contamination by soluble cytoplasmic enzymes was  increased.  It i s 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 i n Avena c o l e o p t i l e s . 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 i o n i c interactions with contaminating proteins.  No evidence supports the alternative that there  are a greater proportion of DIFP s e n s i t i v e enzymes i n the hook than i n  139 the basal region of the hypocotyl. If the reactive serine observed i n 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 i n young c e l l walls which would be glycosylated i n s i t u and e x i s t as glycosyl serine i n the f u l l y extended c e l l wall.  K l i s (1976) examined the quantity of glycosylated (hydrazine  l a b i l e ) and non-glycosylated  (hydrazine stable) serine i n segments  excised from e t i o l a t e d 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 w a l l while non-glycosylated serine increased from 14.6 to 20.9 nmol mg ^ c e l l w a l l .  From these data, i f a l l of the  non-glycosylated serine existing i n the c e l l w a l l became glycosylated during c e l l extension (which would s t i l l not account f o r a l l of the glycosylated serine) and i f the bean hypocotyl behaved s i m i l a r l y , then at least 14.6 nmol reactive serine would have been expected per mg of hook c e l l w a l l .  The maximum value  magnitude less than t h i s .  (Table V) was a c t u a l l y 3 orders of  K l i s concluded  that the glycosylation s i t e  is not l i k e l y to be i n the c e l l w a l l because the hydroxyproline: glycosyl-serine r a t i o remained constant during elongation.  The results  of this study s i m i l a r l y refute the p o s s i b i l i t y that the serine glycosylation s i t e i s i n the c e l l w a l l . It may be possible to use the radioactive DIFP labeling strategy as a rapid method to detect i o n i c a l l y bound proteins, whether native or adventitious, and thereby e s t a b l i s h a c r i t e r i o n of p u r i t y , i f c e l l walls which have been p u r i f i e d 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 i n Methods and the washing procedure to remove 32 excess  P-DIFP  (or any other radioactive isotope of D I F P ) .  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