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Purification and characterization of chloroplast coupling factor from wheat Moase, Elaine Helen 1980

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PURIFICATION  AND CHARACTERIZATION OF CHLOROPLAST  COUPLING FACTOR FROM WHEAT by  ELAINE HELEN MOASE B.Sc,  U n i v e r s i t y of  A THESIS SUBMITTED  New B r u n s w i c k ,  1977  IN PARTIAL FULFILMENT OF  THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department o f  Botany  We a c c e p t t h i s t h e s i s as conforming to the  required standard  THE UNIVERSITY OF BRITISH COLUMBIA June (5)  1980  E l a i n e Helen Moase,  1980  In  presenting this  thesis in partial  an advanced d e g r e e a t the L i b r a r y I further for  shall  the U n i v e r s i t y  make i t  agree that  freely  of  extensive  s c h o l a r l y p u r p o s e s may be g r a n t e d  this  written  thesis for  It  Department nf  f i n a n c i a l gain shall  Botany  The U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 W e s b r o o k P l a c e V a n c o u v e r , Canada V6T 1W5  Date  20 J u n e 1980  I agree  r e f e r e n c e and copying of  this  that  not  copying or  for  that  study. thesis  by t h e Head o f my D e p a r t m e n t  i s understood  permission.  the requirements  B r i t i s h Columbia,  available for  permission for  by h i s r e p r e s e n t a t i v e s . of  f u l f i l m e n t of  or  publication  be a l l o w e d w i t h o u t  my  i i  ABSTRACT  Chloroplast aestivum,  var  sucrose  density  coupling factor  Thatcher)  and e ,  of  of  prevented  the  gradient  proteolysis  proteolysis  CF.j by c h l o r o f o r m ,  thylakoid However,  by t h e  39,  wheat  25  smallest  enzyme,  {Triticum  and  13  and  kd  steps of  subsequent  i n common  subunits, designated  in a l l  molecular  event  weight  inhibitors  they  a,  3, y,  6  respectively. the  purification  subunits during  of  sucrose  by h y p o t o n i c  in any any  did  completely  hypotonic  CF^ r e l e a s e  proteolytic  this  two  T h e wheat  five  inhibitors  the  of  the  sucrose  step.  Although of  of  55,  thylakoids  by c h l o r o f o r m e x t r a c t i o n  contains 57,  weights  loss  from  centrifugation.  factors,  molecular  Inclusion  was p u r i f i e d  gradient  w i t h most c o u p l i n g  (CF^)  not  interfere  prevented  method o f sucrose  CF^ s u b u n i t ,  with  CF^ r e l e a s e  Strotmann  did  the  not  et  as no d i f f e r e n c e  CF^ s u b u n i t was o b s e r v e d when  from  al.  appear  to  in  extraction the  (1973)-  require  a  apparent  CF^ was  released  by  method. A double  SDS-PAGE was subunit,  band o r  apparent  identified  as  mw's  37  and 39  kd f o r  the  y subunit  r e d u c e d and u n r e d u c e d f o r m s o f  s u g g e s t i n g an" i n t e r n a l  disulfide  bridge  in  the  the y  in  same  subunit  of  wheat CF.j. The  latent  but  not  by h e a t ,  the  rate of  a substrate. and V (10 of  max  mM)  CaATPase o f un1ike  A T P , but A 1:1  ratio  of in  M  of  is activated Wheat  nucleotide  the  triphosphate  presence of r  amount  0.125  of  by t r y p s i n  CF^ h y d r o l y z e s  Ca:ATP gave o p t i m a l  an e q u i m o l a r  excess calcium, a K  CF^  spinach CF^.  no o t h e r  were d e t e r m i n e d and w i t h  wheat  a  msv  32%  of  as  activity.  excess of  c a l c i u m and A T P .  mM CaATP and a \l  GTP a t  is e f f e c t i v e  ATPase  large  digestion  In 18.9  calcium the  presence  units/mg  i i i  protein the  were  obtained.  K.. was 0.018 M  order  of  Under c o n d i t i o n s o f  mM CaATP a n d t h e  difference  alteration  of  absence of  excess calcium.  showed a  structure. exhibits  affinity  hexagonal  Markham  wheat  into  a  CF.j and  rabbit.  showed t h a t  subunits  of  wheat  is  enzyme f o r  units/mg •  thought  to  substrate  negatively-stained Escherichia  supported the  CF^ w e r e  The  inhibited  phoresis  K^'s  c a l c i u m and A T P , protein.  b e due t o by t h e  wheat  The  r  CF^  an  presence.or  particles gave a  coli  conclusion that  similar  wheat  CF^  symmetry.  to wheat  similarity  in  particle.  CF,.  latent,  by  antiserum  injecting both  the  two  antiserum  enzymes. reacts  the  purified  precipitated  trypsin-activated  s p i n a c h C F ^ , showing that  between this  generated  resulting  its  serum c r o s s - r e a c t e d w i t h antigenic  the  rotation  a six-fold  Antibodies enzyme  of  microscopy of  solid  was 6.77 max  magnitude  Electron  V  equimolar  CaATPase.  there  Crossed  is  The  considerable  immunoelectro-  s p e c i f i c a l l y with  the  a and £  i. V  TABLE OF CONTENTS Page INTRODUCTION  1  MATERIALS AND METHODS  12  Mater i a l s  12  Plant material  13  CF.j e x t r a c t i o n  and p u r i f i c a t i o n  13  Electrophoresis  15  Assays  16  Electron microscopy  18  P r e p a r a t i o n and c h a r a c t e r i z a t i o n o f antibodies  monospecific  t o w h e a t CF^  19  RESULTS Purification  22 o f w h e a t CF^  22  The e f f e c t o f p r o t e o l y s i s i n h i b i t o r s on e x t r a c t i o n o f w h e a t CF^ The y s u b u n i t o f w h e a t CF^ c o n t a i n s an i n t e r n a l * d i s u l f i d e bridge ATPase a c t i v i t y  o f wheat  CF^  26 ;1 31 37  E l e c t r o n m i c r o s c o p y o f w h e a t CF^  kS  Immunological  hS  s t u d y o f wheat  CF^  DISCUSSION  5h  SUMMARY  64  V  LIST  OF TABLES Page  Table  I.  Purification  Table  II.  Apparent molecular and s p i n a c h C F ^ ' s  Table  Table  III.  IV.  of  wheat  25  CaATPase  weights  of  subunits  Substrate wheat  specificity CF  i  wheat 25  H e a t - a c t i v a t e d CaATPase a c t i v i t y t r e a t e d at various temperatures  of  of  of  the  of  w h e a t CF.j  CaATPase  38 activity  vi  LIST  OF FIGURES  Page  Figure  1.  Diagram  Figure  2.  P r o f i l e of wheat CF.|  Figure  3-  SDS-PAGE from  Figure  Figure  Figure  Figure  Figure  k.  5.  6.  7.  8.  9-  a  crossed  Immunoelectrophoresis  sucrose density  gradient  21  gel  used  to  purify 23  of  crude  CF-| s a m p l e  sucrose density  and  protein  fractions  gradient  2k  C o m p a r i s o n o f c r u d e CF-| e x t r a c t s p r e p a r e d by t h e c h l o r o f o r m method w i t h t h a t p r e p a r e d by t h e h y p o t o n i c s u c r o s e m e t h o d , and t h e e f f e c t o f i n c l u s i o n o f p r o t e o l y s i s i n h i b i t o r s upon e a c h  27  E f f e c t of the i n c l u s i o n of p r o t e o l y s i s d u r i n g CF-| e x t r a c t i o n and p u r i f i c a t i o n c o m p o s i t i o n o f wheat CF^  28  inhibitors upon s u b u n i t  Proteolysis inhibitors present during sucrose d e n s i t y gradient c e n t r i f u g a t i o n prevent loss of and e s u b u n i t s o f wheat CF^ S D S - P A G E o f w h e a t and s p i n a c h CF-j , s h o w i n g d o u b l e band i n t h e y s u b u n i t p o s i t i o n Limited the  Figure  of  y  proteolysis  subunit  of  dimer  each of  and o f  the  the  a  two  and  the  6 30  a 32  bands  of  g subunits  E f f e c t o f r e d u c i n g a g e n t upon t h e s u b u n i t o f wheat C F i n SDS-PAGE  mobility  E f f e c t o f h e a t upon t h e number t h e y s u b u n i t upon SDS-PAGE  bands  of  the  3k y 35  1  Figure  Figure  Figure  10.  11.  12.  13-  Effect  of  \k.  the of  ratio  of  of  wheat  kinetics CF^  in  of the  in 36  calcium  trypsin~activated  Michaelis-Menten CaATPase  Figure  found  T i m e c o u r s e o f t r y p s i n a c t i v a t i o n o f wheat i n t h e p r e s e n c e and a b s e n c e o f p r o t e o l y s i s inhibitors  activity Figure  of  to  CaATPase  ATP upon  39 ATPase  CF^ f r o m wheat the  k2  trypsin-activated  presence of  10 mM C a C ^ .  M i c h a e l i s - M e n t e n k i n e t i c s of the trypsin-activated C a A T P a s e i n t h e p r e s e n c e o f e q u i m o l a r amounts o f c a l c i u m and ATP  k3  kk  vi i  Figure  Figure  Figure  15-  16.  17-  E l e c t r o n m i c r o g r a p h s o f wheat CF-j , s t a i n e d w i t h two d i f f e r e n t s t a i n s  Selected  CF.. p a r t i c l e s ,  hexagonal  showing  18.  Markham  Figure  19-  Ouchterlony double d i f f u s i o n a n t i b o d y t i t e r in serum from a g a i n s t wheat CF^  Figure  Figure  21.  22.  various  rotation  of  I n h i b i t i o n of the a c t i v i t y o f wheat  -  t h r e e wheat  Crossed Immunoelectrophoresis s u b u n i t s o f wheat CF^ a g a i n s t  CF  1  particles  48 50  plate estimation of a rabbit immunized 51 of the s e p a r a t e d a n t i - w h e a t CF^ s e r u m . .  trypsin-activated CF^ by a n t i - w h e a t  CaATPase CF^ serum  P r o p o s e d model f o r t h e a r r a n g e m e n t o f t h e o f wheat C F w i t h i n t h e enzyme c o m p l e x n  47  degrees  structure  Figure  20.  46  E l e c t r o n m i c r o g r a p h s o f Escherichia coli F-| , n e g a t i v e l y - s t a i n e d w i t h ammonium m o l y b d a t e  of  Figure  negatively-  51  53  subunits 60  vii i  ACKNOWLEDGEMENTS  This  r e s e a r c h would  supervision  of  gratitude.  Thanks  Mr.  Adib  their  with  Beverley also  have R.  go t o  been  Green, Dr.  R.  Rowhani  of  the  Agriculture  assistance  in  the  immunization  assistance study  Dr.  not  is Dr.  of  Mr.  L a s z l o Veto  in  acknowledged.  P.D.  (U.B.C.  and  G.H.N.  Dr.  Edith  Bragg  Towers Camm f o r  N.S.E.R.C.C  for  (U.B.C. her  Dept.  Dept.  of  I wish  Canada of  I  rabbits  of  have  for  to  of  in  electron  Special  period  study.  helpful and  Drs.  thanks  to  for  The  microscopy  discussions A.D.M. go  My t h a n k s 1977  and  Vancouver  the  and  sincere  L o i s Johns  this  encouragement. the  my  for  had many  Botany).  guidance  express  Ms.  Biochemistry)  assistance for  the  laboratories  aspects  a s s i s t a n c e and  financial  and  Stace-Smith,  all  gratefully  p o s s i b l e but  to to  1979.  Glass  1  INTRODUCTION  T h e "common c u r r e n c y " adenosine  triphosphate  Y phosphates can  of  provide  19^6).  energy  in  this  living  mitochondria  it  to  drive  was  and  certain  energy for  is  In  to  Lehninger,  in  of  the  of  a membrane.  side  of  the  of  the  chain This  membrane,  across  the  protons  the  molecule  that  the  the  its  living  S and  cleavage  cell  organization  of  similar  (Lipmann,  enzymes  among  the  synthesis  plants  and  and  at  , 195*0  al .  sites  of  These  and an e l e c t r o n chains,  was p o s t u l a t e d  these  and  photosynthetic  animals.  transport  It  sites  in  it the  formation  membrane.  The  back a c r o s s  the  force  for  ATP s y n t h e s i s .  Critical  which  the  electrochemical  potential  Mitchell  ATP.  the  with  results the  the  compartments  Peter  of  that  carried  are  et  the  that  donated  transport  passage a  high  its  energy  1953).  (Slater,  he p o s t u l a t e d  side  gradient  ATP  scheme f o r  transport  in  produced  English biochemist  electron  energy  remarkably  electron  1961,  1961),  the  19^8)  ATP  of  (Mitchell,  be  t o ATP s y n t h e s i s .  intermediate  the  and  is  bond b e t w e e n  many p r o c e s s e s o f  synthesis  different  leakage  sufficient  membrane-bound  points  tied  chemical  the  of  t h a t c h l b r o p l a s t s (Arnon  phosphorylation  chain.  electrons  cells  phosphodiester  ATP s y n t h e s i s  known  each contain  of  the  living  studied.  organelles At  is  in  p r o c e s s now a p p e a r  (Kennedy  and o x i d a t i v e  The  molecule  systems  I960  By  energy  (ATP).  The mechanism o f  involved many  this  of  In  flow of  was  of  this  proposed  protons  hypothesis  gradient  is  is  by  be t h e  the  generated,  on  one one  potential  gradient to  the  them t o  an e l e c t r o c h e m i c a l  membrane this  of  hypothesis  through  delivering  accumulation of  radically  chemiosmotic  electrons  protons,  dissipation  to  the  proposed a  the driving  membrane  and  the  across  assym-  2  metrical  arrangement  resulting In  in  the  of  accumulation  chloroplasts,  ATP s y n t h e s i s came Chloroplasts, synthesized gradient  components o f  the  first  1966  in  transferred  from  protons evidence  from  ATP when ADP and  a c r o s s the  of  the  the high  was  on one for  work o f pH t o  inorganic  thylakoid  electron  transport  side of  a proton  the  chain, membrane.  gradient  Jaegendorf  and  driving  Uribe.  low pH s o l u t i o n s  in  the  phosphate were p r o v i d e d .  sufficient  driving  force  for  dark, A pH  ATP  generat ion. Over support  the  the  past  twenty y e a r s ,  chemiosmotic hypothesis of  m e c h a n i s m by w h i c h ATP i s made the  electrochemical  For  six  views  potential  on t h i s  The c o u p l i n g o f electrochemical dubbed " c o u p l i n g has  been  protein  the  factor"  shown t h a t  extraction  membranes  When in  transport-dependent  Penefsky  et  I960)  The s o l u b i l i z e d  activity  is manifest  activity  of  the  under  the  is  through  In  both  the  chloroplast  coupling  presence of  factor  in  is  of  understood.  this  field,  transport-generated  an enzyme c o m p l e x w h i c h  chloroplasts  (Jaegendorf factor  is  and  resumed  a 1961;  and S m i t h ,  an a p p r o p r i a t e is  mitochondria,  removes  added  is  back  to  divalent  (Avron,  cation,  1963;  conditions.  will  s o l u b l e enzyme enzyme  active  w i t h an EDTA s o l u t i o n  appropriate  the  dissipation  incompletely  electron  i s , ATP h y d r o l y s i s . in  the  to  al.(1977)•  ATP s y n t h e s i s  coupling  t o ATP s y n t h e s i s , t h a t  scientists  uncoupling  this  the  electron  al.,  in  with  still  to  "F^".  resulting  al.,1960).  deficient  gradient or  is  by B o y e r e t  ATP s y n t h e s i s  potential  component,  Penefsky et  review  by s i x  has been a c c u m u l a t e d  ATP s y n t h e s i s , a l t h o u g h  in c o n j u c t i o n gradient  subject  see the m u l t i a u t h o r e d  it  much e v i d e n c e  catalyze For  the  the  mitochondrial  (Pullman  exhibited  reverse  only  et  al.,  after  reaction F^, ATPase  I960), a short  but  the  digestion  3  with  trypsin,  or  by h e a t i n g  to  65*C  for  two m i n u t e s  (Vambutas  and  Racker,  1965). A number o f plast of  coupling  the  factor  activity  reassociation  different  (CF^),  from  of  ATPase a c t i v i t i e s  the  this  as d e m o n s t r a t e d  thylakoid  protein  results  with  the  demonstrated Vambutus  and R a c k e r  a CaATPase a c t i v i t y heating the  65°C.  at  properties  in maleate activity  in It  thylakoid  showed t h a t  trypsin  by EDTA  Mg-dependent  solution.  fraction  digestion,  two  showed minutes  characterized  activiation  at  pH  6.0  soluble CF^. CF.j  is  attached  identified have  removed  freeze-fracture to  as t h e  membranes,  outer  of  1976).  that  F^  membrane,  with  remove is  (Lein  treatments  1967)  not  attached  projecting  In  or  the  to into  to  the the  inner  surface  of  1971a). T h e s e  hypotonic  s u c r o s e wash  (1976) d e m o n s t r a t e d , thylakoid  In  projections native  membrane.  uncouple c h l o r o p l a s t s ,  on t h e  stacked.  knob-like  thylakoid  and R a c k e r ,  and S t a e h e l i n  are  the  s e e n on t h e  known  CF^ p r o j e c t i o n s  F^.  of  projections  stained  Miller  regions which  to  surface  and M o u d r i a n a k i s ,  identified  by p r o c e d u r e s known  the  knob-like  by a number  (Howell  those  to  been n e g a t i v e l y  techniques,  been  mitochondrial  By h e a t  which  or  (1970)  and R a c k e r  CaATPase.  a  (1962, 1963)  Avron  thylakoids  a short  Ba1tscheffsky  8 mM M g C ^ , N e l s o n e t a 1. (1972a) o b t a i n e d a MgATPase  and S t a e h e l i n ,  chondrial  either  it.  and  and  that  has a l s o  first  (1970) and F a r r o n  Farron  s u c h as EDTA wash  F.j  restores  (1965) p r e p a r e d a s t a b l e p r o t e i n after  coupling,  buffer  vivo,  limited  from  of  separation  heat-activated  k n o b s c a n be  are  loss  that  the  has been  (Miller  extracted  fact  chloro-  of  from  thylakoids  a  was a s s o c i a t e d w i t h t h y l a k o i d s .  a MgATPase  associated with  by t h e  in  (1959) and W e s s e l s and B a l t s c h e f f s k y (i960) ATPase a c t i v i t y  are  the that  mitochondrial  of  surface  mitochondrion, c a n be  conformation surface  by  of  the  matrix.  removed  mitoinner Protons  are of  pumped by t h e  electron  chloroplasts, while  The d i f f e r e n c e  in  orientation  these  of  proton in  channel  tuted was  When  to  i n ' r e s p o n s e to  et  al.,  reported  1978),  into  (Younis  although  by t h e  1 . ,,1(977)  .  The o t h e r  CF^-attachment  T h e CF^ c o m p l e x  is  sucrose  (Strotmann et  1971a),  and e m u l s i f y i n g  1977).  Once r e l e a s e d in  al.,  in  a number  of  both  dissipated  and  these  reconsti-  vesicles,  (Pick  gave  and  four  Racker,  functioned  1977).  8300,  Racker,  dicyclohexylcarbodiimide CF  Q  1979;  when  as a p r o t o n  the  was  CFQ p o l y -  P i c k and mw  ATP  vesicles  and W i n g e t , or  of  in  the  bacteriorhodopsin  1977;  polypeptide,  from  the  EDTA  (Lien  1973)>  the  to  complex,  electrochemical  purified  Three  polypeptides  or  a thylakoid  from  matrix.-  pore (DCCD)  c o m p l e x may  act  components.  released  i n c l u d i n g washing  the  (Younis  and W i n g e t ,  uncoupler  is  transition  unclear.  one  membrane  CF^ b i n d i n g  ATP f o r m a t i o n  CFg i s  the  different  gradient  chromatophore  phospholipid v e s i c l e s ,  c o u l d be b l o c k e d  or  in  of  contains  utilizes  an a c i d - t o - b a s e  light-dependent  Ellenson  cryoprotected  intrinsic  When CF^ was a d d e d  CFg-containing vesicles,  been  treatments,  an  C F ^ has been  proton-translocating  into  regulatory  to  enzyme w h i c h  synthesize ATP.  have  as  binds  the  space  complexes.  the  peptides  a  pumping e x p l a i n s  and  polypeptides  et  pumped o u t  manner,  catalyzed  (•Nelson  are  F Q " ^ comprises a complex which  T h e number o f  that  protons  cross-membrane proton  the  reconstituted  intrathylakoid  factor  phospholipid vesicles.  incorporated which  complex  the  the  synthesized  1979).  proton  coupling  factor  gradient  into  of  into  by w h i c h  a controlled  potential  two  Together  F^.  chain  in m i t o c h o n d r i a ,  direction  The c o u p l i n g denoted  transport  and  suspension CF^  Lability  in is  is  by a number o f  Racker,  si1icotungstic  thylakoid,  ways.  thylakoid  acid  1971b), (Lien  chloroform cold  labile  characterized  different  hypotonic and  Racker,  (Younis  et  al.,  unless by a  loss  of  5  'CaATPase a c t i v i t y , at CF.j  k° C o n l y  in a saturated  to g i v e  appears  CF.|  to  probably its  that  both  a c t by the  useful.  the  Beechey e t  f r o m b o v i ne h e a r t Presumably  this  in attachment appears except  Penin et method  a  1.,  F^  to  i s no  t h e membrane.  complex  Younis  spinach  p r o p e r t i e s of  complexes,  of  h a s been w e l l in the  denoted  ? ^nd  3, Y»  cations, for  F^  them  c o u l d be in  binding  and  it  CF^  and  the  complex. has  extracted  chloroform.  neutralizing  forces  by c h l o r o f o r m  involved extraction  p r e p a r e d by o t h e r  (Spitsberg  and B l a i r ,  modified  and  CF^  the  s o l u b i l i z a t i o n of  as F^  (1977)  the  methods  1977;  chloroform  here are c h a r a c t e r i s t i c of  choice for the  the  studied. range of  e in order  study  study  composition of  molecular weight a,  thylakoid  required for  prepared  labile  noted  organism for  polypeptide  the  by c h l o r o f o r m t r e a t m e n t  F^  al.  with  the  enzyme  S i n c e s p i n a c h i s an e a s y p l a n t w i t h w h i c h t o  plant  s e r v e d a s a model  et  CF^  and s u c r o s e  CF^.  CF^  from s p i n a c h .  has b e e n t h e  The  F^  same p r o p e r t i e s  longer cold  1979).  to e x t r a c t  The  the  foundthat  (1975)  2 mM ATP  stored  freezing,  i n t e r a c t i o n s between  a c t s a s an o r g a n i c s o l v e n t ,  of  it  permitting  al.  Upon  is a chaotrope,  mi t o c h o n d r i a by e m u l s i f y i n g  t o h a v e much t h e that  cations  hydrophobic  s o l u b i 1 i z a t i o n of  c a n be  s u c r o s e wash m e t h o d s  those c r i t i c a l  link,  CF^  requires divalent  Si 1 i c o t u n g s t i c a c i d  membrane-associated  proven  Reassociation of  EDTA and h y p o t o n i c  removing  complex.  amnion i u m ' s u l f a t e .  photophosphorylation  thylakoid.  for  the  in the presence of  1972).  a c t s by a l t e r i n g t h e  A new method  it  al . ,  r e s t o r a t i o n of  extraction of  (Lien et  of  s o l ut i on' o f  is c o l d - l a b i l e unless stored  or glycerol  it  due t o d i s s o c i a t i o n  CF^, CF^  CF^,  and  indeed  coupling factors  and many o t h e r  It  has  in general.  coupling  is a large complex,  350,000.  of  of  of  with  i s composed o f  work,  factor a  five  decreasing molecular weight  subunits, (Racker  6  e t a l . , 1971; L i e n e t a l . , 1972).  The observed p a t t e r n o f p o l y p e p t i d e s  o b t a i n e d i n SDS-PAGE has proven t o be c h a r a c t e r i s t i c o f c o u p l i n g f a c t o r s , having been found f o r the c o u p l i n g f a c t o r s o f m i t o c h o n d r i a S e n i o r , 1971; C a t t e r a l l and P e d e r s o n , 1971), Escherichia S m i t h , 1977), Streptococcus lysodeikticus  faecalis  (Brooks and  coli  (Abrams et a l . , 1976),  Micrococcus  (Huberman and Sa1 t o n , 1979) , the t h e r m o p h i l i c b a c t e r i u m PS3  (Ypshida e t a l . , 1975), and the t h e r m o p h i 1 i c b l u e - g r e e n a l g a ( B i n d e r and B a c h o f e n , 1979)•  laminosus  (Wilson and  Mastigocladus  A l t h o u g h t h e number o f d i f f e r e n t  p o l y p e p t i d e s c o m p r i s i n g c o u p l i n g f a c t o r complexes i s g e n e r a l l y accepted as f i v e , the s t o i c h i o m e t r y o f these s u b u n i t s i s by no means c l e a r . S t o i c h i o m e t r i e s o f 3a: 3 3 : l y : 1<$: 1 e ( S e n i o r and B r o o k s , 1971; Yoshida  et a l . , 1979), 2 a : 2 3 : 2 y : ?<5: 2e (Sen i o r , -1975) and 2 a : 2 g : 1y: 1 6 : 2e ( B i n d e r e t a 1. , 1978) have been r e p o r t e d f o r CF^ and o t h e r c o u p l i n g f a c t o r s . Exceptions e x i s t  in the l i t e r a t u r e .  There a r e s e v e r a l r e p o r t s o f  p r o t o n - t r a n s l o c a t i n g ATPases t h a t have a s m a l l e r number o f p o l y p e p t i d e s , such as the two p o l y p e p t i d e F ^ s o f Bacillus 1  B a r l o w , 1973) and B a c i l l u s s u b t i l i s  magaterium  (Mirsky and  ( S e r r a h i m a - Z e i g e r and M o n t e i l , 1978),  and the t h r e e p o l y p e p t i d e F^ o f Clostridium  pasteurianum  (Clarke et a l . ,  1979). Roles  in p h o t o p h o s p h o r y l a t i o n have been a s s i g n e d t o some o f t h e  s u b u n i t s o f C F ^ . Removal o f the 6 s u b u n i t p r e v e n t s b i n d i n g o f the complex t o the t h y l a k o i d  (Younis e t a l . , 1977), but though c o u p l i n g i s p r e v e n t e d ,  t h i s d e f i c i e n t complex s t i l l  has ATPase a c t i v i t y .  C o u p l i n g a c t i v i t y can  be r e s t o r e d by a d d i t i o n o f a minor s u b u n i t f r a c t i o n c o n t a i n i n g t h e <5 and e subunits. E. coli  R e c e n t l y Cox e t a 1. , (1978) demonstrated t h a t e x t r a c t i o n o f  F^ by a low i o n i c s t r e n g t h wash was prevented by an i n h i b i t o r  p r o t e o l y s i s , p-aminobenzamidine.  of  Bragg and Hou (1979) found t h a t the 6  7  subunit  o f ECF^ u n d e r g o e s  bacterial ECF.J .  membrane.  This  involved  Inhibition  evidence  e subunit  acts  A T P a s e o f s p i n a c h CF^ lost  upon d i g e s t i o n  that  the e subunit  membrane,  as w e l l  the soluble  Activation  deficient to  activity synthesis  (Bakker-Grunwald  catalytic  site  subunits  a n d ATP h y d r o l y s i s o c c u r and Racker  These newly-exposed t o form  (Andreo  showed an i n c r e a s e  illumination  (McCarty  conformational phorylation Nelson  change  b y CF^ when  by r e m o v a l  ;  enzyme,  e t a l . (1973)  CF^ a f t e r  i n ATP s y n t h e s i s  i s attached  o f ATPase  is  proposed to the  activity  heat  1973).  o f the e subunit.  to l o c a t e ,  site  the assumption  i n t h e number  activation  t h a t ATP  could  bridges,  be o x i d i z e d  one each  experiments binding  activation  to determine  with  titrable  with o -  i n t h e 8 and y t h e membrane-bound  to the y subunit  o f photophos-  subunits  C F with 1  upon  suggest a  o f the latent  which  by r e a c t i n g  of  o f the latent  o f CF^ upon s t i m u l a t i o n  and h y d r o l y s i s  o f ATPase  site.  These o b s e r v a t i o n s  o r upon h e a t  a s CF^  phosphorylation  o f the a c t i v e  o n e makes  groups  Similar  attempted  it  coupling of  a t t h e same  in the y subunit  by i l l u m i n a t i o n  h a s been  difficult  the location  N-ethylmaleimide  and F a g e n ,  activity  It  or digestion  not give  two new d i s u l f i d e  in  inhibitory  f o r the latency  sulfhydryl  e t a l . , 1979).  is  heat-activated  e t a l . , 1975)-  (1970) o b s e r v e d a n i n c r e a s e  g r o u p s on t h e n a t i v e  factors  a n d Van Dam, 1974; N e l s o n e t a l . , 1975).  does  activated  from t h e  release of  of coupling  Its  o f CF^ h a s been  in the s o l u b l e ,  iodosobenzoate  involved  proceeds  In s t u d y i n g  sulfhydryl  GF^  responsible  prevents  of the l a t e n t ,  ATP h y d r o l y s i s  transport.  Farron  subunits  (Nelson  release  t o t h e membrane.  1., 1972b).  et a  by t r y p s i n  i n o n e o r more  electron  ATPase.  (Nelson  o f CF^ p r o b a b l y  The a c t u a l  the 6 subunit  a s an i n h i b i t o r  prevents  1  that  during  proteolysis  o f t h e complex  as being  CF  cleavage  of this  indicates  i n the attachment  The  of  a proteolytic  CaATPase.  o f CF^ w e r e  anti-  8  bodies generated able  to inhibit  anti-y  against  the ATPase a c t i v i t y  serum i n h i b i t e d  MgATPase a c t i v i t y that  (1978)  against likely This  a n t i body  about  inhibition  labelling  However,  all  or a l l  The  it  suggestive  ability  between  antisera  in r e s u l t s  antibody  i s most  preparations. making  information  Individual  t o say whether  t o g i v e more  or a mixture information  sites are of both.  on t h e mechanism  binding sites  on c o u p l i n g  (1978). o f Y o s h i d a e t al.(1977)  on t h e r o l e  of individual  have  provided  subunits in  s u b u n i t s o f F^ f r o m t h e t h e r m o p h i l i c and t h e r e c o n s t i t u t e d  to hydrolyze ATP.  No A T P a s e a c t i v i t y  was o b t a i n e d  unles the  c o m p l e x , and a l 1 ' c o m b i n a t i o n s  of  t h e 3 and y s u b u n i t s e x h i b i t e d  a n d amount o f A T P a s e a c t i v i t y  bacterium  c o m p l e x was a s s a y e d f o r  in the r e c o n s t i t u t e d  subunits containing  these  (Carlier  o f t h e component p o l y p e p t i d e s .  on n u c l e o t i d e  by H a r r i s  t h e a and 3 s u b u n i t s  in c h a r a c t e r ,  on t h e f u n c t i o n  CF^ d e m o n s t r a t e d ATP b i n d i n g  3 s u b u n i t was i n c l u d e d  properties  with  c a u t i o u s about  on b o t h  is difficult  PS3 w e r e a d d e d b a c k t o g e t h e r the  sites  r e c o n s t r u c t i o n experiments  ATPase a c t i v i t y .  o f ATPase a c t i v i t y The d i f f e r e n c e  a n d ADP s i t e s  of information  see the review  some v e r y  i n p h o t o p h o s p h o r y l a t i o n , K o e n i g and  of heat-activated  regulatory  p h o s p h o r y l a t i o n than  factors,  light-triggered  s o l e l y upon t h e e v i d e n c e o f m o n o s p e c i f i c  b i n d i n g a n a l y s e s tend  an o v e r v i e w  anti-a or  of:enzyme act iv i t y . .  et.al.,1979)•  Nucleotide  sites  Either  Although the authors concluded  o n e s h o u l d be e x t r e m e l y  on t h e 3 s u b u n i t ,  catalytic,  antigenic  active  inhibit!on  Photochemical sites  obtained  due t o d i f f e r e n t  conclusions  For  chlorop1asts.  in i s o l a t e d  that  CF^.  p h o t o p h o s p h o r y l a t i o n and t h e  any o f t h e s u b u n i t s o f C F ^ .  demonstrated  No s i n g l e a n t i s e r u m was  of isolated  t h e a and y s u b u n i t s a r e i n v o l v e d  co-workers  of  each o f the s u b u n i t s .  identical  to those o f the native  catalytic PS3 F  n  .  9  However,  reconstitution  of  mesophilic  bacteria  amino a c i d  sequences of  to  the  the  complex  was d o n e w i t h  I98O). 3  no  33:  0.17y  but  removal  the  3 subunit  of  but  3 subunit  the  It  the  CFj  the  remove  the  them  because  less  the  resistant  necessary to  subunit  subunits  from  f r o m Micrococcus  removes  reconstruct  or  subunits  F^  by t r y p s i n  the  than  ATPase w i t h  twice  F^  the  inactive is  of  a  digestion,  it  required  l e s s e r amounts  et  1.67a:  of  native  Thus  for  and y s u b u n i t s ,  ratio  the  ATPase.  absolutely in  the  a subunit  activity  necessary  (Mollinedo  lysodeikticus  c o n s i d e r a b l e amounts o f  lysodeikticus  studies  is  probably  the  is  tempting  to  from  of  of  this  latent  structural  of  active  coupling factor  site  of  enzyme, appears  for  as  that  ATPase  regulators  this  is  with  a valid to  chloroplast.  and e n z y m a t i c  of  properties  those of  the  C F ^ , but  only  To t h i s  a method was  all  gave  end, its  information  such  a study  1  is  the  subunits.,  was o b t a i n e d  CF  ATPase  subunits  some o f  data  of  of  that  and  characterize  microscopic analyses  The v a l u e  functions  is  further  comparison.  CF^ c o n t a i n i n g  Functional  complexes  ADP p h o s p h o r y l a t i o n  compare c a t a l y t i c  wheat  CF^.  ATPase.  other  r e s e a r c h was  the wheat  wheat  of  coupling factors  prove whether  and e l e c t r o n  structure the  from  purification  phoretic  of  make  manipulation  a and y may be n e c e s s a r y  The o b j e c t  for  enzyme  above mentioned  research w i l l  of  to  more  i n M.  probably  and  effectors.  activity.. of  the  more a and y g a v e an  The e v i d e n c e the  and  A form o f  exhibited  activity, or  the  subunit.  polypeptides  determining  been  Trypsin  but  from o r g a n e l l e s  be more d i f f i c u l t ,  their  approach to has  coupling factors  1978).  (Kagawa,  ATPase a c t i v i t y  al. ,  seems t o  d i s s o c i a t i n g agents  Another  as  the  from  properties devised  Electro-  about  the  enzymatic  two-fold.  from a monocotyledon  The have  analysis  10  never  been c h a r a c t e r i z e d .  cotyledon spinach,  has i d e n t i c a l lettuce  structure add  model  about This  of  wheat  problem  of  research  chloroplast by i s o l a t i n g system,  then  o f wheat outside  CF^.  the c h l o r o p l a s t  organelle. intact  Of s p e c i a l  of  CF.j .  in  the cytoplasm.  1977;  From t h i s  Bouthyette  evidence  that  isolated  intact  synthesized Studies F.j  from they  concluded that  and J a e g e n d o r f ,  larger  1978).  (1976)  originally  Chua and S c h m i d t ,  made a s l o n g e r  the organelle,  only  They  in such a  to the subunits  synthesized  that  into the  the isolated  t h e a, 6 and e s u b u n i t s are synthesized  by two o t h e r  groups  Nelson et a l .  reported  that  polypeptide subunits  show t h a t  (1980)  a r e trimmed  down  in is  mitochondrial  chloroplast  and E l l i s ,  A t some p o i n t  found  in the c y t o p l a s m .  of  these  (Ellis,  the 6 subunit  of yeast  subunit  (Highfield  polypeptides.  the polypeptides  this  a s a, 6 a n d e a r e s y n t h e s i z e d  and t h e small  1978)  o f CF^  showed  Recently,  precursor  carboxylase  unifying  protein  post-translationally  on t h e c y t o p l a s m i c a l l y - s y n t h e s i z e d e t a l . , 1979)  will  factors.  to approach  i n an in vitro  were c o n f i r m e d  as well  a  coupling  t h e y and 6 s u b u n i t s  spinach c h l o r o p l a s t s .  as a s l i g h t l y  a l . , 1979;  it  spinach synthesize  the y subunit,  (Maeccecchini  into  was d e c i d e d  a r e the subunits  et a l .  These f i n d i n g s  species  c o m p o n e n t s o f CF^ t r a n s l a t e d  and t r a n s p o r t e d  ribulose-1,5-bisphosphate et  interest  on t h e  formulating  using monospecific antibodies  Mendiola-Morgenthaler  chloroplasts  It  RNA, t r a n s l a t i n g  s y s t e m by i m m u n o p r e c i p i t a t i o n ,  such as  from an u n s t u d i e d  t o d r a w when  a mono-  included a study o f the b i o s y n t h e s i s  factor.  isolating  from  any i n f o r m a t i o n  of energy-transducing  originally  coupling wheat  factor  from which  an enzyme  from d i c o t y l e d o n s  In a d d i t i o n ,  and f u n c t i o n  project  assume t h a t  to that  of a coupling  information  structure  synthesizing  properties  or snapdragon.  and f u n c t i o n  to the bulk  One c a n n o t  1978;  Cashmore  polypeptides are during to their  their final  transport size,  11  probably a study  by s p e c i f i c e n z y m e s . of  the  immunization of  the  wheat  of  antigenic  one  Time  CF ^ s u b u n i t s . rabbit  properties  against of  the  did  not  permit  Presented  here  wheat  C F ^ , and  wheat  antibodies  the  completion  are  the  the  results  of  such  of  characterization  produced.  the  12  MATERIALS AND METHODS The  following  sodium d o d e c y l Tris,  tris  abbreviations  sulfate;  SDS-PAGE,  (hydroxymethyl) EDTA,  ATP,  triphosphate;  , coli of  coupling  attachment  CaATPase, diamine; kd,  of  of  mw, in  the  membrane; ATPase;  referring  of  one  inhibitors"  is  used  is  umole.of to  ATPase,  to  the  the  inorganic  signify  the  hO mM e - a m i n o - n - c a p r o i c  1  of  Escherichia  complex,  the  site  triphosphatase;  1  factor  enzyme  phosphate  and  dithiothreitol;  phenylmethylsulfonylf1uoride;  0.1  and A T P a s e a r e  of  the  that  per  inclusion of  acid  DTT,  salt;  ECF^ ,  adenosine  activities  amount  (hydroxymethyl)  disodium  protein  Coupling  two  SDS,  N,N,N ,N -tetramethylethylene-  PMSF,  weight.  acid  text:  electrophoresis;  N-tris  factor;  membrane  TEMED,  acid;  gel  diphosphate;  coupling  intrinsic  molecular  ATPase a c t i v i t y  release  di-HCl,  to  an  TCA, t r i c h l o r o a c e t i c  interchangeably  the  F^,  calcium-dependent  kilodalton;  A unit  F^  Tricine,  ADP, a d e n o s i n e  CF^, chloroplast  factor;  this  SDS p o l y a c r y l a m i d e  ethylenediamine-tetraacetate  factor;  coupling  used throughout  aminomethane;  methylglycine; adenosine  are  same enzyme  will  complex.  catalyze  minute.  6 mM  used  "Proteolysis  p-aminobenzamidine  mM PMSF.  M a t e r i a 1s Miracloth provided  (Chi;copee M i l l s ,  e-amrno-n-caproic  polyethylene  glycol  (PEG),  inhibitor.  Molecular  (ovalbumin)  or  A).  Aldrich  (1976)  Adjuvant  was  other  acid,  PMSF,  trypsin  weight  was  kinase,  for  protein  supplied  a s s a y was  by D i f c o .  c h e m i c a l s were o f  by C a l b i o c h e m .  (low  (DPCC-treated)  standards  (pyruvate  supplied  agarose  and  from  Bio-Rad.  Sigma PX,  trypsin  from  Pharmacia  and  The c o l o u r  Sodium d e o x y c h o l a t e grade.  Lubrol  soybean  SDS-PAGE were  di-HCl.  purchased  reagent  EEO) ,  carbonic anhydrase  supplied p-aminobenzamidine  Bradford  All  Sigma  Inc.)  ribonuclease reagent  Freund's  came f r o m  for  the  Complete  Schwarz/Mann.  13  Plant  Material Young w h e a t  seedlings  (Triticum  f r o m s e e d s u p p l i e d by B u c k e r f i e l d s 1  Wheat  Pool,  chlorite washed  Calgary , A l t a .  solution  thoroughly  supplement seedlings  CF,j  overnight.  as n e c e s s a r y .  by b u b b l i n g a i r  (Spinacea  Spinach  leaves  B . C . o r by A l b e r t a  through  "*>% h y p o -  s e e d was sown  the seeds suspended in  vermiculite  16 L : 8 D  were c u t from  light  four-week-old  var Longstanding Bloomsdale)  oleracea,  grown  i n t h e same f o r 30 m i n u t e s ,  soaked  Germinated  were  i n f i l t r a t e d with  i n a g r e e n h o u s e f o r 8 t o 15 d a y s , w i t h  a n d grown  soil  minutes,  and g e r m i n a t e d  in d i s t i l l e d water  L t d . , Vancouver,  Seeds were vacuum  for five  var Thatcher)  aestivum,  grown  in  in a greenhouse. Extraction All  and  steps  Purification  up t o t h e e x t r a c t i o n  o f CF^ f r o m  thylakoids  were  done  o at  k C.  water,  The blades then  distilled  g f r e s h weight 1 mM E D T A , seconds.  water,  in c o l d  s e e d l i n g s were  chopped w i t h  The b r e i  was f i l t e r e d  pelleted  chloroplast  Medium a n d p e l l e t e d  through  cold  as b e f o r e .  Stromal  t o w h i c h was a d d e d p r o t e o l y s i s  total  of four  between  times  washes.  determined  at  During  acid,  i n 1.5  pH 7.k  f o r ten  of Miracloth  and t h e  et al. , :  1973)  T h i s wash was r e p e a t e d p e l 1eted a t  the c h l o r o p h y l l  (19^*9) •  1 ml/g fresh  in about  (Strotmann  being  The  Isolation  (6 mM p - a m i n o b e n z a m i d i n e  mM P M S F ) .  H ° C , the thylakoids  by t h e method o f A r n o n  blendor  ,5"bisphosphate  f o r 30 m i n u t e s  the f i n a l w a s h ,  i n 10 m g /  50 mM T r i c i n e ,  volumes o f  ribulose-1  inhibitors 0.1  layers  in tap  6,000 x g f o r t e n s e c o n d s .  at  10 mM s o d i u m p y r o p h o s p h a t e  kO mM e - a m i n o - n - c a p r o i c  four  was r e s u s p e n d e d  c a r b o x l y a s e was removed by s t i r r i n g weight  s c i s s o r s , , and b l e n d e d  pH 8.0) s i n a c h i l l e d  by c e n t r i f u g i n g  peMet  r e m o v e d , washed  (0.3 M m a n n i t o l ,  I s o l a t i o n i .Medium  1 mM 8 - m e r c a p t o e t h a n o l ,  chloroplasts crude  o f young wheat  di-HCl, for a  10,000 x g  c o n c e n t r a t i o n was  14  S u b s e q u e n t work was p e r f o r m e d a t room t e m p e r a t u r e . extraction (1977).  was p e r f o r m e d  plus  v i a a m o d i f i c a t i o n o f t h e method o f Y o u n i s e t a l . s u s p e n d e d a t 2.0 t o 2 . 5 mg c h l o r o p h y 1 1 / m l  T h e t h y l a k o i d s were  0.25 M sucrose,  in  proteolysis  10 mM T r i s / S O ^  inhibitors.  was a d d e d and t h e m i x t u r e  pH 7 - 6 ,  To this  was e m u l s i f i e d  f o r 15 s e c o n d s .  and  t h e a q u e o u s p h a s e was r e c e n t r i f u g e d  Molecular To sucrose  Separator.  gradient  This  by  a  the crude  Sucrose  refTactometer.  (1976).  Bradford  determined  content  1  In certain in  to  inhibitors)  1 mM E D T A , a t 26,000  and c e n t r i f u g e d  The g r a d i e n t  of gradient  thus  fractions  was f r a c t i o n a t e d fractions  was d e t e r m i n e d  using  by t h e d y e - b i n d i n g method o f  p r e p a r e d was g r e a t e r  in a s i m i l a r  Medium t o f r e s h w e i g h t  various experiments,  than  98% p u r e , a s  and f i g u r e s  proteolysis  as they  that  the r a t i o o f  inhibitors above.  were o m i t t e d  at  These changes a r e noted  occur.  s e c o n d method f o r t h e p r e p a r a t i o n  the procedure o f Strotmann  way e x c e p t  o f y o u n g l e a v e s was 3 : 1 .  stages o f the procedure o u t l i n e d  purposes.  pH 7 - 6 ,  by S D S - P A G E .  the text A  I m m e r s i b l e CX  s a m p l e was l o a d e d o n t o a 37 ml  P r o t e i n was d e t e r m i n e d The CF  The  t h e bottom and c o l l e c t i n g 30-drop  into  stir  centrifugation  a t 48,000 x g 30 m i n .  f o r 28 h o u r s .  S p i n a c h CF^ was p r e p a r e d Isolation  with a magnetic  i n 10 mM T r i s / S O ^  proteolysis  i n a Beckman SW27 r o t o r  the t o p .  by s t i r r i n g  is designated "crude CF^".  (8 t o 25% s u c r o s e  pumping d e n s e r s u c r o s e  off  i volume o f c h l o r o f o r m  t o 1 t o 3 ml w i t h a M i l l i p o r e  sample  CF^,  purify  2 mM A T P , 5 mM D T T , p l u s rpm  2 mM A T P , 5 mM DTT  T h e e m u l s i o n was b r o k e n by low s p e e d  was c o n c e n t r a t e d  further  1 mM E D T A ,  suspension,  bar  supernatant  Chloroform  o f a c r u d e CF^ e x t r a c t ,  according  e t a l . (1973) was u s e d f o r c o m p a r a t i v e  T h y l a k o i d s w e r e washed f o u r  times  in c o l d  10 mM s o d i u m  15  pyrophosphate  pH 7 - 4  by w a s h i n g  a hypotonic  pH 7 - 8 )  in  containing  purified  to  without  proteolysis  sucrose solution  no p r o t e o l y s i s  homogeneity  inhibitors. (0.3  inhibitors.  by s u c r o s e d e n s i t y  CF^ was  M sucrose, This  crude  gradient  then  released  2 mM T r i c i n e - K O H CF^ e x t r a c t  centrifugation  was  as  above.  according  to  the  contained  10%  (w/v)  0.1%  (w/v)  Electrophoresi s S D S - P o l y a c r y 1 a m i d e g e l e l e c t r o p h o r e s i s was method  of  Kirchanski  acrylamide, SDS,  0.47  nent gel  0.27%  (v/v)  and P a r k  (w/v)  contained  pH 6 . 1 ,  0.1%  fate.  The SDS.  (v/v)  glycerol,  Slab gels  r o u t i n e i y run a t At  the  30 m i n u t e s 7% ( v / v )  acetic  acid,  7%  (v/v)  acetic  acid.  to  s p e e d up t h e Molecular  by  the  kinase  s l a b f o r four to s i x  0.2%  A tuft  of  M Tris-HC1 persul-  T r i s and  pH 6 . 8 ,  g-mercaptoethanol  weight  (mw 57 k d ) ,  analysis  Webber  polypeptide  and O s b o r n e  ovalbumin  A (mw 1 3 - 7  of  10% or  kd).  (1969),  for  50%  (v/v)  methanol,  20%  (v/v)  methanol,  was a d d e d d u r i n g  (mw 45 k d ) ,  u s i n g as  10  temperature.  s l a b s were s t a i n e d  bands  0.1  E l e c t r o p h o r e s i s was  changes o f  u n b l e a c h e d wool  M  compo-  stacking  ammonium  0.025  C o o m a s s i e B l u e R,  and d e s t a i n e d i n s e v e r a l  0.1  h o u r s a t room  polyacrylamide  (w/v)  The  65mM T r i s - H C 1  (v/v)  last  destaining  process.  method o f  ribonuclease  of  in  were used t h r o u g h o u t .  electrophoresis,  in a s o l u t i o n  (w/v)  M glycine,  0.2%  This  bisacrylamide,  and 0 . 0 2 5 %  0.192  S D S , and e i t h e r  (2 mM t h i c k )  pH 9 - 8 ,  polymerization.  (w/v)  TEMED,  consisted of  15 t o 20 mA p e r  end o f  initiate  samples were d i s s o c i a t e d  2% (w/v)  gel  ammonium p e r s u l f a t e .  0.14%  (v/v)  resolving  1.1347 M T r i s - H C I ,  to  acrylamide,  buffer  Protein  (w/v)  de-aeration,  S D S , 0.16%  running  (w/v)  mM D T T .  and 0.1%  5% (w/v) (w/v)  The  bisacrylamide,  TEMED,  was a d d e d a f t e r  (1976).  performed  in  SDS-PAGE was  standards  carbonic anhydrase  done  pyruvate  (mw 30 kd)  and  .16  In-gel of  l i m i t e d p r o t e o l y s i s was p e r f o r m e d  Cleveland  visualized where for  e t a l . (1977).  ing  bands w e r e v i s i b l e .  1mM E D T A ) .  in  in  with a four  (no EDTA) was made.  aureus  buffer. V8 p r o t e a s e  was o v e r l a i d .  t h e s a m p l e and t r a c k i n g d y e had s t a c k e d  The c u r r e n t  was t u r n e d  o f f f o r h0 m i n u t e s  The c u r r e n t  was t u r n e d  b a c k on a t t h e end  p e r i o d and e l e c t r o p h o r e s i s  t h e same a s f o r o t h e r  in soaking  b l u e and s o a k i n g b u f f e r  o f 15 mA was r u n u n t i l  to occur.  but c o n t a i n -  I n t o e a c h s l o t on t h e s l a b was p l a c e d  p r o c e e d e d a t 20 mA u n t i l  dye had r u n o f f t h e end o f t h e 15% g e l . done  pH 6.8, 0 . 1 %  cm l o n g s t a c k i n g g e l c o n t a i n i n g 3%  s o l u t i o n o f Staphylococcus  t h e m i d d l e o f t h e 3% g e l .  this  (0.125 M T r i s - H C 1  band a n d 10 u l o f 20% g l y c e r o l  10 u l o f 50 ug/ml  to a l l o w d i g e s t i o n of  to the point  These were e x c i s e d w i t h a r a z o r b l a d e and soaked  10% g l y c e r o l , 0 . 1 % B r o m p h e n o l  A current  by SDS-PAGE w e r e  A 15% SDS-PAGE s l a b g e l ( a s d e s c r i b e d a b o v e ,  the e x c i s e d soaked Over t h i s ,  separated  then d e s t a i n i n g  i n 10 ml o f s o a k i n g b u f f e r  1 mM EDTA i n a d d i t i o n )  acrylamide  bands  by s t a i n i n g f o r f i v e m i n u t e s ,  30 m i n u t e s  SDS,  Protein  a c c o r d i n g t o t h e method  Staining  the tracking  and d e s t a i n i n g  were  SDS g e l s .  ASSAYS The  latent  C a A T P a s e o f CF^ was a c t i v a t e d  a s s a y e d a c c o r d i n g t o t h e method a sample c o n t a i n i n g 0 . 0 2 ml EDTA  ( 0 . 2 M pH 7-6 s t o c k ) ,  o f a 5 mg/ml  eded of  0 . 0 5 ml s o y b e a n  (1971b).  0 . 0 3 ml T r i c i n e - K O H  t o 0 . 9 3 ml w i t h  stock prepared  f o r 6 minutes  and Racker  d i g e s t i o n and Briefly,  50 ug p r o t e i n was added 0 . 0 1 ml ATP ( 0 . 1 M pH 7  and t h e v o l u m e was b r o u g h t ml  of Lien  by t r y p s i n  stock),  (1 M pH 8 s t o c k ) ,  water.  Trypsin  i n 1 mM H^SO^) was a d d e d and d i g e s t i o n  a t room t e m p e r a t u r e . trypsin  distilled  to  inhibitor  (0.02 proce-  D i g e s t i o n was h a l t e d by t h e a d d i t i o n  (5 mg/ml  i n 5mM T r i c i n e - K O H pH 7 ) .  17  Aliquots  o f t h e a c t i v a t e d enzyme c o n t a i n i n g 2 . 5 ug CF^ w e r e  ) t o 0 . 2 ml w i t h 2 0 mM T r i c i n e - K O H  pH 8.  T o t h i s was a d d e d 0.8 ml o f  s u b s t r a t e m i x c o n t a i n i n g kO mM T r i c i n e - K O H CaCI^.  After  a 10 m i n u t e  zero time  (Chen e t a l . ,  was o m i t t e d  determination  o f K^ and V m a x >  Prior  1 mM EDTA, The  protein (O.t  against  heat  activated  t o t h e method o f L i e n  containing  t o assay ATPase a c t i v i t y  at  for very  trypsin,  proteolysis  inhibitors  t w o c h a n g e s o f 10 mM T r i s - S O ^  pH l.k,  EDTA b u f f e r  C a A T P a s e o f CF^ was a c t i v a t e d and R a c k e r  (1971b).  minutes,  and then  K0H pH 8 . 0 , 2 mM EDTA)  A T P a s e a c t i v i t y was a s s a y e d a s d e s c r i b e d  by s u b s t i t u t i n g  To a 0 . 2 ml a l i q u o t  (1 M pH 8) was a d d e d .  bath f o r four  ( 4 0 mM T r i c i n e  Nucleotide  and a s s a y e d of  150 t o 2 0 0 ug C F 1 , 0 . 0 5 ml ATP ( 0 . 2 M pH7) , 0 . 0 1 5 ml DTT  i n a 60°C water  added.  in experiments  1 mM A T P , 5 mM DDT.  M) a n d 0 . 0 3 ml T r i c i n e - K O H  heated  a n d a c o r r e c t i o n was made  substrate.  by d i a l y s i s  latent,  according  activity,  enzyme.  in order  t o enzyme a c t i v a t i o n w i t h  removed  for  In a l l a s s a y s f o r A T P a s e  from t h e t r y p s i n d i g e s t i o n s t e p  low c o n c e n t r a t i o n s o f  were  1956).  of the unactivated  t h e r e a c t i o n was h a l t e d  Samples were a n a l y z e d  i n o r g a n i c p h o s p h a t e was d e t e r m i n e d ,  f o r ATPase a c t i v i t y ATP  pH 8, 1 mM A T P , and 10 mM  i n c u b a t i o n a t 37°C,  by t h e a d d i t i o n o f 1 ml 0 . 5 M c o l d T C A . i n o r g a n i c phosphate  brought  T h e m i x t u r e was 1.7  ml  Tricine-KOH-  a t room t e m p e r a t u r e was above.  s p e c i f i c i t y o f t h e t r y p s i n - a c t i v a t e d A T P a s e was a s s a y e d 1 mM o f t h e v a r i o u s  substrates  f o r A T P , and a c t i v a t i n g  in  the absence of ATP. For  determination  o f Ku and V M  enzyme r e a c t i o n was d e t e r m i n e d i n t e r v a l s over  a three minute  .  graphically, max  r  the v e l o c i t y of the  '  by s a m p l i n g a r e a c t i o n m i x t u r e reaction.  V e l o c i t y was p l o t t e d  ' a t one m i n u t e against the  18  c o n c e n t r a t i o n o f Ca  2+  ATP c o m p l e x ,  rather  than  the c o n c e n t r a t i o n of  ATP.  2+ The  c o n c e n t r a t i o n o f Ca  of Ahlers  et a l .  [Ca2+ATP]  ATP was d e t e r m i n e d  using the f o l l o w i n g  equation  (1975) :  =  [Ca]t  [Ca]t  fATP]t  +  [ATP] .  +  t  +  K  ^  ^  KCaATp  +  l  ^1  +  +  J£J  j  [ H ^ [Ca]t  [ATP^  (1) with  K  C a A  - j - p = 0.725 mM ( N a n n i n g a ,  1957; o b t a i n e d f r o m S i l l e n and M a r t e l 1 ,  1964) and K, = 3 x 10" ( S i l l e n and M a r t e l 1 , 1964). [ c a ] 7  refer  t0 t h e t o t a l  Electron  c o n c e n t r a t i o n of each o f these  mesh e l e c t r o n m i c r o s c o p y g r i d s ,  overnight  negative  staining.  determined Bragg,  protein/ml seconds,  components.  against  2 mM T r i s - H C l  Samples o f wheat  by SDS-PAGE)  o r Escherichia  UBC D e p a r t m e n t were mixed w i t h  and s p r a y e d o n t o  ammonium m o l y b d a t e a c i d - K O H pH 7.0.  CF^  an e q u a l  CF^ s a m p l e s  F^  provided  coli  (kindly  w i t h about  volume o f n e g a t i v e  (1950).  0.1  by were to  by D r .  t o 0.2 mg  s t a i n f o r 30  the spray  Negative  200  salts prior  t h a n SS% p u r i t y a s  the carbon f i l m w i t h  acetate  seconds.  (greater  i n 12 mM s o d i u m o x a l a t e Uranyl  collodion-coated  pH 7-5 t o remove  of Biochemistry)  d e s c r i b e d by B a c k u s a n d W i l l i a m s  onto  and t h e p l a s t i c b a c k i n g was removed  d i p p i n g the cfrid i n t o acetone f o r f i v e  dialyzed  P.D.  [H+]t  Microscopy  A l i g h t c o a t i n g o f c a r b o n was e v a p o r a t e d  gently  [ATP]t.and  t >  gun a p p a r a t u s  s t a i n s u s e d w e r e 2%  pH 7.2 o r 2% p h o s p h o t u n g s t i c  (2% s o l u t i o n , pH 4.3) was n o t a s a t i s f a c t o r y  19  sta i n. Negative  s t a i n e d CF^  and ECF^  were examined  e l e c t r o n m i c r o s c o p e , at m a g n i f i c a t i o n s of voltage prints  setting of-80*kV. and t h e a c t u a l  magnification calibration of  the  of  of  the  the  instrument  s i z e of print  EM-10  al.  Preparation  the  of  hind  of  Adjuvant  a New Z e a l a n d  white  i n j e c t i o n s were g i v e n  titer  Ouchterlony-(1968). t h e method o f purified  (a)  buffer  A: (b)  deoxycholate agarose  the m a g n i f i c a t i o n s  used,  were w i t h i n  2.2%  Monospecific Antibodies  Complete  After  0.8  to  of  was u s e d .  90 mM T r i s 20% ( v / v )  in buffer  A.  (0.1%  i n an e q u a l  later  the  into  intervals,  second  rabbit  bled  from  sodium a z i d e )  was  the  blood  was a d d e d  by t h e  d o u b l e d i f f u s i o n method  i m m u n o e l e c t r o p h o r e s i s was p e r f o r m e d  The  (1979)  water; The  except  following  pH 8 . 6 w i t h  Lubrol  in d i s t i l l e d  t e n day  CF^  to  all  4°C.  was d e t e r m i n e d Crossed  At  t o Wheat  intramuscularly  s e r u m was s e p a r a t e d  Preservative  stored at  injected  and two weeks  Chua and B l o m b e r g  IgG,  and  was e m u l s i f i e d  rabbit.  clotting,  by c e n t r i f u g a t i o n .  1 mM EDTA;  of  Freund's  than  the  e l e c t r o n m i c r o g r a p h s was done by t h e method  t h a n 98% p u r e )  Antibody  (w/v)  that  (greater  s e r a and t h e y w e r e  to  gave m a g n i f i c a t i o n s  CF.|  from the ear v e i n . cells  For  a  photographic  p a r t i c l e was c a l c u l a t e d f r o m  and t h e n e g a t i v e .  and C h a r a c t e r i z a t i o n  leg of  and t h i r d  with  (1963).  200 ug o f volume  40,000 X or g r e a t e r ,  d i a m e t e r s w e r e m e a s u r e d on the  EM-10  settings.  Image e n h a n c e m e n t Markham e t  CF^  using a Zeiss  PX (d)  that  in d i s t i l l e d 50% PEG  g e l was c a s t  hO mM s o d i u m  water;  (w/v)  (c)  10%  in buffer  between  according  whole serum,  s t o c k s o l u t i o n s were  acetic acid,  of  A;  rather  prepared: acetate,  (w/v) (e)  two g l a s s p l a t e s  sodium 1.2% with  20  3 mm t h i c k s p a c e r b a r s on t h e together  in a v e r t i c a l  were c a s t  from bottom  position during to  top  was d i s s o l v e d by h e a t i n g were kept prepared  s i d e s and b o t t o m .  as diagrammed  bath p r i o r  by m i x i n g 2 . 9 ml o f  was t h e n c a s t by p o u r i n g  careful  not  l a y e r s were 1.3  ml o f  1 ml o f cast  p o u r e d on t o p (d)  antiserum that  from e x t r a  anodal  18.7  (c)  b a r was removed cathodal The  The  (e),  and J - C l o t h ends o f  flatbed  hOO ml o f  100 V f o r  ml o f  18 t o 20 h .  Immunoprecipitates  cathodal  (Johnson the gel  The  short  water,  flask.  The  plates,  being  successive  the antibody  unfixed  and J o h n s o n )  1 ml  gel.  (e)  The  bottom  as a  The  s t a r t i n g c u r r e n t was a b o u t  20 mA.  agarose g e l s ,  Coomassie Blue  in d i s t i l l e d  water  of  with  voltage  i n b o t h d o u b l e d i f f u s i o n p l a t e s and c r o s s e d  one d a y and  the  binder.  r e s e r v i o r was f i l l e d a constant  for  spacer  supports  E l e c t r o p h o r e s i s was r u n a t  M NaCl  inter-  solution  fused to  across the  SDS-  the  of  to  was  lane from a  w i c k s were  by s t a i n i n g w i t h  gel,  and added  and p l a c e d a b o v e  t h e SDS  (e).  intermediate gel  g e l , composed o f  Each  was  solution  A.  the f r a g i l i t y of  shaking.  gel  were m i x e d ,  placed h o r i z o n t a l l y  in 0.15  without  (e)  u s i n g warm s o l u t i o n  soaking  Due t o  the  To p r e p a r e  was c a s t a r o u n d  g e l s were v i s u a l i z e d  days.  anodal  ml o f  (e)  solutions  l a y e r had s o l i d i f i e d ,  An u n s t a i n e d ,  electrophoresis the gel  The  layers  Solution  and a l l  between  solution  e l e c t r o p h o r e s i s chamber. buffer  1.  35-1  same m a n n e r .  in d i s t i l l e d  c o m p o s i t e g e l was t h e n  a Gelman about  solution  and a n o d a l  this  gel mixture.  as diagrammed.  and 9 ml o f  with  had been p r e w a r m e d .  PAGE g e l was e x c i s e d , r i n s e d mediate gel  (b)  was c l a m p e d  individual  s w i r l i n g i n a pre-warmed  After  in the  and  bath,  t h e warm m i x t u r e  t o make b u b b l e s .  solution  in f i g .  to c a s t i n g .  solution  M i x i n g was a c c o m p l i s h e d by g e n t l e gel  c a s t i n g , and  in a b o i l i n g water  i n a 65 C w a t e r  The a p p a r a t u s  for  of  Immunoafter two  s t a i n i n g and d e s t a i n i n g w e r e  done  21  Figure  1:  Diagram of Materials  crossed Immunoelectrophoresis and  Methods.  g e l , as d e s c r i b e d  in  21a  Cathodal Gel SDS  Gel  Intermediate Gel  T 1  25  8  T  mm  mm  Antibody Gel  50  mm  Anodal G e l  25  mm  A  22  RESULTS Purification  o f Wheat CF.^  A s a t i s f a c t o r y method was d e v e l o p e d wheat of  for obtaining  homogeneous  CF.j, using a m o d i f i c a t i o n of the chloroform e x t r a c t i o n  Youni s e t al.(1977).  envelope  and s t r o m a l  emulsifying purified  except  the i n i t i a l gave  was r e l e a s e d  suspension  i n h i b i t o r s were  g r i n d i n g medium.  the p r o f i l e  stripped of the c h l o r o p l a s t  i n t o t h e a q u e o u s medium by  in chloroform.  t h a n 38% h o m o g e n e i t y  Proteolysis  gradient  from t h y l a k o i d s  proteins,  the thylakoid  to greater  gradient.  CF1,  The e x t r a c t was  on a l i n e a r 8 t o 25% s u c r o s e  included  in a l l solutions  Fractionation  illustrated  method  of a t y p i c a l  i n f i g . 2.  sucrose  CF^ b a n d s a t 20 t o  21% s u c r o s e . A n a l y s i s o f the major SDS-PAGE r e v e a l e d The  that  smaller protein  polypeptides  protein  a l l five  peak  in the sucrose gradient  s u b u n i t s o f CF1 were p r e s e n t  peak a t t h e b o t t o m o f  corresponding  in apparent  and s m a l l  subunits of ribulose-1,5"bisphosphate  the major  protein  in the c h l o r o p l a s t stroma  so t h a t  further  p u r i f i c a t i o n by s u c r o s e g r a d i e n t a 1.5  Results of a typical  to 2-fold  experiment  (EC  ( l a n e 6, f i g . 3).  i n l a n e 1 o f f i g . 3, t h e " c r u d e C F ^ " i s v e r y  in only  to the  carboxylase  be s e e n  resulted  ( f i g . 3).  the sucrose gradient  molecular weight  nearly  contains large 4.1.1.39), As c a n  homogeneous,  centrifugation  i n c r e a s e i n CaATPase a c t i v i t y are given  by  in Table  I.  at  best.  23  Figure 2:  P r o f i l e of CF.| .  sucrose density  gradient  A crude chloroform extract  c o n t a i n i n g 4 . 9 mg p r o t e i n was (8 t o 25% s u c r o s e i n b u f f e r inhibitors)  with  was c e n t r i f u g e d The g r a d i e n t  activated  of wheat  26,000  protein  wheat  thylakoids gradient  proteolysis  a 50% s u c r o s e c u s h i o n , and 28 h a t  purify  l o a d e d o n t o a 34 ml  containing  was f r a c t i o n a t e d  Sucrose content,  used t o  the  gradient  rpm i n a Beckman SW 27 i n t o 30 d r o p  c o n c e n t r a t i o n and  aliquots. trypsin-  ATPase a c t i v i t y were a s s a y e d as d e s c r i b e d  i n M a t e r i a l s and  Methods.  rotor.  •  n  • % SUCROSE  • ACTIVITY (units-mg protein" ) 1  2k  F i g u r e 3'  SDS-PAGE o f  c r u d e CF^  a sucrose density F i g u r e 2.  L a n e 1,  19 ug o f  gradient  fraction  17;  18 ug o f  gradient  fraction  23.  subunits of  s a m p l e and p r o t e i n  gradient  s i m i l a r to  fractions  t h e o n e shown  20 ug c r u d e c h l o r o f o r m e x t r a c t ; fraction  16;  l a n e k, 33 ug o f fraction  19;  LS and SS r e f e r  l a n e 3, 35 ug o f gradient  the  fraction  ribulose-1,5""bisphosphate  l a r g e and  in lane  2,  gradient 18;  l a n e 6, 9.6 ug o f to  from  lane  gradient  small  carboxylase.  5,  25  Table  I.  Sample  P u r i f i c a t i o n o f wheat  Cone. Protein, mg/ml  Total Volume, ml  Crude CF1  2.96  4.6  Sucrose gradient peak  0.69  1.3  "units/mg  wheat  CF.j  It  was f o u n d  0.90  Specific Activity*  Total Activity, units  Degree of Purification  71.4  5.42  10.42  1  9.4  to  by t h e same p r o c e d u r e f o r  1.99  somewhat  (Table  comparative  h a v e t h e same s e d i m e n t a t i o n v e l o c i t y  in sucrose g r a d i e n t s , the apparent  II.  13.62  was p r e p a r e d  although  Table  Total Protein, mg  protein  S p i n a c h CF^ purposes.  CaATPase  and t h e same p o l y p e p t i d e  molecular weights of  the s u b u n i t s  as  composition, differ  II).  A p p a r e n t m o l e c u l a r w e i g h t s o f s u b u n i t s o f w h e a t and s p i n a c h CF.j , a s d e t e r m i n e d by t h e method o f Webber and O s b o r n e (1969).  Subunit  Apparent Wheat  CFt  molecular weight Spinach  a  57  58  6  55  54  Y  37-39  36-37  <5  25  19  e  13  13  (kd) CF1  26  The  E f f e c t of In  P r o t e o l ys i s  initial  CF^  I n h i b i t o r s on E x t r a c t i o n o f  extractions,  proteolysis  CF^  r e l e a s e and p u r i f i c a t i o n .  manner  the  three  i n h i b i t o r s were added purification stage  the  of  wheat CF^,  presence of  preservation  to a l l  of  the  e-amino-n-caproic  (fig.  inhibitors inhibitors, (fig.  It  5).  appears  these minor  subunits  temperature  density  In a d d i t i o n methods  b u t when  for  peak  s u c r o s e method  (Strotmann et  of  medium c o m p l e t e l y of  CF.j  with  hypotonic  more p o l y p e p t i d e s fig.  k,  s p e c i f i c method  thylakoid,  for  prevent  lane D). CF,  the  proteolysis  loss  the prolonged  CF^  with  1973) to  the  release  as m e n t i o n e d extracted t h e method  hypotonic  (fig.  of room  extraction  than  thus  in  by t h e  the hypotonic  described  sucrose  inhibitors  r e l e a s e d by c h l o r o f o r m Chloroform  other  treatment  method.  Extraction  r e l e a s e d many (compare  seems t o be a much  the s u c r o s e  here.  extraction  l a n e s C and D ) .  h,  s u c r o s e minus p r o t e o l y s i s  than are  lane A with  a l . ,  inhibitors  prevented  The  subunits  there are several  was d e c i d e d t o c o m p a r e CF^  proteolysis  kO mM  centrifugation.  from the  It  by  proteolysis  l a c k e d t h e <5 and e  to c h l o r o f o r m e x t r a c t i o n ,  r e l e a s i n g CF^  what  the  without  also without  inhibitors  at  composition  prepared  f r o m t h e enzyme c o m p l e x d u r i n g  Introduction.  Addition  t h e c r u d e CF^  proteolysis  gradient  for  test  was p r e p a r e d  in polypeptide  in the g r a d i e n t  that  To  6 mM p - a m i n o b e n z a m i d i n e ,  identical  this  and  mM p h e n y l m e t h y l s u l f o n y l f 1 u o r i d e .  were  in  proteolysis  is c r i t i c a l  w h e a t CF^  was p u r i f i e d on a s u c r o s e g r a d i e n t t h e CF^  When  included  extracted  in the e x t r a c t i o n  inhibitors  enzyme c o m p l e x ,  a c i d and 0.1  CF^  subunits c o - p u r i f i e d .  w i t h and w i t h o u t  l a n e s A and B ) ,  k,  five  proteolysis  crude chloroform e x t r a c t s  subunits.  s o l u t i o n s used  all  intact  chloroform extraction  largest  CF^  i n h i b i t o r s were not  in s o l u t i o n s used f o r contained only  Wheat  more  in  27  F i g u r e k:  Comparison of method w i t h  c r u d e CF^  prepared  by  the  chloroform  t h a t p r e p a r e d by t h e h y p o t o n i c  and t h e e f f e c t  of  the  i n c l u s i o n of  proteolysis  upon e a c h .  A.  Crude c h l o r o f o r m e x t r a c t ,  presence of  all  proteolysis  B.  inhibitors.  Crude c h l o r o f o r m e x t r a c t ,  inhibitors  were o m i t t e d  from both  Hypotonic  s u c r o s e wash  i n h i b i t o r s were seen  in t h i s  6.25  ug p r o t e i n .  prepared 73 ug  included.  The  l a n e was w a s h e d o v e r D.  Hypotonic  in the absence of  protein.  medium.  small  inhibitors  25 ug  in  the  protein.  proteolysis  the sodium  in which  method,  prepared  for which a l l  w a s h and t h e c h l o r o f o r m e x t r a c t i o n C.  sucrose  pyrophosphate 25 ug  protein.  proteolysis amount o f  a and B  from the adjacent  s u c r o s e wash o f  proteolysis  lane.  thylakoids,  inhibitors.  27a  28  F i g u r e 5-  Effect CF.j  of  the  i n c l u s i o n of and  purification  w h e a t CF^.  A.  CF^  and p u r i f i e d ,  all  of  extraction  proteolysis  extracted  in the CF^  upon by  presence of  inhibitors.  B.  extracted  and p u r i f i e d  in the absence of  inhibitors  subunit the  during  composition  chloroform  method  proteolysis  by t h e c h l o r o f o r m proteolysis  method  inhibitors.  28a  29  The  prevention  inhibitors  o f CF^  r e l e a s e by h y p o t o n i c  a r e included r a i s e s the q u e s t i o n of whether  event  is required  event  h a s been  f o r CF^  reported  r e l e a s e by t h i s m e t h o d .  f o r ECF^  B r a g g a n d Hou (1979) c o m p a r e d of  proteolysis  in  the absence of p r o t e o l y s i s  6 subunit  ECF.^  inhibitors with  that  released  by a l o w i o n i c s t r e n g t h w a s h .  with  low i o n i c s t r e n g t h To  CF.j  is required  test  whether  compared. comparison.  chloroform method,  They proposed  case,  washes,  p u r i f i e d CF^ w e r e c o m p a r e d  or in the e x t r a c t i o n  i n one o f t h e  o f CF^  sucrose  release  Thus  thylakoids  inhibitors.  were  inhibitors.  by t h e  the  hypotonic  p u r i f i e d on s u c r o s e  When t h e two s a m p l e s  ( f i g . 6 ) , no d i f f e r e n c e  i s seems t h a t  this  In t h e  in either  o f CF^ w i t h  thus o b t a i n e d were  s u c r o s e does not i n v o l v e  these  two methods  i n h i b i t o r s were p r e s e n t ,  i n any o f t h e s u b u n i t s was e v i d e n t ,  although  of  membranes  r e l e a s e by h y p o t o n i c  in the presence of p r o t e o l y s i s  containing a l l proteolysis  by h y p o t o n i c  cleavage  CF^ was r e l e a s e d f r o m  c r u d e CF^ e x t r a c t s  in both.  proteolysis  f r o m t h e same b a t c h o f w h e a t p l a n t s w e r e u s e d f o r  gradients  preserved  that  ECF^  buffer.  Both  weight  larger  than  :  sucrose.  of  buffer  the chloroform-  f o r t h e r e l e a s e o f A T P a s e f r o m E. coli  no p r o t e o l y s i s  sodium pyrophosphate  in the presence  t o a lesser extent  f o r t h e i n h i b i t i o n o f CF^  In t h e f i r s t  second method,  membranes  inhibitors are included,  Thylakoids  proteolytic  a slightly  that  the i n h i b i t i o n of a p r o t e o l y t i c  subunits accounts  when p r o t e o l y s i s  They found  and showed  ECF.J  6 subunit  Such a  r e l e a s e d by l o w i o n i c s t r e n g t h  inhibitors.  t o E .coli  proteolytic  r e l e a s e d by c h l o r o f o r m  i s r e l e a s e d by c h l o r o f o r m , rebinds  a  proteolysis  (Cox e t a l . , 1978, B r a g g and H o u , 1979).  released  the  s u c r o s e when  in apparent  molecular  and t h e 6 and e s u b u n i t s  t h e r e l e a s e o f CF^ any d e t e c t a b l e  r e s u l t s do n o t r u l e o u t a p r o t e o l y t i c  from  the  were thylakoid  proteolysis of event  a t t h e CF  CF^,  30  Figure 6:  Proteolysis  inhibitors  present  gradient  centrifugation  prevent  subunits  of  A.  without  wheat CF^.  proteolysis  pyrophosphate crude  CF^  extract  sucrose containing purified  prepared  in the  and p u r i f i e d proteolysis  B.  of  all  kk ug  prepared  in  inhibitors.  and a hypotonic CF^  was  centrifugation  proteolysis  all  6 and e  sodium  by w a s h i n g  extracted  inhibitors. by  proteolysis  on a s u c r o s e d e n s i t y inhibitors,  were in  density  the  inhibitors,  gradient  Wheat CF^  presence of  loss  washed  no p r o t e o l y s i s  containing  62 ug p r o t e i n .  the  proteolysis  by s u c r o s e d e n s i t y  in a gradient  method  was  sucrose  Thylakoids  inhibitors,  without  during  gradient  protein.  the  chloroform  inhibitors containing  all  30a  MIGRATION  31  attachment strength,  s i t e on C F ^ .  i s t h e r e a s o n why CF^  proteolysis loss  inhibitors with  contains  the hypotonic  t r a c e s of protease  In m a n y ,  o f Wheat  Contains  in apparent  mitochondrial  F^.  run a t  However,  18 C f o r 28 h o u r s  enzyme p r e p a r e d by b o t h  an I n t e r n a l  mw was s e e n  a p a i r o f bands o f  (fig.7)  and G a l l a g h e r  enzyme,  the authors  found  that  i s a c t u a l l y a p r o t e o l y t i c fragment  F^  preparation  in small  t o a l l s o l u t i o n used t o p r e p a r e wheat  p o l y p e p t i d e s were s t i l l  (1977).  W i t h Staphylococcus  proteolysis  fragments  showed no c l e a r the dimer. subjected  To  proteolysis  CF^.  product  test  In  Both order  o f a o r 3,  a c c o r d i n g t o t h e method o f C l e v e l a n d e t a 1.  aureus  V8 p r o t e a s e ,  of the y doublet with  similarities  of the  SDS-activated  CF^,  p r e s e n t on SDS-PAGE.  t o d e t e r m i n e w h e t h e r o n e o f t h e bands was a b r e a k d o w n p e p t i d e a n a l y s i s was p e r f o r m e d  thought  quantities.  p r o t e o l y s i s o c c u r s d u r i n g t h e p r e p a r a t i o n o f wheat  the y subunit  subunit  a kO kd  P r o t e o l y s i s o f t h e a s u b u n i t was d u e t o a l a t e n t ,  protease contaminating t h e i r  about  (1979) i n t h e y e a s t  a subunit.  of  methods  I t was f i r s t  p u r i f y i n g w i t h F^  i n h i b i t o r s w e r e added  without  Bridge  polypeptide  whether  the  be t h e r e s u l t o f p r o t e o l y s i s o f t h e  by R y r i e  With that  Disulfide  in the region of the y  upon SDS-PAGE  o n e o f t h e s e t w o bands m i g h t as found  sucrose method.  preparations,  and s p i n a c h CF^  a or 3 s u b u n i t ,  s u c h as o s m o t i c  activity.  b u t n o t a l l CF^  2 kd d i f f e r e n c e b o t h wheat  CF^  factor,  i s not r e l e a s e d in the presence of  i n h i b i t o r s does suggest t h a t  The y S u b u n i t  that  some o t h e r  o f 8 and e i n t h e s u c r o s e g r a d i e n t s  proteolysis  of  I t may be t h a t  between  a comparison of the l i m i t e d  t h o s e o f t h e a and 3 s u b u n i t s  any o f t h e two l a r g e s t s u b u n i t s and  H o w e v e r , when t h e two bands o f t h e d o u b l e t t o t h e same p r o c e d u r e ,  the r e s u l t i n g peptide  were  separately  fingerprint  shows  32  Figure  7-  SDS-PAGE o f wheat in  the y s u b u n i t  and s p i n a c h C F ^ , position.  i n 65 mM T r i s - H C 1 0-mercaptoethanol, dissociated glycerol  pH 6.8,  A.  S p i n a c h CF^,  2% S D S ,  50 ug p r o t e i n .  i n 65 mM T r i s - H C 1  and 2 . 5 mM DTT,  38 ug  showing a double  dissociated  10% g l y c e r o l and B.  pH 6.8,  Wheat  CF^,  2% S D S ,  10%  protein.  band  0.2%  33  that  the upper  and  lower y bands c o n t a i n  i d e n t i c a l fragments  t h e r e had been a 2 kd mw d i f f e r e n c e b e t w e e n shown up as a t strongly  l e a s t one d i f f e r e n t  suggests the  Electrophoresis dissociated glycerol, difference of  up t o t h i s  increasing that with  i n m o b i l i t i e s was due t o  amounts  of  i n c r e a s i n g amounts  the doublet  disulfide  bonds  polypeptide.  It  o b s e r v e d when CF^ Andreo et formed  in the  To  test  if  gives of  DTT  (fig.  of  the  10,  the  than  (1979)  showed t h a t  lower apparent of  samples  2% S D S ,  to determine  if  10% the  reduction  DTT.  This  F i g u r e 9 shows the y doublet  suggests that  a shorter effective  increase in apparent r a t i o of  s p i n a c h CF^  DTT  upon h e a t  an o x i d i z i n g  10).  CF^  the y subunit  independent  with  mw was to  protein.  agent,  activation  polypeptide of  heating,  of  of  o-iodosobenzoate. the  doublet for  in the presence  or  d i s s o c i a t e d i n 2% SDS w i t h o u t as t h a t  heated  i n SDS.  d u r i n g d i s s o c i a t i o n y i e l d s o n l y o n e band f o r  l a n e s b and c )  more  new d i s u l f i d e b r i d g e s c a n be  was d i s s o c i a t e d w i t h o u t  same two b a n d s f o r  lower  length  r e d u c t i o n was o b t a i n e d  molecular weight  (fig.  is  the  the h e a t i n g s t e p used t o d i s s o c i a t e p o l y p e p t i d e s CF^  This  t h e u p p e r one b e c a u s e o n e o r  complete  s i n c e no f u r t h e r  have  identical.  upon  pH 6 . 8 ,  l o w e r band o f  increased.  r e d u c e d , making  reducing agents  t o CF.J  is  in the p r e s e n c e of  electrophoresis, absence  band  DTT  3 and y s u b u n i t s o f  the  i s an a r t e f a c t  of  should  If  samples were d i s s o c i a t e d i n  was d i s s o c i a t e d i n a h i g h e r  a 1.  ATPase a c t i v i t y  In o r d e r  reducing agent,  was c o n c l u d e d t h a t  33 n m o l e s DTT/mg CF^  y p o s i t i o n are  i n c o m p l e t e d i s u l f i d e bond  migrates faster  are not  the  had been p e r f o r m e d  g-mercaptoethanol, a stronger  it  8).  limited proteolysis.  c o n t a i n i n g 65mM T r i s - H C 1  d e c r e a s e d w h i l e the upper band o f  point  p-mercaptoethanol.  the y subunit w i t h  in the  two p o l y p e p t i d e s a t  in a buffer  and 0 . 2 %  band  the two b a n d s ,  (fig.  heating  the  sample.  the y Thus  heat  Addition subunit it  3h  Figure 8:  Limited  p r o t e o l y s i s of  subunit  d i m e r and o f  of  purified  in  buffer  2% SDS  CF^  separated minutes,  according to  et  Methods.  A.  al .  pH 6 . 8 ,  and t h e  of  polypeptide  point  where  sample of  in-gel  pattern  the y dimer.  glycerol,  bands  were  to  limited  of  in M a t e r i a l s from  Pattern C.  t h e a and 8 s u b u n i t s .  were  five  generated B.  10%  for  method  as d e s c r i b e d  the y dimer. of  y  sample  subunits  g e l was s t a i n e d  the  the  Proteolytic  polypeptide  a combined  (1977)  the  hk ug p r o t e i n was d i s s o c i a t e d  The to  A  T h e s e w e r e e x c i s e d and s u b j e c t e d  Cleveland  lower  two b a n d s o f  8 subunits.  8-mercaptoethanol  and d e s t a i n e d  proteolysis  the  the  65 mM T r i s - H C 1  by S D S - P A G E .  visible.  upper  t h e a and  containing  containing  and 0 . 2 %  each of  and the  from  Pattern  from  34a  MIGRATION  >  35  F i g u r e 9:  Effect  of  r e d u c i n g a g e n t upon  Y subunit  o f w h e a t CF^  38 ug p r o t e i n . containing mg C F , .  C.  A.  11 n m o l e s  the m o b i l i t y  i n SDS-PAGE.  CF^ d i s s o c i a t e d DTT/mg C F ^ .  33 n m o l e s  DTT/mg  CF,.  B.  All  of  the  lanes  contain  i n a 2% SDS 15.4 n m o l e s  solution DTT/  35a  MIGRATION  >  36  Figure  10:  Effect  of  h e a t upon t h e  number o f  a.  CF.j  dissociated  i n 2% S D S ,  2.5  b.  CF1  dissociated  i n 2% S D S ,  12.5  mM DTT,  and  i n 2% S D S ,  12.5  mM DTT,  no  c.  CF.  contain  37.5  in the y  i n SDS-PAGE.  100  lanes  found  subunit  60 s e c a t  All  bands  mM DTT,  ug  no  protein.  heating. heated  C.  dissociated  heating.  36a  a  b  c  37  appears into  that  heat  the y subunit  one can s u g g e s t in  does not  this  o f wheat  that  subunit.  the s u b u n i t  ATPase A c t i v i t y  its  of  CF^.  there  The  with  artificially  two  Wheat  bridge  S i n c e two d i s c r e t e bands a r e  is at bands  internal  introduce a d i s u l f i d e  l e a s t one  internal  c o u l d be t h e  disulfide  completely  obtained, bridge  reduced  subunit  and  crosslink(s).  CF. ]  Spinach c h l o r o p l a s t coupling f a c t o r ,  the  best c h a r a c t e r i z e d of  the  2+ plant  CF^'s,  be a c t i v a t e d or  by h e a t  (Farron,  exhibits  by a s h o r t  treatment  1970).  whether Table  the  i s o l a t i o n of  the wheat  III  by h e a t i n g  gives at  initial  that  CF^  the  the  intact  has a h e a t  r e s u l t of  various  -dependent  digestion  presence of  was r o u t i n e l y  i t was d i s c o v e r e d  improved  Ca  trypsin  in the  In t h e  CaATPase a c t i v i t y when  a latent  wheat  (Vambutas  CF^  and R a c k e r ,  preparations  for  of  proteolysis  complex,  activated  an e x p e r i m e n t  DTT study,  but  inhibitors  i t was d e c i d e d t o  CaATPase a c t i v i t y to  can 1965)  this  by t r y p s i n i z a t i o n ,  inclusion  temperatures.  that  a low c o n c e n t r a t i o n o f  unmasked  CF^  ATPase a c t i v i t y  determine  also.  i n d u c e a CaATPase  activity  38  TABLE  III.  Heat a c t i v a t e d CaATPase a c t i v i t y o f wheat CF1 t r e a t e d a t v a r i o u s t e m p e r a t u r e s and a s s a y e d a c c o r d i n g t o t h e method o f L i e n and R a c k e r ( 1 9 7 1 b ) . 2+ Ca - d e p e n d e n t A T P a s e a c t i v i t y o f w h e a t CF^ a c t i v a t e d at the f o l l o w i n g temperatures (units/mg protein)  Length of time heated (minutes)  49°C  61°C  0  0  0  0.5  0.10  0  0  1  0.22  0  0  2  0.46  0  0  3  0.48  0  0  4  0.40  0  0  6  0.30  0  0  0  In c o m p a r i s o n ,  activation  units/mg  protein  activity  obtained  The  56°C  by t h e t r y p s i n method  of CaATPase a c t i v i t y . (0.48 units/mg  i n a b i l i t y t o heat  activate  s i n c e s p i n a c h CF^ a c t i v a t e d mg p r o t e i n decided  CaATPase a c t i v i t y .  t o remain w i t h  CaATPase of wheat Since solutions inhibitors  gives  heat-activated  c a n be c o n s i d e r e d  is a property  o f t h e wheat  negligble.  CF^  complex,  f o r 4 m i n a t 6 0 ° C showed  On t h e b a s i s o f t h e s e  t h e t r y p s i n method  results  for activating  7 t o 20  the  10 u n i t s /  i t was latent  CF1 .  proteolysis  inhibitors  included  i n t h e wheat  (p-aminobenzamidine , e - a m i n o - n - c a p r o i c of serine proteases,  d e t e r m i n e what e f f e c t activation  Thus t h e maximal  protein)  by h e a t i n g  routinely  including trypsin,  the presence  o f CaATPase a c t i v i t y .  acid  of t h e s e Figure  11  CF^  preparation  a n d PMSF) a r e s p e c i f i c  i t was n e c e s s a r y  i n h i b i t o r s might shows t h a t  have  a longer  to  upon t h e  trypsin  39  Figure  11:  Time c o u r s e of the  trypsin  activation  p r e s e n c e and a b s e n c e o f  #  CF.j  sample d i a l y z e d  before  trypsin  in  presence of  the  activation  0.H3  activation.  of  2.9  wheat CaATPase  proteolysis remove A  proteolysis  was done  concentration  to  of  inhibitors.  proteolysis  Trypsin  protein  mM e - a m i n o - n - c a p r o i c  mM p - a m i n o b e n z a m i d i n e  inhibitors  activation  inhibitors.  in a sample of  in  of  CF^  Trypsin with acid,  and 0 . 0 0 7 2 mM PMSF.  a  final  39a  TIME ACTIVATED (min)  ko  digestion  is required  proteolysis absence  of  inhibitors, to  the  the  activate  However,  tion mix,  reach maximal  i n h i b i t o r s than  was a d e q u a t e shown.  to  in t h e i r  depending  latent  upon  concentration of  out  proteolysis  inhibitors prior  the  s i x minutes  digestion. specificity  in Table  a c t i v i t y w i t h ATP nucleotide TTP  Wheat  IV.  presence of  In e i t h e r  the  digestion  routine  wheat  CF^  the n u c l e o t i d e  is thus  triphosphates  specificity  of  than  very  used experiment  the  routinely  enzyme a s s a y s ,  w i t h GTP,  activa-  and  remain  and t h e  results  with  purine CTP,  specific,  i n t o t h e enzyme a c t i v e  UTP  of  wheat  % of  activity  ATP  100  GTP  32  UTP  0  CTP  ].k  TTP  0  with  ATP  or  accepting  site(s),  ATP.  the CaATPase a c t i v i t y  with  triphosphatase  the other  substrate  dialyze  CF^.  L a t e n t A T P a s e a c t i v i t y was a c t i v a t e d by t r y p s i n d i g e s t i o n in the absence o f ATP. A l l s u b s t r a t e s w e r e 1mM n u c l e o t i d e t r i p h o s p h a t e i n kO mM T r i c i n e - K O H pH 8 . 0 , 10 mM C a C 1 _ . Substrate  or  i n any o n e enzyme  N e g l i g i b l e a c t i v i t y was o b t a i n e d CaATPase  to  C a A T P a s e was d e t e r m i n e d , of  these  presence  in the  enzyme added  inhibitors  to c r i t i c a l  to a lesser extent  Substrate  in the  i t was d e c i d e d t o  of wheat  p u r i n e base n u c l e o t i d e  Table  Thus  About o n e - t h i r d  triphosphate.  c l e a v i n g GTP  of  as s u b s t r a t e was o b t a i n e d  as s u b s t r a t e s .  only but  IV.  CaATPase o f  proteolysis  vary  are given  greatly.  trypsin  the volume  activation will  Substrate  absence.  s i x minutes  the  activity  41  For  a s s a y i n g the  used as s u b s t r a t e . dependent,  the  concentration  latent  To  t e s t whether  the wheat  o f wheat ATPase  c a l c i u m c o n c e n t r a t i o n was v a r i e d , constant  at  i s o b t a i n e d w i t h a 1:1 complex  CaATPase a c t i v i t y  is the  true  1 mM.  As shown  r a t i o of  substrate  for  1 mM ATP  is s t r i c t l y  holding  in f i g .  c a l c i u m to ATP,  CF^,  12,  the  calcium-  ATP  optimal  activity  implying that  the ATPase r e a c t i o n .  was  the  Excess  cation-ATP  calcium  2+ (10  Ca  :  1 ATP)  does  K.. and V for M max two d i f f e r e n t  i n h i b i t enzyme a c t i v i t y t h e CaATPase o f wheat  sets of  conditions.  In t h e  CF,  about 1  25%.  were d e t e r m i n e d  first,  initial  under  v e l o c i t i e s of  the  2+ r e a c t i o n were determined excess,  at  a constant  concentration of r e s u l t of of  ^  g i v e s a K., o f w a s  m a x  found  t o be  In t h e that  of  plex,  M found but  in the  a K.. o f  i s shown  0.125  under  0.1  (1)  that  order of  of  magnitude  and a p p e a r s  the  lower  3  preparation  t o be a d i r e c t e f f e c t  the  The analysis  to ,  kept  equimolar  shown  in f i g .  13. '  but  CaATP c o m obtained.  not  the  The enzyme u s e d  in  batch of  values  are  K^, this  seed, not  max c a l c i u m and ATP  a large excess of  the d i f f e r e n t  to  r  so V  equimolar  obtained with of  p r o t e i n were  and a d i f f e r e n t  •  that  as  u n i t s / mg  enzyme a c t i v i t y ,  t o enzyme p r e p a r a t i o n .  in the case of than  18.9  e x c e s s Ca  units/mg  6.77  the maximal  the experiment  However,  (1).  max f r o m enzyme p r e p a r a t i o n 2+  r  comparable.  was p l o t t e d  t h e c a l c i u m l e v e l was  of  was f r o m a d i f f e r e n t  used f o r  in  to c a l c u l a t e the c o n c e n t r a t i o n of  that  f r o m enzyme p r e p a r a t i o n  Ca  t o 0 . 3 mM C a A T P .  conditions,  was o b s e r v e d  with  Eadie-Hofstee  t h e same c o n d i t i o n s o f  18 uM CaATP and a V  It  determination than  13.  max  14).  varies  in f i g .  greatly  M (fig.  Substrate  mM CaATP a n d a V  range o f  Using equation  10 mM.  ATP  as c a l c u l a t e d u s i n g e q u a t i o n  to vary  second s e t of  ATP.  concentrations of  concentration of  CaATP c o m p l e x ,  enzyme p r e p a r a t i o n , was  various  one such e x p e r i m e n t  t h i s data  protein.  at  l e v e l s of  i s an  calcium,  calcium.  Figure  12:  Effect  of  of  trypsin-activated  the  the  concentration  r a t i o of  c a l c i u m t o ATP CF^  upon A T P a s e  from wheat.  The  ATP  i n t h e a s s a y m i x was h e l d c o n s t a n t  and t h e c a l c i u m c o n c e n t r a t i o n was  varied.  activity  at  1 mM  42a  43  Figure  13:  Michae1is-Menten of  wheat CF^  concentration according of  in the (S)  the  presence of  trypsin-activated 10 mM C a C I ^ .  was d e t e r m i n e d  to equation  this data,  This  k i n e t i c s of  used f o r  (1).  Inset  CaATPase  Substrate  by c a l c u l a t i n g CaATP i s an E a d i e - H o f s t e e  the d e t e r m i n a t i o n  l i n e has a c o r r e l a t i o n c o e f f i c i e n t  of of  K..  plot  and V  M 0.93-  max  43a  kk  Figure  Ik:  Michaelis-Menten CaATPase o f in equimolar  k i n e t i c s of  wheat CF^. amounts  the  trypsin-activated  C a l c i u m and ATP to  the assay m i x ,  were and  added  substrate  c o n c e n t r a t i o n was c a l c u l a t e d a c c o r d i n g t o e q u a t i o n Inset for  i s an E a d i e - H o f s t e e  the d e t e r m i n a t i o n  has a c o r r e l a t i o n  of  plot  of  K.. and V  M coefficient  of  this data, . max 0.87-  This  used line  (1).  45  Preliminary activity do n o t  experiments  showed  that  observed, calcium  Electron  rather  to  Microscopy of  microscope  acid,  inhibitory.  the  A when  Furthermore,  presence of  Wheat  gave  CF^,  of  s e t t l e onto  CF^  experiments  c a l c i u m was  either  s t a i n , and a c i d gave  its  ECF^  to  s l i g h t l y better  phosphoand  acetate  subunits. to  CF^  when  stained  has a p a r t i c l e d i a m e t e r o f stained with  ammonium  the e l e c t r o n microscopy g r i d at a hexagonal  random.  by  100 A"  molybdate. stained,  It,is  as  possible  structure, with a central  s u c h an o r i e n t a t i o n  be t i l t e d o u t  of  the p l a n e ,  such high m a g n i f i c a t i o n s ,  it  is d i f f i c u l t  r e s o l u t i o n of  electron  be 108 A  Uranyl  at  However,  or  resolution,  show many o r i e n t a t i o n when n e g a t i v e l y  p a r t i c l e s w h i c h have  a central  in the  110 A + 6.3 A w i t h  similar structure  16).  seems t o  excess  ammonium m o l y b d a t e  and w h a t may be s i x s u b u n i t s  with  inhibitor  ADP.  with  p a r t i c l e s are negatively  Particles  these  i d e n t i c a l p a r t i c l e s when v i e w e d  shows a v e r y (fig.  results  CF^  to d i s s o c i a t e into  same p r o c e d u r e  these  is a competitive  r e s u l t s t h a n d i d ammonium m o l y b d a t e .  F^  CaATPase  i n h i b i t i o n s e e n may h a v e been due t o  Phosphotungstic  c a u s e d CF^ E. coli  ADP  upon  However,  high concentration of  ammonium m o l y b d a t e  more c o n s i s t e n t  o f ADP  P a r t i c l e d i a m e t e r s were e s t i m a t e d  15)-  acid.  to p i c k out  the  of  s t a i n i n g o f wheat  (fig.  + 9-5 A\ w i t h  they  of  than  phosphotungstic  tungstic  the e f f e c t  and p a r t  Negative  + 5.1  indeed  the e f f e c t  some a l l o s t e r i c m e c h a n i s m .  w e r e done b e f o r e  the  ADP was  test  g i v e a c l e a r p i c t u r e as t o w h e t h e r  o r a c t s by  stain  to  the v e r t i c i e s  (fig.  Many  17).  giving  core  times particles  depression. at  enzyme p a r t i c l e s b e c a u s e o f  the  to obtain  l a r g e background  grain  good size.  46  Figure  15:  Electron  micrographs of  with  two d i f f e r e n t  acid  KOH  pH 7 - 0 .  sodium o x a l a t e micrographs  wheat  stains. B.  pH 7 - 2 .  is 516,000  A.  CF^, 2%  negatively-stained phosphotungstic  2% ammonium m o l y b d a t e Magnification x.  in  i n 12 mM  both  46a  hi  Figure  16:  Electron  micrographs  2% ammonium m o l y b d a t e Magnification  o f E. c o l i  F^,  negatively-stained  i n 12 mM s o d i u m o x a l a t e  i s 413,000  x.  pH  7.2.  with  47a  48  Figure  17:  S e l e c t e d CF^  particles,  phosphotungstic degrees of  739,000 x .  negatively  a c i d - K O H pH 1.2,  hexagonal  structure.  stained with  showing  2%  various  Magnification  is  For  this  reason,  symmetrical particles  i t was d e c i d e d t o  molecules printed  three  et- a l . ,  and  differentiate  between  t h e most  fig.  part  rotation,  with  Figure  inherent  be a s i x - f o l d  18 shows  From t h e o r i g i n a l five-,  18 shows  two-  to  any  and  six-,  image  it  most  structure. were  rotation  symmetry,  CF^ blown  rotation of  is d i f f i c u l t  r e s o l u t i o n of  rotations  the  by Markham  r e s u l t of  and s e v e n - f o l d  the best  three-fold  the  of  symmetry  image a n a l y s i s was p e r f o r m e d  1963).  such p a r t i c l e s .  image e n h a n c e m e n t  b r i n g out  e x h i b i t i n g what a p p e a r e d  up p h o t o g r a p h i c a l l y (Markham  to  try  to  but  for  apicies with a  also giving  six-fold  hexagonal  particles.  Immunological  Study of  Anti-wheat from a r a b b i t taken it  from  CF^  after  the  Wheat  CF^  serum, w i t h three  rabbit  an a n t i b o d y  i n j e c t i o n s of  prior  to  immunization  similar  antiserum also precipitated  antigenic sites  Complete  CF^  was  on t h e CF^  injected  In o r d e r  to determine  reacted,  two d i m e n s i o n a l  three  anti-CF^  the  a g a i n s t w h i c h CF^  smaller subunits. serum r e a c t s a g a i n s t  bind  against It  is  both  s p i n a c h CF^ ,  from the  rabbit  two  to generate  subunit  or  t h e a and 8 s u b u n i t s ,  antibodies. the  antibodies  (fig. but  or only  i s s y m m e t r i c a l , so  largest  plates.  subunits of  wheat C F , .  20).  not  i m p o s s i b l e to a s c e r t a i n whether  immunoprecipi t a t ion r o c k e t the  since  indicating  subunits  t h e a and 8 s u b u n i t s ,  Serum  19).  plants.  However,  both of  (fig  in double d i f f u s i o n  resolved  with  obtained  to CF^,  b e c a u s e t h e s e two s u b u n i t s a r e n o t w e l l the  was  1/80,  i m m u n o e l e c t r o p h o r e s i s was p e r f o r m e d  A p r e c i p i t i n rocket developed the  d i d not  purified  complexes  into  of  0.2 mg p u r i f i e d CF^  g a v e no p r e c i p i t i n l i n e a g a i n s t w h e a t CF^  Wheat CF^  titer  against the  one  subunit,  i n a 10% SDS-PAGE g e l . it  probably  Thus t h i s a n t i s e r u m  reacts  would  50  Figure  18:  Markham  r o t a t i o n of  numbers d e n o t e photographic exposures.  t h r e e wheat  the d i v i s i o n s  p a p e r was  of  rotated  CF^  particles.  a circle in making  The  through which the  multiple  the  50a  51  Figure  19:  Ouchterlony titer  double d i f f u s i o n  i n serum from  antibody  rabbit  immunized  a g a i n s t wheat  W h o l e s e r u m was d i l u t e d as  indicated  in the  wells,  and p r e c i p i t i n l i n e s w e r e d e v e l o p e d  overnight.  These p r e c i p i t i n l i n e s were  by s t a i n i n g w i t h and  F i g u r e 20:  p l a t e e s t i m a t i o n of  peripheral against  then  CF^  visualized  C o o m a s s i e b l u e as d e s c r i b e d  in  Materials  Methods.  Crossed of  CF^.  Immunoelectrophoresis  w h e a t CF^  t h e method  against  of  anti-wheat  separated  contained  k~J ug p r o t e i n .  gel.  to  b.  unfractionated visualized  The  according  a.  Wheat  against  rabbit  gel  Methods.  into  the  is a  immunoprecipitin  contained  1 ml  to  CF1 sample lane  antibody  t h e a and 3 s u b u n i t s  antiserum.  by s t a i n i n g w i t h  The  l a n e shown h e r e  showing  antibody  i n M a t e r i a l s and  serum,  subunits  by SDS-PAGE on a 10% g e l .  Antibody g e l ,  The  separated  (1979).  t h e one e l e c t r o p h o r e s e d  w h i c h has d e v e l o p e d w h e a t CF.j .  the CF^  Chua and B l o m b e r g  subunits,  adjacent  of  rocket of  of  Precipitin lines  were  C o o m a s s i e b l u e as d e s c r i b e d  51a  52  be u s e f u l  i n p r e c i p i t a t i n g t h e a and $ s u b u n i t s  translation The  o f w h o l e wheat  antiserum obtained  trypsin-activated completely  CF1  (fig.  i n h i b i t ATPase  in a c e l l - f r e e  RNA. also 21).  i n h i b i t s the Very small  activity.  latent amounts  CaATPase (15  ul)  of of  antiserum  53  Figure  21:  Inhibition of  wheat  of  CF^  the  trypsin-activated  by a n t i - w h e a t  CF^  CaATPase  serum.  The  latent  C a A T P a s e was a c t i v a t e d  by t r y p s i n d i g e s t i o n ,  s e r u m was added  ug a l i q u o t s o f  enzyme. to  to 2.5  C a A T P a s e was a s s a y e d  t h e method  of  Lien  the  immediately,  and R a c k e r  (1971b).  activity  and  anti-  activated according  1  % INITIAL ATPase ACTIVITY  5k  DISCUSS ION In t h e p r o c e d u r e d e v e l o p e d was  removed  from  wheat c h l o r o p l a s t c o u p l i n g  the surface of the t h y l a k o i d  by a s i m p l e t w o - s t e p sucrose density  here,  gradient  during  centrifugation.  Better  This  from  l a r g e amounts  Sephadex et  It  lost  in preparations  of thylakoids.  seems t h a t  this  inhibitors  t o some o s m o t i c o r i o n i c e f f e c t Characterization  interesting  results.  significantly subunit  higher  (25 kd v e r s u s  discrete  present.  have noted  the 6 subunit  of  through  a DEAE-  to lose the 6 subunit fragile,  (Younis above and  t h e p r o t e c t i o n o f CF^  compounds  is  obtained proteolysis  unknown.  o f t h e s u b u n i t s o f w h e a t CF^ by SDS-PAGE g a v e  than  19 kd f o r s p i n a c h ) .  to e l e c t r o p h o r e s i s .  found, It  several  o f t h e y and 6 s u b u n i t s a r e  those o f s p i n a c h CF^,  forms o f t h e y s u b u n i t were  the  separated  i s due t o i n h i b i t i o n o f  of these  Molecular weights  the  subunits  is inherently  Whether here  both  w h e r e CF^ was c h l o r o f o r m - r e l e a s e d  complex  reported  in which  Inclusion  authors  that  proteolysis  step.  P a s s a g e o f s p i n a c h CF^  properties.  o f a CF^  inhibitors,  i t h a s been  B i n d e r e t a l . (1978) f o u n d  its eoTdlability  with proteolysis or  Several  A50 c o l u m n a l s o c a u s e d t h i s c o m p l e x  a l . , 1976).  beyond  of f a c t o r s .  a l l five  o f t h i s enzyme t o l o s e s u b u n i t s o n c e  the t h y l a k o i d .  In e x p e r i m e n t s  the sucrose gradient  i n h i b i t o r s y i e l d e d a CF^ w i t h  s p i n a c h CF^ was o f t e n from  lost during  c o u l d be d u e t o a number  tendency  preservation  in the absence of p r o t e o l y s i s  6 and e s u b u n i t s were proteolysis  homogeneity  by t h e i n c l u s i o n o f  i s o l a t i o n and p u r i f i c a t i o n .  w h e a t CF.| was i s o l a t e d  of  to  p r o c e d u r e c o n s i s t i n g o f c h l o r o f o r m e x t r a c t i o n and  c o m p l e x c o n t a i n i n g a l l s u b u n i t s was o b t a i n e d inhibitors  and p u r i f i e d  factor  e s p e c i a l l y that  In a d d i t i o n , dependant  a number  upon  i s not s u f f i c i e n t  of the 6 of  the degree  reduction  prior  wheat CF1  i n a s t o c k s o l u t i o n o f 2% SDS p l u s 0.2% 8 - m e r c a p t o e t h a n o l  of  to d i s s o c i a t e  55  o r 5 mM  DTT.  A critical  amount o f r e d u c i n g a g e n t  c o m p l e t e r e d u c t i o n o f t h e y s u b u n i t , a t l e a s t 33 Care should  be  taken  when m o l e c u l a r in  weight  r e d u c e and  determinations  n m o l e s DTT/mg  dissociate  a r e made on  to  insure  CF^.  a l l polypeptides  a basis of  mobility  SDS-PAGE. B e c a u s e two  in  d i s c r e t e y b a n d s a r e o b t a i n e d when CF^  low amounts o f  is at  l e a s t one  Racker  heat  reducing agent,  internal  disulfide  These a r e not  activation  of the  number o f t i t r a b l e proposed subunit  The  been  i s s t r i k i n g , and d) one  sucrose  specificity, than  discovery  can  protease  see  internal  that  (arrows)  prevent  that proteolysis  specific  but  they  One CF^  Murata,  thus argue t h a t the  prevent  the  of  loss of  from  In f i g . k  not  prevent  extracted  is  the  by  any  inhibitors,  by  prevented,  In a d d i t i o n ,  gradient centrifugation  in the  mobility  release of  r e l e a s e because p r o t e o l y s i s  inhibitors  It is  r e l e a s e o f CF^  i n h i b i t o r s do  effect.  or  1979).  complex.  inhibit  CF^  i n the  bridge  polypeptide  polypeptides normally  osmotic  trypsin  1970).  disulfide  f o r t h e CF^  completely  could  Racker,  kd  and  in the spinach  in an.increase  in preventing  that proteolysis  during sucrose CF,.  i n t h e 33  there  Farron  c y s t i n e which a l t e r s  ( K u w a b a r a and  other unidentified  extracts with  bonds  result  internal  inhibitors  b e c a u s e o f some g e n e r a l  s u b u n i t s o f CF^  disulfide  one  identified  in spinach  o f t h e p o l y p e p t i d e s o f CF^.  rather  least  A similar  II p a r t i c l e s  r e l e a s e o f two  hypotonic  i s at  e f f e c t of p r o t e o l y s i s  ( l a n e s c and  to conclude  i n the y s u b u n i t .  l a t e n t ATPase d i d not  o f w h e a t CF^.  the t h y l a k o i d  two  is electrophoresed  b r o k e n d u r i n g A T P a s e a c t i v a t i o n , as  here that there  Photosystem  their  bridge  h a l f c y s t i n e s ( a l s o F a r r o n and  i n e l e c t r o p h o r e s i s has  the  i t i s tempting  ( 1 9 7 0 ) showed t h a t t h e r e a r e  molecule.  y  to completely  i s necessary  the  minor  suggests  that a  56  All  attempts  to heat  were u n s u c c e s s f u l . t o a maximum o f that  there  Farron  activate  Under  protein.  is a basic difference  did  eight  not r e s u l t s p i n a c h CF .  of  the enzyme,  However,  four  additional  the activated  titrable  strong oxidizing  iodoacetamide  agent  inhibit  ( F a r r o n and R a c k e r , latent  possible  It  These  o-iodosobenzoate  wheat  The o p t i m a l from s p i n a c h  h a s been shown  implying that  CF^  should  Heat  SH g r o u p s  CF^  activation bridges  a r e n o t on t h e s u r f a c e  solvents,  only  Heat a c t i v a t i o n newly-exposed  two SH  made  SH g r o u p s  that  heat  b u t n o t by  t h e two t y p e s mechanisms.  show t r y p s i n  activation  N-ethylmaleimide  by h e a t  activation  of  or trypsin  of a c t i v a t i o n Thus  it  of the  is  of  quite  latent  activation.  CarATP r a t i o  i s 1:1  heat  and u n a c t i v a t e d  during  t h e a c t i v a t i o n o f CF^  1970),  CaATPase but n o t heat  reported  SH g r o u p s .  C a A T P a s e p r o c e e d by d i f f e r e n t  that  the process of  i n u r e a o r SDS.  most o f t h e e i g h t  enzymes.  i n t r a p e p t i d e d i s u l f i d e b r i d g e s by t h e a d d i t i o n  ( A n d r e o e t a l . , 1979).  the  (1970) s t u d i e d  s i n c e in the absence o f denaturing  c a n be i n d u c e d t o f o r m the  l e a d s one t o c o n c l u d e  p e r m o l e c u l e o f CF^ c o u l d be t i t r a t e d .  accessible  activated  i n t h e s t r u c t u r e o f t h e s e two  In b o t h  SH g r o u p s w e r e  CF^  t h e s p i n a c h CF^  i n t h e c l e a v a g e o f e i t h e r o f t h e two d i s u l f i d e  of  groups  This  and Racker  a c t i v a t i o n o f s p i n a c h CF^. complexes,  CaATPase o f wheat  identical conditions,  10 u n i t s / m g  (1970) and F a r r o n  the latent  (Vambutas  h e r e f o r wheat  CF^.  f o r ATPase a c t i v i t y and R a c k e r , This  1965).  suggests  that  of the a c t i v a t e d Similar  CF^  results are  the actual  substrate  2+ for  t h e ATPase r e a c t i o n The  dependent to  formation upon  i s t h e Ca  ATP  of the cation-ATP  complex.  complex  the d i s s o c i a t i o n constant  the r e l a t i o n s h i p given  in equation  i s pH d e p e n d e n t  f o r the complex,  (1).  It  as w e l l as  according  i s important  to c a l c u l a t e  57  substrate very  low ATP  values. for  c o n c e n t r a t i o n as complexed  The  concentrations complex  t h i s very  a Ca:ATP r a t i o o f substrate,  non-1inear  shown  these unusual  that  and f a i l u r e complex. the  In e x p e r i m e n t s  Lineweaver-Burke  study  13 and  A number o f literature.  Tricine-maleate of  et  for  al.  buffer  0.8 mM A T P .  by p l o t t i n g  at In  (1972), a s s a y i n g t h e pH 8 a n d  a  presented calcium but  lower  This  2.7  pH medium  in t h i s d i s s e r t a t i o n ,  obtained  is probably  complex  (pH  those  Hochman  s u b s t r a t e of  2+ Mn  the  has substrate  cation-ATP  velocities  s p i n a c h ATPase  in  is  the  in the presence of  6)  was o b s e r v e d ,  in the  to values  et  for  ^  al.  In t h e  2 mM  m a x  was  (1976)  results  presence of in the  lettuce  excess literature,  c a l c i u m and ATP  spinach or  (1976)  et  reported  i n t h e enzyme f r o m al.  though  Hochman  obtained  reported  found  (18 uM ATPases.  wheat.  indicates that  ATPase a c t i v i t y  of  measured d i r e c t  binding of  the  l e t t u c e CF^.  cation-ATP By  2+ for  against  the  8 mM C a C l ^ ,  l e t t u c e enzyme.  i s comparable  than  k i n e t i c data of  substituting  mM ATP  due t o a d i f f e r e n c e  i s a l s o the  the  choice of  in  in experiments w i t h equimolar  i s much l o w e r  The  0.1  mM CaATP f o r  (0.125 mM CaATP)  the  CaATP)  of  as  reported  in the presence of  In pH 8 Hepes-NaOH b u f f e r ,  obtained  Ahlres  Ahlers  the  initial  t h e C a A T P a s e h a v e been  very  pH.  (1975)  a Michaelis-Menten relationship  a KM o f  this  faulty  c o n c e n t r a t i o n of  M g C l ^ a MgATPase w i t h low a t  two  14).  K.. v a l u e s M  Neison  by  at  w h e r e C a r r e i r a and Munoz u s e d  p l o t s were o b t a i n e d .  here,  c a l c u l a t e d CaATP c o n c e n t r a t i o n , (figs.  the  into question  k i n e t i c c u r v e s a r e due t o  reported  for  between  r e a c t i o n r a t e s w i t h ATP  to c a l c u l a t e the e f f e c t i v e  In t h e  obtained  a  were c a l l e d  in determining  0.5:1  f r e e ATP,  p u b l i s h e d by C a r r e i r a and Munoz  o f E. c o l i  reason.  than  is a large difference  k i n e t i c curves  the ATPase a c t i v i t y  (1976) f o r  there  CaATP r a t h e r  Ca  ,  they  MnATP  by  58  the amplitude results tight  show t w o t i g h t  cation binding  the K  values  propose while of  of the M n i T  that  s i t e s have a  these high a f f i n i t y  was f o u n d .  here,  ratio  while with equimolar  in these  several  two types  excess calcium binds at other the a f f i n i t y  by c a l c i u m .  working with  about  sites, the  s i t e , which has a  rate  in the  i n t h e p r e s e n c e of a v a s t With excess Ca, a  amounts  s i t e s than  the a c t i v e  excess  of  reasons f o r the d i f f e r e n t It  ATPase  o f 0.1  o f Ca a n d ATP a  of d e t e r m i n i a t i o n s .  t o 0.3 18 uM  affinity  is possible  that  s i t e s on t h e A T P a s e , Alternatively,  f o r t h e same h i g h a f f i n i t y  call  Hochman  of the r e s u l t s presented of substrate  Negative-stained  for a competitive  et a l .  the heat-activated  the a f f i n i t y  viewed  authors  the  i n K.. o b s e r v e d w i t h e x c e s s c a l c i u m c o u l d be due t o t h e e x c e s s  second p o s t u l a t e would  nature  These  They c o n c l u d e t h a t  o f t h e enzyme f o r s u b s t r a t e .  calcium binding competitively  reaction  cations.  of the t r y p s i n - a c t i v a t e d  of Ca:ATP.  One may s u g g e s t  decreasing  the  u s i n g CaATP a s s u b s t r a t e  mM was o b t a i n e d ,  The  s i m i l a r to  c a t i o n s i t e s are the c a t a l y t i c  by t h e l o w a f f i n i t y  reported  Ca and w i t h a 1:1  increase  range,  The  range.  was d e t e r m i n e d  constants  i n h i b i t i o n of free  Their  s i t e s on C F ^ .  in the micromolar  sites are regulatory.  c a t a l y s i s i s regulated  resonance s i g n a l .  loose cation binding  for the competitive  In e x p e r i m e n t s  of  and t h r e e  the low a f f i n i t y  millimolar  e l e c t r o n paramagnetic  w h e a t CF^  (1976)  ATPase o f here  give  binding has a  i n h i b i t i o n o f t h e ATPase a K.  l e t t u c e CF^.  prevents  orientation.  show a h e x a g o n a l  structure  The e l e c t r o n m i c r o g r a p h s  of  7  mM f o r C a ,  The p r e l i m i n a r y  making any c o n c l u s i o n s  i n l o w and h i g h Ca c o n c e n t r a t i o n s . p a r t i c l e diameter  i n t h e e l e c t r o n m i c r o s c o p e u s i n g two d i f f e r e n t  micrographs  s i t e as CaATP.  of  108-110 A when  negative  stains.  The  i n p a r t i c l e s o f one p a r t i c u l a r presented  here a r e s t r i k i n g l y  similar  59  to  published micrographs of  and b e e f  heart  yeast  mitochondria  F^  mitochondrial  (Kagawa  g a v e 80-90 A* s p h e r e s w h i c h c o n s i s t o f Markham  rotation  However,  of  i t must  technique,  Particles will  microscopy g r i d . as t h e a c t u a l  orientation  most  these hexagonal  core of  subunit  On t h e a model  of  regions of (fig. that  or  wheat  CF^  six discrete  introduces  (fig.  found  that  these are  preparations  symmetry.  suggest the  solid,  a particular  electron  s t a i n i n g as w e l l that  symmetry.  as  if  this  one  For  there  17). and t h e w o r k o f  (fig.  <5 and e s u b u n i t s a r e p l a c e d a t  22).  authors  The  because of  (Younis  are e a s i l y  s t u d i e s of the o n l y  the  is a central  my o b s e r v a t i o n s  these polypeptides  the c r o s s - 1 i n k i n g  of  shows a s i x - f o l d  p a r t i c l e s appear  F^  subunits.  18 one c a n c o n c l u d e o n l y  et  others,  I am p r o p o s i n g  evidence presented  a l . ,  Binder et  1977;  exposed  here  a l . ,  1978)  l o s t d u r i n g enzyme p u r i f i c a t i o n .  and e s u b u n i t s a r e d r a w n a t o p p o s i t e e n d s o f of  upon  one enhances a r t e f a c t s  CF^  1967)  b i a s when u s i n g  i n many o r i e n t a t i o n s  o f wheat  al . ,  1966). T h e s e  by s e l e c t i n g p a r t i c l e s t h a t  s o l u b i l i z e d complex  5) and by o t h e r  about  one  From f i g .  subunits  basis of  the  that  fall  structures.  and R a c k e r ,  p a r t i c l e s enhances a s i x - f o l d  In a d d i t i o n ,  particular part,  CF^  be remembered  image e n h a n c e m e n t symmetry.  wheat  (Schatz et  t h e a^B^Y  c o r e on t h e  B a i r d and Hammes (1976),  subunits  not  basis  in which  i n c l o s e enough  The 6  it  proximity  was to  be c h e m i c a l l y c r o s s - l i n k e d . Stoichiometry Introduction, factor  and s t r u c t u r e  the question of  complexes  is s t i l l  s t o i c h i o m e t r y would of  the  inequality  can account  for  not  are c l o s e l y  s t o i c h i o m e t r y of  a controversial  be e x p e c t e d  in s i z e of  a regular  linked.  issue.  subunits of Any 2^2*2 ° a  to g i v e a hexagonal  these s u b u n i t s .  hexagonal  the  As n o t e d  particle,  r  in the a  small  coupling  2^2  particle  A core p a r t i c l e of s i n c e the  the  Y  because a^B^Y  size  60  Figure  22:  P r o p o s e d model o f wheat  CF1  for  the arrangement of  the  w i t h i n t h e enzyme c o m p l e x .  subunits  60a  a  and j8  SIDE V I E W  a  and  /5  TOP  VIEW  61  of  t h e 6 and e s u b u n i t s  gross  shape o f t h e p a r t i c l e .  reported. of  probably  showing a hexagonal used a F o u r i e r filtered  for a near-perfect  c r y s t a l s of  from  structure  (Wakabayashi  transform  image o f t h i s  spacing with  programme  F1.  factors  optical  little hexagon  h a s been  diffraction  patterns  e t a l . , 1977).  t o computer  but not p e r f e c t ,  in determining  They  construct crystal  re-evaluated  further  the o p t i c a l l y showed a 90 K  s i x - f o l d and t h r e e - f o l d  the subunit  stoichiometry  may be d u e t o u n r e l i a b l e m o l e c u l a r w e i g h t  e t a l . (1979)  to the  t h e t h e r m o p h i l i c b a c t e r i u m PS3  The t w o - d i m e n s i o n a l  considerable,  Much o f t h e p r o b l e m  Yoshida  Evidence  they c o n t r i b u t e  Kagawa a n d h i s c o - w o r k e r s o b t a i n e d  two-dimensional  coupling  means t h a t  the molecular weight  symmetry.  for  determinations.  o f F^  from t h e  b a c t e r i u m P S 3 , c h l o r o p l a s t s and m i t o c h o n d r i a  by e q u i l i b r i u m  cemtrifugation  and g e l f i l t r a t i o n ,  by e q u i l i b r i u m  centrifugation  and g e l f i l t r a t i o n over-estimate when of  and t h a t o f t h e s u b u n i t s by h i g h s p e e d  of the molecular weights  chromatography. o f F^  subunits  They r e p o r t  an  i n a l l c a s e s , and  t h e new m o l e c u l a r w e i g h t s w e r e u s e d t o c a l c u l a t e t h e s t o i c h i o m e t r y  t h e enzyme c o m p l e x , In s u p p o r t  specific  a n a^^y&e,  of t h i s  of  model  c o u l d be a c c o m o d a t e d .  E s c h and A l l i s o n  (1979) f o u n d o n e  of p-fluorosulfonylbenzoyl-5'-adenosine,  F ^ - A T P a s e , on each o f t h e 3 s u b u n i t s o f beef h e a r t  total  of three  three  3 subunits  attachment  or "hexagonal"  stoichiometry,  s i t e f o r arrachment  inhibitor  three  liquid  specific  sites  p e r F^  per complex.  per a s u b u n i t were  a per complex  complex were f o u n d ,  In a d d i t i o n , found,  with  F^ .  o f 1.5  A  suggesting  0.5 n o n - s p e c i f i c s i t e s  a total  an  p e r F^,  of  giving  also.  3 Conflicting beef heart of  data a l s o e x i s t .  with a stoichiometry  6 subunits  i s unknown  H-N-ethylmaleimide  of 2a:23:2y:?6:2e  because t h i s  reagent  binds  (Senior,  t o F^  from  1975; t h e number  does not b i n d t o  this  subunit)  62  but  binding of t h i s molecule requires free  l o w o r l a c k i n g i n SH m o i e t i e s w i l l work  f r o m t h e same l a b o r a t o r y  not bind  (Senior  Accuracy  a good v a l u e several  in determining  authors  have  shown t h a t  of  the smaller subunits w i l l  of  F1 . One  but taken  e l e c t r o n micrographs the major easy  subunits,  presented with  as w e l l  generated  i s very  fragile,  recent  t o support  upon  earlier,  and t h e  for the molecular  t h e more  t h e 6 and e s u b u n i t s  a g a i n s t wheat  as i n h i b i t e d t h e l a t e n t  Immunoelectrophoresis largest  loss  weight  electron literature,  a model  of  the  & ^ for  r e l a t i v e l y exposed  CF^  precipitated  CaATPase a c t i v i t y  h a s shown t h a t  s u b u n i t s o f wheat  However, is  here tend  i n SDS-PAGE  As n o t e d  s o l e l y on t h e b a s i s o f  in conjunction with  6 E  by t h i s method d e p e n d s complex.  lower v a l u e s  Earlier  for  remova1. Antiserum  two  give  s h o u l d n o t p r o p o s e a model  microscopy,  of the  t h e complex  so any s u b u n i t  1971) g a v e an a ^3  staining of subunits  stoichiometry  f o r the molecular weight  groups,  it stoichiometrically.  and B r o o k s ,  s t o i c h i o m e t r y on t h e b a s i s o f r e l a t i v e bands.  sulfhydryl  site  is c r i t i c a l  3 s u b u n i t s o f wheat  t o enzyme a c t i v i t y ,  interfere with  t h e enzyme f u n c t i o n .  serum t o  s u b u n i t o f Antirrhinum  case,  the y  the a n t i - y  serum  serum f r o m a n o t h e r same a c t i v i t y  (Koenig  Rocket  reacts only with the  This  immunized  e t a 1. , 1976).  that  t h e enzyme a c t i v e  CF^.  Antibody  and d e p e n d i n g  the antibody  majus  upon  against  CF^.  with In  anti-  one  e t a l . , 1978),  the y subunit  Antibody  whether  may o r may n o t  (snapdragon)  (Koenig  site  b i n d i n g may  h a s been d e m o n s t r a t e d  i n h i b i t e d t h e MgATPase  rabbit  complex  CF^.  o c c u r a t a n y s i t e on an a n t i g e n i c p o l y p e p t i d e , this  o f CF^.  this antiserum  t h i s does not c o n c l u s i v e l y prove  l o c a t e d on t h e a a n d / o r  t h i s enzyme  stimulated  while the  b i n d i n g a t one s i t e may  63  cause a conformational in a c t i v a t i o n o r Koenig et ment of  of  al.  (1978) w a r n s  a n t i s e r a to the  serum from o n l y  a distant  i n h i b i t i o n , depending  a component  be made a b o u t  change at  one  that  same component  the a c t i v e  the  l o c a t i o n of  " . . . c o n c l u s i o n s concerning  c a n be drawn w i t h  rabbit  upon  s i t e on t h e e n z y m e ,  have  certainty been  only,  CF,.  the the  antibody. uninvolve-  a large  investigated."  has been c h a r a c t e r i z e d  s i t e o f wheat  if  resulting  number  Since  no c o n c l u s i o n s c a n  yet  6k  SUMMARY Wheat factor  CF.|  exhibits  complexes  from o t h e r  and m i c r o b i a l subunits,  extraction  A number o f this  study.  extraction here. CF^. is for  However,  properties,  may be due t o  demonstration the  internal  of  that  this  for  Unlike  s p i n a c h CF^,  CaATPase a c t i v i t y . and  The  values  amounts, differ  i s not  p u r i f i c a t i o n h a s been extraction  of  CF^  in  its y  t h e wheat  CF^  described  a protease  by h y p o t o n i c  coupling  wheat  with  s u c r o s e wash  inhibitors, while  shown t h a t  described  factor.  proteolysis  CF^  contains  of an  subunit. enzyme d o e s  trypsin-activated  by an o r d e r o f  of  elucidated  Two  not  exhibit  ATPase a c t i v i t y different  a low  a  heat-activated  i s both c a l c i u m -  values  for  when c a l c i u m and ATP  when c a l c i u m i s magnitude.  More  reflects a competitive  in excess.  research  the  are  in  These  is necessary  to  i n h i b i t i o n o r an a l l o s t e r i c  by c a l c i u m .  Electron a structure six-fold  inhibitors during  due t o an i n h i b i t i o n o f  I have  and a h i g h e r  this  enzyme h a v e b e e n  b e e n shown w i t h a p l a n t  i n h i b i t e d by c a l c i u m .  determine whether control  proteolysis  proteolysis  CaATPase a c t i v i t y were o b t a i n e d , equimolar  of  s p e c i f i c i t y , and a s i m i l a r  the wheat  extraction  In a d d i t i o n ,  d i s u l f i d e bridge  dependent  unique to  the concomitant  before  CF^.  mitochondrial  s i m i l a r i t i e s i n c l u d e t h e number  substrate  i n c l u s i o n of  has n e v e r  as  coupling  stained.  A requirement  u n l i k e ECF^ ,  a subunit  These  properties  i n h i b i t e d by ECF.J ,  s o u r c e s , as w e l l  by c h l o r o f o r m and s u b s e q u e n t  This The  plant  complexes.  s t r u c t u r e when n e g a t i v e  in  many c h a r a c t e r i s t i c s i n common w i t h  microscopy of  simi 1 ar  symmetry  negatively  to other  F^'s  s t a i n e d wheat  viewed  i n one o r i e n t a t i o n ,  in the  a symmetry  CF^  particles  shows  same m a n n e r .  CF^  which  shown  is best  shows with  a  65  the  image e n h a n c e m e n t  a n a l y s i s of The reported  ECF^  technique of  by n e g a t i v e  Markham r o t a t i o n .  s t a i n i n g showed a v e r y  beginnings of  a study of  here.  a n t i b o d i e s t o t h e enzyme w e r e  Rabbit  With a n t i b o d i e s a g a i n s t  r e c e n t work o f the  intact  results Ellis,  Nelson et  CF^  al.  from three o t h e r  the s i t e of  Bouthyette  informative.  The  between  As  t e c h n i q u e s and  enzyme w h i c h w i l l  CF^  s y n t h e s i s and method o f  studied.  r e g u l a t i o n and b i o s y n t h e s i s a r e a l l today.  would  polypeptides  RNA.  t h e y and 6 s u b u n i t s o f  i s an  n u c l e a r and c h l o r o p l a s t  p r o t e i n complexes yet  one  In v i e w o f  Their  transport  by  with  a 1.,  would  the  1976;  study  of  be most of  those  i n t r i g u i n g example  of  genomes. t h e most  structure,  important  mode o f  groups  action,  questions which f a s c i n a t e s c i e n t i s t s  insights are gradually  r e c e i v e much  et  an a d d i t i o n a l  1978)  The c o u p l i n g f a c t o r s a r e p r o b a b l y o n e o f of  CF^,  in d i r e c t c o n f l i c t  p o l y p e p t i d e s made o u t s i d e t h e c h l o r o p l a s t cooperation  are  characterized.  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