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The ATPase complex of Escherichia coli : studies on the DCCD-binding protein Loo, Tip Wah 1983

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THE ATPase COMPLEX OF E s c h e r i c h i a  coli:  STUDIES ON THE DCCD-BINDING PROTEIN  by  TIP WAH[LOO B.Sc,  The U n i v e r s i t y o f B r i t i s h Columbia, 1977  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department o f B i o c h e m i s t r y  We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA June 1983  Q  T i p Wah Loo, 1983  In p r e s e n t i n g requirements  this thesis f o r an  of  British  it  freely available  agree t h a t for  understood that for  Library  shall  for reference  and  study.  I  f o r extensive copying of  h i s or  be  her  copying or  f i n a n c i a l gain  shall  g r a n t e d by  not  be  BIOCHEMISTRY  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall Vancouver, Canada V6T 1Y3 10  DE-6  (3/81)  June  1983  of  Columbia  make  further this  thesis  head o f  this  my  It is thesis  a l l o w e d w i t h o u t my  permission.  Department o f  the  representatives. publication  the  University  the  p u r p o s e s may by  the  I agree that  permission  department o r  f u l f i l m e n t of  advanced degree a t  Columbia,  scholarly  in partial  written  (ii) ABSTRACT  The ATPase complex of E_. c o l l consists of two functional u n i t s . E C F ^ i s an e x t r i n s i c membrane protein having the active s i t e ( s ) for ATP synthesis and hydrolysis. F Q i s i n t r i n s i c and catalyzes the reversible transfer of protons across the membrane.  E C F ^ consists of f i v e  polypeptides (<*-e) ranging i n molecular weight from 13 000 - 57 000. F  Q  has three polypeptides (9 000, 18 000, 24 000), the smallest of which  i s the dicyclohexylcarbodiimide (DCCD)-binding protein postulated to be a transmembrane pathway f o r proton translocation.  An  ECF^FQ  complex was  s o l u b i l i z e d from the membranes of E_. c o l i with N-lauroyl sarcosine and p u r i f i e d by chromatography on Phenyl-Sepharose CL-4B followed by sedimentation of the enzyme at 250 000 xg f o r 16-17 h. ECF^FQ  The p u r i f i e d  complex consisted of the eight polypeptides described above, as  well as associated polypeptides of molecular weights 30 000, 28 000 and 14 000. Removal of E C F ^ from the membranes of the wild-type" IS. c o l i resulted i n the membranes becoming leaky to protons so that they could not be energized. The unc mutants, E . c o l i AN382, CBT-302 and  could  maintain a proton gradient across the membrane i n the absence of E C F ^ .  A  normal DCCD-binding protein was present i n the F Q complex of each mutant.  However, the 18 000 dalton polypeptide of F Q was absent i n the  membranes of E_. c o l i N^^, suggesting that i t was required f o r a functional F Q .  The involvement  of the 18 000 dalton polypeptide i n the  proton-translocating a c t i v i t y was also suggested t h i s polypeptide was absent i n the  ECF^FQ  complex  by the observation that immunoprecipitated  from trypsin-treated "stripped" v e s i c l e s , which had been reconstituted with  (iii) ECF^. ECF^,  Although these trypsin-treated "stripped" v e s i c l e s could rebind the membranes could not be energized during ATP hydrolysis. Leakiness of the membranes to protons could be repaired by the  reaction of the E C F ^ s t r i p p e d membranes with DCCD or ECF^. antibody raised against the DCCD-binding protein prevented protons.  Similarly, this leakage of  The antibody also inhibited the rebinding of ECF^ to the  "stripped" everted membrane v e s i c l e s .  These results indicated that the  DCCD-binding protein was exposed on the cytoplasmic surface of the c e l l . Attempts to show whether the DCCD-binding protein was transmembranous were not successful.  Radioimmunoassay techniques were used to show In v i t r o ,  the involvement of the a r g i n y l residue(s) of the DCCD-binding protein i n the binding of ECF^. Binding of ECF to the DCCD-binding protein 1  appeared to involve the <* and/or B subunits of ECF^. Chemical modification of the methionyl  residue(s) of the DCCD-binding protein did  not a l t e r i t s capacity to bind ECF^, but destroyed the antigenic s i t e ( s ) of the polypeptide.  In summary, these results are consistent with the I  proposed "loop" arrangement of the DCCD-binding protein i n which the polar (  central region of t h i s molecule i s at the cytoplasmic surface of the c e l l membrane.  (iv)  TABLE OF CONTENTS  TITLE-PAGE  U)  ABSTRACT  (ii)  TABLE OF CONTENTS  (  LIST OF TABLES  i v  )  (xi)  LIST OF FIGURES  (xii)  ABBREVIATIONS  (xvi )  ACKNOWLEDGEMENTS  ( x  i  x  )  INTRODUCTION  ( 1.  Location of the F-^FQ Complex  3.  Role of the F^F^ Complex i n Energy Transduction  4.  Properties of F^  5.  Subunit Composition of F^  5.  Tightly-Bound Nucleotides..  7.  Function of the Subunits of E C F  1  The Delta (6) Subunit  8.  The Epsilon (e) Subunit  9. 11.  The Gamma (Y) Subunit The Alpha (a) and Beta (6) Sununits Studies on the Active Site of ECF  1  '  12. 13.  Cross-Reconstitution Studies  15.  The Arrangement of the Subunits of ECF.^  16.  The F Q Complex  19.  S o l u b i l i z a t i o n of the F F n  n  Complex  19.  (v) C r i t e r i a f o r Determining the Intactness and Purity of the F ^ F Q Complex  19  The' F F  20  1  Q  Complex  The Isolation of the F  . Q  Complex  26  Biochemical Genetics  28  The DCCD-Binding Protein  33  I d e n t i f i c a t i o n and Isolation  33  Reconstitution of Proton Translocating A c t i v i t y  35  The Amino Acid Composition  36  The Amino Acid Sequence  38  DCCD-Resistant Mutants  40  Objectives of t h i s Study  42  MATERIALS AND METHODS  44  Chemicals  44  Maintenance of B a c t e r i a l Strains  46  Growth of Cells  46  Media  48  Preparation of Membranes  48  Preparation of EDTA-Lysozyme Spheroplasts  49  Preparation of K -Loaded Spheroplasts  50  Isolation of ECF^.  50  P u r i f i c a t i o n of ECF^ on AH-Sepharose 4B  51  P u r i f i c a t i o n of ECF^ and TPCK-Trypsin Treated ECF^ Sucrose Density Gradient Centrif ugation  by 1  52  Preparation of ECF^-Depleted Membranes  52  Preparation of Rat-Liver Mitochondrial Membranes  53  Preparation of the Subunits of ECF  53  1  (vi) TPCK-Trypsin Treated ECF .  53  a and J3 Subunits of ECF  54  1  1  S o l u b i l i z a t i o n of Membrane Vesicles with Detergents P u r i f i c a t i o n of the  ECF^Q  Complex Solubilized  54  with  N-Lauroyl Sarcosine  •• •  Gel F i l t r a t i o n on Sepharose 6B  55 55  Hydrophobic-Interaction Chromatography P u r i f i c a t i o n of the ECF^Fg Complex by Sucrose Density  56  Gradient Centrif ugation  57  DEAE Ion-Exchange Chromatography  58  Preparation of DCCD-Binding Protein  58  P u r i f i c a t i o n of DCCD-Binding Protein  •.  Thin Layer Chromatography  60 .  60  Chromatography on CM-Cellulose  60  Chromatography on Sephadex LH-60  62  SDS-Polyacrylamide Gel Electrophoresis  •  62  Sample Preparation  62  Depolymerization  63  of Samples  Slab-Gel Electrophoresis (i) (ii)  64  Preparation of Separating Gel  i  Preparation of Stacking Gel  Gel Electrophoresis i n Tubes...  64 65  .  65  Gradient Gel Electrophoresis  66  Electrophoresis  68  (i)  Slab Gels  68  (ii)  Tube Gels  68  Two-Dimensional I s o e l e c t r i c Focusing Gel Electrophoresis Preparation of Sample  /68 68  (vii) First-Dimension I s o e l e c t r i c Focusing  69  Determination of pH Gradient  70  Second-Dimension SDS-Polyacrylamide  Gel  70  Crossed Immunoelectrophoresis  71  First-Dimension Gel Electrophoresis  71  Second-Dimension Gel Electrophoresis  72  Staining and Drying Gels SDS-Polyacrylamide  72  Gels  ..  72  Crossed Immunoelectrophoresis Gels  73  P u r i f i c a t i o n of Goat Anti-Rabbit Immunoglobulin by A f f i n i t y Chromatography  74  Preparation of A f f i n i t y Column  74  P u r i f i c a t i o n of Goat Anti-Rabbit Immunoglobulin  75  Radioiodination of Goat Anti-Rabbit Immunoglobulin  75  Peptide .Mapping of CNBr-Cleaved DCCD-Binding Protein  76  One-Dimensional Thin Layer Separation  76  Two-Dimensional Thin Layer Separation  , 76  Detection of Peptides  77  Chemical Modification of Membranes  78  Labelling of Membrane Vesicles of E. c o l i with [ "C]DCCD  78  Treatment of Membrane Vesicles with Phenylglyoxal  78  l  Chemical Modification of the DCCD-Binding Protein  X  79  Hydrolysis of the DCCD-Binding Protein  79  Treatment of the DCCD-Binding Protein with Cyanogen Bromide  80  Treatment of the DCCD-Binding Protein with Performic Acid  80  Treatment of the DCCD-Binding Protein with 2,3-Butanedione or Phenylglyoxal Treatment with Proteases  81 82  (viii)  ECFj^ and DCCD-Binding P r o t e i n  82  P r e p a r a t i o n o f A n t i g e n s f o r Immunization  ECF  82  82  ±  DCCD-Binding P r o t e i n .  83  Immunization o f t h e Rabbit  83  Bleeding the Rabbit  84  S e p a r a t i o n of Serum  85  P a r t i a l P u r i f i c a t i o n o f Immunoglobulins  85  Binding of E C F  86  1  t o Membrane V e s i c l e s  Assays  87  Determination of P r o t e i n  .  K  ,  D e t e r m i n a t i o n o f ATPase A c t i v i t y (i) (ii)  88  R a p i d Assay  88  Slow Assay  88  S u b s t r a t e Oxidation-Dependent 9-Aminoacridine  Quenching o f F l u o r e s c e n c e o f 89  Measurement o f P r o t o n Conduction i n K - l o a d e d Membrane V e s i c l e s . . .  90  D e t e r m i n a t i o n o f Cytochrome Content  90  D e t e r m i n a t i o n of C a t a l a s e A c t i v i t y  91  Determination of R a d i o a c t i v i t y i n Gel S l i c e s  91  S o l i d Phase Radioimmune Assays  92  +  95  RESULTS Part I  P u r i f i c a t i o n of the  S e l e c t i o n o f an E. c o l i  ECF^FQ  Complex  Strain  S o l u b i l i z a t i o n o f the E C F ^ Complex S e l e c t i o n o f Detergent \  87  95 95 99 99  (ix)  Effect  of D e t e r g e n t s on the Membrane-Bound ATPase A c t i v i t y . . . .  S t a b i l i t y of the S o l u b i l i z e d Molecular  Enzyme  105  S i z e of the D e t e r g e n t - S o l u b i l i z e d  105 105  P u r i f i c a t i o n of the E C F - ^ F Q Complex by I n t e r a c t i o n Chromatography  113  Intactness  of the  ECF^Q  Hydrophobic-  Resins  F u r t h e r P u r i f i c a t i o n of the E C F ^ F Q  118  Complex  on Phenyl Sepharose CL-4B  139 in  of the Membrane-Bound ATPase A c t i v i t y  143 to  by DCCD i n the PresenceJ^Cations  Measurement of the P r o t o n Gradient  using  L a b e l l i n g of Membranes of E. c o l i w i t h  137  Protein-Detection  II S t u d i e s on Mutants of E. c o l i D e f e c t i v e Proton-Translocating A c t i v i t y  Inhibition  120 132  Comparison of the G e l E l e c t r o p h o r e s i s and Systems  Sensitivity  1  Complex  R e p r o d u c i b i l i t y of P u r i f i c a t i o n  Part  Enzyme  G e l F i l t r a t i o n on Sepharose 6B o r B i o - G e l A-0.5m  Other H y d r o p h o b i c - I n t e r a c t i o n  Part  103  145  9-Aminoacridine  ['"CJDCCD  145 151  P u r i f i c a t i o n of the DCCD-Binding P r o t e i n  152  Amino A c i d Composition of the DCCD-Binding P r o t e i n  161  A n a l y s i s of the Membranes of E. c o l i by Two-Dimensional I s o e l e c t r i c Focusing Gel E l e c t r o p h o r e s i s  161  III S t u d i e s on the DCCD-Binding P r o t e i n of the Complex of E_. c o l i . •>  168  Fo  E f f e c t of A n t i s e r u m to the DCCD-Binding P r o t e i n on the E n e r g i z a t i o n of the Membrane o f U r e a - S t r i p p e d Everted Membrane V e s i c l e s  168  E f f e c t of A n t i s e r u m t o the DCCD-Binding P r o t e i n on the of ECF-^ to U r e a - S t r i p p e d E v e r t e d Membrane V e s i c l e s  170  Binding  E f f e c t of A n t i s e r u m to the DCCD-Binding P r o t e i n on the Proton P e r m e a b i l i t y of the R i g h t - S i d e Out V e s i c l e s of E_. c o l i .  .171  (x)  I  Effect of Antiserum to the DCCD-Binding Protein on the Energization of the Membrane of Native Everted Membrane Vesicles Energization of the Membrane of Trypsin-Treated  Urea-Stripped  Everted Vesicles i  179  181  Binding of ECF^ to Protease-Treated Membrane Vesicles  185  Effect of DCCD on the ATPase A c t i v i t y of the ECFj^ Bound to Trypsin-Treated Vesicles Immunoprecipitation of the ECF-^FQ Complex with Antiserum  188  Detection by Solid Phase Radioimmune Assay of the Reaction of Antibody with Membrane Vesicles  191  198  Reaction S i t e ( s ) f o r the Antibody on the DCCD-Binding Protein..... 203 Binding of ECF-^ by P u r i f i e d DCCD-Binding Protein  205  Reaction S i t e ( s ) on ECF-^ f o r the DCCD-Binding Protein  209  E f f e c t of Chemical Modification of the DCCD-Binding Protein on i t s Reaction with ECFj  214  Effect of Phenylglyoxal on the Binding of ECF-^ to UreaStripped Everted Vesicles  219  f  DISCUSSION.  225  P u r i f i c a t i o n of the E C F ^ Q Complex  ;  225  Some Mutants of E,. c o l i Defective i n Proton Translocation  229  Orientation of the DCCD-Binding Protein i n the Membrane  234  Interaction of the .DCCD-Binding Protein with ECF  237  REFERENCES  ;  f  1  243  (xi) LIST OF TABLES  Table 1.  2.  Polypeptide Composition Various Sources  of F^Fg-ATPase Complexes from  Properties of Various Preparations of B a c t e r i a l  22 ETJFQ  Complexes  25  3.  Properties of Various Preparations of B a c t e r i a l FQ Complexes...  27  4. 5.  Polypeptides Coded by the "unc" Genes Amino Acid Composition of the DCCD-Binding Protein from Mitochondria, Chloroplast and Bacteria  31  6.  Properties of DCCD-Resistant Mutants of E. c o l i  41  7.  B a c t e r i a l Strains used i n ' t h i s Study  47  8.  Specific A c t i v i t y of the Membrane-Bound ATPase of Different B a c t e r i a l Strains  96  9. 10. 11.  12. 13. 14. 15.  37  S o l u b i l i z a t i o n of the Membrane-Bound ATPase A c t i v i t y of E_. c o l i with Sodium Cholate  102  Effect of Detergent of E. c o l i .  104  on the Membrane-Bound ATPase A c t i v i t y  Estimation of the Molecular Weight of the Solubilized E C F ^ Q Complex by Gel F i l t r a t i o n Chromatography  Ill  Some Properties of the unc Mutants of IS. c o l i used i n t h i s Thesis  144  Amino Acid Composition of the DCCD-Binding Protein from Different Strains of E_. c o l i .  163  Energization of the Membrane of Trypsin-Treated Everted Membrane Vesicles of E. co l i  182  Subunit Composition Purifications  of FQ from Various ECF^Fo and  Fo 227  (xii)  LIST OF FIGURES  Figure  — — — —  1.  t  \  Schematic representation of oxidative phosphorylation and generation of a proton gradient  2  2.. Three models f o r the arrangement of the subunits i n the Fj-ATPase of E. c o l i .  18  3.  4.  5.  Amino acid sequences of the DCCD-Binding Protein from Neurospora crassa bovine heart, Saccharomyces cerevisiae, spinach chloroplasts, Mastigocladus laminosus, Escherichia c o l i , and the thermophilic bacterium PS-3  39  E f f e c t of cations on the membrane-bound ATPase a c t i v i t y of d i f f e r e n t strains of E_. c o l i .  97  S o l u b i l i z a t i o n of the membrane-bound ATPase a c t i v i t y of E_. c o l i by various detergents....  101  6.  S t a b i l i t y of s o l u b i l i z e d ATPase a c t i v i t y on storage at 4 ° C . . .  106  7.  Chromatography of the detergent-solubilized ATPase complex on Sepharose 6B i n the presence of various detergents  8.  E f f e c t of DCCD on the detergent-solubilized ATPase a c t i v i t y . . .  9.  Chromatography of the detergent-solubilized ATPase complex on Phenyl-Sepharose CL-4B P u r i f i c a t i o n of the E C F ^ F Q complex by sucrose gradient c e n t r i f ugation SDS-polyacrylamide gel electrophoresis of the E C F ^ F Q complex p u r i f i e d by chromatography on Phenyl-Sepharose CL-4B and sucrose gradient c e n t r i f ugation  10. 11.  12.  13.  14.  15.  SDS-polyacrylamide g e l electrophoresis of the E C F ^ F Q complex obtained a f t e r chromatograpy on Phenyl-Sepharose CL-4B and sucrose gradient c e n t r i f ugation SDS-polyacrylamide g e l electrophoresis of the E C F ^ F Q complex obtained by chromatography on Phenyl Sepharose J CL-4B and sedimentation at 250 000 xg f o r 16-17 h E f f e c t of DCCD on the ATPase a c t i v i t y of the complex  108 112  115 121  125  128  131  ECF^Q  SDS-polyacrylamide gel electrophoresis of ECF^ and ECF^Fo complex labelled with ['"CJDCCD  134  135  (xiii) 16.  17.  18.  19. 20.  21.  SDS-polyacrylamide g e l electrophoresis of the E C F ^ F Q complex obtained a f t e r chromatography on Phenyl-Sepharose CL-4B: Reproducibility of the p u r i f i c a t i o n  138  Comparison of the SDS-gel electrophoresis and proteindetection systems  140  S e n s i t i v i t y of the membrane-bound ATPase a c t i v i t y to i n h i b i t i o n by DCCD i n the presence of cations  146  Measurement of the proton gradient i n everted membrane v e s i c l e s using the fluorescent dye, 9-aminoacridine  148  E f f e c t of stripping everted membrane v e s i c l e s from the parent (E. c o l i WS1) and mutant (E. c o l i N J 4 4 ) on the energization of the membrane  150  SDS-polyacrylamide g e l electrophoresis of,[ "C]DCCDl a b e l l e d membranes and of ether-precipitated proteins of chloroform-methanol extracts of the l a b e l l e d membranes x  of E. c o l i  153  22.  Thin layer chromatography of the DCCD-binding protein  155  23.  Chromatography of the DCCD-binding protein on CM-cellulose....  157  24.  SDS-polyacrylamide g e l electrophoresis of DCCD-binding protein obtained by chromatography on CM-cellulose Chromatography of the CM-cellulose-purified DCCD-binding protein on Sephadex LH-60  25. 26.  27.  28.  29/.  30.  31.  160 162  Two-dimensional thin-layer chromatography of cyanogen ' bromide cleaved fragments of the DCCD-binding protein of E. c o l i CBT-302  164  Two-dimensional i s o e l e c t r i c focusing g e l electrophoresis of membranes of parent (WS1) and mutant ( N J 4 4 ) strains of E. c o l i  166  E f f e c t of the antiserum to the DCCD-binding protein and of ECF^ on the ascorbate-oxidation-dependent quenching of fluorescence of 9-aminoacridine by urea-stripped everted membrane v e s i c l e s  169  E f f e c t of antiserum to the DCCD-binding protein on the binding of ECF^ to urea-stripped everted membrane v e s i c l e s . . . .  172  Crossed Immunoelectrophoresis of antiserum to the DCCDbinding protein  175  Schematic representation of the proton-pathway provided by the "right-side out" v e s i c l e s of E_. c o l i DL-54  176  (xiv)  32.  33.  34.  35.  36.  37. 38. 39.  40.  ,  Effect of DCCD and of antiserum to the DCCD-binding protein on the proton permeability of "right-side out" membrane v e s i c l e s  177.  E f f e c t of DCCD and of antiserum to the DCCD-binding protein on the proton-permeability of "right-side out" membrane v e s i c l e s of E. c o l i DL-54  178.  E f f e c t of antiserum to the DCCD-binding protein on the energization of untreated everted membrane vesicles  180.  E f f e c t of antiserum to the DCCD-binding protein on the ,ascorbate-oxidation-dependent quenching of fluorescence of 9-aminoacridine by trypsin-treated everted membrane vesicles  184.  Binding of ECF^ to trypsin-treated everted membrane vesicles  186.  Binding of, ECF^ to Staphylococcus aureus Vs proteasetreated everted membrane v e s i c l e s  189.  _ E f f e c t o f DCCD on the ATPase a c t i v i t y of the ECF^ bound to trypsin-treated everted membrane vesicles  192.  x  SDS-polyacrylamide g e l electrophoresis of the complex immunoprecipitated with antiserum  ECF^FQ  193.  Two-dimensional g e l electrophoresis of the ECF^Fo complex' obtained by immunoprecipitation with antiserum to ECF^  196.  T i t r a t i o n of the DCCD-binding protein with antiserum to t h i s polypeptide  199.  I n h i b i t i o n of antibody binding to immobilized DCCD-binding protein by membrane v e s i c l e s of E_. c o l i , PS3 and rat l i v e r mitochondria, and by phospholipid vesicles  200.  I n h i b i t i o n of antibody binding to immobilized DCCD-binding protein by protease-treated or chemically-modified DCCDbinding protein.........  204.  I n h i b i t i o n of antibody binding to immobilized DCCD-binding protein by protease-treated or chemically-modified everted membrane v e s i c l e s  206.  45.  Schematic representation of the radioimmune binding assay  207.  46.  Binding of ECFi to the DCCD-binding protein  47.  E f f e c t of ECFi on the binding of anti-DCCD-binding protein serum to the DCCD-binding protein and the e f f e c t of DCCDbinding protein on the binding of anti-ECF^ serum to ECF^  41. 42.  43.  44.  ... 208.  210.  (ix) E f f e c t of Detergents on the Membrane-Bound ATPase A c t i v i t y . . . .  103  S t a b i l i t y of the Solubilized Enzyme  105  Molecular  Size of the Detergent-Solubilized Enzyme  Gel F i l t r a t i o n on Sepharose 6B or Bio-Gel A-0.5m  105 105  P u r i f i c a t i o n of the ECF^FQ Complex by HydrophobicInteraction Chromatography Other Hydrophobic-Interaction  113 Resins  Further P u r i f i c a t i o n of the ECF^FQ Complex Intactness of the  ECF-JFQ  Complex  Reproducibility of P u r i f i c a t i o n on Phenyl Sepharose CL-4B Comparison of the Gel Electrophoresis and Protein-Detection Systems Part II Studies on Mutants of E_. c o l i Defective i n Proton-Translocating A c t i v i t y  118 120 132 137 139  143  S e n s i t i v i t y of the Membrane-Bound ATPase A c t i v i t y to I n h i b i t i o n by DCCD i n the Presence of Cations  145  Measurement of the Proton Gradient using 9-Aminoacridine  145  Labelling of Membranes of E. c o l i with [^CJDCCD  151  P u r i f i c a t i o n of the DCCD-Binding Protein  152  Amino Acid Composition of the DCCD-Binding Protein  161  Analysis of the Membranes of E_. c o l i by Two-Dimensional I s o e l e c t r i c Focusing Gel Electrophoresis  161  Part I I I Studies on the DCCD-Binding Protein of the FQ Complex of E_. c o l i  168  E f f e c t of Antiserum to the DCCD-Binding Protein on the Energization of the Membrane of Urea-Stripped Everted Membrane Vesicles  168  Effect of Antiserum to the DCCD-Binding Protein on the Binding of ECF^ to Urea-Stripped Everted Membrane Vesicles  170  E f f e c t of Antiserum to the DCCD-Binding Protein on the Proton Permeability of the Right-Side Out Vesicles of E_. c o l i  171  (xv) 48.  SDS-polyacrylamide g e l electrophoresis of subunits of ECF^....  49.  Binding of ECF^ to the DCCD-binding protein:  211  Effect of  protease treatment  212  50.  Binding of DCCD-binding protein to subunits of ECF^  215  51.  Effect of chemical modification of the DCCD-binding protein on the binding of ECF-^ Effect of modification of the a r g i n y l residue(s) of the DCCD-binding protein on the binding of ECF-L  52.  216 217  53.  Binding of ECF^ to phenylglyoxal-treated v e s i c l e s  220  54.  SDS-polyacrylamide g e l electrophoresis of the DCCD-binding protein of E. c o l i labelled with [7 "C]phenylglyoxal  222  Amino acid sequence of the DCCD-binding protein of E. c o l i  238  1  55.  (xvi) ABBREVIATIONS  ADP  Adenosine-5 -diphosphate.  AH-Sepharose 4B  Amino Hexyl Sepharose 4B.  Aminoxid WS-35  Acyl  ATP  Adenosine-5'-triphosphate.  Ammonyx Lo  Lauryl dimethylaminoxide.  B r i j 35  Polyoxyethylene (23) l a u r y l ether.  BSA  Bovine serum albumin.  Chloramine T  N-chloro-4-methylbenzenesulphonamide, salt.  CMC CM-cellulose DCCD DEAE-Sepharose CL-6B DNase DTT EDTA  1  aminopropyldimethylaminoxide.  sodium  C r i t i c a l m i c e l l a r concentration. Carboxymethyl  cellulose.  N,N'-dicyclohexylcarbodiimide. Diethylaminoethyl-Sepharose CL^6B. Deoxyribonuclease. Dithiothreitol. (Ethylenedinitrilo)-tetraacetic  acid.  EGTA  [Ethylenebis(oxyethylenenitrilo)]-tetraacetic > acid.  F-L -ATPase  ATP phosphohydrolase ( c a t a l y t i c portion of the proton-translocating adenosine triphosphatase); CF chloroplast F -ATPase; ECFj, E_. c o l i Fj^-ATPase; MF-^, mitochondrial Fj-ATPase; TF , F-j-ATPase from the thermophile, PS3. l9  1  1  F-^Fg-ATPase complex  Proton-translocating adenosine triphosphatase; ECF-J^FQ, _E. c o l i F-LFQ complex; T F J F Q , F^FQ complex from the thermophile, PS3.  HEPES  N-2-hydroxyethylpiperazine N'-2-ethanesulphonic acid.  HPLC  - High performance (pressure) l i q u i d chromatography.  (xvii) Lubrol 17A-10  Polyoxyethyleneglycol (n=?) c e t y l - s t e a r y l alcohol.  Lubrol PX  Polyoxyethyleneglycol (n=?) c e t y l - s t e a r y l alcohol.  Lubrol WX  Polyoxyethyleneglycol (17) c e t y l - s t e a r y l alcohol.  MOPS  Morpholinopropanesulphonic  NADH  Reduced nicotinamide adenine dinucleotide.  NAP4-ADP  3'-0-(4-[N-(4-azido-2-nitrophenyl) butyryl) ADP.  amino]  NAP4-ATP  3'-0-(4-[N-(4-azido-2-nitrophenyl) butyryl) ATP.  amino]  Nbf-Cl  4-chloro-7-nitrobenzofuran.  NEM-Hg  Mercuriated N-pyrrolo-isomaleinimide.  Ninhydrin  1,2,3-triketohydrindene  Nonidet P-40  Polyoxyethyleneglycol (9) p-t-octylphenol.  Pi  inorganic phosphate.  PBS  Phosphate-buffered saline (0.137 M NaCl, 2 mM KC1, 1.47 mM KH P0 , 8.09 mM Na HP0 , pH 7.5). 2  2  acid  hydrate.  4  4  PEG  Polyethyleneglycol.  PMS  Phenazine methosulphate.  PMSF  Phenylmethysulphonylfluoride. J  psi RNase SDS Sephadex LH-20 Sephadex LH-60 TAMM TCA  Pounds per square inch. Ribonuclease. Sodium dodecyl sulphate. Hydroxypropyl  Sephadex G-25.  Hydroxypropyl  Sephadex G-50.  Tetrakis (acetoxymercuri) methane. Trichloroacetic acid.  (xviii) TEMED  - N,N,N',N'-tetramethylethylenediamine.  TPCK-trypsin  - Trypsin treated with L-(tosylamido ethyl chloromethyl ketone.  Tris Triton X-100 T r i t o n X-114 Tween 60  2-phenyl)  - T r i s (hydroxymethyl)-aminoethane. - Polyoxyethyleneglycol (9-10) p-t-octylphenol. - Polyoxyethyleneglycol (7-8) p-t-octylphenol. - Polyoxyethyleneglycol (20) s o r b i t o l monostearate. - Polyoxyethyleneglycol (20) s o r b i t o l monooleate.  Tween 80  - Membrane p o t e n t i a l . - Difference i n pH across the membrane.  A  pH  - Electrochemical potential difference of protons across the membrane, proton-motive force.  (xix)  ACKNOWLEDGEMENT S  I thank Dr. P.D. Bragg f o r his advice, patience and understanding throughout my graduate studies and during the writing of this thesis. The completion  of this thesis would not have been possible without the  help of the many members of the Department of Biochemistry.  In p a r t i c u l a r ,  I thank Drs. R.S. Molday, R. Barton and J.C. Brown (Physiology) f o r access to the equipment i n t h e i r laboratories. I especially thank Mr. D.J. Mackenzie f o r help i n setting up the radioimmunoassay experiments and f o r the photography.  I am very g r a t e f u l to my colleagues, Mr. David M. Clarke,  Ms. Cynthia Hou and Dr. Helga Stan-Lotter f o r providing a pleasant atmosphere which was conducive to research and collaboration. I am also ' grateful to the Medical Research Council of Canada for supporting work.  this  The typing of t h i s manuscript by Ms. Judith Smith i s greatly  appreciated. F i n a l l y , I would l i k e to acknowledge the support of my family during this project.  1.  INTRODUCTION  The  r e v e r s i b l e , p r o t o n - t r a n s l o c a t i n g adenosine  (F^F^-ATPase complex) p l a y s a major r o l e i n t h e reactions of the c e l l .  triphosphatase  energy-transduction  S t r u c t u r a l l y s i m i l a r forms o f t h e enzyme a r e found  i n t h e membranes o f e u k a r y o t e s and p r o k a r y o t e s ( 1 - 4 ) .  The enzyme c a t a l y z e s  t h e s y n t h e s i s o f ATP by o x i d a t i v e o r p h o t o - p h o s p h o r y l a t i o n , h y d r o l y s i s o f ATP ( 5 ) a c c o r d i n g  and t h e  to the reaction.  nH?" + ATP + H 0 in 2 o  ADP + P. + n H l out +  The ATPase complex c o n s i s t s o f two f u n c t i o n a l u n i t s , F^ and F Q . F^ i s a complex o f e x t r i n s i c membrane p r o t e i n s c o n t a i n i n g t h e a c t i v e s i t e ( s ) o f ATP s y n t h e s i s and h y d r o l y s i s ( 6 - 8 ) .  The F  interacts with  FQ, w h i c h i s a complex o f i n t e g r a l membrane p r o t e i n s n o t h a v i n g ATPase activity.  The FQ complex i s thought t o extend t h r o u g h t h e membrane and  f u n c t i o n s as a pathway f o r t h e r e v e r s i b l e t r a n s l o c a t i o n o f p r o t o n s t h r o u g h the membrane ( 9 , 1 0 , 1 4 ) . The  e x i s t e n c e o f r e v e r s i b l e , p r o t o n - t r a n s l o c a t i n g pumps i s an  i m p o r t a n t p o s t u l a t e o f M i t c h e l l ' s chemiosmotic h y p o t h e s i s of o x i d a t i v e phosphorylation  (11).  According  on t h e mechanism  t o t h i s hypothesis, the  components o f t h e e l e c t r o n t r a n s p o r t c h a i n a r e a r r a n g e d i n t h e membrane s u c h t h a t t h e r e i s v e c t o r i a l t r a n s l o c a t i o n o f p r o t o n s a c r o s s t h e membrane during r e s p i r a t i o n or light-dependent  e l e c t r o n f l o w ( F i g 1) (5,12,13).  The  v e c t o r i a l t r a n s l o c a t i o n o f p r o t o n s g e n e r a t e s a p r o t o n - m o t i v e f o r c e (Au +), w h i c h c o n s i s t s o f two components: t h e membrane p o t e n t i a l (A\|>) , g e n e r a t e d as a r e s u l t o f charge s e p a r a t i o n , and t h e c h e m i c a l c o n c e n t r a t i o n (ZApH).  The r e l a t i o n i s g i v e n by t h e f o l l o w i n g  equation:  gradient,  2.  FIG 1:  Schematic representation of oxidative phosphorylation and generation of a proton gradient. "mH " and "nH " represent protons which are translocated, but whose exact number has not been agreed on. +  +  Ajfjjf = Aip-ZApH  (where Z =  ^ — = 59 mV a t 25°C)  The energy stored within t h i s proton gradient i s used to drive other energy-requiring processes.  For example, the synthesis of ATP i s coupled  to the return of protons down the gradient through,the F^Fg-ATPase complex.  S i m i l a r l y , the proton gradient can be regenerated  through the  hydrolysis of ATP. A stoichiometry of two or three protons translocated per molecule of ATP hydrolyzed or synthesized has been measured (5,15). Some microorganisms such as Streptococcus f a e c a l i s ,  Streptococcus  l a c t i s (2) and the s t r i c t anaerobe Clostridium pasteurianum (16), growing i n the presence of a l i m i t i n g amount of glucose or i n the absence of oxygen or other terminal electron acceptors, cannot carry out oxidative phosphorylation.  This i s because t h e i r respiratory chains are either  non-functional or non-existent.  These organisms rely solely on the ATP  produced by substrate-level phosphorylation ( g l y c o l y s i s ) and the -^FQ f  complex to generate a proton-motive force, which i s capable of driving other energy-requiring  processes.  LOCATION OF THE F J F Q COMPLEX ATPase a c t i v i t y was shown to be l o c a l i z e d i n the plasma membrane of Streptococcus  f a e c a l i s (17) , B a c i l l u s cereus and Escherichia c o l i (18).  F e r r i t i n - l a b e l l e d antibody against p u r i f i e d F^-ATPase was used to show that the F^ was located on the inner surface of the b a c t e r i a l plasma membrane (19).  Negatively-stained preparations of everted membrane v e s i -  cles of E_. c o l i revealed the presence of "knobs", each with a diameter of about 100 A, attached to the inner surface of the plasma membrane v i a a "stalk".  A similar morphology e x i s t s i n the submitochondrial p a r t i c l e s and  4.  i n the thylakoid membranes (6,20).  ROLE OF THE FjFp COMPLEX IN ENERGY TRANSDUCTION The r o l e of the F^F^ complex of 15. c o l i i n energy-transduction reactions has been demonstrated  through reconstitution studies and the  i s o l a t i o n of mutants. The F^ component of the F^F^ complex can readily be released from the everted membrane v e s i c l e s of E_. c o l i by washing them i n buffers of low ionic strength containing EDTA (21-23,33).  The ECF^depleted mem-  branes no longer exhibit energy-transducing properties such as r e s p i r a t i o n induced proton uptake, ATP-driven and respiration-driven transhydrogenation of NADP by NADH, or oxidative phosphorylation. +  Restoration of these  a c t i v i t i e s to normal levels can be achieved by the addition of p u r i f i e d 2+ ECF^-ATPase to the depleted membranes i n the presence of Mg i o n i c strength buffer (21-25,33).  or high  These results suggest that the loss of  the energy-transducing properties of ECF^-depleted membranes i s due to an increased permeability of the membranes to protons upon the removal of ECF^, such that the proton-gradient cannot be b u i l t up or maintained by respiration. Proton impermeability can also be restored i n the ECF^-depleted membranes by treatment with either ECF^, isolated from an unc A mutant, which has no ATP hydrolytic a c t i v i t y or with DCCD.  DCCD i s a potent  i n h i b i t o r of the ATPase a c t i v i t y of the F ^ Q complex and under F  appropriate conditions i t reacts with a s p e c i f i c component of F Q and i n h i b i t s proton-translocating a c t i v i t y (discussed under "The DCCD-binding protein"). In the E. c o l i mutants, DL-54 and NR-70, the F, i s either defective  or absent (26,27).  Membrane v e s i c l e s or whole c e l l preparations of these  mutants show decreased l e v e l s of respiration-driven transhydrogenation and transport of proline than i n the corresponding parent-strains.  K -loaded +  spheroplasts of E_. c o l i DL-54 also show a larger change i n the pH of the external medium upon addition of valinomycin, than i s seen with the w i l d type s t r a i n (28).  The increased permeability of these mutant membranes to  protons i s due to an exposed F Q since the addition of DCCD or ECF^ causes the membranes to become less permeable to protons. These r e s u l t s suggest (24) that  ( i ) the ECF^ i s responsible not  only f o r the energization of the membrane through ATP hydrolysis, but also f o r maintaining the impermeability of the membranes to protons by i n t e r acting with F Q , and  ( i i ) the F Q acts as a proton-specific channel or  pore.  ( The concept of F Q being a proton-specific pore i s complicated by the  observations that i t s proton-conducting properties i n E. c o l i DL-54 and NR-70 varies s i g n i f i c a n t l y when the growth conditions are varied i n aerobic culture (29-32).  PROPERTIES OF F Subunit Composition of F^ The molecular weight of ECF^ has been determined by a variety of methods, with the following values: (33);  g e l f i l t r a t i o n , 360 000 - 390 000  sedimentation equilibrium, 360 000 - 390 000 (34);  scattering, 362 000 (35);  laser l i g h t  small angle X-ray scattering, 35 8000 (36);  sedimentation c o e f f i c i e n t , d i f f u s i o n c o e f f i c i e n t and p a r t i a l s p e c i f i c volume, 350 000 (36);  l i g h t scattering, 345 000 (36) and small angle  neutron scattering, 315 000 (37).  Three problems e x i s t which make i t d i f f i c u l t  t o determine t h e c o r r e c t  m o l e c u l a r weight o f ECF^ ( 6 ) . F i r s t , t h e r e i s the tendency o f t h e enzyme to  d i s s o c i a t e , r e s u l t i n g i n the l o s s o f some s u b u n i t s o f ECF^.  Secondly,  the extent o f t h e l o s s o f the S s u b u n i t d u r i n g p u r i f i c a t i o n o f ECF^ i s v a r i a b l e and t h i s i s dependent t o some e x t e n t on t h e c h o i c e o f t h e p u r i f i c a t i o n procedure.  F i n a l l y , the amount of $ subunit i n ECF^  p r e p a r a t i o n s i s a l s o dependent on t h e source, f o r the amount of 6 subunit i n F^ p r e p a r a t i o n s i s o l a t e d from _E. c o l i than t h a t from t h e E_. c o l i ML s t r a i n s . weight of the ECF^ t o be underestimated. ECF^  s t r a i n s i s more v a r i a b l e These f a c t o r s cause the m o l e c u l a r The a c t u a l m o l e c u l a r weight o f  may be c l o s e r t o t h e m o l e c u l a r weight o f the more s t a b l e TF^,  which has been r e p o r t e d t o be 380 000 ( 3 8 ) . The F^ from a l l sources pasteurianum  (with the exception of C l o s t r i d i u m  and L a c t o b a c i l l u s c a s e i ) g e n e r a l l y c o n t a i n f i v e  p o l y p e p t i d e s ( a , J3, Y,  different  6 and e) ( 4 ) . MF^ has an a d d i t i o n a l  subunit  ( T a b l e 1) which i s a n a t u r a l i n h i b i t o r o f the ATPase a c t i v i t y , but i s unnecessary  f o r ATP s y n t h e s i s ( 2 4 ) .  c o n s i s t s of only three d i f f e r e n t  The F^ from C_. pasteurianum  (16)  s u b u n i t s w i t h m o l e c u l a r weights o f 65 000,  57 000 and 43 000, w h i l e t h a t from L_. c a s e i (39) c o n t a i n s o n l y one p o l y p e p t i d e o f 43 000 d a l t o n s . The m o l e c u l a r weights o f each o f the s u b u n i t s o f ECF^, TF^, CF^ and MF^ a r e v e r y s i m i l a r ( 4 0 ) , w i t h those o f ECF^ r e p o r t e d t o be a, 56 800;  B, 51 800; Y, 32 000; 6, 20 700;  and e, 13 200 (7,41).  a r e i n good agreement w i t h t h e m o l e c u l a r weights determined sequence (42) o f each p o l y p e p t i d e : 6, 19 310; The  a,  These  from t h e DNA  55 264; 6, 50 157; Y, 34 100;  and e, 14 194.  r a t i o o f these s u b u n i t s i n F., remains c o n t r o v e r s i a l .  Most o f  the data on F^ from b a c t e r i a and y e a s t m i t o c h o n d r i a stoichoimetry  (8,41,43-44,72,155).  mammalian m i t o c h o n d r i a  3  J 3  3  Y 5 e  s  t  o  i  c  h  i  o  Tightly-Bound CF^,  m  e  t  r  y  an a^B^ySe  Although the d a t a on the F^^ from  and c h l o r o p l a s t suggest 2 ^ 2 ^ 2 ^ 2 2 a  s t o i c h i o m e t r i e s , r e s p e c t i v e l y (45-48); a  support  e  a n c  *  a  more r e c e n t e s t i m a t e s  2^2 ^ (l-2) Y  6  support  the  (4"9).  Nucleotides  ECF^,  MF^  and  TF^ a l l c o n t a i n n o n - c o v a l e n t l y  bound n u c l e o t i d e s ,  which cannot be removed even a f t e r e x t e n s i v e p u r i f i c a t i o n of the enzyme (50,51,73).  In E_. c o l i ,  t h r e e m o l e c u l e s of t i g h t l y - b o u n d n u c l e o t i d e s  m o l e c u l e of F^ were d e t e c t e d . were ATP  and ADP.  r e p o r t e d , but  o n l y n u c l e o t i d e s found t o be  t h i s c o u l d be a t t r i b u t e d to the d i f f e r e n t procedures used f o r and  f o r nucleotide detection.  f u n c t i o n ( s ) of these t i g h t l y - b o u n d n u c l e o t i d e s has not  established.  The  turnover  r a t e of the t i g h t l y - b o u n d ATP  t o be i n v o l v e d as an i n t e r m e d i a t e  i n oxidative phosphorylation  " r e g u l a t o r y " s i t e r a t h e r than i n the a c t i v e s i t e .  In  At p r e s e n t ,  (52).  It i s  the there i s  i n f o r m a t i o n about the r e g u l a t i o n of the enzyme In v i v o .  FUNCTION OF THE  SUBUNITS OF  ECF^  U n d e r s t a n d i n g the f u n c t i o n and ECF^  been  i s too slow f o r i t  more l i k e l y t h a t these t i g h t l y - b o u n d n u c l e o t i d e s a r e present  very l i t t l e  present  D i f f e r e n t molar r a t i o s of these n u c l e o t i d e s were  the p r e p a r a t i o n of ECF^ The  The  per  arrangement of the s u b u n i t s of  i s e s s e n t i a l t o the d e t e r m i n a t i o n  phosphorylation.  the  of the mechanism of o x i d a t i v e  S e v e r a l approaches t o the study of the f u n c t i o n and  p r o p e r t i e s of the s u b u n i t s of ECF^  have been used.  c h e m i c a l m o d i f i c a t i o n of s p e c i f i c r e s i d u e s , a f f i n i t y  These have i n c l u d e d labelling,  8.  immunological  techniques, genetics, and subunit i s o l a t i o n and holoenzyme  reconstitution. Recently, methods have been developed f o r the d i s s o c i a t i o n and i s o l a t i o n of the subunits of ECF^ i n non-denatured form such that a functional ATPase could be reconstituted from i t s subunits (6).  The Delta (6) Subunit ECF^ preparations can be obtained which are deficient i n the 6 subunit (53-57).  This 6-deficient, four-subunit enzyme ( i . e . a^ByrO,  has an ATPase a c t i v i t y equal to that of the native, five-subunit enzyme. However, the former i s incapable of reconstituting respiration-driven and ATP-driven transhydrogenase  a c t i v i t y i n F^-depleted membranes.  Also,  ATPase a c t i v i t y was not detected i n the depleted membranes, which had been reconstituted with the 6-deficient ATPase. Smith and Sternweiss (58,59) have p u r i f i e d the 6 subunit, by treatment of ECF with 50% pyridine followed by chromatography of the 1  f r a c t i o n containing the 6 and e subunit on Sephadex G-75.  Addition of  the p u r i f i e d <5 subunit to the <S-deficient enzyme restored the membranebinding c a p a b i l i t y of the enzyme.  Besides rebinding, ATP-driven  transhy-  drogenase a c t i v i t y and the formation of ATP by oxidative phosphorylation were restored i n these depleted membranes (57). These results suggest that the 6 subunit i s involved i n the binding of ECF^ to the membrane and that the binding of 6 restores the ATPase to i t s native state.  The role  of the <5 subunit i n attaching the F^ to the membrane has also been confirmed f o r (62).  (60), TF.^ (61) and F  1  from Streptococcus f a e c a l i s  9.  The E p s i l o n (e)  Subunit i  By p a s s i n g the $ - d e f i c i e n t , f o u r - s u b u n i t enzyme ( i . e .  a^Q^ye)  t h r o u g h an a f f i n i t y column c o n t a i n i n g i m m o b i l i z e d a n t i b o d i e s t o t h e e s u b u n i t , a t h r e e - s u b u n i t enzyme ( i . e . ot^B^Y) w h i c h i s d e f i c i e n t i n the e s u b u n i t has been o b t a i n e d ATPase a c t i v i t y w h i c h was s u b u n i t enzyme.  (63).  T h i s t h r e e - s u b u n i t enzyme had  an  10-15% g r e a t e r t h a n t h a t of the n a t i v e , f i v e -  The p u r i f i e d 6 or e s u b u n i t c o u l d b i n d t o the t h r e e -  s u b u n i t enzyme t o f o r m f o u r - s u b u n i t complexes ( i . e . a^B^Y^  o r  a^B^Ye).  N e i t h e r of t h e s e complexes c o u l d b i n d ECF^-depleted membranes and r e c o n s t i t u t e o x i d a t i v e p h o s p h o r y l a t i o n a c t i v i t y u n l e s s b o t h 6 and  e were p r e s e n t .  T h i s s u g g e s t s t h a t b o t h 6 and e a r e i n v o l v e d i n b i n d i n g ECF^ membrane and  to the  t h a t t h e " s t a l k s " seen-, i n n e g a t i v e l y - s t a i n e d p r e p a r a t i o n s  e v e r t e d E. c o l i membrane v e s i c l e s may  of  be composed of the 6 and e  subunits ( 6 ) . Thus, i t might be expected t h a t t h e 6 and e s u b u n i t s i n t e r a c t d i r e c t l y w i t h the FQ component i n the membrane.  should However, the  b i n d i n g of e i t h e r p u r i f i e d 6 o r e s u b u n i t t o ECF^-depleted membranes c o u l d not be d e t e c t e d by u s i n g a n t i b o d i e s r a i s e d a g a i n s t e i t h e r o f subunits.  these  F u r t h e r m o r e , a m i x t u r e o f p u r i f i e d Y , $ and e s u b u n i t s d i d  not reduce t h e p r o t o n p e r m e a b i l i t y o f the d e p l e t e d membranes as measured by r e s p i r a t i o n - d r i v e n transhydrogenase (6,63). e s u b u n i t s of TF^  By c o n t r a s t , b o t h the 6 and  c o u l d b i n d t o the TFQ w h i c h had been r e c o n s t i t u t e d  i n t o l i p o s o m e s (61) and t o t h i s TFQ - 6e complex c o u l d be bound e i t h e r the p u r i f i e d Y s u b u n i t o r a 3 s u b u n i t enzyme of TF^,  containing  a B Y. 3  3  A second f u n c t i o n has been a s s i g n e d t o the e s u b u n i t . non-competitive  i n h i b i t o r o f the ATPase a c t i v i t y of p u r i f i e d  It is a ECF-.  10.  Addition of the p u r i f i e d e subunit to either the five-subunit enzyme (a^B^Y^e), (a^Y)  t  n  e  four-subunit enzyme (a^B^Y^) or the three-subunit enzyme  resulted i n 70-90% i n h i b i t i o n of the ATPase a c t i v i t y (58,64).  The  i n h i b i t i o n of the a c t i v i t y of the five-subunit enzyme can be explained by the dissociable nature of the e subunit.  The ATPase a c t i v i t y of an  ECF^ preparation was increased by more than f o u r - f o l d following d i l u t i o n of the enzyme preparation.  This suggested that by d i l u t i n g the ECF^  preparation, the endogenous e subunit dissociated from the enzyme and was i n equilibrium with the ECF^-e complex.  Addition of anti-e-serum to  an ECF^ preparation also stimulated the a c t i v i t y of the enzyme by more than two-fold.  Immunoprecipitation of a reconstituted five-subunit enzyme,  i n which the e subunit was labelled with  1 2 5  1 , with anti-e-serum,  resulted i n quantitative p r e c i p i t a t i o n of r a d i o a c t i v i t y (65). However, no ATPase a c t i v i t y was detected i n the p r e c i p i t a t e . The anti-e-serum, therefore promoted the d i s s o c i a t i o n of e from the F^ either d i r e c t l y or by the removal of free e which was i n equilibrium with the ECF^-e complex. It appears that the relationship of the e subunit to ECF^ i s similar to that of the mitochondrial ATPase i n h i b i t o r protein to MF^, i n that both may be involved i n determining  whether the ATPase i s operating  i n the d i r e c t i o n of ATP hydrolysis or synthesis.  I t has been suggested  (66,67) that substrate oxidation or a low molar ratio of ATP/ADP tends to decrease the i n t e r a c t i o n between the i n h i b i t o r and MF^.  The k i n e t i c data  (64) on ECF^ also suggest that ATP accelerates the release of the e subunit from ECF^, whereas ADP prevents t h i s a c t i v a t i o n , perhaps by s t a b i l i z i n g the i n t e r a c t i o n of e with ECF^. The e subunit of ECF^ d i f f e r s from the mitochondrial protein (6) i n that  inhibitor  ( i ) the mitochondrial ATPase i n h i b i t o r protein i s not  11.  necessary f o r attaching MF^ to the membrane, and  ( i i ) the mitochondrial  ATPase i n h i b i t o r protein i n h i b i t s the ATPase a c t i v i t y of the membrane-bound enzyme, whereas an excess of e subunit has no effect on t h i s a c t i v i t y i n J£. c o l i membranes.  The Gamma (Y) Subunit The a c t i v i t y of p u r i f i e d ECT^ i s stimulated by up to 100% by brief treatment  of the enzyme with t r y p s i n (41).  Examination of the t r y p s i n -  treated enzyme on SDS-polyacrylamide gels revealed that the 5 and e subunits, and to a small extent the Y subunit, were completely (53,63).  destroyed  The resulting enzyme following trypsin-treatment i s e s s e n t i a l l y a  two subunit enzyme ( i . e . 2 3^ a  Q  with three copies of each subunit.  Dunn  and coworkers (68) have shown that trypsin-treatment removes the f i r s t 15 residues from the NI^ terminus of each of the a subunits and that a fragment of the Y subunit of 10 000 dalton remained bound to the enzyme. Although the p u r i f i e d e subunit i n h i b i t e d the ATPase a c t i v i t y of the three-subunit enzyme ( i . e . a^B^Y), i t had no e f f e c t on the a c t i v i t y of the trypsin-treated enzyme.  Incubation of the trypsin-treated enzyme following  c o l d - d i s s o c i a t i o n with a Y r i c h f r a c t i o n prepared from native ECF^ -  restored the s e n s i t i v i t y of the trypsin-treated enzyme to i n h i b i t i o n by the e subunit (69).  These results suggest that the Y subunit i s required  to bind the e subunit to ECF^ and i s supported by the observation (70) that there i s a high a f f i n i t y i n t e r a c t i o n between p u r i f i e d Y and e subunits, i n a 1:1 r a t i o .  This i n t e r a c t i o n i s s p e c i f i c since interactions  of the p u r i f i e d e with either p u r i f i e d a or Q subunits was not detected. Also, the addition of p u r i f i e d Y subunit to a p u r i f i e d e preparation prevented  the l a t t e r from i n h i b i t i n g the ATPase a c t i v i t y of a three-subunit  12.  enzyme ( a ^ B ^ ) ' Thus the y subunit not only binds the e subunit but i t , or a portion of i t , also holds the a and fi subunits together.  The Alpha (a) and Beta (13) Subunits ECF^ has the property of being c o l d - l a b i l e and cold-dissociable. Taking advantage of t h i s , Vogel and Steinhart (77) dissociated the enzyme by freezing i t i n a solution of high i o n i c strength. chromatography, the dissociated enzyme was  separated into three f r a c t i o n s ,  none of which possessed any c a t a l y t i c a c t i v i t y . a, y and e subunits, f r a c t i o n 2, the a, y 3, only the B-subunits.  t  Through ion-exchange  Fraction 1 contained the  6 and e subunits, and f r a c t i o n  ATPase a c t i v i t y could be reconstituted by  combining either fractions 1 and 3 or 2 and 3 but not 1 and 2.  This was  the f i r s t i n d i c a t i o n that the active s i t e ( s ) for ATP hydrolysis was probably on the a and B subunits. Recently, Dunn and Futai (72) have been able to purify the i n d i v i d u a l subunits from the cold-dissociated enzyme. reconstituted to give enzyme a c t i v i t y .  The p u r i f i e d subunits could be  The highest s p e c i f i c a c t i v i t y  obtained when the molar r a t i o of the a, fi and y subunits was  was  3:3:1  ( i . e . ot^fi^Y)., The Y subunit was e s s e n t i a l f o r expression of ATPase a c t i v i t y since a reconstituted a and fi (1:1) preparation had only  0-10%  of the maximum a c t i v i t y . ECF^ of the unc A mutant has no hydrolytic a c t i v i t y , but i s indistinguishable from the parent enzyme i n subunit composition, bound nucleotides and possession of an i n h i b i t o r - s e n s i t i v e (Nbf-Cl) tyrosine residue on the fi subunit (24,73).  The a c t i v i t y of the mutant ECF^ could  be restored by replacing the cold-dissociated enzyme f r a c t i o n containing  13.  the o f , Y and e subunits with the corresponding  f r a c t i o n obtained from  the wild-type s t r a i n (74). S i m i l a r l y , the ATPase a c t i v i t y could be restored i n the  from the unc A mutant, E_. c o l i AN120, by d i a l y z i n g the  t o t a l cold-dissociated enzyme together with an excess of p u r i f i e d o-subunit from the wild-type s t r a i n .  The 13 and Y subunits, p u r i f i e d  from the wild-type ECF^ d i d not restore ATPase a c t i v i t y i n the mutant enzyme.  These r e s u l t s indicate that the l e s i o n responsible f o r the lack of  ATPase a c t i v i t y i n the unc A mutant resides i n the « subunit (75,76).  STUDIES ON THE ACTIVE SITE OF ECFj^ It was previously mentioned that the tightly-bound nucleotides of ECF^ are u n l i k e l y to be involved as intermediates of oxidative phosphoryl a t i o n and are probably involved i n the regulation of enzyme a c t i v i t y . Therefore, the intermediates of oxidative phosphorylation must be at other nucleotide-binding s i t e s .  Detection of these binding s i t e s have involved  the use of photo-affinity labels as well as compounds which l a b e l s p e c i f i c amino acids and i n h i b i t the ATPase a c t i v i t y of the isolated ECF^ (6). Under s l i g h t l y a c i d i c conditions (pH 6.5), the compound DCCD reacts with a carboxyl residue on the B subunit of ECF^ and i n h i b i t s the ATPase a c t i v i t y (77,78).  The reaction with DCCD also affected the a b i l i t y of the  ECF^ to bind to ATP, suggesting that there i s a loss of an ATP-binding site. The compound Nbf-Cl, which can react with cysteine and tyrosine residues, i n h i b i t s the ATPase a c t i v i t y of ECF^. ECF  1  Prolonged incubation of  (24 h) with [^CJNbf-Cl (53), resulted i n the B subunit  being  p r e f e r e n t i a l l y l a b e l l e d , but a f t e r a shorter period of incubation (30 min) (79), most of the l a b e l was associated with the « subunit.  The binding  14.  of Nbf-Cl to ECF (80). ADP  1  also resulted i n the loss of a nucleotide-bindlng s i t e  P h o t o - a f f i n i t y l a b e l l i n g of ECF  1  with arylazido analogs of ATP  and  (NAP^-ATP and NAP^-ADP) showed that the i n a c t i v a t i o n of ATPase  a c t i v i t y was associated with the incorporation of 2 mol NAP^-ADP per mol ECF  1  (80).  At low concentrations (5 uM) of either analog, the a  subunit was p r e f e r e n t i a l l y l a b e l l e d , but at high concentrations (75 both ot and fi subunits were labelled to the same extent.  This  uM),  suggests  the presence of a high a f f i n i t y nucleotide-binding s i t e ( s ) on the ct subunit and a lower a f f i n i t y binding s i t e on the J3 subunit and that- the i n t e r a c t i o n of both s i t e s may  be e s s e n t i a l f o r expression of a c t i v i t y .  Other l a b e l s which also bind to the a subunit are [2,8- H]-ATP, [2,8- H]3  ADP  (72) and 8-azido ATP  (79).  3  In the ECF^ of the unc A mutant, E. c o l i  AN120, the ct subunit was not l a b e l l e d with 8-azido  ATP.  / Bragg et a l . (81,82) have used the 2',3'-dialdehyde derivates of ADP(oADP) and ATP(oATP) to probe f o r nucleotide-binding s i t e s involved i n ATP hydrolysis i n ECF^. respective binding s i t e s .  These compounds react covalently at t h e i r Only the binding of oADP resulted i n the  i n h i b i t i o n of ATPase a c t i v i t y . located on the ct subunit.  Both the oADP and oATP binding s i t e s were  The ct subunit of the unc A mutant, IS. c o l i  AN120, did not possess the oADP binding s i t e ( s ) . that the oADP-binding s i t e on the ct subunit may  These results suggest be the active s i t e ( s ) f o r  ATP hydrolysis, while the oATP-binding s i t e ( s ) may  be regulatory s i t e s .  The compounds DCCD and Nbf-Cl also affected the binding of oADP to the ct subunit.  The binding of oATP was influenced to a lesser extent.  i n h i b i t i o n of binding of oADP to the active s i t e may  The  be due to a conforma-  t i o n a l change induced i n the ct subunit as a result of reaction of the Q subunit with DCCD or Nbf-Cl.  However, i t i s also possible that the  15.  presence of these substituents on thefisubunit could s t e r i c a l l y prevent the binding of oADP.  The results suggest that the oADP-binding s i t e on the  a subunit must l i k e l y be adjacent to the active carboxyl, tyrosyl or c y s t e i n y l residue on the B subunit.  Therefore, the active s i t e would  l i k e l y be at the interface of the a and fi subunits.  Such an arrangement  would explain why i n d i v i d u a l a orfior both subunits have low ATPase a c t i v i t y and that the maximum a c t i v i t y i s obtained when the a,fiand y subunits are present.  Presumably the y subunit i s necessary to hold the  a and fi subunits i n the proper conformation, such that the active s i t e i s preserved.  CROSS-RECONSTITUTION STUDIES Reconstitution of energy-transducing reactions i n F^-depleted membranes with F^ i s not r e s t r i c t e d to ATPases from the same species. Schatz et a l . (83) f i r s t reported hybrid reconstitution between F^ from Baker's yeast and F^-depleted submitochondrial p a r t i c l e s of beef heart, with the restoration of oxidative phosphorylation a c t i v i t y . and ATP-driven transhydrogenase  Respiration  a c t i v i t y was also demonstrated i n  Fj--depleted membranes of E. c o l i , which were reconstituted with F^ from Salmonella typhimurium (41).  S i m i l a r l y , hybrid reconstitution between rat  l i v e r MF^ and F^-depleted submitochondrial membranes from human l i v e r carcinoma has also been demonstrated (84).  More recently (85), ATP-driven  proton uptake was demonstrated i n the membranes by cross-hybrid reconstitution between F^ and F^-depleted membranes of either 15. c o l i or rat l i v e r mitochondria. Hybrid F^ has also been reconstituted by using subunits of F^ i s o l a t e d from d i f f e r e n t sources (86,87).  The following combinations, i n  16.  the molar r a t i o s indicated were found to contain ATPase a c t i v i t y : E ET a^^Y  (I.e. a andflsubunits from ECF^  X X E agBgY  and the Y subunit from TF^),  E X E and ^BgY • The Y subunit seems to be mutually interchangeable.  These reconstituted hybrid enzymes had physical properties, such as thermostability and optimum pH, which were d i f f e r e n t from either ECF^ or ATPase a c t i v i t y could not be reconstituted by randomly combining the subunits.  XEE EXX XEX For example, the combinations: "3^3^ and o^J^Y or "3^3^  d  i  d  not reconstitute ATPase a c t i v i t y .  THE ARRANGEMENT OF THE SUBUNITS OF E C ^ The arrangement of the subunits of ECF^ has been examined by two general methods.  The f i r s t method has involved the reconstitution with the ,  isolated subunits, or various forms of ECF^, depleted membranes as was discussed  and t h e i r binding to ECF^-  earlier.  In t h i s respect, one has to  be f a i r l y cautious i n interpreting the data because these reconstitution experiments do not take into consideration the possible conformational change induced i n the enzyme, when a p u r i f i e d subunit i s added. The  second approach has been through the use of cleavable, v  cross-  l i n k i n g reagents, such as d i t h i o b i s (succinimidyl propionate) and cupric 1,10-phenanthroline, which w i l l c r o s s - l i n k suitably placed amino and sulphydryl groups, respectively.  The results of such experiments (88,89)  indicated cross-linking between aa, aJ3, a6, J3B, B Y , B6, Be, Y and possibly E  ay.  There was no formation of Y  pointed  6  or  pairs, but i t has been  out that the absence of cross-linked products does not exclude the  p o s s i b i l i t y of other subunits being i n close proximity.  The results of  these cross-linking experiments are often compatible with those obtained  17.  through reconstitution studies. ctB, ay and BY i n t e r a c t i o n are suggested by the observations that the reconstitution of ATPase a c t i v i t y from the individual subunits require the Y subunit.  A ye i n t e r a c t i o n has been demonstrated by Dunn (70)  using p u r i f i e d y and e subunits.  The ct6 interaction i s also suggested i n  the experiments of Dunn et a l . (68) i n which they have shown that the p u r i f i e d & subunit does not bind to trypsin-treated ECF^ because of the removal of 15 amino acids from the Nl^-terminal region of the ct subunits. Electron micrographs of p u r i f i e d ECF^ which has been negativelystained with uranyl acetate suggest that the enzyme i s a planar hexagon of six subunits about a central core (7).  The presence of three subunits of  both OL and fi suggest that the s i x peripheral subunits of the hexagon are l i k e l y to be composed of ct and B subunits. • I t i s d i f f i c u l t to propose a unique arrangement of the subunits of ECF^ because the data do not completely support any one p a r t i c u l a r model.  Three models ( F i g . 2) have been proposed (7,89):  i ) Model 1  consists of a s l i g h t l y puckered hexagon of alternating a and B subunits around the c e n t r a l y subunit. opposite sides of the hexagon.  The e and 6 subunits are placed on This arrangement of the <S and e subunits  contradict the r e s u l t s from the reconstitution experiments i n which both 6 and e were required to bind ECF^ to the membrane.  In support of t h i s  arrangement i s the finding that anti-o"-serum detached the entire ECF^ from the membrane without affecting the ATPase a c t i v i t y , whereas a n t i - e serum s e l e c t i v e l y removed only the e subunit (65).  i i ) Model 2 i s  similar to Model 1 except that the ot and B subunits are stacked as pairs,  i i i ) Model 3 i s d i f f e r e n t from models 1 and 2 i n that the ot and B  do not alternate and the 6 and £ subunits are on the same side of the  18.  Lower  FIG 2:  Three models f o r the arrangement of the subunits i n the F^-ATPase of E. c o l i (7, 89).  19.  hexagon.  This arrangement of the subunits i s supported by the  reconstitution experiments discussed f o r Model 1 and by results of cross-linking experiments (89).  More recently, i t was shown that the 6  and e subunits contain additional antigenic s i t e s i n the isolated  ECF^  which are inaccessible when the ECF^ i s bound to the membrane (65).  THE FQ COMPLEX S o l u b i l i z a t i o n of the F^FQ Complex One approach to the study of the FQ polypeptides has been through the p u r i f i c a t i o n of the F^FQ complex.  This requires the presence of  high ionic strength buffers and the use of detergents to s o l u b i l i z e the F^FQ complex (4,24,25).  Buffers of high ionic strength are needed  during s o l u b i l i z a t i o n and p u r i f i c a t i o n of the F^FQ complex to keep the F^ attached to the FQ, as the a c t i v i t y of the F^ provides a means of following the presence of the F^FQ  c o m  P^  e x  *  Non-ionic and ionic  detergents, such as T r i t o n X-100, sodium cholate and deoxycholate are most commoniy used to s o l u b i l i z e membrane proteins (90,91).  Zwitterionic  detergents such as Ammonyx Lo and Aminoxid WS 35 have recently become available and these are becoming the detergents of choice, since they have powerful s o l u b i l i z i n g properties and often do not possess the denaturing effects shown by ionic detergents (92).  C r i t e r i a f o r Determining  the Intactness and Purity of the F ^ F Q Complex  The f i r s t c r i t e r i o n often used to determine whether the F^ and the FQ have remained associated during the p u r i f i c a t i o n i s the retention of s e n s i t i v i t y to i n h i b i t o r s of ATPase a c t i v i t y (5).  Membrane-bound ATPase  a c t i v i t y i s sensitive to DCCD i n prokaryotes, while i n eukaryotes, i t i s  20.  sensitive to both DCCD, and oligomycin.  Both i n h i b i t o r s bind to a s p e c i f i c  polypeptide of F Q (discussed under DCCD-binding.protein) to i n h i b i t the ATPase a c t i v i t y when F^ i s coupled to F Q . Ryrie ( 9 3 ) has shown that the s e n s i t i v i t y of the p u r i f i e d F ^ F Q to these i n h i b i t o r s i s often a poor guide to determine i t s intactness.  The s e n s i t i v i t y of the F ^ F Q  complex from yeast mitochondria to these i n h i b i t o r s varied considerably and depended on the type of a c t i v i t y being measured (e.g. ATPase or A T P - P i 32  J exchange) and whether the complex was f i r s t reconstituted into phospholipid vesicles. with the  Similar results were also found by Foster and Fillingame ( 9 4 ) ECF FQ 1  complex.  The second c r i t e r i o n used i s to reconstitute the p u r i f i e d F ^ F Q complex into phospholipid vesicles and to demonstrate energy-transducing reactions such as A T P - P i exchange a c t i v i t y , ATP-driven proton-uptake, and A'vf-f -driven ATP synthesis. These are properties shown by the n 32  membrane-bound ATPase  (5,25).  The c r i t e r i o n f o r purity of the functional F ^ F Q complex i s through SDS-polyacrylamide g e l electrophoresis. However, t h i s method of determining the p u r i t y of a F ^ F Q preparation can also be misleading, since the number of polypeptides present i n the gel w i l l depend on the resolving power of the gel system used and on the choice of the proteindetecting reagents.  For example, the yeast mitochondrial F ^ F Q complex  which was o r i g i n a l l y determined to contain 9 d i f f e r e n t subunits,  was  resolved into 1 2 bands on a more resolving g e l system ( 9 5 ) .  The  FJFQ  Complex  The F ^ F Q complexes have been p u r i f i e d from various organisms.  The  numbers of polypeptides i n each of these preparations are summarized i n  21.  Table 1.  The mitochondrial -^ 0 P F  F  r e  P  a r a t i o n s  contain more polypeptides  either because the enzyme i s more complex than i n other organisms or because these preparations are contaminated with other proteins or protease digestion has occurred.  The polypeptides l i s t e d i n Table 1 are those which  the authors have determined to be the major components of the F^FQ complex.  In many of these preparations, varying numbers of minor bands  were present to which no function was assigned. The least complex preparation of the ^ o F  c  ^ o  m  Pl  e  x  w a s  from the thermophilic bacterium, PS3 (Tables 1 and 2 ) .  obtained Densitometric  tracing of the p u r i f i e d F^FQ complex, analyzed on SDS-polyacrylamide gels which had been stained with Amido Black, revealed the presence of eight major polypeptides.  Five of these polypeptides were from TF^.  The  remaining three polypeptides with molecular weights of 19 000, 13 500 and 5 400  were designated as  TFQ  of the energy-transducing  polypeptides.  The  TF-^FQ  complex was  reactions i summarized i n Table 2.  A , J  g  capable +  -driven  ATP synthesis could only be demonstrated using phospholipids from PS3 f o r reconstitution.  Soybean phospholipids were i n e f f e c t i v e .  Furthermore, a  of greater than 170 mV was necessary f o r the demonstration  of  ri  appreciable formation of ATP The  ECF^FQ  (112,113).  complex also appears to consist of eight subunits.  Foster and Fillingame ( 9 4 ) s o l u b i l i z e d the F^FQ complex from the membrane of E_. c o l i with deoxycholate s o l u b i l i z e d f r a c t i o n was  i n the presence of 1M KC1.  The  subjected to ammonium sulphate p r e c i p i t a t i o n  and  the F^FQ complex was p u r i f i e d by sucrose density gradient c e n t r i fugation.  The p u r i f i e d complex was capable of the  reactions summarized i n Table 2.  energy-transducing  Analysis of the -^FQ preparation on f  SDS-polyacrylamide gels, revealed the presence of eight major subunits,  TABLE 1  Source  Polypeptide Composition of FiFn-ATPase Complexes from Various Sources  Rat Liver Mitochondria  S_. cerevisiae  N. crassa  Reference  (96)  (97)  (95)  (98)  (99)  (102)  (94)  Fl a  54 000  52 000  59 000  59 000  56 000  55 000  48 000  56 000  55 000  53 000  50 000  31 000 14 500 10 700  36 000 15 000 12 000  37 000 17 500 13 500  32 000 15 500 11 000  37 000 20 000 12 000  -  -  -  -  -  -  Y 6 e  33 000 19 500 7 700  62 500 000 48 000 33 800 12 500 7 000  I.P*  12 300  13 000  7 000  OSCP**  20 800  22 000  23 000  20 000  46 31 26 9  28 24 21 16 12 9  21 19 16 8  48 000  25 20 20 14 12 11 8 6 * ** a b  000 500 000 700 300 800 500 500  1  800 600 000 200  500 500 500 700 700 000  a  000 000 000 000  Chloroplast  Bacillus PS3  Beef Heart Mitochondria  17 15 13 7  500 500 500 500  b  19 000 13 500 5 400  E_. c o l i  b  24 000 19 000 8 400  Inhibitor polypeptide Oligomycin-sensitivity conferring protein Inhibitor polypeptide did not precipitate with p u r i f i e d enzyme Recent studies suggest that this component may be an impurity (100, 101, 103)  S3  23.  f i v e of which corresponded  to the subunits of ECF^.  had molecular weights of 24 000, 19 000 and 8 400.  The FQ polypeptides The smallest subunit of  FQ was i d e n t i f i e d as the DCCD-binding protein (discussed under "The DCCDbinding p r o t e i n " ) . When the  ECF^FQ  complex was isolated from c e l l s  grown i n a medium containing succinate, malate and acetate, rather than glucose as the carbon source, a d d i t i o n a l bands with molecular weights of 76 000,  68 000,  preparation.  34 000, 26 000, 15 000 and 14 000 were also present i n the Of these, only the 14 000 dalton polypeptide copurified with  an invariant stoichiometry when d i f f e r e n t fractions of the sucrose gradient were analyzed on SDS-polyacrylamide g e l s .  Thus, the 14 000 dalton poly-  peptide cannot be excluded as a possible subunit of the F^FQ complex when the c e l l s are grown on a mixture of succinate, malate and acetate.  It  has been observed i n _E. c o l i that some c h a r a c t e r i s t i c s of the F^FQ complex do change depending on the growth conditions (29-32). Rosen and Hasan (105) have also p u r i f i e d the F-^FQ complex from E_. coli.  Following s o l u b i l i z a t i o n of the membrane with deoxycholate  presence of 140 mM KC1,  i n the  the F^FQ was p u r i f i e d by chromatography on  DEAE-cellulose and g l y c e r o l gradient centrifugation.  This preparation  consisted of s i x subunits and was d e f i c i e n t i n the 6 subunit.  Although  the ATPase a c t i v i t y of t h i s preparation was inhibited by DCCD, other energy-transducing  a c t i v i t i e s could not be demonstrated with t h i s  preparation (Table 2).  The polypeptides with molecular weights of 10000  and 8300 were designated as the subunits of FQ. Recently, the membranes of E_. c o l i were s o l u b i l i z e d with the zwitterionic detergent, Aminoxid WS 35, and the F^FQ complex p u r i f i e d by chromatography on DEAE-Sepharose CL-6B (106). capable of the energy-transducing  The p u r i f i e d complex was  reactions summarized i n Table 2 and  24.  consisted of eight d i f f e r e n t polypeptides.  Five of these polypeptides were  subunits of F^ and the FQ polypeptides had molecular weights of 28 19 000 and 8 500.  000,  About 4% of the t o t a l protein of the F-^FQ preparation was  residual contamination,  the majority of which had spectral c h a r a c t e r i s t i c s  of cytochrome b^ and a molecular weight of 65 000.  Minor components with  molecular weights of 24 000 and 14 000 were also present and these were concluded to be possible degradation products of the larger subunits.  A  s i m i l a r subunit composition f o r the E C F ^ F Q complex was obtained by Schneider and Altendorf (107) who  used the same method of p u r i f i c a t i o n , but  included c e n t r i f u g a t i o n of the a c t i v e ATPase fractions from the DEAESepharose CL-6B column at 220 000 xg f o r 15 h (Table 2). differences were s t i l l present.  It was observed  However,  that the E C F ^ F Q  prepa-  ration obtained a f t e r chromatography on DEAE-Sepharose CL-6B contained the 28 000 and 19 000 dalton polypeptides i n approximately  equal amounts, and  the 8 500 dalton subunit was present i n only a t h i r d of the amount of the e subunit.  By including the c e n t r i f u g a t i o n step, the 28 000 dalton  subunit was present i n about twice the l e v e l as the 19 000 dalton subunit, and the 8 500 daIton/and e subunits were present i n about equal amounts. Further modification of t h i s p u r i f i c a t i o n procedure (108) has included the p r e c i p i t a t i o n of the E C F ^ F Q complex, obtained a f t e r DEAE-Sepharose CL-6B, with polyethylene g l y c o l 6000 and 400. observed  Differences which were  (Table 2) i n t h i s E C F ^ Q preparation were:  i ) the 28  000  dalton subunit so prominent i n the previous preparations was present i n almost n e g l i g i b l e amounts,  i i ) the 24 000 dalton subunit was  major polypeptides of F , and Q  /  one of the  i i i ) the complete absence of the 14  000  dalton polypeptide. Table 2 l i s t s only the major subunits of F.F  n  which were present  TABLE 2  P r o p e r t i e s o f Various P r e p a r a t i o n s of B a c t e r i a l F. F. Complexes Method of P u r i f i c a t i o n of  Source  E.  E.  Detergent  coli  coli  Z. c o l i .  F F 10  Deoxycholate  Sucrose  Deoxycholate  DEAE-Cellulose and sucrose g r a d i e n t  Deoxycholate  Sucrose  gradient  Subunit of  F  o  n.d.  Aminoxid WS-35  Aminoxid WS-35  coli  29;  6  S e n s i t i v i t y of ATPase a c t i v i t y to DCCD. f  a  19; 8.4  a  24;  19; 8.4  a  28;  19; 8.3  a  19;  14; 8.3  M.A.  24;  19; 8.3  s  24;  19; 8.3  N.A.  19;  13.5;  5.4  a  19;  13.5; 5.4  a  Treatment  of  DEAE-Sepharose CL-6B and PEG p r e c i p i t a t i o i  DEAE-Cellulose and Sepharose 6B  N.A. s  n.d Treatment of F - F with EDTA and KSCN.  Treatment of F,F 1 0  with 7M Urea  s  S.  C.  faecalis  pasteurianum  a. b. c. d.  X-100  Sucrose  gradient  6  M.A. +  I  ATP - d r i v e n proton uptake.  Ref.  n.d.  104  -  105  +  94 9  N.A. +  106," 107  13.5;  5.4  N.A.  +  . N.A.  +  N.A. •  +  108 +  102  c 109 c  a  +  s  c  24;  18; 8  s  n.d.  n.d.  n.d.  27;  15; 6  s  n.d.  n.d.  s  n.d.  '+  Deoxycholate  Triton  DEAE-Sepharose CL-6B Treatment of F ^ F Q and Sephadex LH-60 with EDTA  T h i s subunit i d e n t i f i e d as t h e DCCD-binding p r o t e i n u s i n g [ Measured a f t e r r e c o n s t i t u t i o n i n t o p h o s p h o l i p i d v e s i c l e s . Demonstrated when the a p p r o p r i a t e F j - ATPase was added. N.A., not a p p l i c a b l e - see Table 3.  15  107  108  n.d.  Ammonium sulphate p r e c i p i t a t i o n and non-denaturing g e l .  X-100  P  s  Q  Triton  d  2  Exchange.  >  s  3  b  FJFQ  Treatment of F with 4M Urea and CM-cellulose M. p h l e i  -  n.d.  24;  n.d.  ATP  s  gradient  DEAE-Sepharose CL-6B and c e n t r i f u g a t i o n  Activity  n.d.  n.d.  ' *  0  s  10; 8.3  n.d.  PS3  9  FjF  n.d.  0  Triton X-100  Q - 3  with 7M Urea  E.  composition  (M^ x 1 0 ) .  Treatment of FJFQ with EDTA  E. c o l i .  F  a  a  c]DCCD  103  110  111  16  e. n.d., not done. f . , sensitive. . + ] d e t e c t e d , -, not d e t e c t e d . s  g  N3  N  on the SDS-acrylamide  gels.  26.  In a l l cases, small amounts of minor  contaminants were also present and these could have been p r o t e o l y t i c degradation products.  Ryrie and Gallagher ( 9 5 ) have demonstrated  the  existence of proteases i n the F ^ F Q complex from yeast mitochondria. Thus, i t would do well to keep i n mind the presence of proteases during the p u r i f i c a t i o n of the F ^ F Q complex as t h i s could affect the i d e n t i f i c a t i o n of the F Q subunits.  I t i s also c l e a r from Table 2 that, although i t i s  possible to p u r i f y the  ECF^FQ  complex and reconstitute energy-transducing  reactions, the authenticity and the minimum number of subunits of F Q required to reconstitute these reactions are s t i l l controversial.  The I s o l a t i o n of the F Q Complex, The second approach to the study of the polypeptides of F Q has been through the i s o l a t i o n of the intact F Q complex from the p u r i f i e d or p a r t i a l l y p u r i f i e d F-^FQ complex. ' Isolation of the F Q complex has involved the extraction of the F ^ F Q complex with either urea, EDTA, or EDTA and  KSCN  i n order to remove the  F  1  subunits.  As with the  F^FQ  complex, the intactness of the F Q complex can be determined by demonstrating proton-translocating a c t i v i t y i n reconstituted v e s i c l e s . These results are summarized i n Table 3. The F Q complex was f i r s t i s o l a t e d i n b a c t e r i a l systems from the thermophile,  PS3  (109).  I t consisted of three subunits with molecular  weights of 19 000, 13 500 and 5 400. urea and subsequent  Treatment  of the F  Q  complex with  p u r i f i c a t i o n on CM-cellulose ( 1 0 3 ) resulted i n an F Q  preparation containing two subunits with molecular weights of. 13 500 and 5 400.  This F Q complex was capable of mediating proton conduction i n  reconstituted v e s i c l e s .  Energy-transducing reactions could also be  Table  3  Properties  of Various Preparations of Bacterial  F „ Complexes  Method  of Purification of  Subunit of  "  (M  _E.  coli  Deoxycholate  _E.  coli  Deoxycholate  E.  coli  Deoxycholate  1 0 F  Sucrose  F  gradient  FQ  Activity *  D E A E - C e l l u l o s e and sucrose  gradient  Sucrose  gradient  translocation  6  9  n.d.  10;  8.3  n.d.  24;  19; 8 . 4  a  DEAE - S e p h a r o s e  WS-35  and  24;  19; 8 . 4  a  28;  19; 8 . 3  a  19;  centrifugation  Aminoxid  DEAE - S e p h a r o s e  WS-35  and  PEG  Treatment 7M  of  n.d.  CL-6B  precipitation  Treatment  of FJFQ  DEAE -  X-100  and  Cellulose  Sepharose  N.A.  N.A.  105  N.A.  N.A.  94  +  s  e  f  9  N.A.  N.A.  1 4 ; 8.3  +  s  107  24;  1 9 ; 8.3  N.A.  N.A.  108  24;  1 9 ; 8.3  +  s  108  19;  13.5;  5.4  a  N.A.  N.A.  102  19;  13.5;  5.4  a  +  s  109  +  s  103  N.A.  N.A.  110  N.A.  N.A.  111  106,107  with  n.d.  Triton  104  d  FJFQ  Urea  EDTA a n d KSCN  PS3  Ref.  N.A.  N.A.  a  Sensitivity of Proton Conduction t o DCCD  CL-6B  with  coli  1  of FJFQ  EDTA  n.d. Aminoxid  J  29;  Treatment  coli  Proton  x 10" )  r  0  n.d.  with  E.  composition  Q  Detergent  Source  F  J2.  F  Treatment  6B  with  7M  of Urea  Treatment 4M  FJFQ  '  of F  Q  with  Urea and  13.5;  5.4  a  CM-cellulose M . phlei  T r i t o n X-100  Sucrose Ammonium  S.  C.  faecalis  denaturing  subunit  and  a.  This  b.  Measured  identified  c.  n . d . , n o t done  after  Sephadex  and n o n -  CL-6B  18; 8  n.d.  27;  15; 6  into  Treatment  LH-60  a s t h e DCCD - b i n d i n g  reconstitution  24;  a  gel .  DEAE-Sepharose  T r i t o n X-100  n.d.  sulphate  precipitation  Deoxycholate  pasteurianum  gradient  with  protein  phospholipid  of  F.F.  EDTA  using  vesicles  [  c]DCCD  15  +  3  3  d.  N.A., n o t a p p l i c a b l e  e.  +, d e t e c t e d ; - , n o t d e t e c t e d .  f .  S  j  sensitive  - see Table 2  16  28.  reconstituted by the addition of p u r i f i e d TF^ to the TF -reconstituted Q  vesicles.  It was concluded  that the T F Q consisted of two subunits and  that the 1 9 0 0 0 dalton polypeptide was most l i k e l y a contaminant.^ When the E C F ^ F Q complex, which contained the  28  000,  19  000  and  8 3 0 0 dalton polypeptides as the major subunits of F Q , and the 2 4 0 0 0 and 1 4 0 0 0 dalton polypeptides as the minor contaminants, was extracted with urea, an F Q preparation containing e s s e n t i a l l y the 1 9 0 0 0 , 1 4 0 0 0 and 8  300  dalton polypeptides was obtained  (106,107).  This F Q complex could  reconstitute proton translocating a c t i v i t y i n reconstituted v e s i c l e s (Table 3).  It was suggested  that the 2 8 0 0 0 dalton subunit was probably a dimer,  such that conversion to the monomer was obtained a f t e r urea In contrast, treatment  treatment.  of the E C F ^ F Q complex, which contained the  2 4 0 0 0 , 1 9 0 0 0 and 8 3 0 0 dalton polypeptides as the major subunits of F , Q  with EDTA or EDTA and KSCN resulted i n an F the same three polypeptides  The  (9,108).  Q  24  preparation consisting of 000,  19  000  and  8  300  dalton  polypeptides were shown to be genuine subunits of F Q , through the use of the defective transducing phage, *  _  d.sn  the E C F ^ F Q polypeptides  (114,115).  which c a r r i e s the genes f o r  J  /  This i s also confirmed by the  r e s u l t s obtained from the genetic studies.  Biochemical  Genetics  The t h i r d approach to the study of the subunits of F Q and therefore F ^ F Q , has been through the i s o l a t i o n and generation of mutants of E_. c o l i which are defective i n oxidative phosphorylation mutants can be generated  (25,116,117).  These  by the use of mutagens such as N-methyl-N'-  nitrosoguanidine, ethyl methyl sulphonate, hydroxylamine, u l t r a - v i o l e t i r r a d i a t i o n and the bacteriophage, Mu.  The mutants defective In oxidative  29. y  phosphorylation  cannot grow on non-fermentable substrates such as  acetate,  malate or succinate, and have low y i e l d s when grown on a l i m i t i n g amount of glucose.  The membranes of these mutants also cannot be energized with  and are therefore referred to as "unc"  ("uncoupled") mutants.  I n i t i a l l y , two classes of mutants were described. designated unc A (e.g. unc A401)  The  The  first,  did not possess ATPase a c t i v i t y , whereas  the second group, which retained a c t i v i t y was B402).  ATP  designated, unc B (e.g.  lesions i n these mutants were further characterized  unc  as  a f f e c t i n g e i t h e r the F ^ or F Q portion of the E C F ^ F Q complex, through reconstitution experiments;  Oxidative phosphorylation  a c t i v i t y could  be  reconstituted i n ECF^-depleted membranes from the unc A s t r a i n with p u r i f i e d E C F ^ e i t h e r from the wild-type s t r a i n or from the unc B s t r a i n . The ECF^-depleted membranes from the unc B s t r a i n were also r e l a t i v e l y impermeable to protons upon energization of the membrane through substrate oxidation.  Thus i t was  established that the unc A mutation affected the  subunit(s) of E C F ^ , whilst the unc B mutation affected the FQ portion of the E C F F Q complex (116,118-120). 1  A l l of the known unc mutations map c o l i chromosome (25,121).  at the 83.5 min region i n the E_.  Since the E C F ^ F Q complex probably consists  of 7-9  d i f f e r e n t polypeptides,  and a l l the mutations map  at the same locus,  i t was  not possible to d i s t i n g u i s h mutations i n the d i f f e r e n t genes by  simple genetic-mapping. The exact composition of the FQ complex and the characterization of the order and number of genes responsible f o r oxidative phosphorylation required more refined biochemical and genetic experiments. complementation system was the unc mutants.  '  A genetic  developed to characterize the defective gene i n  This involved the construction of p a r t i a l d i p l o i d s which  /  30.  contained two d i f f e r e n t unc a l l e l e s : one on the host chromosome and the other on a plasmid (F-plasmid) (25,121-124).  When the mutation affected  the same gene on the plasmid as on the host chromosome, the resulting p a r t i a l d i p l o i d remained defective i n oxidative phosphorylation and expressed the phenotype of being unable to grow i n the presence of non-fermentable carbon sources.  On the other hand, when the mutation  affected d i f f e r e n t genes, there was a normal copy of each of the affected genes present i n the c e l l . the wild-type s t r a i n .  The r e s u l t i n g p a r t i a l d i p l o i d was similar to  Biochemical tests, such as ATP-driven membrane  energization and two-dimensional  i s o e l e c t r i c focusing gels were used to  confirm the presence or absence of genetic complementation. Seven d i s t i n c t complementation groups were i d e n t i f i e d (121) i n the unc region of the chromosome and these were designated unc A, B, C, D, G, F and G coding f o r the polypeptides shown i n Table 4. Since the unc genes a l l mapped i n the same region of the E_. c o l i chromosome, the bacteriophage Mu was used to determine the gene i n t e r - r e l a t i o n s h i p and whether these genes formed an operon (125) .  The  bacteriophage Mu has a polar effect on an operon, i n that the i n s e r t i o n of the phage i n an early gene prevents the t r a n s c r i p t i o n of a l l subsequent genes.  Genetic complementation tests on these Mu-induced mutants  demonstrated that the unc genes did form an operon. information obtained from cloning experiments,  Together with the  the gene order was  postulated by Downie et a l . (126,127) to be unc BFEAGDC. subsequent DNA  However, the  sequencing of the unc operon not only showed that the order  unc BFE was incorrect, but also that the gene coding f o r the 6 subunit (unc H) was located between unc F and unc A (42, 128-133).  Therefore, the  correct order of the genes i n the unc operon i s unc BEFHAGDC, with the unc  TABLE 4  Polypeptides Coded by the "uric" Genes  Polypeptides of  Gene  Molecular Weight  b  Molecular Weight  c  Gene 1  1  14 183  unc B  a  30 258  24 000  E  c  8 365  8 300  F  b  17 233  19 000  H  6  19 310  20 700  A  a  55 264  56 800  G  Y  34 100  32 000  D  B  50 157  51 800  C  e  14 194  13 200  ?  a  The general nomenclature f o r the FQ polypeptides are a, b and c with being the DCCD-binding protein  b  Determined from the DNA sequence  c  Determined by SDS-polyacrylamide g e l electrophoresis  32.  B gene being closest to the promoter (Table 4). In v i t r o protein synthesis experiments with plasmids (126,127,134) or specialized X transducing phages (135) carrying the unc genes confirmed that the unc B, E and F genes code f o r polypeptides of FQ with molecular weights of 24 000, 8-9 000 and 18 000, respectively. The molecular weight of the unc B gene product determined by SDS-polyacrylamide g e l electrophoresis has been underestimated.  This  appears to be common to i n t e g r a l membrane proteins (136,137).  Steffens et  a l . (138) have p u r i f i e d the 24 000 and the 18 000 dalton polypeptides from the p u r i f i e d  ECF^FQ  complex and found that the amino acid  agreed with those deduced from the corresponding DNA The DNA  compositions  sequences.  sequence of the unc operon also indicates the existence of  another gene (Gene 1) coding f o r a polypeptide of molecular weight 14 (128).  This gene l i e s between the promoter and the unc B gene.  Whether  gene 1 i s transcribed i n vivo i s not c l e a r , but the presence of a 14 dalton polypeptide i n some (94,106,107).  ECF^FQ  preparations may  183  000  suggest that i t i s  In v i t r o coupled t r a n s c r i p t i o n - t r a n s l a t i o n experiments with  plasmids containing the whole unc operon also resulted i n the production of a 14 000 dalton polypeptide (126).  The function of this polypeptide i s  unknown, although i t has been postulated/that i t may the assembly of the FQ polypeptides (128).  function i n regulating  In v i t r o studies (139) with  plasmids containing the unc genes, but not the promoter or Gene 1, showed that the membrane-association of the F Q polypeptides occurred v i a the i n s e r t i o n of the proteins into the membranes and t h i s process  was  independent of the synthesis of each FQ polypeptide or of other F^ polypeptides.  Whether the polypeptides of F Q are inserted into the  membrane i n the correct orientation, or whether a functional F Q complex  ,  33.  was formed, was not clear. By contrast, Cox et a l . ( 1 4 0 ) have shown that the i n s e r t i o n of some of the F Q polypeptides into the membrane, and therefore the assembly of a functional F Q complex, required the presence of a and J3 subunits of ECF . 1  THE DCCD-BINDING  PROTEIN  The function of each subunit of F Q has not been characterized because of the d i f f i c u l t i e s encountered i n the i s o l a t i o n of the i n d i v i d u a l i  subunits i n  non-denatured form.  However, there i s considerable evidence  i n prokaryotic and eukaryotic systems suggesting that the smallest subunit of FQ i s intimately involved i n the proton-translocating properties of the F Q complex.  This polypeptide i s the best characterized subunit of  F Q i n mitochondria, chloroplasts, and bacteria with respect to i t s structure and function.  I d e n t i f i c a t i o n and  Isolation  The compound DCCD i s able to react n o n - s p e c i f i c a l l y with amino, carboxyl, hydroxyl and sulfhydryl residues to form stable adducts  (141).  But under basic conditions (pH 8 . 5 ) , i t reacts s p e c i f i c a l l y with a carboxyl residue of a subunit of F Q and Inhibits the ATPase a c t i v i t y of either the membrane-bound ATPase or of the p u r i f i e d  ECF^Q  complex  (78,142).  The  reaction of DCCD with t h i s subunit of F Q also decreases the permeability of the F^-depleted membranes to protons, as discussed before.  Beechey et  a l . ( 1 4 3 ) were the f i r s t to demonstrate that the i n h i b i t i o n of the ATPase a c t i v i t y i n beef-heart mitochondria was associated with the s p e c i f i c incorporation of [ "C]DCCD into a 9 0 0 0 dalton polypeptide. 1  In E. c o l i  34.  (141,144), the l a b e l was also associated exclusively with a 8 000 - 9 000 dalton polypeptide.  This polypeptide was termed the "DCCD-binding protein".  DCCD also i n h i b i t s the energy-transducing reactions of the  F-JFQ  and FQ complexes which have been reconstituted into liposomes (Table 2). I n h i b i t i o n of these reactions was associated with the l a b e l l i n g of the smallest polypeptide of F  Q  with ['"CJDCCD.  The DCCD-binding protein i s unique i n that i t can be extracted from the membrane with organic solvents (145,146).  I t i s due to t h i s property  that i t was o r i g i n a l l y c a l l e d a " p r o t e o l i p i d " .  The DCCD-binding protein  from the mitochondria of Neurospora crassa and yeast can be extracted i n almost pure form with a mixture of chloroform-methanol  (2:1) i f the  mitochondrial membranes are prewashed several times with a mixture of chloroform:methanol:diethylether (2:1:12).  Similarly, the DCCD-binding  protein i s o l a t e d from beef heart and'rat l i v e r mitochondria, lettuce or pea chloroplast and the sarcoplasmic reticulum, by extraction with n-butanol followed by p r e c i p i t a t i o n with d i e t h y l ether, i s also i n pure form (146). i By contrast, the extraction of membranes or whole c e l l s preparations with a mixture of chloroform:methanol  (2:1) without p r i o r washing with  solvent, also extracts together with the DCCD-binding protein, several other hydrophobic proteins and phospholipids.  P u r i f i c a t i o n of the  DCCD-binding protein i s achieved by repeated p r e c i p i t a t i o n of the extract with d i e t h y l ether followed by e i t h e r :  thin layer chromatography (147),  adsorption chromatography on Sephadex LH-20 or LH-60 (145),  reversed-phase  HPLC (148) or In the case of E. c o l i (149,150), by ion-exchange chromatography on e i t h e r DEAE or CM-cellulose followed by adsorption chromatography on Sephadex LH-60. The p u r i f i e d DCCD-binding protein of 15. c o l i (149,150) migrates with  35.  an apparent molecular weight between 8 000 and 9 000 on SDS-polyacrylamide gels.  Reconstitution of Proton Translocating A c t i v i t y The DCCD-binding protein, i s o l a t e d from chloroplasts (151) or beef heart mitochondria (152) by extraction with n-butanol was  capable of  proton-translocating a c t i v i t y when reconstituted into liposomes.  With the  DCCD-binding protein from beef-heart mitochondria, the rate of proton i n f l u x was  dependent on the amount of the protein incorporated into the  liposome.  In both cases, proton-translocating a c t i v i t y was  sensitive to  DCCD but oligomycin-sensitivity was not observed with the protein from beef heart mitochondria.  S i m i l a r l y , the protein isolated by extraction of yeast  mitochondria with a mixture of chloroformrmethanol (2:1) was also capable of proton-translocating a c t i v i t y when reconstituted into liposomes (153,154).  The rate of proton i n f l u x was proportional to the amount of  protein incorporated.  Oligomycin, which i s a s p e c i f i c i n h i b i t o r of  oxidative phosphorylation  i n eukaryotes and which also binds to the DCCD-  binding protein, i n h i b i t e d proton conduction. a c t i v i t y was  The  proton-translocating  i n s e n s i t i v e to oligomycin when the DCCD-binding protein from  an oligomycin-resistant s t r a i n was  used.  Similar types of reconstitution experiments with the DCCD-binding protein from b a c t e r i a l systems, did not result i n any a c t i v i t y (5).  This may  proton-translocating  be due to the method of i s o l a t i o n and  of this protein, which may  purification  result i n the loss of the native oligomeric  structure and subsequent loss of function.  Evidence f o r the existence of  this protein i n an oligomeric form i n E. c o l i or PS3 comes from the determination  of the stoichiometry of the subunits of the F,F-. .complex.  36.  The F-jFfj 3 5  c  o  m  Pl  e  x  i s o l a t e d from c e l l s grown i n the presence of either  S 0 " , [U-^CJD-glucose (155) or L-[U- C] amino acid mixtures 2  (156)  ll,  4  r e s u l t i n stoichiometrics of a^Q^y& 2 10 eab  and TF^FQ, respectively.  an(  * 2^2 ^2 5 a  (&ea  C  E C F  1 0 F  In addition, the i n h i b i t i o n of membrane-bound  ATPase a c t i v i t y of E. c o l i was DCCD-binding protein was  C  complete when only one t h i r d of the t o t a l  l a b e l l e d with [^CJDCCD, suggesting that the  protein existed as a trimer (144). The second reason f o r the lack of protein conduction upon r e c o n s t i tution may be due to the requirement of additional polypeptides  to d i r e c t  the proper i n s e r t i o n of the DCCD-binding protein into the membrane (103,140).  The Amino Acid Composition The DCCD-binding protein has been p u r i f i e d from various sources and the amino acid composition of each i s l i s t e d i n Table 5.  In a l l cases, the  amino acid composition i s derived from the amino acid analysis and confirmed by either complete or p a r t i a l sequence analysis (146). The DCCD-binding proteins from the various sources contain  unusually  high amounts of nonpolar amino acids and therefore are very hydrophobic i n nature.  The protein from E. c o l i i s the most hydrophobic of those which  have been studied (Table 4).  Only 16.5%  of i t s amino acids are polar. The  predominating nonpolar amino acids are alanine (15%), leucine  (16%),  glycine (13%) and methionine (10%). The DCCD-binding protein from a l l sources except Aspergillus nidulans (157) lack h i s t i d i n e .  S i m i l a r l y , tryptophan i s present only i n the protein  from the chromatophbres of Rhodospirillum  rubrum (158).  In addition to  h i s t i d i n e and tryptophan, the protein from E_. c o l i (149) also lacks the  Table 5  Amino A c i d  C o m p o s i t i o n o f t h e D C C D - B i n d i n g P r o t e i n f r o m M i t o c h o n d r i a , C h l o r o p l a s t and B a c t e r i a  3  (146)  Bacillus Aspergillus  Amino a c i d  nidulans  Neurospora  Saccharomyaes  Dovine Heart  Spinach  Mastigocladus  acidocal-  Escherichia  PS-3  coli  darius  Halobacteriwn  Rhodospirillum  Lysine  2  2  2  2  1  1  1  -  3  1  Histidine  1  -  -  -  -  -  -  -  -  Trace  Arginine  3  2  1  1  2  2  2  4  2  1  1  6  4  3  3  2  3  5  1  3  4  4  Threonine  1  ' 2  3  3  3  3  1  3  1  7  3  Serine  7  5  5  5  3  4  '•-  3  4  3  3  6  5  2  3  7  7  4  5  5  5  3  Proline  5  1  2  1  4  4  3  3  2  6  1  Glycine  12  11  10  11  11  10  10  11  12  10  10  Alanine  .12  14  10  13  17  16  13  9  14  18  16  Valine  5  6  6  4  7  4  6  8  9  6  6  Methionine  4  4  3  3  2  2  8  2  4  1  3  Isoleucine  5  6  9  7  6  8  8  9  5  5  9  Leucine  8  11  12  9  12  13  12  10  9  10  8  Tyrosine  3  2  1  2  1  1  2  1  2  1  1  Phenylalanine  4  6  6  7  3  3  4  3  4  3  3  Cysteine  -  1  1  -  -  -  -  -  -  Tryptophan  -  -  -  -  -  -  -  -  • -  ND  81  . 76  75  81  81  79  72  79  81  Tyr  f-Met  Asp  Aspartic  Glutamic  acid  acid  Total residues End  83 . f-Met  group  30  Polarity  ND, n o t done. b  from (157)  24.7  21.7  22.7  f-Met  f-Met.  22.2  24.7  V a l u e s a r e g i v e n i n u n i t s o f moles per mole  f-Met  f-Met  f-Met  16.5  22.2  22.8  2  - .  C  ND 25.9  2 75 f-Met 25  38.  amino acids serine and cysteine. In most cases, the amino acid content i s i n agreement with the apparent molecular weight determined by SDS-polyacrylamide gel e l e c t r o phoresis.  The exception i s the DCCD-binding protein from PS3, whose  molecular weight of 5 400 determined by SDS-gel electrophoresis was underestimated (102).  The results from the amino analysis suggests that  the molecular weight i s 7 300.  The Amino Acid Sequence If the native DCCD-binding protein i s indeed a proton channel, then the knowledge of i t s amino acid sequence i s a pre-requisite f o r the understanding of i t s structure and function (146). knowing i t s primary structure are:  The advantages of  i ) t h e o r e t i c a l calculations can be used  to predict the arrangement of the polypeptide i n the membrane and  ii) a  better understanding of how mutations i n the polypeptide can a f f e c t i t s r e a c t i v i t y with DCCD (discussed under DCCD-resistent mutants) and oligomycin, or affect the proton permeability of the membrane, can be obtained. The complete amino acid sequence of the DCCD-binding protein has been determined from a variety of organisms and these are summarized i n F i g . 3. The amino acid sequence of the protein from E. c o l i has been confirmed by i t s DNA  sequence (128).  In almost a l l organisms residue ( F i g . 3). (asp ). 6 1  ['''CJDCCD reacted with a s p e c i f i c glutamyl  But i n E_. c o l i , i t reacted with an aspartyl residue  The proteins from d i s t a n t l y related organisms have a high  degree of homology i n t h e i r amino acid sequence and t h i s r e f l e c t s t h e i r being involved i n a s i m i l a r function i n the c e l l .  39.  N. c r .  lyr-Scr-Scr-C1u-nc-AU-Cln-AU-Mct-V<t-Clv-V<1-.S«r-lys-Asn-lcu-Cly-r1tt-C1y-S<r>Alt-Al<-nc-Cl/-lcu-  Bovine  Aip-IW-Ajp-Thr-Ala-Ala-tyi-Phc-Jlt-Gly-Ala-Cly-Ala-Ala-'lhr-Val-Gly-Yal-  J. t e r .  f-Het-Cln-Uv-m-l«u-AU-A1<-l s-tyr-!1c-C1y-AU-Cl/-n«-Wr-'thr-llc-Cty-ltur  Spinach  f-rVt-Ajn-Pro-Uu-ilt-Ala-Ala-Ala-S«r-Val-nr-Ala-Ala-C!y-Uu-Ala-»aI-Gly-Uu-Ala-Str-  H. l a » .  f-Mct-A»p-Pro-Uu-llt-W-Ala-Ala-S«r-Val-ltu-Ala-Ala-AW-leu-Ala-llt-Gly-ltu-Ala-Ala-  (.  f-Mcl-Clu-Asn-l»u-Ajn-rVt-Aip-teu-l«u-Tyr-het-Al»-AU-AU-Vjl-rVt-Met-CI)-ttu-Al«-AW-  coll  PS-3  f-hcl-Str-llu-Gly-Val-lru-Ala-Ala-Ala-llt-Ala-Yal-Cly-ltu-Gly-Ala-  N. c r .  30 40 SO lhr-C)jr-AU-Cl/-ll<-Cl/-]l<-Cly-Lcu-V<l-Phc-Al4-Al<-:«u-leu-Asn-C1y-V<l-Al<-Ar9-Asn-Pro-Al«-lcu-Ars-  Bovine  An-C1y-S«r-CI/-Ali-Cl)r-ll»-Cl/-Ihr-Vl1-Ph«-C)y-S«r-l*u-He-lt«-Cly- t/r-AU-Ar9-Aiii-Prc-Ser-t»u-ly»,  5. c e r .  lru-Clj-»n-Cly-lU-CI/-l1c-Alj-lU-»»l-Pht-Al«-A)»-ltu-H«-A5n-Cly-V»l-Str-Ar -Asn-Pro-Str-lU-ly»-  Spinach  IU-Clj-Pro-Cl/-Vil-C1/-Gln-Cly-lhr-Alj-AU-Cl/-C)r>-A)j-V«l-Clu-Cly-IU-Ar«-Ar9-Cln-Pro-Glu-AU-Clu-  K. I n .  llc-Cly-Pro-Cly-nt-Cly-Cln-Cly-Asn-An-Ala-Cly-Cln-Al<-V<l-Clu-Clyllc-Al<-Ar9-Cln-Pro-Clu-AU-Clu-  t.  coM  PS-3  9  n»-01/-AI;-AU-lU-CI '-r.t-CI/-lle-ltu-Cl/-Cly-l/»-Pht-ltu-Cln-ClyAll-Alj-Ar9-Cln-Pro-Asp-ltu-lUJ  ltu-Cly-AU-Cly-1 lt-Cly-Asn-Cly-lcu-llfVll-Srr-Arf-lhr-IU'Clu-Cly-nt-AU-Arg-Cln-Pro-Glu-Ltu-Arg-  eo  ;o  H. c r .  Cly-Gln-l(u-Phc-Scr-Tyr-AU-tlfltu-Gly-Pr«-A1<-Phc-V<l-Glu-Al<-Uc-Gly-leu-Phc-Asp-lcu-Het-Val-Ala-  Bovine  Gln-Gln-lcu-Phr-Scr-iyr-Aia-nt-leu-Gly-rht-Ala-ltu-Scr-Glu-AU-rvt-Gly-ltu-Phr-Cys-leu-rVl-Val-AU-  S. e r r .  Aip-ltir-v<l-Phc-Pro-Kct-Ala-llr-lru-G1y-Phf-Ala-L(u-$<r-Glu-Ala-lhr-G1y-lru-Phe-Cys-l«u-Hrt-Val-Ser-  SO.WK  Gly-lyl-l Ic-Arj-Gly-lhr-lcu-leu-leu-Srr-leu-AIa-Pht-Mtl-Glu-Ala-ltu-lhr-1 lc-Tyr-Gly-Uu-Val • Val-Ala-  H. l a m .  G l y - l y s .1 I r - A r g . G l y - l ( i r - l r - l r u - L e u - 1 h r - l e u - A l a - P » i t - H c t - G l u - S e r - l c u - l h r - H e - t y r - G ) y - l c u - V a l - I I t - A l a -  t.  u  coli Pro-teu-ltu-Arj-lt.r-Gln-Phe-Pht-lie-Va1-Kcl-Gly-lcu-Val-Asp-Ala-1le-Pro-Kel-Ile-AIa-Val-Gly-lfu-Gly-  PS-3  Pro-Val-l«u-Gln-lhr-Ihr-«el-rhc-IU-Gly-Val-A'la-l*u-Val-Glu-Ala-lcu-Pro-lU-Il*-Gly-Val-Val-Phc-Scr80  *. cr. Bovine  Phc-lcu-Ile-leu-The-Ala-Hot  S. c c r .  Phr-lcu-lcu-teu-rhc-Gly-Val  Spinach  lcu-Ala-lcu-lcu-Phc-Ala-A*n-Pro-Phe-Val  M. l a m .  Ico-Val-lcu-Lcu-rhc-Ala-Ain-Pro-Phc-Scr  I.  leu-lyr-Val-ni-rtc-Ala-Val-Ala  coli  PS-3  FIG. 3  Icu-Kct-Ala-lyi-Phc-lhr  Phc-Ilc-lyr-lcu-Gly-Arg  Amino a c i d sequences of the DCCD-binding protein from Neurospora crassa (N. c r . ) bovine heart (Bovine), Saccharomyces c e r e v i s i a e , (S. c e r . ) , spinach chloroplasts (Spiiiach), Mastigocladus laminosus (M. lam.), E s c h e r i c h i a c o l i ( E . c o l i ) , and the thermophilic bacterium PS-3. The numbering I s according to the Neurospora sequence. Reprinted from Sebald and Hoppe (146).  •  40.  DCCD-RESISTANT MUTANTS DCCD-resistant mutants have been i s o l a t e d from E_. c o l i . these mutants (25) have been i d e n t i f i e d :  Two types of  i ) Mutants of the unc B phenotype.  These mutants have a functional ECF^, with a c t i v i t i e s comparable to those of the wild-type s t r a i n , but the a c t i v i t y of the membrane-bound ATPase i s i n s e n s i t i v e to DCCD.  Also, the membranes of these mutants cannot be  energized through ATP hydrolysis suggesting a defect i n the F Q component, i i ) Mutants i n which the a l t e r a t i o n causes an i n s e n s i t i v i t y of the membranebound ATPase a c t i v i t y to DCCD. two classes:  These mutants can further be divided into  class I and class I I (159).  The wild-type membrane-bound  ATPase a c t i v i t y i s i n h i b i t i e d half-maximally at 3-5 nmol DCCD per mg membrane protein, but the mutants of class I and I I are inhibited h a l f maximally, at 30 and 200 nmol DCCD per mg membrane protein, respectively.. These mutants are d i s t i n c t from the unc B?phenotypes,  insthat  the removal  of ECF^ from the membrane r e s u l t s - i n the membrane, becoming leaky to protons. Sequencing  studies on the-DCCD-binding protein^isolated from these  DCCD-resistant mutants, revealed that point-mutations i n the polypeptide are responsible f o r DCCD-insensitivityand proton*impermeability.  These  results are summarized i n Table 6. In the unc B phenotypesj i t i s observed that the replacement  of the  residue responsible f o r reacting with DCCD ( i . e . asp) with either glycine or asparagine caused the loss of DCCD-binding and the membrane became impermeable to protons. affected.  The membrane-bound ATPase a c t i v i t y was not  This suggests that ATP hydrolysis and proton-translocation are  uncoupled and that the mutation causes a conformational change i n F Q or i n the DCCD-binding protein, which does not favour proton conduction.  The  Properties of DCCD-Resistant Mutants of :E. c o l i  TABLE 6  Strain  S e n s i t i v i t y of the Proton-Permeability of F^-depleted Membrane-bound Membranes ATPase A c t i v i t y to DCCD  Binding of [ -C]DCCD to DCCD-binding Protein l  Amino Acid affected i n the DCCD-binding b  Protein  sensitive  permeable  binds  DG 7/1; DG 7/10  insensitive  impermeable  does not bind  Asp  61  * Gly  DG 18/3; DG 3/2  insensitive  impermeable  does not bind  Asp  61  * Gin  sensitive a  permeable  binds  lie  2 8  - Val  sensitive  permeable  binds  lie  2 8  * Thr  Control  „ References  none  159  "Unc B" 159, 160 161  Class I DC 1; DC 13  160, 162  DC 19; DC 24  Class I I DC 25; DC 54  a  3  only at very high concentrations of DCCD  3  162  )  42.  importance of t h i s a c i d i c residue (aspartic acid) i n proton translocation i s also seen i n the DCCD-binding proteins from other organisms i n which this a c i d i c residue i s conserved  (glutamic a c i d ) .  Also, the mutants of  classes I and I I , i n which the aspartic acid at position 61 i s conserved, have membranes which are permeable to protons upon removal of  ECF^.  In the mutants of classes I and I I , DCCD-resistance i s due to the replacement of isoleucine at position 28 with either value or threonine. Since a change at i l e  2 8  can cause such a large change i n DCCD-sensitivity,  i t has been proposed that t h i s residue interacts at the DCCD-binding s i t e (asp ). 6 1  Although the mutations are 33 residues away from the DCCD-  binding s i t e , these two residues could be I n close proximity to each other i f the DCCD-binding protein exists as a "hairpin" structure (150), similar to that proposed f o r bacteriorhodopsin (163).  OBJECTIVES OF THIS STUDY From the information presented i n the INTRODUCTION, i t i s evident that more i s known about ECF^ than about the F Q complex.  The elucidation of  the subunit composition of FQ, of the mechanism of proton translocation through FQ, and of the i n t e r a c t i o n of FQ with ECF^ has been hindered by the lack of a p u r i f i e d , intact  ECF^FQ  complex.  Therefore, one of  the aims of this thesis was to purify the  ECF^FQ  complex i n order to  i d e n t i f y the subunits of the F Q complex.  Secondly, mutants of E_. c o l i  were available i n which the membranes were r e l a t i v e l y impermeable to protons.  Since, an  ECF^FQ  complex with an altered or missing subunit(s)  i n the FQ complex could give an insight into the function of the i n d i v i dual subunits of the FQ complex, these mutants were characterized by a variety of biochemical methods.  F i n a l l y , understanding  the orientation  43.  and/or organization of the FQ polypeptide(s) i n the membrane i s a prerequisite f o r studying the mechanism of proton-translocation. DCCD-binding protonophoric  The  protein of the FQ complex has been implicated i n the a c t i v i t y of FQ.  Therefore, the orientation and/or  organization of t h i s polypeptide  i n the membrane, as well as i t s  interaction with ECF, were studied by immunological  techniques.  44.  MATERIALS AND METHODS  CHEMICALS A l l chemicals were of reagent-grade purity and were purchased from the following sources:  A l d r i c h Chemical Company:  9-aminoacridine hydrochloride.  Amersham Corporation: ACS (Aqueous counting s c i n t i l l a n t ) , NCS tissue s o l u b i l i z e r , [7- *C]phenylglyoxal. 1  BioRad Laboratories:  Acrylamide, agarose, ammonium persulphate, Bio-Gel  P6DG, Bio-Gel HTP (hydroxylapatite), Cellex-CM, 2-mercaptoethanol, N,N'-methylene-bis-acrylamIde,  SDS, TEMED, urea.  Calbiochem Corporation: Complete and incomplete Freund's adjuvant, ct-chymotrypsin (bovine pancreas), o c t y l B-D-glucopyranoside, PMS, pronase, valinomycin.  '  Chemical Dynamics Corporation: e-aminocaproic acid, p-aminobenzamidine dihydrochloride. Difco Laboratories:  Bacto-agar, Bacto-Penassay broth, Bacto-peptone,  Bacto-tryptone, casamino acids, nutrient broth, yeast extract^ E. Merck. AG (Germany): Eastman Kodak Company:  Amido Black 10B. Chloramine T, DCCD, Merthiolate.  Fisher S c i e n t i f i c Company:  S i l i c o t u n g s t i c acid.  Goldschmidt (Essen): Aminoxid WS-35. LKB-Produkter AB (Sweden):  Ampholytes.  Mann Research Laboratories:  Tween 60, Tween 80, thyroglobulin (Pig).  Mandel S c i e n t i f i c Company:  Whatman CM-32. i  45. i  Matheson, Coleman and B e l l Manufacturing Chemists:  Cyanogen bromide,  sodium d i t h i o n i t e . Miles-Yeda Ltd.: w-Amino Butyl Agarose, Butyl Agarose, Decyl Agarose. Miles Laboratories:  Staphylococcus aureus Vg protease.  Onyx Chemical Company:  Ammonyx Lo. )  P a r t i c l e Data Laboratories Ltd.: Nonidet P-40. Pharmacia Fine Chemicals, Inc.:  AH-Sepharose 4B, Blue Dextran,  DEAE-Sepharose CL-6B, Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, Sepharose CL-6B, Sephadex G-50,  Sephadex LH-60, Molecular weight  markers f o r g e l electrophoresis. Pierce Chemical Company:  Amino acid standards f o r analyzer, ninhydrin.  Research Products International Corporation: Sigma Chemical Company:  ['"CpCCD.  ATP, bovine serum albumin, Brig 35,  2,3-butanedione, Coomassie B r i l l i a n t Blue G, Coomassie B r i l l i a n t Blue R, catalase (bovine), DNase 1 (bovine pancreas), DTT, guanidine hydrochloride, HEPES, L-amino acids, lysozyme (egg), Lubrol PX, Lubrol WX, Lubrol 17A-10, L-a-phosphatidylcholine (egg), MOPS, NADH, phenylglyoxal, poly-L-lysine (M , 400 000), PMSF, r  phosphatidylcholine (soybean, 22%), RNase (bovine pancreas), sodium cholate, sodium deoxycholate, sodium N-lauroyl sarcosine, sodium succinate, sucrose, T r i s base, Triton X-100, Triton X-114. Worthington Biochemical Corporation:  TPCK-trypsin, soybean trypsin  inhibitor.  Rabbit y-globulin and N a ( Molday.  1 2 S  I ) were generous g i f t s from Dr. R.S.  TPCK-trypsin treated ECF was a kind g i f t from Cynthia Hou.  46.  MAINTENANCE OF BACTERIAL STRAINS Table 7 l i s t s the strains of bacteria used i n t h i s study.  These  strains were maintained as slants and stabs containing Penassay broth-agar. The slants were prepared by b o i l i n g 1.75 g Bacto-Penassay broth and 1.5 g agar i n 100 ml d i s t i l l e d water.  The mixture was then dispensed i n 5  ml portions into screw-cap tubes and autoclaved f o r 20 min at a pressure of 15 p s i . The tubes were allowed to cool such that the nutrient broth s o l i d i f i e d as s l a n t s .  Exponentially growing b a c t e r i a l cultures were  streaked onto the slants, incubated overnight at 37°C and then stored at 4°C u n t i l needed.  These strains were subcultured every 4-6 months a f t e r  checking f o r n u t r i t i o n a l markers and then transferred to fresh slants. Cultures to be stored f o r longer periods of time (9 months) were inoculated into nutrient broth prepared as f o r slants, but containing 0.7% (w/v) agar and dispensed i n 2.5 ml aliquots into screw-cap v i a l s .  GROWTH OF CELLS A 10 ml volume of Penassay broth was inoculated with a culture from a slant and grown overnight at 37°C.  This was then transferred to 400 ml of  -the appropriate medium i n a 2 1 conical f l a s k and grown overnight at 37°C with shaking (at 250 rpm) i n a New Brunswick Rotary Incubator.  The c e l l s  were either harvested and used immediately or the growing culture was used as 10% (v/v) innocula f o r growing larger quantities of c e l l s (4.5 1). Batches of 4.5 1 were grown at 37°C (except f o r PS3 which was grown at V  60°C) with vigorous aeration (at 25 1/min) i n a Lab-Line/S.M.S. Hi-Density Fermentor.  C e l l growth was monitored by measuring the absorbance of the  culture at 420 nm. The c e l l s were harvested at the l a t e exponential phase of growth by  Bacterial Strains used i n t h i s Study  TABLE 7  Origin  Genotype  Strain  i " , Z", y+, a+  H.R. Kaback  DL-54  i~,  R.D. Simoni  AN 180  F~, arg, t h i , mtl, x y l , s t r  AN 120  F~, arg, t h i , mtl, x y l , s t r , unc A401  AN 382  F~, arg, t h i , mtl, x y l , s t r , t f r - 3 , X , unc B402  WS1  F~, pro, l a c , J^g , g a l , ara, h i s , x y l , man, t h i , s t r  E_. c o l i ML 308-225  Z~, y , a , unc +  +  F. Gibson  R  F. Gibson  R  R  R  R  D.L. Gutnick D.L. Gutnick  I44  same as WS1 but neo  CBT-1  thi  T.C.Y. Lo  CBT-302  t h i , unc  T.C.Y. Lo  N  Thermophile PS3  R  and unc  B.J. Bachmann  -  Y. Kagawa  48.  centrifugation at 4 500 xg f o r 15 min, then washed twice i n 0.9% (w/v) NaCl and sedimented by centrifugation at 17 600 xg f o r 10 min.  The c e l l s were  stored at -20°C.  MEDIA A l l the strains l i s t e d i n Table 7, except PS3, were grown on a minimal salts-glucose media (164) containing 0.7% (w/v) K HP0 .3H 0, 0.3% (w/v) 2  4  2  KH P0 , 0.05 % (w/v) sodium c i t r a t e .2H 0, 0.02% (w/v) MgS0 .7H 0, 0.1% (w/v) 2  4  2  4  2  (NH ) S0 , 0.4% (w/v) glucose and supplemented with 0.1% (w/v) casamino 4  2  4  acids.  In some experiments, the glucose concentration was increased to 0.8%  (w/v).  F e r r i c c i t r a t e (12 PM) was added to media of volumes exceeding 1  litre.  Where required, amino acids and thiamine were added at 50 pg per  ml and 1 P g per ml, respectively. In order to obtain a higher y i e l d of c e l l mass when growing mutants, the media was also supplemented with 0.1% (w/v) yeast extract, 0.1% (w/v) Bacto-tryptone, 3.3 uM C a C l , 6 PM CaS0 , 6 PM MnCl , 7 PM C o C l , 2  0.6 UM Z n C l  2  4  2  2  and 64 PM EDTA.  In experiments requiring large quantities of E. c o l i ML 308-225, the c e l l s were purchased from a commercial source (University of Alberta). These c e l l s were grown to the late exponential phase on minimal - s a l t s glucose (0.4% (w/v)). PS3 was grown on media (165) containing 13.3% (w/v) Nutrient broth, 6.7% (w/v) Bacto-peptone, 5% (w/v) NaCl and 5% (w/v) K HP0 .3H 0 at pH 7.4. 2  4  2  PREPARATION OF MEMBRANES The method described by Bragg et a l . (164) was used.  Cells were  suspended i n buffer containing 50 mM T r i s - H S 0 , 10 mM MgCl ?  4  2  pH 8.0  49.  (TM buffer) at a r a t i o of 1 g wet weight/3 ml.  About 5-10  pg of DNase  was added and the c e l l s broken by, one or, i n some cases, two passages through an Aminco French Pressure c e l l precooled to 0°C, at a pressure of 2 1400 Kg/cm .  C e l l debris and unbroken c e l l s were removed by centrifuga-  tion at 12 000 xg f o r 10 min and the membranes sedimented at 250 000 xg f o r 2 to 2.5 h.  by centrifugation  Unless indicated otherwise, a l l centrifuga-  tion steps were carried out at 0-5°C.  The membranes were washed twice by  resuspension i n TM buffer and resedimented  as before.  They were processed  i n a number of ways to be described below. PREPARATION OF EDTA-LYSOZYME SPHEROPLASTS These were prepared by the method of Singh and Bragg (166).  When  possible, freshly-grown c e l l s were used. E_. c o l i c e l l s were grown to the late exponential phase and harvested by centrifugation at 4 500 xg f o r 15 min.  The c e l l s were washed three  times by resuspension i n 50 mM Tris-HCl buffer, pH 8.0 and centrifugation at 12 000 xg f o r 10 min.  The washed c e l l s (2.75 g) were suspended at 20°C  i n 37 volumes (w/v) of 50 mM Tris-HCl buffer, pH 8.0 containing 0.5 M sucrose.  EDTA and lysozyme were added to f i n a l concentrations of 2.5  mM I  and 0.1 mg/ml, respectively. The rate of spheroplast formation was. followed by removing a 50 u l sample of the c e l l suspension at various' i n t e r v a l s , d i l u t i n g  i t to 2 ml  with d i s t i l l e d water, and then measuring the absorbance at 420  nm.  After 30 min of incubation, the suspension was centrifuged at 12 xg f o r 10 min.  The p e l l e t was suspended i n 275 ml of 20 mM  000  potassium  phosphate buffer, pH 8.0 and MgSO^ was added to a f i n a l concentration of 1 mM.  This was followed by addition of RNase and DNase at 20 yg/ml and  50.  the suspension s t i r r e d f o r 20 min and then centrifuged at 6 400 xg f o r 15 min.  The supernatant was centrifuged at 125 000 xg f o r 45 min. When  K -loaded v e s i c l e s were not required, the spheroplasts were washed twice +  by resuspension  i n a buffer containing 0.4 M Sucrose and 10 mM MgC^, and  resedimented as before.  The spheroplasts were f i n a l l y taken up i n the same  buffer, stored at 4°C and were used within 12 h.  PREPARATION OF K -L0ADED SPHEROPLASTS +  The p e l l e t containing the spheroplasts was taken up i n 10 volumes (w/v) of 0.5 M potassium phosphate buffer, pH 7.0 and gently heated at 40°C f o r 0.5 h (28,167).  The suspension was then rapidly c h i l l e d i n an ice-bath  and "after 10 min of incubation, MgSO^ was added to a f i n a l concentration of 10 mM.  After a further 10 min incubation, the spheroplasts were  sedimented by centrifugation at 125 000 xg f o r 45 min. were washed twice by resuspension centrifuged as before.  The spheroplasts  i n 0.4 M Sucrose, 10 mM MgC^ and  The p e l l e t containing K -loaded spheroplasts was +  f i n a l l y taken up i n the same buffer, and stored at 0°C.  It was used within  12 h.  ISOLATION OF E C ^ ECF^ was i s o l a t e d from 60 g of c e l l s as described by Bragg and Hou (89).  Membrane v e s i c l e s from 60 g of E_. c o l i ML 308-225 were prepared i n  TM buffer as previously described.  They were washed twice by suspension i n  a buffer (200 ml) containing 5 mM Tris-HCl, pH 7.3, 10% (v/v) g l y c e r o l , 20 mM e-aminocaproic acid, 6 mM p-aminobenzamidine, and 0.5 mM DTT, followed by centrifugation at 250 000 xg f o r 2 h. The washed v e s i c l e s were suspended i n 60 ml of 1 mM Tris-CHl buffer pH 7.5 containing 0.5 N  mM EDTA, 0.1  51.  mM  DTT and 10%  (v/v) g l y c e r o l ("dialysis buffer") and dialyzed against 3 1  d i a l y s i s buffer f o r 16 h at 4°C.  The dialyzed material was  ml with d i a l y s i s buffer and then centrifuged as above. recentrifuged at 250 000 xg for 3 h.  diluted to  The supernatant  After t h i s step, the supernatant  200 was was  removed and concentrated to 18 ml by u l t r a f i l t r a t i o n using an Amicon XM-100A filter.  Methanol (4.5 ml) was  solution followed by 0.46 for  ml of 1M C a C l . 2  20 min at 20°C, i t was  After incubation of the mixture  centrifuged at 12 000 xg f o r 20 min and  supernatant, containing ECF^,  PURIFICATION OF E C ^  then added drop-wise to the concentrated  was  the  applied to a column of AH-Sepharose 4B.  ON AH-SEPHAROSE 4B  AH-Sepharose 4B was brated for 4 h at 20°C.  suspended i n an excess of 0.5 M NaCl and The suspension was  2 cm diameter column to a height of 7.5  cm.  equili-  degassed and then poured into a The column was washed with  several volumes of 0.5 M NaCl, d i s t i l l e d water and f i n a l l y equilibrated with d i a l y s i s buffer containing 1 mM 0.5 mM  EDTA and 0.1 mM  DTT.  the column at 20°C which was buffer.  Tris-HCl, pH 7.5,  10% (v/v) g l y c e r o l ,  The sample containing ECF^ was  adsorbed onto  then washed with 2-3 volumes of d i a l y s i s  E l u t i o n of the enzyme was  ( t o t a l volume, 200 ml) of 0.25  carried out with a l i n e a r gradient  to 0.75  M KC1  i n d i a l y s i s buffer.  The  fractions containing ATPase a c t i v i t y were pooled and concentrated to one two ml by passage through an Amicon XM-100A f i l t e r . material was  or  The concentrated  e i t h e r used immediately or divided into small aliquots,  rapidly frozen, and stored at -70°C. The column was volumes of 1 M KC1 and stored at  4°C.  regenerated between experiments by washing with several i n d i a l y s i s buffer, re-equilibrated with d i a l y s i s buffer,  52.  In some cases, the ECF obtained a f t e r AH-Sepharose 4B was further 1  p u r i f i e d by sucrose density gradient centrifugation.  PURIFICATION OF ECFj^ AND TPCK-TRYPSIN TREATED ECF BY. SUCROSE DENSITY 1  GRADIENT CENTRIFUGATION A discontinuous gradient (23) consisting of successive 1 ml layers of 25%, 22.5%, 20%, 17.5% and 15% (w/v) sucrose i n 50 mM T r i s - H S 0 2  pH 7.8 containing 0.5 mM EDTA and 0.1 mM DTT was poured.  4  buffer,  The sample (0.4  ml, 3-5 mg) protein was, loaded and the gradient centrifuged at 260 000 xg for 16 h.  Twenty drop fractions were collected by using a Beckman  fractionator attached to a f r a c t i o n c o l l e c t o r .  PREPARATION OF ECF^-DEPLETED MEMBRANES Several methods were used to s t r i p the membranes of ECF^. a)  The membrane f r a c t i o n remaining a f t e r d i a l y s i s against 1 mM Tris-HCl  buffer, pH 7.5 containing 0.5 mM EDTA, 0.1 mM DTT and 10% (v/v) g l y c e r o l ("dialysis buffer") as described under "Isolation of ECF^", was essent i a l l y devoid of ECF^. The membranes were washed once by resuspension i n d i a l y s i s buffer and centrifuged as before, and then stored at 4°C. b)  More d r a s t i c treatments with urea, guanidine hydrochloride, s i l i c o -  tungstic acid, or proteases were, also used. Dialyzed membranes (described- i n ( a ) ) , or membranes prepared i n TM i  buffer, pH 8.0, suspended i n d i a l y s i s buffer, pH 7.5 at a protein concent r a t i o n of 10 mg/ml were incubated at 20°C f o r 30 min with either 2 M urea, 2 M guanidine hydrochloride, 1% or 2% (w/v) s i l i c o t u n g s t i c acid, Staphylococcus aureus Vg protease or TPCK-trypsin at an enzyme to protein r a t i o of 1:40 and 1:15, respectively.  In the case of TPCK-trypsin, the reaction  53.  was stopped by the addition of soybean trypsin i n h i b i t o r at a r a t i o to trypsin of 3:5 with incubation f o r another 10 min.  The incubated mixtures  were diluted 4-fold with buffer and then centrifuged at 250 000 xg f o r 2.5 h.  The sedimented  vesicles were washed twice by resuspension i n d i a l y s i s  buffer followed by resedimentation as above. i The ECF^-depleted membranes prepared by either method (a) or (b) were f i n a l l y suspended i n the appropriate buffer at 4°C. The pH of the v e s i c l e suspension was readjusted to pH 7.5 with d i l u t e KOH when s i l i c o tungstic acid was used. PREPARATION OF RAT-LIVER MITOCHONDRIAL MEMBRANES Phosphate-washed p u r i f i e d inner mitochondrial membranes were prepared from r a t - l i v e r mitochondria as described by Soper and Pedersen (168). The p u r i f i e d membranes at 5 mg protein/ml of PBS-10 mM KgCl^ were sonicated at a power of 50 W i n a Branson W185D s o n i f i e r f o r 15 s periods f o r a t o t a l of 2.5 min.  The temperature was kept at 0°. Large fragments were removed  by centrifugation at 12 000 xg f o r 10 min.  The supernatant was either  centrifuged at 250 000 xg f o r 2 h to sediment "sonicated washed mitochond r i a l membranes" or incubated with 4 M urea f o r 30 min at 22°C p r i o r to centrifugation as above.  The "urea-treated washed mitochondrial membranes"  were washed by recentrifugation of a suspension i n PBS-10 mM MgC^. A l l membranes were resuspended  i n PBS-10 mM MgC^.  PREPARATION OF THE SUBUNITS OF ECF^  TPCK-Trypsin ECF  Treated ECFj^  lacking the 6 and e, and perhaps part of the Y subunit, was  54.  prepared  by c o n t r o l l e d treatment  of the ?  1  with TPCK-trypsin,  as d e s c r i b e d  by Bragg and Hou ( 8 9 ) .  '  1.25 mg o f s u c r o s e g r a d i e n t - p u r i f i e d EZY^ i n 0.385 m l of 22.5% (w/v) sucrose i n 20 mM t r i e t h a n o l a m i n e - H C l was t r e a t e d w i t h 37 yg T P C K - t r y p s i n  b u f f e r , pH 7.5 c o n t a i n i n g 0.5 mM EDTA i n 10 y l b u f f e r a t 37°C f o r 10 min.  B o v i n e pancreas t r y p s i n i n h i b i t o r (146 yg i n 10 y l b u f f e r ) was added and a f t e r 5 min a t 20°C, t h e s o l u t i o n was d i l u t e d w i t h an e q u a l volume of b u f f e r and t h e t r y p s i n - t r e a t e d ATPase r e i s o l a t e d by sucrose  density  i  gradient c e n t r i f u g a t i o n .  a and B S u b u n i t s o f ECF^ These were p r e p a r e d Columbia).  by Dr. H e l g a S t a n - L o t t e r ( U n i v e r s i t y of B r i t i s h  ECF.^ o b t a i n e d a f t e r p u r i f i c a t i o n on AH-Sepharose 4B was  d i s s o c i a t e d i n t o i t s s u b u n i t s w i t h h i g h i o n i c s t r e n g t h b u f f e r as d e s c r i b e d by Dunn and F u t a i ( 7 2 ) . The d i s s o c i a t e d ECF.^ s u b u n i t s were a p p l i e d t o a f r e s h l y made column o f h y d r o x y l a p a t i t e .  The subsequent s e p a r a t i o n o f t h e  a and £ s u b u n i t s was c a r r i e d out on a column o f DEAE-Sepharose CL-6B as d e s c r i b e d by Bragg e t a l . ( 8 2 ) .  SOLUBILIZATION OF MEMBRANE VESICLES WITH DETERGENTS - The s o l u b i l i z a t i o n c h a r a c t e r i s t i c s o f v a r i o u s d e t e r g e n t s  were  determined as f o l l o w s . Membranes o f E_. c o l i ML 308-225 were prepared described previously.  i n TM b u f f e r as  They were suspended i n 0.5 M T r i s - H 2 S 0 ^  pH 8.0 c o n t a i n i n g 0.25 M N a ^ O ^  buffer,  and 10% ( v / v ) g l y c e r o l a t a membrane  p r o t e i n c o n c e n t r a t i o n o f 20-25 mg/ml.  The s u s p e n s i o n was d i v i d e d i n t o  s e v e r a l a l i q u o t s and t o each was added, d r o p - w i s e , d i f f e r e n t amounts o f  55.  detergents from either a 10% (w/v) or 20% (w/v) stock solution i n d i s t i l l e d water, or i n some instances added neat.  After incubation f o r 30 min at  20°C, they were centrifuged at 250 000 xg for 2.5 h and the ATPase a c t i v i t y and protein content determined  PURIFICATION OF THE  ECFJFQ  i n the supernatant and p e l l e t f r a c t i o n s .  COMPLEX SOLUBILIZED WITH N-LAUROYL SARCOSINE  v  Gel F i l t r a t i o n on Sepharose 6B Membranes of E_. c o l i ML 308-225 were prepared i n TM buffer as described previously. They were suspended i n 0.5 M T r i s - ^ S O ^ buffer, pH 8.0 containing 0.25 M Na^O^ and 10% (v/v) glycerol at a protein concentration of 20-25 mg/ml.  Detergent was added drop-wise, from a 20%  (w/v) stock solution i n d i s t i l l e d water, to give a detergent (mg) to protein (mg) r a t i o of 0.5. After incubation at 20°C f o r 30 min, the mixture was centrifuged at 250 000 xg f o r 2 h.  The supernatant was  recentrifuged, then concentrated to about one quarter of i t s o r i g i n a l volume (20-25 mg protein per ml) by u l t r a f i l t r a t i o n using an Amicon XM-100 A filter.  The concentrated material was loaded onto a column of Sepharose  6B. Pre-swollen Sepharose 6B was suspended i n 50 mM T r i s - ^ S O ^ buffer, pH 8.0 containing 10% (v/v) g l y c e r o l at 4°C.  A slurry was poured into a  2.5 cm diameter column to a height of 37 cm, and the column equilibrated with 50 mM T r i s - H ^ O ^ buffer, pH 8.0, containing 0.25 M Na^O^, 10% (v/v) g l y c e r o l and 0.5% (w/v) of detergent.  The sample (2-5% (v/v) of  t o t a l bed volume) was loaded onto the column and eluted with the appropriate buffer.  The column was regenerated between experiments by  washing with 2-3 bed volumes of the apropriate buffer.  56.  Hydrophobic-Interaction Chromatography Membranes of E_. c o l i were prepared i n TM buffer and then washed twice by. resedimentation at 250 000 xg f o r 2 h from 5 mM pH 7.3 containing 10% (v/v) g l y c e r o l , 20 mM p-aminobenzamidine and 0.5 mM DTT. 0.5 M T r i s - H S 0 2  4  drop-wise, from a 20% (w/v) detergent (mg) f o r 30 min,  e-aminocaproic acid, 6 mM  The washed membranes were suspended i n  buffer, pH 8.0 containing 0.25  glycerol at a protein concentration  M Na S0 and 10%' (v/v) 2  of 20-25 mg/ml.  4  Detergent was  added  stock solution i n d i s t i l l e d water, to give a  to protein (mg)  the mixture was  Tris-HCl buffer,  r a t i o of 0.25-0.5.  After incubation at 20°C  centrifuged at 250 000 xg for 2 h.  The  supernatant was  recentrifuged and then subjected to ammonium sulphate  precipitation.  Saturated  NH^OH) was  ammonium sulphate (adjusted to pH 7.5  added to the supernatant at 0°C to give 35% of saturation.  A f t e r 30 min incubation, the solution was min.  with  The supernatant was  centrifuged at 30 000 xg f o r 20  next brought to 50% of saturation.  The  p r e c i p i t a t i n g at 35-50% (0.35-0.5 P) saturation of (NH^SO^ was up at 4°C, i n s o l u b i l i z a t i o n buffer at a protein concentration and N-lauroyl sarcosine added to give a detergentrprotein  pellet taken  of 4-6 mg/ml  r a t i o of 0.8-1.0.  This was applied to a column of Phenyl-Sepharose CL-4B. Phenyl-Sepharose CL-4B (or other hydrophobic-exchange resins) i n 50 Tris-H S0^ buffer, pH 8.0 was 2  poured into a 1.8  cm diameter column to a  height of 25 cm and the column equilibrated at 4°C with 50 mM buffer, pH 8.0  which was was  Tris-H^O^  containing 20% (NH^SO^ (by saturation), 10 mM MgCl  10% (v/v) g l y c e r o l .  The s o l u b i l i z e d f r a c t i o n was  2  and  adsorbed onto the column  then washed with several volumes of the same buffer.  The enzyme  eluted f i n a l l y with the appropriate buffer as described i n the  "RESULTS" section.  mM  57.  The r e s i n was regenerated by washing with 2-3 bed volumes of 2%  (w/v)  T r i t o n X-100 followed i n succession by i) ii) iii) iv) v) vi)  2 bed volumes of d i s t i l l e d water 2 bed volumes of ethanol 2 bed volumes of n-butanol 2 bed volumes of ethanol  N  2 bed volumes of d i s t i l l e d water and e q u i l i b r a t i o n with s t a r t i n g buffer. With Phenyl-Sepharose CL-4B, an additional washing with 2-3 bed  volumes of 2% (w/v) SDS a f t e r the T r i t o n X-100 wash was found to be necessary to remove tightly-bound  proteins.  PURIFICATION OF THE E C F ^ Q COMPLEX BY SUCROSE DENSITY GRADIENT CENTRIFUGATION The enzyme obtained a f t e r chromatography on Phenyl-Sepharose CL-4B was concentrated  by u l t r a f i l t r a t i o n  using an Amicon PM-10  by sucrose density gradient centrifugation.  A l i n e a r 17.5% to 25%  sucrose gradient (12 ml, t o t a l volume) was poured. dissolved i n 50 mM T r i s - ^ S O ^ cholate, 5 mM MgCl  2  f i l t e r , and p u r i f i e d (w/v)  The sucrose was  buffer, pH 8.0 containing 0.5% (w/v) sodium  and 0.25 mM DTT.  In some experiments, 12 uM  p-aminobenzamidine was included i n the gradients as a protease i n h i b i t o r and, on occasion, 0.1% (w/v) L-ct-phosphatidylcholine (soybean) was also , added. The sample (1-1.2 ml, 5-10 mg protein per ml) was loaded and the gradients centrifuged at 280 000 xg f o r 23 h and ten-twenty drop fractions c o l l e c t e d by using a Beckman f r a c t i o n a t o r connected to a f r a c t i o n c o l l e c t o r .  i  58.  DEAE ION-EXCHANGE CHROMATOGRAPHY This was based on the method of F r i e d l et a l . (106). pre-swollen gel i n 50 mM  A slurry of  Tris-HCl buffer, pH 8.0 at 4°C was poured into a  1.5 cm diameter column to a height of 13 cm and the column equilibrated with 50 mM Tris-HCl buffer, pH 8.0 containing 1 mM MgCl , 0.2 mM DTT, 2  mM EGTA, 0.1 mM PMSF, 100 mM KC1, phospholipid, 0.9% effluent was  (w/v)  0.2  20% (v/v) methanol, 50 mg/ml soybean  Aminoxid WS-35, u n t i l the conductivity of the  the same as the buffer.  The sample was adsorbed onto the column and washed with e q u i l i b r a t i o n buffer.  The enzyme was eluted with a l i n e a r gradient of 100-300 mM KC1 i n  e q u i l i b r a t i o n buffer.  The r e s i n was  regenerated by washing with 1 M KC1 i n  50 mM Tris-HCl buffer, pH 8.0 and equilibrated with the appropriate buffer.  PREPARATION OF DCCD-BINDING PROTEIN The method described by Fillingame (149) was used. or ECF^-depleted  Membrane vesicles  membranes at a protein concentration of 40-50 mg/ml i n  e i t h e r TM buffer, pH 8.0 or i n d i s t i l l e d water, were mixed with 20 volumes of chloroform:methanol (2:1) and stored at 4°C f o r 24 h.  The  was passed through a f r i t t e d glass funnel of medium porosity.  suspension To the  f i l t r a t e , i n a glass-stoppered bottle, was added 0.20 volume of d i s t i l l e d water and the contents mixed by gently inverting the bottle.  After  standing at 4°C overnight, the contents of the bottle were warmed to 20°C to completely resolve the two phases. with 0.05  The lower phase was washed twice  volume (of o r i g i n a l f i l t r a t e ) of a mixture of chloroform:  methanol:water (3:47:48).  F i n a l l y , the washed lower phase was  clarified  with a small amount of methanol and the mixture diluted with one volume of chloroform.  The chloroform was added i n small amounts and a requisite  59.  amount of methanol was added whenever the mixture started to turn cloudy. It was then c a r e f u l l y taken to dryness by rotoevaporation at 20°C using the vacuum from a water-aspirator.  On occasion, chloroformrmethanol (10:1) was  added to the evaporation f l a s k whenever the contents started to turn cloudy.  The dried contents of the f l a s k were taken up i n a small volume of  chloroform:methanol (2:1) and any traces i n insoluble material removed by passage through a medium or f i n e porosity f r i t t e d glass funnel.  The  f i l t r a t e was c h i l l e d to -20°C and 4 volumes of precooled (-20°C) d i e t h y l ether added slowly with s t i r r i n g .  The mixture was stored at -20°C f o r 24 h  and the p r e c i p i t a t e removed by centrifugation f o r 45 min at 4 000 xg and -20°C i n a capped stainless s t e e l tube.  The p e l l e t was again taken up i n  the o r i g i n a l volume of chloroforro:methanol (2:1) and p r e c i p i t a t i o n with 4 volumes of pre-cooled d i e t h y l ether was repeated.  The precipitate was  removed by centrifugation and dried at 20°C with a stream of nitrogen gas. It was dissolved i n a small volume of chloroform methanol  (2:1) and  - insoluble material removed by f i l t r a t i o n through a f r i t t e d glass funnel. The f i l t r a t e was then applied to a thin-layer plate or adsorbed onto a column of CM-cellulose as described below. For the i s o l a t i o n of large amounts of DCCD-binding of Altendorf et a l . (150) was followed.  protein, the method  In t h i s procedure, the use of  whole c e l l s as starting material was more convenient than using membrane vesicles. 400 to 500 g (wet weight) of frozen IS. c o l i c e l l was mixed with 20 volumes (w/v) of chloroform:methanol (2:1) at 20°C and the suspension mixed i n a Waring blender at top speed f o r 1 min.  I t was s t i r r e d at 4°C f o r 24 h  and then f i l t e r e d through a medium porosity f r i t t e d glass funnel. i  f i l t r a t e was treated as described above.  i  The  60.  PURIFICATION OF DCCD-BINDING PROTEIN Thin Layer Chromatography  v  A 20 x 20 cm glass plate, precoated with s i l i c a g e l G (1500  pm,  Analtech Inc.) was activated by placing i t into an oven at 100°C f o r 1 h and then allowed to cool to 20°C.  The isolated DCCD-binding  protein (2 mg  protein) i n chloroformmethanol (2:1) was applied as a 0.5 cm wide streak, 9 cm i n length and 2.5 cm from the bottom edge of the plate.  The streak  was thoroughly a i r - d r i e d and the chromatogram developed i n a covered tank at  20°C i n a mixture of chloroformmethanol:water  mM HC1 (147). top  (65:25:4) containing 20  The solvent was allowed to ascend to within 1 cm from the  of the plate (0.5,-0.75 h).  The plate was a i r - d r i e d and placed i n a  tank equilibrated with iodine c r y s t a l s f o r 3-5 min.  The stained spots were  scraped from the plate and treated with enough water to completely wet the powder.  After 30 min at 20°C, the powder was extracted with 5-10 ml of  chloroformmethanol (2:1) containig 20 mM HC1 f o r 30 min. was centrifuged at 2 500 xg f o r 15 min. the  same manner, three times.  fine-porosity f r i t t e d nitrogen.  The suspension  The s i l i c a g e l was re-extracted i n  The combined extracts were passed through a  glass funnel and the f i l t r a t e dried under a stream of  The residue was taken up i n a small volume of chloroformmethanol  (2:1) and c h i l l e d to -20°C.  To this was added 4 volumes of precooled  (-20°C) d i e t h y l ether and the mixture stored at -20° C f o r 24 h. p i t a t e containing the DCCD-binding  The p r e c i -  protein was removed by centrifugation at  4 000 xg and -20°C f o r 45 min and then dried under nitrogen at 20°C.  Chromatography on CM-Cellulose The methods described by Rouser and coworkers (169) and Altendorf and coworkers (150) were modified f o r our purposes.  61.  CM c e l l u l o s e (Whatman CM-32 or BioRad Cellex CM) was suspended i n 15 volumes (w/v) of g l a c i a l acetic acid f o r 1.5 h at 20°C.  The fines were  removed and the resin washed with d i s t i l l e d water u n t i l the pH of eluent was between 5 and 7.  Next, the r e s i n was suspended i n 10 volumes (v/v) of  25% (v/v) ammonium hydroxide.  After 1.5 h at 20°C, the suspension was  d i l u t e d several f o l d with d i s t i l l e d water and centrifuged at 4 500 xg f o r 10 min.  The sedimented resin was washed repeatedly with d i s t i l l e d water  u n t i l the pH of the supernatant was between 7 and 9.  F i n a l l y , the resin  was washed sequentially with 3 volumes (v/v) each of methanol, chloroform: methanol (1:1), and chloroform methanol (2:1). A slury of the resin i n chloroform:methanol (2:1) was poured into a solvent-resistant column and the column bed protected with glass wool held down with marbles.  Two volumes (v/v) of chloroformmethanol (2:1) was  passed through the column and the sample ( i n chloroformmethanol (2:1)) was then applied.  The column was washed sequentially with 5-10 volumes of  chlorof ormmethanol  (2:1), 3-5 volumes of chlorof orm methanol (1:1), and  the DCCD-binding protein eluted with 5'volumes of chlorof ormmethanol: water (5:5:1).  In some experiments, the column was washed with 5 volumes of  chlorof ormmethanol: water (10:10:1) p r i o r to elution of the DCCD-binding protein.  The fractions containing the DCCD-binding protein were pooled and  chloroform and water added so that,the f i n a l ratio of chloroformmethanol: water was 8:4:3 and a two-phase system was obtained (149).  The lower phase  was diluted with 1 volume of chloroform and the requisite amount of methanol was added to keep the solution c l e a r . by rotoevaporation at 20°C.  The mixture was then taken to dryness  The dried contents were immediately taken up  i n a small volume of chlorof ormmethanol  (2:1) containing 20 mM ammonium  acetate and applied to a Sephadex LH-60 column.  Chromatography on Sephadex LH-60 T h i s was based on t h e method o f F i l l i n g a m e ( 1 4 9 ) .  Sephadex LH-60 was  s t i r r e d i n t o 25 volumes (w/v) o f c h l o r o f o r m : m e t h a n o l ( 2 : 1 ) c o n t a i n i n g 20 mM ammonium a c e t a t e and l e f t a t 20°C f o r 6 h.  A s l u r r y o f t h i s s u s p e n s i o n was  poured i n t o a s o l v e n t - r e s i s t a n t column and a s m a l l amount o f washed r e a g e n t grade s e a sand c a r e f u l l y a p p l i e d t o t h e t o p o f t h e bed t o f a c i l i t a t e application. the  sample  The column was e q u i l i b r a t e d w i t h t h e same s o l v e n t a t 20°C and  sample a p p l i e d .  The DCCD-binding p r o t e i n was e l u t e d w i t h t h e same  s o l v e n t system a t a f l o w r a t e of 0.1 t o 0.15 m l p e r m i n .  The f r a c t i o n s  c o n t a i n i n g t h e p u r i f i e d DCCD-binding p r o t e i n were p o o l e d and s t o r e d a t -20°C.  When t h e p r o t e i n was needed, samples were removed and t a k e n t o  d r y n e s s under reduced p r e s s u r e a t 16-18°C i n a r o t a r y e v a p o r a t o r ( B u c h l e r I n s t r u m e n t s , R o t a r y Evapo-mix).  SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS Sample  Preparation  S a l t s and d e t e r g e n t s were removed from t h e samples i n one o f two ways: (i)  Samples from f r a c t i o n s o b t a i n e d a f t e r column chromatography were  d i a l y z e d a t 4°C f o r 20-48 h a g a i n s t 100 volumes o f d i s t i l l e d w a t e r ( c o n t a i n i n g 0.01% ( v / v ) h i b i t a i n e ) and w h i c h was changed a t 8 h i n t e r v a l s . The d i a l y z e d f r a c t i o n s were l y o p h i l i z e d and s t o r e d a t -70°C. (ii)  A more r a p i d method o f removal o f s a l t and d e t e r g e n t f r o m t h e  samples was by t h e c e n t r i f u g a t i o n - c o l u m n chromatography procedure o f Penefsky (170). A s l u r r y o f Sephadex G-50 e q u i l i b r a t e d w i t h 50 mM T r i s - H S 0 2  b u f f e r , pH 8.0 c o n t a i n i n g 10 mM M g C l  2  4  was poured i n t o t h e b a r r e l o f a 1  m l d i s p o s a b l e t u b e r c u l i n s y r i n g e ( B e c t o n - D i c k i n s o n ) w h i c h had been  fitted  63.  with a porous polyethylene and allowed to drain.  disk.  I t was placed i n a 15 x 125 mm test tube  The volume of the g e l was 1 cm . 3  Excess buffer  was removed by centrifuging at 900 xg f o r 2 min at 20°C (International Equipment Company, Model CL45436M).  A 100 u l sample was c a r e f u l l y  onto the column which was then recentrifuged.  loaded  The eluent was depolymerized  before g e l electrophoresis.  Depolymerization of Samples The eluent was depolymerized by adding an equal volume of 125 mM Tris-HCl buffer, pH 6.8 containing 4% (w/v) SDS, 4% (v/v) 2-mercaptoethanol, 20% (v/v) g l y c e r o l and 0.002% (w/v) bromophenol blue, and the mixture heated at 100°C f o r 5 min.  The sample was cooled to 20°C.  Lyophilized samples were dissolved i n a solution containing 62.5 mM Tris-HCl buffer, pH 6.8, 2% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 10% (v/v) g l y c e r o l and 0.001% (w/v) bromophenol blue and heated at 100°C as above. The DCCD-binding polypeptide was depolymerized i n the same buffer as described previously except that the f i n a l T r i s concentration was 50 mM and the sample was heated at 45°C f o r 18 h. The protein concentration of the depolymerized sample was i n the range of 0.25-1 mg/ml. In some instances, centrifugation of the depolymerized sample i n a desk-top centrifuge (International Equipment Company, model CL45436M) at f u l l speed f o r 5 min was needed to remove any insoluble material p r i o r to gel electrophoresis.  64.  Slab-Gel Electrophoresis (i)  Preparation of Separating Gel  SDS-polyacrylamide gels were run using the discontinuous buffer system of Laemmli (171).  A 13% (w/v) acrylamide gel was prepared as follows:  15 ml of separating g e l containing 0.375 M Tris-HCl buffer, pH 8.8, 0.1%  (w/v) SDS, 13% (w/v) acrylamide and 0.35% (w/v) N,N'-methylene-bis-  acrylamide was prepared  (the acrylamide and N,N'-methylene-bis-acrylamide  were made up as a stock solution of 30% (w/v) and 0.8% (w/v), respectively, i n d i s t i l l e d water, f i l t e r e d through a Whatman 1 MM f i l t e r and stored i n the dark at 4°C).  75 u l of freshly-prepared 10% (w/v) ammonium per-  sulphate was added to the mixture and the s o l u t i o n degassed by a s p i r a t i o n . Polymerization of the solution was i n i t i a t e d by addition of 15 u l of TEMED and the solution was quickly poured between two glass plates of a BioRad slab-gel former (172) separated by 0.75 mm spacers.  A pocket-former  consisting of a t e f l o n comb, 0.75 mm thick, and with a row of ten or twenty teeth, was inserted between the upper edges of the plates and the separating gel was poured to within 1.0 - 1.5 cm from the bottom of the comb.  A small  amount of t e r t i a r y butanol was gently layered over the gel so as to obtain a straight boundary during polymerization.  The gel was allowed to poly-  merize at 20°C f o r 1-2 h or i n some cases, overnight, a f t e r which the t e r t i a r y butanol was removed by means of a f i l t e r paper. J  The surface of  the gel was rinsed with d i s t i l l e d water and the excess water removed with a f i l t e r paper.  The pocket-former was reinserted between the plates and the  stacking gel poured to the top of the comb.  In some experiments, the  separating g e l consisted of 10% (w/v), 15% (w/v) or a l i n e a r gradient of 7.5 to 16.5% (w/v) acrylamide.  65.  (ii)  Preparation of Stacking Gel  A 5 ml stacking g e l solution containing 0.125 6.8,  0.1%  (w/v)  SDS,  4% (w/v)  bis-acrylamide was prepared.^  acrylamide  M Tris-HCl buffer pH  and 0.105% (w/v)  N,N'-methylene-  To t h i s was added 15 u l of a f r e s h l y -  prepared solution of 10% (w/v)  ammonium persulphate and the solution  deaerated.  i n i t i a t e d by adding 10 u l of TEMED and  Polymerization was  solution layered over the separating g e l . merize f o r 1.5-2  The gel was  h a f t e r which the pocket former was  the  allowed to poly-  c a r e f u l l y removed and  the pockets rinsed and f i l l e d with electrode buffer, pH 8.4 consisting of 25mM T r i s base, 192 mM glycine and 0.1%  (w/v)  SDS.  The depolymerized  samples (25-100 ug protein) were applied to the gel beneath the electrode buffer by means of a Hamilton syringe.  Gel Electrophoresis i n Tubes Two (i)  types of gels were used. A separating gel solution was prepared as described before under  "Preparation of separating g e l " . gel  solution was  After i n i t i a t i o n of polymerization,  poured into a glass tube to a height of 10 cm.  had a length of 13 cm with an i n t e r n a l diameter of 6 mm. sealed with a piece of parafilm.  the  The tube  The lower end  was  The surface of the gel was overlayed  t e r t i a r y butanol and the g e l allowed to polymerize f o r 1.5-2 removal of the t e r t i a r y butanol, a 4% (w/v)  acrylamide  h.  with  Following  stacking g e l ,  prepared as described above was. layered on top of the separating gel to a height of 1.0-1.5 cm.  Tertiary butanol was  stacking gel solution which was gel  surface was  buffer.  again layered on top of the  allowed to stand at 20°C for 2-3 h.  The  then rinsed with d i s t i l l e d water, then with electrode  The tube was  f i l l e d to the surface with electrode buffer, pH  8.4  66.  and the sample applied to the surface of the gel beneath the buffer as before. (ii)  The second method, described by Fillingame (144), i s a modifi-  cation of that of Laemmli (171) and includes 8 M urea. consisting of 0.375 M Tris-HCl buffer, pH 8.8, 0.5% 10% (w/v) acrylamide, 0.5%  A gel solution  (w/v) SDS,  (w/v) N,N'-methylene-bis-acrylamide  8 M urea, and 0.075%  T  (w/v) ammonium persulphate was deaerated and polymerization i n i t i a t e d by addition of TEMED at a concentration of 125 y l per ml of gel solution. The solution was immediately poured into 13 cm x 0.6 cm (internal glass tubes, to a height of 10 cm.  diameter)  Tertiary butanol was c a r e f u l l y layered  on top of the gel solution and allowed to stand at 20°C for 1.5-2  h.  The  surface of the gel was rinsed with d i s t i l l e d water, and then with electrode buffer, pH 8.4 which contained 25 mM T r i s base, 192 mM glycine and (w/v) SDS.  The stacking gel was omitted.  0.2%  Samples were loaded onto the gel  surface as before.  Gradient Gel Electrophoresis One of two procedures was (i)  v  used:  The f i r s t method used was developed by Cox and coworkers  (173)  and i s a modification of the Laemmli method (171). Two g e l solutions were prepared simultaneously.  6 ml of a gel  solution containing 0.375 M Tris-HCl buffer, pH 8.8, 0.1% (w/v) acrylamide, 0.2%  (w/v) N,N'-methylene-bis-acrylamide  (w/v) SDS, and 0.033%  7.5% (w/v)  ammonium persulphate and a second 6 ml solution containing 0.375 M Tris-HCl buffer, pH 8.8, 0.44%  10% (v/v) g l y c e r o l , 0.1%  (w/v) N,N'-methylene-bis-acrylamide  phate were deaerated.  (w/v) SDS, 16.5% and 0.01%  (w/v)  acrylamide,  (w/v) ammonium persul-  Polymerization was i n i t i a t e d by addition of 6 u l  and 3 Ul o f TEMED t o t h e s o l u t i o n , r e s p e c t i v e l y . B o t h s o l u t i o n s were immediately  t r a n s f e r r e d t o a Buchler Instruments gradient-maker attached t o  a B u c h l e r I n s t r u m e n t s p o l y s t a l t i c pump by means o f a Tygon t u b i n g (2 mm i n t e r n a l diameter).  The o u t l e t o f t h e t u b i n g was p l a c e d between t h e two  p l a t e s o f a s l a b - g e l former s e p a r a t e d  by two 0.75 mm t h i c k l u c i t e  spacers.  The g e l m i x t u r e was a l l o w e d t o d r i p between t h e g l a s s p l a t e s such t h a t a l i n e a r g r a d i e n t o f 7.5% (w/v) a c r y l a m i d e amide a t t h e bottom was o b t a i n e d .  on t h e t o p t o 16.5% (w/v) a c r y l -  T e r t i a r y b u t a n o l was used t o g i v e a  s t r a i g h t boundary d u r i n g p o l y m e r i z a t i o n , a f t e r w h i c h a s t a c k i n g g e l ( 4 % (w/v)  a c r y l a m i d e ) was c a s t .  The p o c k e t s were washed and f i l l e d  with  e l e c t o d e b u f f e r pH 8.4 and sample l o a d e d onto t h e g e l s u r f a c e a s p r e v i o u s l y described. (ii)  The second method was a m o d i f i c a t i o n o f t h e procedure o f Weber  and Osborn (174) and i n v o l v e d a p h o s p h a t e - b u f f e r e d s o l u t i o n s were prepared  simultaneously.  system.  Two g e l  A 6 ml s o l u t i o n c o n s i s t i n g o f 0.1  M sodium phosphate b u f f e r , pH 7.2, 0.1% (w/v) SDS, 7.5% (w/v) a c r y l a m i d e , 0.2%  (w/v) N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e and 0.033% (w/v) ammonium p e r s u l -  phate and a second 6 ml s o l u t i o n c o n t a i n i n g 0.1 M sodium phosphate b u f f e r , pH 7.2, 10% ( v / v ) g l y c e r o l , 0.1% (w/v) SDS, 16.5% (w/v) a c r y l a m i d e , 0.45% (w/v)  N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e and 0.01% (w/v) ammonium p e r s u l p h a t e  were degassed.  P o l y m e r i z a t i o n was i n i t i a t e d by a d d i t i o n o f 6 P i and 3  ul o f TEMED, r e s p e c t i v e l y . A 0.75 mm t h i c k g r a d i e n t s l a b g e l was c a s t as i n t h e d i s c o n t i n u o u s b u f f e r system.  The s t a c k i n g g e l c o n s i s t e d o f 0.1 M  sodium phosphate b u f f e r , pH 7.2, 0.1% (w/v) SDS, 4% a c r y l a m i d e , 0.11% (w/v) N , N ' - m e t h y l e n e - b i s - a c r y l a m i d e and 0.03% (w/v) ammonium p e r s u l p h a t e . P o l y m e r i z a t i o n was i n i t i a t e d by a d d i t i o n o f 2 M l TEMED p e r m l o f g e l solution.  The sample p o c k e t s were r i n s e d and f i l l e d w i t h an e l e c t r o d e  68.  buffer consisting of 0.1 M sodium phosphate, pH 7.2 and 0.1%  (w/v)  SDS.  Samples were then loaded onto the g e l .  Electrophoresis ( i ) Slab Gels The two glass plates containing the g e l were attached to a gel e l e c t r o phoresis c e l l (BioRad, Model 220, dual v e r t i c a l slab gel electrophoresis c e l l ) and the samples loaded onto the gel surface.  The cathode and  the  anode reservoirs were then f i l l e d with the appropriate electrode buffer and electrophoresis was 40 mA  f o r 0.75  mm  carried out at 20°C at a constant  and 1.5 mm  current of 30 mA  thick gels, respectively.  stopped when the bromophenol blue dye was within 0.5-1  and  Electrophoresis  was  cm from the anodic  edge of the gel.. (ii)  Tube Gels  The tube containing the gel was  placed i n a BioRad Model 150A  electrophoresis c e l l and sample loaded onto the gel surface. p r i a t e electrode buffer was electrophoresis was  gel  The appro-  placed i n the cathode and anode chambers and  carried out at 20°C and at a constant current of 5 mA  u n t i l the dye front was within 0.5-1  cm from the anodic edge of the g e l .  TWO-DIMENSIONAL ISOELECTRIC FOCUSING GEL ELECTROPHORESIS Preparation of Sample (i)  This was  based on the method of Cox and coworkers (140).  10 ml of precooled (10 mg/ml).  (4°C) acetone was  added to 15 mg of membrane protein  After 30 min at 4°C, the mixture was  centrifuge at maximum speed f o r 10 min.  centrifuged i n a desk-top  The p e l l e t was  again extracted  with 10 ml of acetone and then dried under a .stream of nitrogen gas at  20°C.  The dried material was taken up i n l y s i s buffer containing 9.5 M  urea, 2% (w/v) Nonidet P-40, 1.6% (w/v) pH 5-7 ampholytes, 0.8% (w/v) pH 3.5-10 ampholytes and 5% (v/v) 2-mercaptoethanol, and centrifuged at 15 600 xg f o r 10 min to remove any insoluble material.  Extraction with acetone  was omitted when either immunoprecipitated or p u r i f i e d ATPase complex preparations were analyzed. (ii)  The second method was that developed by M e r r i l et a l . (175).  To 1 ml of membrane protein (5 mg/ml) was added 1 volume of denaturation solution containing 4% (w/v) SDS, 10% (v/v) 2-mercaptoethanol, 40% (v/v) g l y c e r o l , 4.5% (w/v) Nonidet P-40, 3.2% (w/v) pH 5-7 ampholytes and 0.16% (w/v) pH 3.5-10 ampholytes.  The mixture was sonicated at 50 W  and 0°C i n a Branson W185D Sonifier, f o r 15 second periods f o r a t o t a l of 1 min.  I t was then heated at 95°C f o r 5 min, after which i t was cooled to  20°C and centrifuged at 15 600 xg f o r 10 min.  To the supernatant was added  urea at a concentration of 56 mg per 100 y l (6.6 M urea) and then applied to the g e l .  First-Dimension I s o e l e c t r i c Focusing The first-dimension g e l was prepared as described by O'Farrel (176) with the following modifications. The first-dimension g e l contained 9.5 urea, 3.77% (w/v) acrylamide, 0.215% (w/v) N,N'-methylene-bis-acrylamide, 2% (w/v) Nonidet P-40, 1.6% (w/v) pH 5-7 ampholytes, 0.8% (w/v) pH 3.5-10 ampholytes and 0.03% (w/v) ammonium persulphate.  Polymerization was i n i t i a t e d by addition of 1 y l  TEMED per ml of g e l solution and the g e l cast i n tubes of 2 mm internal diameter and to a height of 10 cm.  Tertiary butanol was layered over the  g e l which was allowed to stand at 20°C f o r 6-8 h.  The surface of the g e l  70.  was then rinsed with l y s i s buffer.  50-250 yg of the prepared sample was  loaded onto the g e l and overlayed with a solution of 8 M urea i n water. The gels were placed i n a BioRad Model 150A gel electrophoresis c e l l the anode and cathode chambers f i l l e d with freshly prepared 0.01 M and deaerated 0.02 M NaOH, respectively.  and H^PO^  The first-dimension was run at  20°C and 400-450 V f o r 16-18 h, followed by 800 V f o r 1-2 h, to give a t o t a l of 9000 to 9500 volt-hours.  Determination of pH Gradient In the first-dimension, a gel loaded with sample preparation buffer rather than the prepared sample was run simultaneously.  After e l e c t r o -  phoresis, the a c i d i c or basic end of the gel was i d e n t i f i e d and the gel cut into 1 cm segments.  Each segment was incubated with 1.5 ml of deaerated  d i s t i l l e d water f o r 1.5-2  h at 20°C a f t e r which the pH was measured.  Second-Dimension SDS-Polyacrylamide The second-dimension 16.5%  Gel  gel was either a 13% (w/v) or a l i n e a r 7.5% to  (w/v) acrylamide gel prepared i n the discontinuous buffer system of i  Laemmli (171).  The slab gels were cast as described before but modified as.  follows. The gel was 1.5 mm  thick rather than 0.75 mm and the pocket former  not used with the stacking g e l . height of 1.5-2  was  Instead, the stacking gel was cast to a  cm and was about 3 mm  from the upper edge of the glass  plate. P r i o r to running the second dimension, the a c i d i c or basic end of the gel  from the f i r s t dimension was i d e n t i f i e d with India ink'.  .incubated f o r 3 h  The gel was  i n 5 ml of 125 mM Tris-HCl buffer, pH 6.8 containing 10%  71.  (v/v)  g l y c e r o l , 4% (w/v) SDS, 4% (v/v) 2-mercaptoethanol and 0.001% (w/v)  bromophenol blue.  When the gel from the first-dimension was not to be run  immediately i n the second dimension, the g e l i n the above solution was frozen rapidly i n a dry-ice ethanol mixture and stored at -70°C. The gel from the f i r s t dimension was mounted above the seconddimension g e l i n 1% (w/v) agarose i n 62.5 mM Tris-HCl buffer, pH 6.8, (v/v)  10%  g l y c e r o l , 2% (w/v) SDS, 2% (v/v) 2-mercaptoethanol and 0.001% (w/v)  bromophenol blue.  .  '  i  Electrophoresis was carried out as previously described except that i t was continued f o r 10-15 min a f t e r the dye front had run o f f the lower (anode) end of the g e l . The gel could then be stained immediately without removal of ampholytes.  CROSSED IMMUNOELECTROPHORESIS This was based on the methods of Nowotny (177), Bjerrum and Lundhal (178) and Mayer and Walker (179).  First-Dimension Gel Electrophoresis 2.4 ml of 1% (w/v) agarose i n Bjerrum buffer, pH 8.8 containing 100 mM glycine, 38 mM T r i s and 1% (w/v) Triton X-100 at 55°C was poured onto a 50 x 50 mm glass plate surrounded by a p l a s t i c mold.  The agarose was allowed  to cool to 20°C and the mold c a r e f u l l y removed such that a 40 x 40 x 1.5 gel  was obtained.  Agarose i n the same buffer was layered around the  periphery of the cast g e l to the edges of the plate (agarose bridges) to give a more uniform conduction of the current.  A row of four wells (3 mm  diameter), about 1 cm from the cathodic edge of the g e l , and about 1 cm apart, was cut out with a BioRad gel-puncher.  The wells were f i l l e d with  mm  various l e v e l s of antigen and the gel placed i n a Pharmacia flat-bed electrophoresis unit at 4°C.  Wicks, 4 cm wide (Ultrawicks, BioRad) were  placed onto the agarose bridges i n such a manner that they did not overlap , onto the wells.  The chambers were f i l l e d with Bjerrum buffer, pH  without any detergent and electrophoresis was 4°C and 100 V.  8.8,  carried out for 1.5-2  After electrophoresis, the gel was  h at  cut into s t r i p s (5 mm  width) such that each contained a sample well at one end.  The i n d i v i d u a l  s t r i p s were stained immediately or run i n the second dimension.  Second-Dimension Gel Electrophoresis 1% (w/v)  agarose i n Bjerrum buffer, pH 8.8 and various l e v e l s of  antiserum ( f i n a l volume, 2.4 ml) were'mixed at 55°C and cast into a 40 x 40 x 1.5 mm  gel as above.  A s t r i p of the gel from the first-dimension  placed at the cathodic end of the g e l and agarose bridges around the g e l . dimension.  The electrode buffer was  Electrophoresis was  of the f i r s t dimension for 16 to 18 h at 10 V and  STAINING AND  constructed  the same as i n the  carried out perpendicular  was  first  to the d i r e c t i o n  4°C.  DRYING GELS  SDS-Polyacrylamide Gels A f t e r electrophoresis, these gels were either stained immediately or fixed p r i o r to staining. (i)  5% (w/v) for  (ii)  TCA,  Gels were fixed i n either a solution of:  5% (w/v)  s u l f o s a l i c y l i c acid and 10% (v/v) methanol  30 min at 60°C (173)  50% (w/v)  TCA,  or  overnight at 20°C (171).  The staining solutions were f i l t e r e d through a Whatman 1 MM before use.  filer  The gels were then stained i n one of the following staining  systems f o r d i f f e r e n t periods at 20°C. (i)  0.12% (w/v) Coomassie Blue ( B r i l l i a n t Blue R) i n ethanolracetic acid:water (25:8:67) (171)  (ii)  0.05% or 0.1% (w/v) Coomassie Blue i n isopropanol:acetic acid:water (25:10:65) (180)  (iii)  0.25% (w/v) Coomassie Blue i n methanol:acetic acid:water (45:10:45) (174)  (iv)  1% (w/v) Amido Black 10B i n 7% (v/v) acetic acid (165). Gels were then destained i n 10% (v/v) acetic acid u n t i l a f a i r l y clear  background was obtained.  The tube gels were scanned at 545 nm and 620 nm  when Coomassie Blue and Amido Black were used, respectively.  Slab gels  were incubated i n a solution of 10% (v/v) acetic acid and 2% (v/v) g l y c e r o l for 4-6 h and then dried onto a piece of Whatman 3 MM paper i n a Gel Slab Dryer, Model 224 (BioRad), equipped with a heating unit and connected to a vacuum pump.  Crossed Immunoelectrophoresis  Gels  The gels were processed by the methods of Weeke (188) and Mayer and Walker (179).  After electrophoresis, the glass plate containing the g e l  was taken and the sample well f i l l e d with 0.9% (w/v) NaCl.  A 5 x 5 cm  piece of Whatman 3 MM paper was c a r e f u l l y placed on top of the g e l , followed by a pad of absorbent paper towels, a glass plate and f i n a l l y a weight of about 0.5 Kg. After 10 min at 20°C, the pressed g e l was washed t h r i c e by incubating each time with 0.9% (w/v) NaCl f o r 20 min.  The gel  was then soaked i n d i s t i l l e d water f o r 20 min and then a piece of 5 x 5 cm Whatman 3 MM paper gently placed on top of the g e l . oven at 70°C and allowed to dry.  This was placed i n an  After the g e l had dried onto the glass  74.  plate, the paper was gently removed and the cooled (20°C) plate was incubated f o r 30 min i n a solution of 0.25% (w/v) Coomassie Blue i n methanolracetic acid:water (45:10:45).  The plate was rinsed with d i s t i l l e d  water and destained i n a solution containing 10% (v/v) acetic acid and 45% (v/v) methanol f o r 5-10 min or u n t i l the stained proteins (rockets) could be seen against a s u f f i c i e n t l y colourless background.  F i n a l l y , the plate  was rinsed with d i s t i l l e d water and dried with a hair-dryer.  I t was kept  i n the. dark.  PURIFICATION OF GOAT ANTI-RABBIT IMMUNOGLOBULIN BY AFFINITY CHROMATOGRAPHY Preparation of A f f i n i t y Column The a f f i n i t y column was prepared by L. Molday (University of B r i t i s h Columbia) using the method of Cuatrecasas (181). 750 mg of CNBr was dissolved i n 10 ml of d i s t i l l e d water and mixed with 20 ml of washed Sepharose 4B (equal volumes of resin and water) which had previously been adjusted to pH 11 with NaOH.  The suspension was  s t i r r e d at 0°C f o r 25 min and the pH kept between 10 and 11 by addition of 10 M NaOH.  The g e l was f i l t e r e d and washed with 200 ml of d i s t i l l e d water,  then with 0.1 M sodium borate buffer pH 9.0.  The r e s i n was washed for 2-3  min since CNBr-activated agarose i s unstable. 80 mg of rabbit Y - g l o b u l i n i n 4 ml of 0.1 M sodium borate buffer pH 9.0 was added to 20 ml of the CNBr-activated g e l (equal volumes of gel and buffer) i n borate buffer and then s t i r r e d at 20°C f o r 16-18 h.  The amount  of protein bound to the resin was determined from the amount s t i l l remaining unbound i n the supernatant following sedimentation of the g e l . This was calculated from absorbance at 280 nm and using the.extinction of £  0.1% 280  = 1  . ' 3 5  The r e s i n was t h e n washed w i t h 0.1 M sodium b o r a t e b u f f e r pH 9.0 and the r e s i d u a l a c t i v e s i t e s quenched by i n c u b a t i n g t h e r e s i n i n s e v e r a l volumes o f 0.1 M g l y c i n e f o r 6-8 h a t 20°C.  F i n a l l y , t h e r e s i n was washed  w i t h p h o s p h a t e - b u f f e r e d s a l i n e , pH 7.5 (PBS: 0.137 M N a C l , 2.68 mM KC1, 1.47 mM K H P 0 , 8.09 mM Na^PO^) and s t o r e d i n t h e same b u f f e r i n t h e 2  4  presence o f 20 mM NaN^ a t 4°C.  P u r i f i c a t i o n o f Goat A n t i - R a b b i t  Immunoglobulin  The r a b b i t Y - g l o b u l i n - S e p h a r o s e 4B a f f i n i t y column i n PBS was poured i n t o a 10 x 1 cm BioRad column t o a h e i g h t o f 4 cm and e q u i l i b r a t e d w i t h PBS.  5 m l o f goat a n t i - r a b b i t serum was a p p l i e d t o t h e column a t 20°C and  washed w i t h s e v e r a l volumes o f PBS.  Goat a n t i - r a b b i t immunoglobulin was  e l u t e d from t h e column w i t h 3M NaSCN i n d i s t i l l e d w a t e r (pH 7 . 5 ) . F r a c t i o n s c o n t a i n i n g goat a n t i - r a b b i t immunoglobulin, as determined by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s , were p o o l e d and d i a l y z e d a g a i n s t 50 volumes o f 0.1 M NH HC0 4  3  b u f f e r pH 7.4 c o n t a i n i n g 20 mM NaN^ f o r 24 h  a t 4°C w i t h exchanges o f t h e e x t e r n a l b u f f e r a t i n t e r v a l s o f 6 h.  The  d i a l y z e d m a t e r i a l was l y o p h i l i z e d and s t o r e d a t -70°C.  RADI0I0DINATI0N OF GOAT ANTI-RABBIT IMMUNOGLOBULIN To 1 mg o f a f f i n i t y - p u r i f i e d goat a n t i - r a b b i t immunoglobulin ( 1 mg/ml) i n phosphate b u f f e r e d s a l i n e , pH 7.4 (PBS) was added 1 mCi o f N a ( f o l l o w e d by 100 u l o f a 0.4% (w/v) Chloramine T s o l u t i o n .  l 2 5  I)  The m i x t u r e  was i n c u b a t e d i n t h e fume-hood a t 20°C f o r 20 min, a f t e r w h i c h , i t was l o a d e d onto a 19 x 1.5 cm column o f B i o G e l P6DG w h i c h had been e q u i l i b r a t e d w i t h PBS.  The column was e l u t e d w i t h t h e same b u f f e r a t a f l o w r a t e o f  0.1-0.15 ml p e r m i n and 0.9 m l f r a c t i o n s c o l l e c t e d .  Fractions  containing  76.  protein (determined by absorbance at 280 nm) and the highest r a d i o a c t i v i t y )  were pooled, made up to 20 mM NaN^, a c t i v i t y of 0.5 to 2 x 10  6  and stored at 4°C.  A s p e c i f i c radio-  dpm/ug was obtained  PEPTIDE MAPPING OF CNBr-CLEAVED DCCD-BINDING PROTEIN One-Dimensional Thin Layer Separation A 20 x 20 glass plate precoated with s i l i c a gel G (250 um, Inc.) was activated by placing i t i n an oven at 100°C for 1 h.  Analtech The plate  was cooled to 20°C and then the sample ( i n 80% (v/v) formic acid) was applied as a spot of 2-3 mm  i n diameter. The sample was applied repeatedly  on the same spot and f i n a l l y a i r - d r i e d at 20°C.  The plate was placed i n a  covered glass tank, which had been equilibrated with the appropriate solvent, and developed at 20°C.  The solvent system consisted of one of the  following: (1)  chloroformmethanol  (9:1 (v/v)).  Run time, 40 min.  (2)  n-butanol:acetic acid:water (4:1:5).  (3)  chloroformmethanol:water  Run time, 3 h.  (65:25:4) containing 20 mM HC1.  Run  time, 65 min. (4)  n-butanol:pyridine:acetic acid:water (60:40:12:48). 4.5-5  Run  time  h.  After the plates were developed, they were dried overnight at 20°C and the positions of the peptides determined.  Two-Dimensional Thin Layer Separation The f i r s t dimension was thin-layer electrophoresis.  Prior to  electrophoresis, a 20 x 20 cm c e l l u l o s e MN300 plate (CEL 300-10, 0.1  mm,  Macherey-Nagel Co.) was developed i n n-butanolrpyridineracetic acid:water (60:40:12:48) perpendicular to the d i r e c t i o n of electrophoresis, for 6 h at 20°C and then thoroughly a i r - d r i e d .  The plate was then sprayed with  thin-layer electrophoresis (TLE) buffer, pH 6.45 consisting of a mixture of pyridine:acetic acidrwater (20:0.7:180) and electrophoresis carried out at 400 V f o r 1 h i n a Camag TLE apparatus.  The sample ( i n 80% (v/v) formic  acid) was spotted (2-3 mm diameter) onto the dried plate at the midpoint between the electrodes and 2.5 cm from the edge of the plate.  The spot was  a i r - d r i e d and protected by gently inverting a clean v i a l over the spot. The plate was resprayed with TLE buffer and through d i f f u s i o n , the area under the v i a l was dampened with buffer.  Electrophoresis was carried out  at 400 V f o r 65 min, a f t e r which the plate was thoroughly dried at 22°C, overnight. Separation i n the second dimension was by ascending chromatography.  A  covered glass chromatography tank was equilibrated at 20°C with a solvent mixture of n-butanol:pyridine:acetic acid:water (60:40:12:48) by l i n i n g the tank with sheets of Whatman 3 MM paper soaked with the solvent mixture. (  The plate from the f i r s t dimension was then developed i n the solvent system such that the d i r e c t i o n of migration of the solvent front was perpendicular to the d i r e c t i o n of electrophoresis.  After 5.5-6 h, at which point the  solvent front was 2-3 cm from the top edge of the plate, the plate was removed, thoroughly dried at 20°C, and the peptides detected.  Detection of Peptides i  The dried thin-layer plate was sprayed with a solution containing 1% (w/v) ninhydrin and 4% (v/v) acetic acid i n acetone, and then developed i n an oven at 100°C f o r 15 min.  An outline of the peptide spots was made  78.  immediately.  In some cases, the spots were fixed by spraying the plate  with solution containing 1 ml saturated Cu(NC> ) , 0.2 ml 10% (v/v) HN0 3  and 100 ml 96% (v/v) ethanol.  2  3  The plate was a i r - d r i e d and stored i n the  dark.  CHEMICAL.MODIFICATION OF MEMBRANES Labelling of Membrane Vesicles of E. c o l i with ^"CjDCCD [ "C]DCCD (50 mCi/mMol) was purchased i n ether i n a sealed ampoule. l  It was opened and the contents transferred to a v i a l . evaporated  The ether was  at 20°C i n the fume-hood and the dried contents taken up i n  absolute ethanol and stored at -20°C. The method of l a b e l l i n g was based on that described by Fillingame (144).  Membranes prepared i n TM buffer, pH 8.0 were suspended i n 10 mM  Tris-H S0 2  4  buffer, pH 7.6 containing 0.25 M sucrose, 5 mM MgS0  4  DTT at a protein concentration of 20 mg/ml.  and 0.2 mM  To 0.5 ml of membrane suspen-  sion was added 0.02 volumes (10 ul) of 5 mM [ "C]DCCD i n absolute l  ethanol and the suspension s t i r r e d at 4°C for 24 h.  The labelled membranes  were washed three times by resuspension i n buffer following centrifugation at 250 000 xg f o r 2 h.  The membranes were used immediately  or stored at  -20°C.  Treatment of Membrane Vesicles with Phenylglyoxal A 200 mM phenylglyoxal solution was prepared by dissolving 53.8 mg i n 1.8 ml of 50 mM HEPES-K0H buffer, pH 7.5 containing 10 mM MgCl (v/v) g l y c e r o l .  2  and 10%  The pH was readjusted to 7.5 by addition of KOH and the  f i n a l volume made up to 2 ml with buffer. Two ml of urea-stripped membrane vesicles i n 50 mM HEPES-K0H buffer,  pH 7.5 containing 10 mM MgCl and 10% (v/v) g l y c e r o l at a protein concen2  t r a t i o n of 12.5 mg/ml was incubated with 1 volume of 200 mM phenylglyoxal i n the dark at 20°C f o r 3 h.  The suspension was then diluted 8-10  with buffer and centrifuged at 250 000 xg f o r 2.5 h.  fold  The membranes were  washed once by resedimentation i n buffer and f i n a l y suspended at a protein concentration of 10 mg/ml.  They were used immediately or rapidly frozen  and stored at -70°C.  CHEMICAL MODIFICATION OF THE DCCD-BINDING PROTEIN Hydrolysis of the DCCD-Binding  Protein  This was based on the method of Moore and Stein (182). Heavy-walled 13 x 100 mm Pyrex culture tubes were washed sequentially i n chromic acid, a l c o h o l i c KOH and 1 M HC1.  They were rinsed with d i s t i l l e d  water a f t e r each step and f i n a l l y dried at 100°C.  100-150 ug of p u r i f i e d  DCCD-binding protein i n chloroformmethanol (2:1) containing 20 mM ammonium acetate, was taken to dryness i n a 13 x 100 mm Pyrex tube at 16-18°C by using a rotary evaporator (Buchler Instruments, Rotary Evapo-mix). protein i n the tube was taken up i n 1.5-2 ml of 6 M HC1.  The  With an oxygen  flame, a section of the tube about 3 cm from the top was p a r t i a l l y constricted.  The lower half of the tube was immersed i n a dry ice-ethanol  mixture f o r 5-10 min, at which point the solution i n the tube started to become viscous.  The tube was then connected to a vacuum pump through a  piece of Tygon tubing and the system evacuated to a pressure of less than 50 microns.  During t h i s period, gently warming the lower half of the tube  by hand caused bubbles to r i s e from the viscous solution. tube into the dry i c e mixture broke the bubbles.  Re-immersing  the  The tube was sealed at  the c o n s t r i c t i o n , when bubble formation had almost ceased.  The vacuum seal  80.  was checked f o r leaks with an ionizing gun.  Hydrolysis was then conducted  at 110°C ± 2°C f o r 24, 31, 42 and 60 h a f t e r which the tubes were cooled to 20°C.  Any l i q u i d on the inner walls of the tube was spun down by  centrifugation i n a desk-top centrifuge (International Equipment Company, model CL 45436 M) at 900 xg f o r a few minutes.  The,tube was scored with a  f i l e near the c o n s t r i c t i o n and cracked open by means of a hot glass rod. Hydrochloric acid was removed under reduced pressure at 70-80°C by placing the sample tube i n an 18 x 150 mm Pyrex i g n i t i o n tube and attaching i t to the rotary evaporator.  Almost a l l of the HC1 was removed i n 45-60 min.  The dried contents were taken up i n the appropriate buffer f o r analysis of i t s amino acid composition.  The amino acid composition was determined by  using the Durram (model D-500) amino acid analyzer.  Treatment of the DCCD-Binding Protein with Cyanogen Bromide The method of Sebald et a l . (183) was followed. 2 mg of p u r i f i e d DCCD-binding protein was taken to dryness and the residue dissolved i n 0.8 ml of 1 M cyanogen bromide i n 98% (v/v) formic acid.  0.2 ml d i s t i l l e d water was added to the mixture which was incubated  i n the dark at 20°C f o r 17-18 h.  The solvent was then removed by rotary  evaporation under reduced pressure at 30-40°C as previously described. ^The dried residues were dissolved i n the appropriate buffer.  Control experi-  ments were simultaneously carried out with DCCD-binding protein, which were treated i n the same manner but i n the absence of cyanogen bromide.  Treatment of the DCCD-Binding Protein with Performic Acid This was done according to the method of Hirs (184). Performic acid was prepared by mixing 9 ml of 90% (v/v) formic acid  81.  with 1 ml of 30% (v/v) H 0 2  h.  2  and incubating the mixture at 20°C f o r 1  The solution was cooled to 0°C and used  immediately.  DCCD-binding protein i n chloroformmethanol (2:1) containing 20 ammonium acetate was  mM  taken to dryness and performic acid added to the dry  protein such that the protein concentration was 1-2%  (w/v).  The tubes were  capped and incubated'in the dark at 0°C for 20 h, a f t e r which the performic acid was  removed by evaporation under reduced pressure at 35-40°C.  t i o n to dryness took about 45 min.  Evapora-  The residue was either subjected to  hydrolysis i n the same tube to determine i t s amino acid composition described before or taken up i n the appropriate buffer.  as  In some cases,  control experiments were simultaneously carried out with DCCD-binding prot e i n , which were treated i n the same manner with 80% (v/v) formic acid only.  Treatment of the DCCD-Binding Protein with 2,3-Butanedione or Phenylglyoxal A 200 mM  solution of 2,3-butanedione and a 150 mM  phenylglyoxal were made up i n 100 mM  solution of  sodium borate buffer containing 2%  (w/v) Triton X-100, and the pH ad jus-ted to 8. 8 withNaOH. 0.3 mg of dried DCCD-binding protein was  dissolved i n I ml of borate-  Triton buffer, pH 8.8 and incubated with an equal volume of either 200 2,3-butanedione or 150 mM phenylglyoxal i n the dark at 20°C for 3 h. of 330 mM L-arginine hydrochloride i n 100 mM was then added. then l y o p h i l i z e d .  The f i n a l mixture was  1 ml  sodium borate buffer, pH  incubated for another 30 min  mM  8.8  and  The dried residue was dissolved i n borate-Triton buffer  at a protein concentration of 0.15 mg per ml.  Control experiments were  simultaneously carried out with DCCD-binding protein, which were treated i n the same manner but i n the absence of either 2,3-butanedione or phenylglyoxal.  82.  TREATMENT WITH PROTEASES ECFj^ and DCCD-Binding Protein ECF^ obtained a f t e r p u r i f i c a t i o n on AH-Sepharose CL-4B was suspended i n 50 mM HEPES-KOH buffer, pH 7.5 containing 10 mM MgCl glycerol and treated with one of the following:  2  and 10% (v/v)  a-chymotrypsin,  Staphylococcus aureus Vg protease or pronase at a protein:protease of 10:1.  The f i n a l protein concentration  ratio  of ECF^ was 0.75 mg/ml.  S i m i l a r l y , DCCD-binding protein i n 100 mM sodium borate buffer, pH 8.8 containing 2% (w/v) T r i t o n X-100 was treated with one of the above proteases at a protein:protease  r a t i o of 10:1.  The DCCD-binding protein  was also treated with TPCK-trypsin at a protein:protease  r a t i o of 10:1.  The reaction mixtures were incubated at 37°C f o r 3 h a f t e r which TPCK-trypsin was i n h i b i t e d by addition of an equivalent amount of soybean trypsin i n h i b i t o r followed by incubation f o r 15 min at 37°C.  The f i n a l  concentration of DCCD-binding protein i n the reaction mixtures was 0.13 mg protein/ml.  25 p i of these mixtures were incubated i n f l e x - v i n y l micro-  t i t r e wells, which had been previously coated with 0.1% (w/v) polyrL-lysine. For controls, 25 P i of these mixtures containing no DCCD-binding protein was also incubated i n polylysine-treated microtitre wells.  The  appropriate  radioimmune assays were then carried out as described i n the legends to the Figures i n the RESULTS' section.  PREPARATION OF ANTIGENS FOR IMMUNIZATION ECF^ ECF^ was prepared f o r i n j e c t i o n by Helga Stan-Lotter  (University of  B r i t i s h Columbia) as follows: 0.7 mg of ECF  (obtained a f t e r AH-Sepharose 4B chromatography) at  83.  1.4 mg/ml was mixed with 1 volume of complete Freund's adjuvant and the mixture emulsified by repeated passing between two syringes connected through a 22 gauge needle.  The mixture was ready f o r i n j e c t i n g into the  rabbit when a drop of the emulsion placed on the surface of the water did not disperse when the beaker was  s l i g h t l y agitated.  DCCD-Binding Protein The DCCD-binding protein was dissolved i n a solution containing 0.9% (w/v) NaCl and 2% (w/v) SDS at a protein concentration of 2 mg/ml.  It was  mixed with two volumes of complete Freund's adjuvant and emulsified. some preparations, incomplete Freund's adjuvant was  In  used.  IMMUNIZATION OF THE RABBIT / A female, New Zealand rabbit of about 3 Kg was obtained from the University of B r i t i s h Columbia Animal Care Unit, several months p r i o r to immunization and maintained as recommended (185).  A month a f t e r i t s  a r r i v a l , the rabbit was bled at 4-week intervals u n t i l enough preimmune serum was obtained. immunized a follows.  Three weeks a f t e r the l a s t bleeding, the animal was The rabbit was wrapped i n a blanket leaving only the  head and the s i t e of i n j e c t i o n exposed.  A small amount of hair was  clipped  off at the area to be injected and the i n j e c t i o n s i t e cleaned with 70% (v/v) ethanol.  A tent of skin was raised with the thumb and index finger  and with the other hand, the needle (22 gauge) was inserted underneath the skin and p a r a l l e l to the underlying muscle.  The antigen was  injected  immediately, the needle withdrawn, and the s i t e of i n j e c t i o n wiped with ethanol.  The rabbit was generally injected at two s i t e s on the thigh of  the hind legs with a t o t a l of either 0.6 mg ECF  1  or 0.3 mg DCCD-binding  84.  protein.  Subsequent injections were at 4-week i n t e r v a l s , with a t o t a l of  either 0.3 mg ECF.. or 0.2 mg DCCD-binding protein and incomplete Freund's adjuvant was substituted f o r the complete Freund's adjuvant i n the case of ECF^.  For the DCCD-binding protein, incomplete Freund's adjuvant wis used  a f t e r the f i f t h i n j e c t i o n as continued use of the complete adjuvant resulted i n the development of hard pea-size lumps at the s i t e s of i n j e c t i o n .  BLEEDING THE RABBIT The method of Herbert (186) was  used.  The rabbit was wrapped i n a blanket, leaving only the head exposed.  A  small area over the outer marginal vein of the ear was shaved and cleaned with soap and water. petroleum j e l l y .  The shaved area was dried and l i g h t l y smeared with  A gauze soaked i n xylene was pressed onto the shaved area  for a few seconds, u n t i l the marginal ear vein was v i s i b l y d i l a t e d .  With a  s t e r i l e razor blade, an i n c i s i o n of about 2 mm was made into the vein i n the longitudinal d i r e c t i o n and about 2 inches from i t s d i s t a l end.  The ear  was held i n the horizontal p o s i t i o n and the thumb and index finger was to occlude the venous return. to c o l l e c t the blood.  used  A centrifuge tube was placed under the ear  When the flow of blood stopped prematurely, the  wound was wiped with a piece of gauze to open the wound.  Usually 25 to 30  ml of blood was collected at each bleeding. The bleeding was stopped by placing a dry gauze on the wound and securing i t with a paper c l i p .  On occasion, the bleeding was  persistent  and a piece of Gelfoam sponge (The Upjohn Co., Michigan) was used to stop the bleeding.  After the bleeding had stopped, any xylene remaining  was  removed with 70% (v/v) ethanol and the ear wiped with soap and water. rabbit was bled alternately from each ear at 7-10 days post-injection.  The  /  85.  SEPARATION OF SERUM This was  based on the method of Garvey (187).  The blood on c o l l e c t i o n c l o t t e d to some degree. separated  The c l o t  was  from the wall of the centrifuge tube with a wooden applicator  s t i c k and the blood allowed to stand at 20°C f o r 2 h, then at 4°C for h.  The serum was  fugation was was  removed and centrifuged at 2 000 xg f o r 30 min.  repeated when the serum was  not c l e a r .  18-24  Centri-  On occasion, the serum  turbid due to the presence of l i p i d s (or haemoglobin) and could not be  cleared by centrifugation at 2 000 xg. centrifuged at 25 000 xg f o r 30 min. which was  In these cases, the serum was A f i l m of l i p i d formed on the  e a s i l y removed with a tissue paper.  a s t e r i l e 0.22  The serum was  surface  passed through  ym M i l l i p o r e f i l t e r and stored at -70°C.  PARTIAL PURIFICATION OF IMMUNOGLOBULINS This was  based on the methods of Nowotny (177) and Mayer and Walker  (179). 3 ml of saturated (NH^SO^ (neutralized to pH 7.0 with NH^OH) was  added dropwise to 5 ml of serum and the mixture s t i r r e d at 20°C.  pH of the mixture was monitored during the addition of (NH^^SO^. 30 min of incubation, the mixture was The p e l l e t was  After  centrifuged at 17 600 xg f o r 30  resuspended to the o r i g i n a l volume (5 ml) i n 10 mM  phosphate buffer, pH 7.0 and to this was  min.  potassium  added 2.6 ml of saturated  (NH^^SO^ and the p r e c i p i t a t e removed by centrifugation. up i n 5 ml of 10 mM  The  It was  then taken  potassium phosphate buffer, pH 7.0 and dialyzed at  against 500 ml of d i s t i l l e d water for 20 h followed by d i a l y s i s against ml of 0.4 M Sucrose and 10 mM MgSO^ for 12 h.  4°C 250  The external medium was  changed at 6 h i n t e r v a l s . The dialyzed material was  centrifuged to remove  86.  insoluble material and the supernatant concentrated to half i t s o r i g i n a l volume (2.5 ml) by u l t r a f i l t r a t i o n using an Amicon XM-50 f i l t e r .  It was  stored at -70°C.  BINDING OF ECFj^ TO MEMBRANE VESICLES The measurement of the extent of rebinding of ECF^ to urea-treated membrane vesicles which^had been treated with proteases or phenylglyoxal was carried out as follows. (i)  2-4 mg of the treated membrane v e s i c l e s i n 50 mM HEPES-KOH  buffer, pH 7.5 containing 10 mM MgCl and 10% (v/v) glycerol were 2  incubated with various levels of ECF^ at 4°C f o r 45 min i n a f i n a l volume of 0.5 ml.  The mixture was then diluted 8-10 f o l d with buffer and the  membranes sedimented at 250 000 xg f o r 2.5 h.  The membranes were  resuspended i n buffer and the ATPase a c t i v i t y measured. (ii)  Urea-treated membrane v e s i c l e s were also treated with antiserum  p r i o r to rebinding of ECF^. Urea-treated membranes (15 mg/ml) i n 50 mM HEPES-KOH buffer, pH 7.5 containing 10 mM MgCl and 10% (v/v) glycerol were incubated at 4°C f o r 2  20 h with d i f f e r e n t amounts of antiserum i n a f i n a l volume of 4.4-4.5 ml. After a 4-fold d i l u t i o n i n buffer, the mixture was centrifuged at 250 000 xg f o r 2.5 h.  The sedimented vesicles were washed once by suspension i n  buffer followed by resedimentation as before.  The washed vesicles were i  suspended i n buffer at 6.5 mg protein per ml and reconstituted with ECF^ as described above.  87.  ASSAYS Determination of Protein One of several procedures was (i)  used.  The protein i n most experiments was determined by the method of  Lowry et a l . (189). range of 0-300 yg.  Bovine serum albumin was used as the standard over a Interference by components i n the sample buffer was  corrected f o r by incorporating i n the standard curve the appropriate volume of sample buffer equivalent to that assayed. T r i t o n X-100  i n high concentrations gave a greenish-yellow p r e c i p i t a t e .  In these cases, the precipitate was removed by centrifugation at 12 000 xg for 10 min and the absorbance of the supernatant measured at 500 (ii)  nm.  The protein content i n samples obtained during the i s o l a t i o n and  p u r i f i c a t i o n of the DCCD-binding protein was determined by a modified procedure of Lowry et a l . (189), as done by Fillingame (144). Membrane samples were dissolved i n 0.5 M NaOH whereas DCCD-binding protein samples were taken to dryness under reduced pressure to remove organic solvents and then s o l u b i l i z e d by adding 2.5% (w/v) SDS i n 0.5 M NaOH.  Both were incubated at 37°C f o r 2 h prior to assay.  In addition, 1% (w/v) SDS was included i n the 2% (w/v) Na C0 2  Lowry reagent used i n these assays.  3  Bovine serum albumin was used as the  standard protein over a range of 0-300 yg. (iii)  The t h i r d method was that of Bradford (190).  This method was  used f o r soluble enzyme preparations (e.g. ECF^ and i t s subunits) where i n t e r f e r i n g substances i n the buffer were minimal and the amount of protein to be assayed was low. a range of 0-50  Bovine serum albumin was used as the standard over  yg.  If an i n t e r f e r i n g substance was present i n the sample buffer, an  88.  equivalent volume was,incorporated curve.  i n the samples used f o r the standard  3 ml of f i l t e r e d reagent containing 0.01% (w/v) Coomassie B r i l l i a n t  Blue G-250, 5% (v/v) 95% (v/v) ethanol and 0.1% (v/v) 85% (v/v) H P0 3  4  was added to 0.1 ml of sample or standard protein and the absorbance at 595 nm measured a f t e r 15 min at 20°C.  Determination of ATPase A c t i v i t y Measurement of ATPase a c t i v i t y was done according to the method of Davies and Bragg (21). The inorganic phosphate released during the 1  reaction was determined (i)  by a modified method of Ames (122).  Rapid Assay  This assay was used when i n t e r f e r i n g substances such as phospholipids and detergents were not present i n the sample and was generally used to determine the a c t i v i t y of the ECF^ preparations. The sample was incubated with 0.5 ml of 100 mM Tris-HCl buffer, pH 8.3 containing 5 mM ATP and 2.5 mM C a C l  2  at 37°C.  After a period of  Incubation, 2.5 ml of "inorganic phosphate assay mixture" containing 1.8 ml ammonium molybdate (0.42% (w/v) ammonium molybdate i n 2.86% (v/v) H^O^), 0.3 ml of 10% (w/v) ascorbic acid, 0.25 ml of 10% (w/v) TCA and 0.15 ml water, was added. The reaction mixture was incubated at 37°C f o r 15 min and the reaction stopped by placing the mixture at 0°C. The absorbance at 660 nm was measured against a reagent blank and the amount of inorganic phosphate liberated was calculated from a standard curve.  MgCl,, was substituted  for C a C l when the a c t i v i t y of TPCK-trypsin-treated ECF^ was measured. 2  (ii)  Slow Assay  This assay was used when i n t e r f e r i n g substances i n the samples were  •  present.  -  89.  The procedure was e s s e n t i a l l y the same as described f o r the  "rapid" assay except that a f t e r incubation of the sample with ATP, the reaction was stopped by addition of 0.5 ml of 10% (w/v) TCA and the reaction mixture centrifuged at 12 000 xg for 10 min  0.5 ml of the  supernatant was removed and the inorganic phosphate which had been released was determined by addition of 2.5 ml of "inorganic phosphate assay mix" as before. One unit of ATPase a c t i v i t y was defined as the amount of enzyme which liberated 1 nmol of phophate per min at 37°C.  Substrate Oxidation-Dependent  Quenching of Fluorescence of 9-Aminoacridine  This was based on the method of Singh and Bragg (191). 0.1 ml of everted membrane vesicles (10 mg/ml) i n 50 mM HEPES-KOH buffer, pH 7.5 containing 5 mM MgCl was mixed i n a fluorescence cuvette 2  i n a f i n a l volume of 2 ml with 10 mM HEPES-KOH buffer pH 7.5 containing 300 mM KC1, 5 mM MgCl and 2-10 UM 9-aminoacridine. 2  The energy sources  were NADH, ascorbate i n the presence of PMS or ATP.  Each was prepared  fresh i n 10 mM HEPES-KOH buffer, pH 7.5 containing 300 mM KC1 and 5 mM MgCl . 2  In the case of ascorbate, PMS was added to the membrane vesicles  before 9-aminoacridine.  DCCD, when required, was prepared as a stock  solution i n absolute ethanol and stored at -20°C.  The maximum l e v e l of  ethanol allowed i n the reaction cuvette was 2% (v/v). Fluorescence was excited by l i g h t at 420 nm and emission was measured at 500 nm (192).  At these wavelengths,  fluorescence of NADH.  there was no interference from the  Fluorescence was measured at 20°C with a Turner  Model 420 Spectrofluorometer connected to a Varian s t r i p chart recorder.  90.  Measurement of Proton Conduction i n K -loaded Membrane Vesicles K -loaded v e s i c l e s i n 0.4 M sucrose, 10 mM MgSO^ (0.85 ml) were +  s t i r r e d i n a glass cuvette at 20°C.  Valinomycin was added to the v e s i c l e  and the pH change which followed was measured with a glass pH electrode connected to a Fisher Accumet Model 325 expanded scale pH meter.  The  output of the meter was amplified so that 0.2 pH units gave a f u l l - s c a l e d e f l e c t i o n on the recorder (John's S c i e n t i f i c Linear Co.).  In some  experiments, the v e s i c l e s were preincubated with DCCD ( i n ethanol) or with ammonium sulphate p u r i f i e d antiserum f o r 45 min. at 20°C.  Each assay was  i n t e r n a l l y calibrated by addition of an aliquot of known concentration of acid ( H S 0 2  4  or HC1).  Determination of Cytochrome Content This was used i n a semi-quantitative manner to detemine whether any form of separation was achieved between the ATPase complex and the cytochromes during ion-exchange, hydrophobic-exchange  or g e l exclusion  chromatography. 1.5 ml of sample at 20°C was placed i n each of two cuvettes of 1 cm l i g h t path.  25-50 u l of 0.3%  (v/v) H 0 was added to the reference 2  2  cuvette, while the other sample was reduced with an excess of sodium dithionite.  The reduced versus the oxidized (difference) spectrum was  then  scanned between 500 and 670 nm i n a Perkin Elmer model 356 Double Beam Spectrophotometer  equipped with a recorder.  Baseline readings were obtained  by scanning two cuvettes of untreated samples i n the same range. Cytochome d content was calculated by measuring one-half the height i n absorbance units from the trough at 648 nm to the peak at 628 nm i n the -1 difference spectra and using the e x t i n c t i o n c o e f f i c i e n t of 8.51 mM  -1 cm  91.  (193).  The content of cytochrome  was determined from the absorbance  above the baseline at 594 nm and using the extinction c o e f f i c i e n t of cytochrome a^ of 4.6 mM "*"cm  (193).  Cytochrome b^ content was determined  from the height of the peak above the baseline at 558 nm and using the extinction c o e f f i c i e n t of 16.0 mM "*"cm ^ (193).  Determination of Catalase A c t i v i t y Catalase was used as a standard of molecular weight 232 000 f o r c a l i b r a t i n g g e l - f i l t r a t i o n columns.  A c t i v i t y was determined by the method  of Beers and Sizer (194). 1.5 ml of 4% (v/v) 1^02 was mixed with 25 ml of 50 mM potassium phosphate buffer, pH 7.0.  To 1.99 ml of t h i s mixture i n a cuvette was  added 10 vl of the enzyme solution to be measured.  The change i n  absorbance at 240 nm was measured with a spectrophotometer equipped with a recorder. One unit of a c t i v i t y was expressed as the rate of change i n absorbance at 240 nm per min at 37°C.  Determination of Radioactivity i n Gel Slices The tube g e l was frozen at -70°C f o r 30-45 min and then s l i c e d into 1 mm thick disks.  Each s l i c e was incubated with 0.5 ml of a mixture of  NCS:water (9:1) at 50°C f o r 2 h, a f t e r which i t was cooled to 4°C and ml of ACS added.  5-10  These were c h i l l e d to 4°C i n the dark and the radio-  a c t i v i t y determined i n a Packard Model 3000 s c i n t i l l a t i o n counter.  The  samples were corrected f o r quenching by using standard *C-quenched 1  series under i d e n t i c a l conditions.  In some experiments, the g e l was  sliced  into 2 mm thick disks, i n which case the volume of NCSrwater (9:1) used was  92.  increased to 1.0  ml.  Solid Phase Radioimmune Assays The procedures of Mackenzie and Molday (195) were used. "competitive  i n h i b i t i o n assay" f l e x v i n y l m i c r o t i t r e plate wells were  pretreated with 25 y l portions of 0.1% 6 hours.  sodium borate buffer, pH 8.8,  0.13  containing 2% (w/v) T r i t o n  incubated i n the wells f o r 8 to 10 hours at 22°C.  peptide was (PBS)  polylysine at 22°C for 4 to  DCCD-binding protein (25 p i ) at a concentration of  mg/ml i n 100 mM was  (w/v)  The wells were subsequently washed with water to remove unbound  polylysine.  X-100  In the  Unbound poly-  removed by extensive washing with phosphate-buffered saline  (0.13 M NaCl, 2.68 mM KC1,  containing 10 mM MgCl » 2  1.47  mM  KH P0 , 8.09 mM 2  4  Na HP0 , pH 2  4  7.5)  Nonspecific binding s i t e s were then quenched by  incubation overnight at 4°C with radioimmune assay (RIA) buffer consisting r  of 2% (w/v) NaN^  bovine serum albumin, 2% (v/v) f e t a l c a l f serum and 0.1%  i n PBS-10 mM MgCl . 2  The assay wells were then incubated with  (w/v) 25  y l portions taken from a mixture of varying concentrations of the free antigen (vesicles, modified DCCD-binding protein, etc.) which had been preincubated  with appropriately diluted antiserum to the DCCD-binding  protein (1:500 i n RIA buffer) f o r 60 min.  After 1.5 h at 22°C, the wells  were washed with PBS-10 mM MgCl , and then incubated with 25 y l of 2  a f f i n i t y purified  1 2 5  1 - l a b e l l e d goat anti-rabbit immunoglobulin (15-40  yg/ml RIA buffer: 1-2 x 10  6  dpm/yg) f o r 60 min.  The wells were rinsed  extensively with PBS-10 mM MgCl , cut out, and the bound r a d i o a c t i v i t y 2  determined i n a Beckman Gamma 8000 counter.  Two  basic versions of the  "binding assay" were used which d i f f e r e d i n the nature of the free antigen. This was e i t h e r E. c o l i F  or the DCCD-binding protein.  Binding of the  93.  free antigen to the fixed antigen was then detected by using an antiserum to the free antigen.  For example, polylysine-treated m i c r o - t i t e r wells  were incubated with 25 u l portions of the antigen to be fixed (ECF^, 0.75 mg protein/ml i n 50 mM HEPES-KOH buffer, pH 7.5 containing 10 mM MgCl  2  and 10% (v/v) g l y c e r o l ;  DCCD-binding protein, 0.13 mg protein/ml  i n 100 mM sodium borate buffer, pH 8.8, containing 10 mM MgCl (w/v) Triton X-100).  2  and 2%  Following quenching of non-specific binding s i t e s as  i n the competitive i n h i b i t i o n assay, the wells were incubated with EGF^ (0.75 mg/ml i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM M g C l ^ 10% (v/v) g l y c e r o l and 3% (w/v) bovine serum albumin) or DCCD-binding protein (0.13 mg/ml i n 100 mM sodium borate buffer, pH 8.8, containing 10 mM MgCl , 3% (w/v) bovine serum albumin and 2% (w/v) Triton X-100) 2  depending on the nature of the fixed antigen.  The wells were washed with  PBS-10 mM MgCl and then reacted with 25 u l of s e r i a l d i l u t i o n s i n RIA 2  buffer of the antiserum to the free antigen. rabbit antiserum was measured with immunoglobulin as described above.  1 2 5  The extent of binding of the  I - l a b e l l e d goat anti-rabbit Controls f o r the non-specific binding  of ECF^ or DCCD-binding protein to the wells were run by omitting the f i x e d antigen i n the procedure.  Controls f o r potential c r o s s - r e a c t i v i t y  between ECF^ and the antiserum to the DCCD-binding protein, or between the DCCD-binding protein and the antiserum to ECF^, were run by omitting the free antigen i n the procedure. For experiments i n which the fixed antigen (ECF^, chemically-modified or normal DCCD-binding protein, subunits of ECF^) was t i t r a t e d with the free antigen (DCCD-binding protein or ECF^), the experiments were carried out as described above except that the concentration of free antigen was varied with a constant amount of fixed antigen.  (The amounts are given i n  94.  the legends to the Figures i n the "Results" section). ECF  1  and the DCCD-binding protein were d i l u t e d 1:300  u l was used i n each w e l l .  The antisera against  i n RIA buffer.  "Net binding" of free to fixed antigen  25  was  always corrected f o r any non-specific binding of the free antigen or for c r o s s - r e a c t i v i t y with heterologous antiserum.  95.  RESULTS  PART I  PURIFICATION OF THE  ECFJFQ  COMPLEX  SELECTION OF AN E. c o l i STRAIN The ATPase a c t i v i t y of the membrane-bound enzyme from several strains of bacteria was measured at 37°C and pH 8.3 using the "slow ATPase" assay procedure as described i n MATERIALS AND METHODS. Table 8 shows the average s p e c i f i c a c t i v i t i e s f o r the ATPase enzyme i n the membrane v e s i c l e s of E_. c o l i AN180, WS1  and ML308-225.  Under these conditions, E. c o l i ML308-225  had a higher average s p e c i f i c a c t i v i t y (560 nmol per min per mg protein) than either E.  c o l i AN180 (250 nmol per min per mg protein) or E_. c o l i  WS1  (370 nmol per min per mg protein). From the r e s u l t s i n Table 8, i t was not known i f the d i f f e r e n t s p e c i f i c a c t i v i t i e s were due to each s t r a i n of E_. c o l i having a d i f f e r e n t cation to ATP r a t i o f o r optimal a c t i v i t y .  In order to determine t h i s , membrane v e s i -  c l e s were prepared from each s t r a i n of E_. c o l i l i s t e d i n Table 8 and ded i n 50 mM T r i s - ^ S O ^ buffer, pH 8.0.  suspen-  ATPase a c t i v i t y was then measured  as previously described, except that CaC^  was replaced with d i f f e r e n t  amounts of one of the following cations: MgC^,  CaC^,  MnC^  or  ZnC^.  2+ The results of such an experiment are shown i n F i g . 4.  Mg  had the  highest capacity f o r stimulating the membrane-bound ATPase a c t i v i t y .  For  2+ each s t r a i n , maximum a c t i v i t y was obtained at a [Mg 0.3 to 0.5;  ] to [ATP] r a t i o of  above and below which, the a c t i v i t y decreased s i g n i f i c a n t l y .  These results are consistent with the findings of others (196,197) i n which 2+ the maximum a c t i v i t y was obtained at a [Mg ] to [ATP] r a t i o of 0.5. 2+ In the presence of Mg , the maximum a c t i v i t y of E. c o l i ML308-225  96. Table 8  S p e c i f i c A c t i v i t y of the Membrane-Bound ATPase of D i f f e r e n t B a c t e r i a l Strains  Specific Activity (units/mg protein)  Strain  Mean ± S.D*  Range  E. c o l i  AN 180 ( 6 ) * *  0.25 ± 0.07  0.12 - 0.31  E. c o l i  WS1  0.37 ± 0.095  0.14 - 0.56  E. c o l i  ML 308-225  0.56 ± 0.117  0.39 - 0.85  (16) (40)  *  Standard  deviation  **  Figures i n parenthesis i n d i c a t e the number of membrane v e s i c l e preparations assayed.  Membrane v e s i c l e s from each s t r a i n of E. c o l i were suspended 50 mM T r i s - ^SO^ buffer, Tris-H S0 2  4  buffer,  pH 8.0 containing 10 mM MgCl  2  i n either  or i n 0.5 M  pH 8.0 containing 0.25M Na^O^ and 10% (v/v) g l y c e r o l .  ATPase a c t i v i t y was assayed by the "slow assay" as described i n MATERIALS AND METHODS.  97.  Fig.  4  Effect of cations on the membrane-bound ATPase a c t i v i t y of d i f f e r e n t s t r a i n s of IS. c o l i .  Membranes from E. c o l i ML308-225, WS1 and AN180 were prepared i n 50 mM T r i s - ^ S O ^ buffer, pH 8.0 containing 1 mM MgCl2- The membrane v e s i c l e s were suspended at a protein concentration of 3.3 mg/ml i n 50 mM Tris-r^SO^ buffer, pH 8.0. ATPase a c t i v i t y was measured by the "slow ATPase" assay as described i n MATERIALS AND METHODS, except that C a C l was replaced with various l e v e l s of either MgCl ( • - • ) , C a C l (O-O), MnCl ( ~ ) or Z n C l ( A - A ) . The concentration of ATP i n the assay mixture was 5 mM and the amount of membrane protein per assay was 16.5 Pg. Enzyme a c t i v i t y i s expressed as u n i t s per mg p r o t e i n . 2  A  2  2  2  A  2  ML 308-225  (980 nmol per min per mg protein) was present i n about twice the amount of that found i n jS. c o l i AN180 (460 nmol per min per mg protein) and IS. c o l i WS1  (420 nmol per min per mg protein). The r e s u l t s suggested that the ATPase enzyme was l i k e l y to be present  i n a higher amount i n E_. c o l i ML308-225 than i n E_. c o l i AN180 and WS1  and  thus t h i s s t r a i n would be a good source of the ECF^FQ complex.  SOLUBILIZATION OF THE ECF-^Q COMPLEX  Selection of Detergent In contrast to water-soluble proteins, membrane-bound proteins must be released from the membrane p r i o r to p u r i f i c a t i o n . been used f o r t h i s purpose (90,91). certain conditions.  Numerous detergents have  An e f f e c t i v e detergent should s a t i s f y  I t should not inactivate the enzyme.  As some  p u r i f i c a t i o n procedures were carried out over a period of 3 to 5 days, i t was important to select a detergent which would preserve the enzyme activity.  Second, the detergent should s e l e c t i v e l y s o l u b i l i z e a s i g n i f i c a n t  amount of the enzyme without s o l u b i l i z i n g a l l other membrane-bound proteins. On t h i s basis, a number of detergents were tested, so that a suitable detergent could be selected f o r the extraction of the ATPase complex from the membranes of IS. c o l i . High i o n i c strength buffers have often been used to keep the F-^-ATPase associated with the FQ. T r i s - H S 0 , pH 8.0, 2  4  Sone et a l . (102) used a buffer containing 0.5  0.25 M Na S0 2  4  M  and 10% (v/v) glycerol to keep  the TF^FQ complex together during s o l u b i l i z a t i o n of the membrane of PS3. The same buffer was used i n my experiments f o r studying the s o l u b i l i z a t i o n characteristics  of d i f f e r e n t detergents.  100.  Membrane v e s i c l e s of E_. c o l i ML308-225 were suspended i n high i o n i c strength buffer and treated with various levels of detergents as described i n MATERIALS AND METHODS.  Membrane proteins and ATPase a c t i v i t y were  considered as being " s o l u b i l i z e d " i f they were not sedimented following centrifugation at 250 000 xg f o r 2 h. The r e s u l t s of the treatment with various detergents are i l l u s t r a t e d i n F i g . 5.  Optimal s o l u b i l i z a t i o n of the ATPase a c t i v i t y was obtained  the detergent, N-lauroyl sarcosine. 0.25,  At a detergent  with  to protein r a t i o of  80% of the a c t i v i t y was s o l u b i l i z e d by N-lauroyl sarcosine, whilst  less than 60% was s o l u b i l i z e d by the other detergents.  N-lauroyl sarcosine  also exhibited some s e l e c t i v i t y , i n that only 38% of the membrane protein was  s o l u b i l i z e d at t h i s detergentrprotein r a t i o .  at higher r a t i o s .  This s e l e c t i v i t y was lost  At a detergentrprotein r a t i o of 0.96, almost a l l the  a c t i v i t y was s o l u b i l i z e d (96%), while at the same time, the amount of protein s o l u b i l i z e d increased to 80%. Ammonyx Lo and sodium deoxycholate also s o l u b i l i z e d amounts of ATPase a c t i v i t y at higher detergent  significant  to p r o t e i n ^ r a t i o s . At at .  r a t i o of 0.4, Ammonyx Lo and deoxycholate s o l u b i l i z e d 80% and 50% of the a c t i v i t y , respectively. With sodium cholate, only 50% of the a c t i v i t y was s o l u b i l i z e d at a r a t i o of 0.8. Sodium cholate and deoxycholate required the presence of high ionic strength buffers, i n order f o r s i g n i f i c a n t amounts of membrane proteins to be s o l u b i l i z e d (94,198).  As shown In Table 9, sodium cholate at a  detergentrprotein r a t i o of 0.69 i n the presence of low ionic buffer (50 mM Tris-H S0 , 2  3.9%  4  pH 8.0, 10 mM MgCl  2  and 10% (v/v) g l y c e r o l ) ,  of the a c t i v i t y and 8.4% of the membrane proteins.  solubilized But at the same  r a t i o , i n the presence of high ionic strength buffer, sodium cholate  D e t e r g e n t -- m g P r o t e i n -- m g  Fig.  5  S o l u b i l i z a t i o n of the membrane-bound ATPase a c t i v i t y of E_. c o l i by various detergents.  Membrane v e s i c l e s of E. c o l i ML308-225 were suspended at a protein concentration of 15-20 mg/ml i n 0.5 M Tris-H S04 buffer, pH 8.0 containing 0.25 M Na S04 and 10% (v/v) g l y c e r o l , and s o l u b i l i z e d , as described i n MATERIALS AND METHODS, with one of the following detergents: PANELS A and B: N-lauroyl sarcosine ( ) , Ammonyx Lo ( ) , sodium deoxycholate (•-•), sodium cholate (O-O), octyl-B-D-glucopyranoside (•-•), B r i j 35 (•-•). PANELS C and D: T r i t o n X-100 (•-•), T r i t o n X-114 (O-O) , Lubrol WX (*-A) , Lubrol 17A-10 ( A - A ) , Tween 60 or Tween 80 (•-•), Lubrol PX (•-•). 2  2  A - A  A - A  Table  9  Detergent: Protein  S o l u b i l i z a t i o n of the Membrane-Bound ATPase A c t i v i t y of E. c o l i with Sodium Cholate  Percent  Total Protein (mg)  Total Units  Solubilized  ATPase A c t i v i t y  Protein  A 33.5  1.6  2.7  0.34  34.2  2.1  4.5  0.69  37.2  3.9  8.4  1.42  39.7  11.3  13.1  37.2  11.8  30  0.22  37.2  34.8  32.9  0.46  37.6  43  36.5 .  0. 69  36.9  45  40.5  0.94  37.7  50.8  48  37  0  B 0  35.5  Membranes v e s i c l e s of E. c o l i  ML308-225 were suspended at a protein  concentration of 15-20 mg/ml i n buffer containing H S0 2  4  pH 8.0, lOmM MgCl  pH 8.0, 0.25M N a S 0 2  4  2  either  (A) 50mM T r i s -  and 10% (v/v) g l y c e r o l or (B) 0.5 T r i s - H ^ O ^  and 10% (v/v) g l y c e r o l .  The v e s i c l e s were  s o l u b i l i z e d with various amounts of sodium cholate as described MATERIALS AND METHODS section.  i n the  103.  s o l u b i l i z e d 45% and 40.5% of the a c t i v i t y and protein, respectively. The non-ionic detergents, T r i t o n X-100, Triton X-114 and Lubrol WX showed some p o t e n t i a l i n extracting the membrane-bound ATPase a c t i v i t y ( F i g . 5). At a detergent to protein r a t i o of 0.96, T r i t o n X-100, T r i t o n X-114 and Lubrol WX s o l u b i l i z e d 70%, 55% and 45% of the ATPase a c t i v i t y , while the corresponding  amounts of protein s o l u b i l i z e d were 42%, 45% and  55%, respectively. At the same detergentrprotein r a t i o , B r i j 35, Tween 60, i  S  Tween 80, Lubrol PX and Lubrol 17A-10 extracted less than 30% of the a c t i v i t y and protein from the membrane.  Effect of Detergents on the Membrane-Bound ATPase A c t i v i t y The detergents also stimulated the membrane-bound ATPase a c t i v i t y (Table 10).  In general, greater stimulation of the a c t i v i t y was observed  with higher detergent to protein r a t i o s .  N-lauroyl sarcosine, at ratios of  0.12 and 0.48, stimulated the ATPase a c t i v i t y by 1.3- and 4.5-fold, respectively. There also appeared to be some c o r r e l a t i o n between the extent of stimulation and the s o l u b i l i z i n g capacity of the detergent. i o n i c detergents, sodium cholate and deoxycholate,  Comparing the  i t was found that at a  r a t i o of 0.98, the a c t i v i t i e s were increased by 1.1- and 2.45- f o l d s , respectively.  S i m i l a r l y , the non-ionic detergent, T r i t o n X-100, at a r a t i o  of 0.96 stimulated the a c t i v i t y by 2.5 f o l d whereas Lubrol PX at a r a t i o of 2.48 stimulated the a c t i v i t y by only 1.1 times. The a c t i v i t i e s of the s o l u b i l i z e d fractions could also be stimulated by up to 100%, when assayed i n the presence of L - a - l y s o l e c i t h i n (data not shown). detergent  I t was not known i f the increase i n a c t i v i t y i n the presence of lyso-phospholipid was due to a c t i v a t i o n of latent ATPase enzymes  Table  10  E f f e c t o f D e t e r g e n t on t h e Membrane-Bound of E. c o l i  Ionic  Detergents  Non-Ionic Detergents  Percent Control Value  Detergent  0  100  -  Ammonyx Lo  0.34 0.68 1.4  340 410 425  N-lauroyl sarcosine  0.12 0.24 0.48  130 270 450  Octyl-fr-Dglucopyranoside  0.21 0.42 0.84  115 170 260  Sodium cholate  0.48 0.98 2.08  105 110 170  Sodium deoxycholate  0.48 0.98 2.08  145 245 350  Detergent  D:P*  -  * Detergent  ATPase A c t i v i t y  D:P  1  Percent Control Value  0  100  B r i j 35  0.52 1.06  120 125  L u b r o l PX  0.59 2.48  105 110  L u b r o l WX  0.14 0.28  160 165  Lubrol  0.14 0.28  150 180  T r i t o n X-100  0.47 0.96  200 250  T r i t o n X-114  0.47 0.96  280 325  Tween 60  0.33 0.67  135 140  Tween 80  0.33 0.67  140 155  17A-10  104.  to protein r a t i o  Membrane v e s i c l e s o f JE. c o l i ML308-225 were suspended a t a p r o t e i n l e v e l c o n c e n t r a t i o n o f 15 - 20 mg/ml i n 0.5M Tris-H^SO^ b u f f e r , pH 8.0 c o n t a i n i n g 0.25 M N a S 0 , and 10% ( v / v ) g l y c e r o l . D i f f e r e n t l e v e l s of various deterg e n t s were added t o a l i q u o t s o f t h e membrane v e s i c l e s u s p e n s i o n . A f t e r 30 min. a t 20 C, samples were withdrawn and assayed f o r ATPase a c t i v i t y by t h e " s l o w a s s a y " a s d e s c r i b e d i n MATERIALS AND METHODS. 2  105.  as i n Mycobacterium  p h l e i (199) and Micrococcus lysodeikticus  proteolytic digestion of ECF  1  (200) or to  (6,7,24).  S t a b i l i t y of the Solubilized Enzyme Since the detergents stimulated the ATPase a c t i v i t y , i t was of interest to determine whether the a c t i v i t y of the solubilized f r a c t i o n  was  stable over the period of time spanned by some of the p u r i f i c a t i o n procedures used i n t h i s study. The solubilized- fractions of membrane vesicles of E. c o l i ML308-225, solubilized with either Ammonyx Lo, N-lauroyl sarcosine or sodium cholate (at detergent to protein ratios of 0.68, stored at 4°C.  0.48 and 0.48,  respectively) were  Samples were removed at timed intervals and the ATPase i  a c t i v i t y measured. In the presence of Ammonyx Lo, the a c t i v i t y decreased by 30% after 140 h ( F i g . 6).  This decrease In a c t i v i t y could be due to denaturation  or s o l u b i l i z a t i o n of the other, membrane components, necessary f o r the functional  conformation of the enzyme.  By contrast, the a c t i v i t y i n the  presence of sodium cholate had increased to 112% forty hours a f t e r s o l u b i l i z a t i o n , and decreased to i t s o r i g i n a l l e v e l (100%) only a f t e r another 200 h.  S i m i l a r l y , the a c t i v i t y i n the presence of N-lauroyl  sarcosine or i n control membranes decreased by only 5-10% over 10 days.  MOLECULAR SIZE OF THE DETERGENT-SOLUBILIZED ENZYME Gel F i l t r a t i o n on Sepharose 6B or Bio-Gel A-0.5m The s o l u b i l i z e d enzyme was chromatographed on gel f i l t r a t i o n columns i n order to estimate the molecular weight of the s o l u b i l i z e d ATPase enzyme.  In addition, g e l f i l t r a t i o n was thought to be suitable as an  106.  Fig. 6  S t a b i l i t y of s o l u b i l i z e d ATPase a c t i v i t y on storage at 4°C.  Membrane v e s i c l e s of IS. c o l i ML308-225 were suspended i n 0.5 M T r i s buffer, pH 8.0 containing 0.25 M N a S 0 4 and 10% (v/v) g l y c e r o l at a protein concentration of 15-20 mg/ml. The v e s i c l e s were s o l u b i l i z e d with e i t h e r Ammonyx Lo ( ) , N-lauroyl sarcosine ( O - O ) , or sodium cholate (•-•) at detergent:protein r a t i o s of 0.68, 0.48 and 0.48, respectively. The conditions of s o l u b i l i z a t i o n are described i n MATERIALS AND METHODS. The ATPase a c t i v i t y of the s o l u b i l i z e d f r a c t i o n was measured immediately and at various times following storage at 4°C. A control, consisting of a suspension of untreated membrane v e s i c l e s of E_. c o l i ML308-225 (•-•) , was also stored at 4°C and the a c t i v i t y measured at timed i n t e r v a l s . H2SO4  2  A - A  107.  i n i t i a l p u r i f i c a t i o n step.  Since the ATPase complex (molecular weight  380 000) i s much larger than most other membrane-bound proteins i n J £ . c o l i , i t should be separated from the proteins with smaller molecular weights on a gel f i l t r a t i o n column. Membrane p a r t i c l e s of E. c o l i ML308-225 i n 0.5 M T r i s - H S 0 2  pH 8.0 containing 0.25 M Na S0 2  4  4  buffer,  and 10% (v/v) glycerol were s o l u b i l i z e d  with N-lauroyl sarcosine at a detergent to protein r a t i o of 0.5.  The  s o l u b i l i z e d material was concentrated and then applied to a Sepharose 6B column, which had been equilibrated with 0.5%,  (w/v) of various detergents.  P r o f i l e s of the separations on Sepharose 6B i n the presence of (w/v) Lubrol WX and 0.5% 7, panels A and B. the presence of 0.5%  0.5%  (w/v) N-lauroyl sarcosine are i l l u s t r a t e d i n F i g .  F i g . 7C i s the separation p r o f i l e on Sepharose 6B, i n (w/v) N-lauroyl sarcosine, of the s o l u b i l i z e d material  which was made up to 3% (w/v) N-lauroyl sarcosine and 2% (w/v) sodium cholate, p r i o r to loading onto the column (201). In a l l three cases, the ATPase a c t i v i t y migrated as a single peak, close to the void volume (V  = 75 ml).  Cytochromes a-^, b^ and d comigrated  with the ATPase a c t i v i t y peak as did the majority of the protein which was applied to the column.  Similar e l u t i o n p r o f i l e s were obtained i n the  presence of the other detergents l i s t e d i n Table 11. When the s o l u b i l i z e d f r a c t i o n was passed through a column of Bio-Gel A-0.5 m, which was equilibrated with 0.5%  (w/v) N-lauroyl sarcosine, the  enzyme a c t i v i t y also co-eluted with a l l the cytochromes and the majority of the protein close to the void volume (V r  o  = 85 ml).  The recovery of a c t i v i t y depended on the detergent used during chromatography (Table 11).  Recoveries ranged from 55 - 60% with N-lauroyl  sarcosine to 125% with sodium cholate.  Although the recovery was low i n  108. Fig. 7  Chromatography of the detergent-solubilized ATPase complex on Sepharose 6B i n the presence of various detergents.  Membrane v e s i c l e s of E_. c o l i ML308-225 were s o l u b i l i z e d with N-lauroyl sarcosine at a detergentrprotein r a t i o of 0.5. The s o l u b i l i z e d f r a c t i o n was concentrated by u l t r a f i l t r a t i o n through an Amicon XM-100A f i l t e r as described i n MATERIALS AND METHODS. The concentrated material (100-150 mg protein) was applied to a column of Sepharose 6B (2.5 x 37 cm) e q u i l i b r a t e d with 50 mM Tris-H S04 buffer, pH 8.0, containing 0.25 M Na S04, 10% (v/v) g l y c e r o l and 0.5% (w/v) of one of the following detergentsr PANEL A, Lubrol WX; PANELS B and C, N-lauroyl sarcosine. In PANEL C, the concentrated material was made up to 3% (w/v) N-lauroyl sarcosine and 2% (w/v) sodium cholate p r i o r to loading onto the column. The columns were then eluted with the same buffer containing the appropriate detergent. Fractions (10 ml) were c o l l e c t e d and assayed f o r ATPase a c t i v i t y (•-•), protein (O-O) and t o t a l cytochromes (aj_, b^ and d) ( ) as previously described. ' 2  2  A - A  109.  Volume-ml  110.  the presence of N-lauroyl sarcosine, i t was higher (105%) i f the s o l u b i l i z e d material was made up to 3% (w/v) N-lauroyl sarcosine and 2% (w/v) sodium cholate before applying to the column. The p r o f i l e s i n a l l cases showed that the enzyme a c t i v i t y eluted as a broad peak.  This suggested  i n d i f f e r e n t aggregated  that the s o l u b i l i z e d enzyme most l i k e l y existed  forms.  The molecular weight of the s o l u b i l i z e d enzyme was estimated i n the presence of d i f f e r e n t detergents. 11.  These values are summarized i n Table  The molecular weights were calculated to be i n the range of 450 000 to  890 000.  In general, chromatography i n the presence of non-ionic detergents  resulted i n higher molecular weights f o r the ATPase enzyme than i n the presence of i o n i c detergents.  With the non-ionic detergents, the highest  molecular weight f o r the enzyme was obtained with either Lubrol WX or T r i t o n X-114 (890 000 daltons), and the lowest with Triton X-100 (640 000 daltons).  With the i o n i c detergents, the highest molecular weight was  obtained i n the presence of Ammonyx Lo (580 000 daltons) and the lowest with N-lauroyl sarcosine (450 000 to 510 000 daltons). The molecular weights estimated by g e l f i l t r a t i o n suggested that the ECF^ and FQ remained associated during s o l u b i l i z a t i o n of the membrane and subsequent chromatography.  In order to test f o r t h i s , the s e n s i t i v i t y  to i n h i b i t i o n by DCCD of the f r a c t i o n from the Sepharose 6B column containing the highest ATPase a c t i v i t y was determined enzyme.  The results are shown i n F i g . 8.  f o r two preparations of the  The ATPase a c t i v i t y i n the  presence of either 0.01% (w/v) B r i j 35 or 0.025% (w/v) N-lauroyl sarcosine was inhibited by 50% at 0.4 mM and 1 mM DCCD, respectively. ECF  1  was i n h i b i t e d by 5-10% at 1 000 yM DCCD.  These results  that the s o l u b i l i z e d ATPase enzyme was the intact ECF F  n  By contrast, suggested  complex.  111. Table  11  Estimation of the Molecular Weight of the S o l u b i l i z e d ECF.F Complex by Gel F i l t r a t i o n Chromatography  1  Detergent Concentration [% (w/v)]  Estimated Molecular Weight ,(M x 10 ) r  Recovery of activity (%)  Gel f i l t r a t i o n resin  Detergent i n equilibration buf f er  Sepharose 6B  B r i j 35  0.5  8.5  110  Sepharose 6B  Lubrol WX  0.5  8.9  80  Sepharose 6B  T r i t o n X-100  0.5  6.4  64  Sepharose 6B  T r i t o n X-114  0.5  8.9  69  Sepharose 6B  Ammonyx Lo  0.5  5.8  73  Sepharose 6B  Sodium cholate  0.5  5.2  125  Sepharose 6B  N-lauroyl sarcosine  0.5  5.1  61  N-lauroyl sarcosine  0.5  4.7  107  N-lauroyl sarcosine  0.5  4.5 - 4.8  Sepharose 6B Bio-Gel A 0.5m  a  55  The s o l u b i l i z e d material was made up to 3% (w/v) N-lauroyl sarcosine and 2% (w/v) sodium cholate before loading onto the column.  a  The d e t a i l s of the experiment are described i n the legend to F i g . 7. Thyroglobulin (669 00 daltons), catalase (232 000 daltons) and haemoglobin (64 500 daltons) were used to c a l i b r a t e the g e l f i l t r a t i o n columns under the various conditions.  112.  Fig. 8  E f f e c t of DCCD on the detergent-solubilized ATPase a c t i v i t y .  Membrane v e s i c l e s of E. c o l i ML308-225 were s o l u b i l i z e d with N-lauroyl sarcosine at a detergentrprotein r a t i o of 0.5. The s o l u b i l i z e d f r a c t i o n was chromatographed on a column of Sepharose 6B i n the presence of 0.5% (w/v) detergent, as described i n the legend to F i g . 7, PANELS A and B.. Samples of the most active f r a c t i o n from the Sepharose 6B column, i n the presence of 0.5% (w/v) B r i j 35 (O-O) or N-lauroyl sarcosine (•-•) were incubated with various l e v e l s of DCCD i n 0.1 M T r i s - H C l , pH 8.0 f o r 45 min at 37°C. ECF of E. c o l i ML308-225 (O-O), obtained a f t e r d i a l y s i s of the membranes i n low i o n i c strength buffer, was also treated with DCCD. Following incubation with DCCD, ATPase a c t i v i t y was measured as described i n MATERIALS AND METHODS. The s p e c i f i c a c t i v i t i e s of the ATPase enzyme i n the presence of B r i j 35 ( f i n a l concentration, 0.01% (w/v)), N-lauroyl sarcosine ( f i n a l concentration, 0.025% (w/v)) and of ECFj^ were 2.06, 2.52 and 3.04 u n i t s per mg protein and the amount of protein per assay was 3, 13.5 and 14 wg, r e s p e c t i v e l y . 1  113.  Since, the ATPase a c t i v i t y was not separated from other s o l u b i l i z e d proteins (cytochromes) by chromatography on Sepharose 6B, other methods of purifying the  ECF^FQ  complex were investigated.  These are discussed i n  the following sections .  P u r i f i c a t i o n of the ECF^FQ Complex by Hydrophobic-Interaction Chromatography Hydrophobic-interaction chromatography has been used mainly f o r the p u r i f i c a t i o n of water-soluble proteins (202).  Membrane-bound proteins  interact very strongly with these resins and can only be released from the r e s i n with the use of detergents.  Usually l i t t l e separation i s achieved.  However, hydrophobic-interaction chromatography on Phenyl Sepharose CL-4B was used successfully to purify the membrane-bound enzyme, fumarate reductase (203).  This enzyme consists of a membrane-bound and a water-  soluble component and was eluted from the r e s i n by lowering the i o n i c strength. ECF^FQ  The enzyme obtained was e s s e n t i a l l y i n a pure form.  Since the  complex resembles fumarate reductase i n having a water-soluble  (ECF^) and a membrane-bound  (FQ) component, experiments were carried  out to test the f e a s i b i l i t y of using Phenyl-Sepharose CL-4B as a means of purifying the ECF^FQ complex. Membrane v e s i c l e s of E. c o l i ML308-225 were s o l u b i l i z e d with N-lauroyl sarcosine at a detergent to protein r a t i o of 0.5, as described e a r l i e r . The s o l u b i l i z e d material was applied to a column of Phenyl-Sepharose CL-4B, which was equilibrated with 50 mM T r i s - H S 0 2  4  buffer, pH 8.0 containing  0.25 M Na S0 , 10 mM MgCl , 10% (v/v) g l y c e r o l and 0.5% (w/v) sodium 2  cholate.  4  2  The column was washed with the same buffer and then a l i n e a r  decreasing gradient of 0.25 M to 0 M Na„S0, applied. F i n a l l y , the  114.  column was washed with 2%, (w/v) T r i t o n X-100. shown i n F i g . 9A.  The e l u t i o n p r o f i l e i s  The ATPase a c t i v i t y eluted as two peaks during the  i n i t i a l washing of the column.  The f i r s t a c t i v i t y peak was eluted during  the a p p l i c a t i o n of the f i r s t two column volumes of wash buffer and represented 37% of the t o t a l a c t i v i t y recovered.  The second a c t i v i t y peak  was eluted during the l a s t two column volumes of wash buffer and 63% of the t o t a l a c t i v i t y was recovered i n t h i s peak.  No enzyme a c t i v i t y was detected  i n the f r a c t i o n s from the gradient e l u t i o n .  Only a small amount of  cytochrome d co-eluted with the ATPase a c t i v i t y . (a^,  Most of the cytochromes  b^ and d) were t i g h t l y bound to the resin and were eluted with 2%  (w/v) T r i t o n X-100. An attempt was made to increase the i n t e r a c t i o n of the ATPase enzyme with the r e s i n , by omitting sodium cholate from the e q u i l i b r a t i o n buffer only.  The experiment was repeated as described above and the e l u t i o n  p r o f i l e i s shown i n F i g . 9B.  Again, the ATPase a c t i v i t y was detected only  i n the f r a c t i o n s obtained during the i n i t i a l washing. enzyme a c t i v i t y eluted as two peaks.  As before, the  However, i n contrast to F i g . 9A, 96%  of the t o t a l a c t i v i t y was associated with the second peak.  Although most  of the cytochromes (a^, b^ and d) bound very t i g h t l y to the r e s i n , some co-eluted with the two ATPase a c t i v i t y peaks. Further attempts to improve the i n t e r a c t i o n of the ATPase enzyme with the r e s i n was achieved by. using ions which had "salting-out" properties. This was achieved by replacing the ^ £ 8 0 ^ with 20% ammonium sulphate (by saturation) i n the buffers described i n the legend to F i g . 9B.  The  gradient consisted of a l i n e a r decreasing concentration of 20% to 0% ammonium sulphate.  The experiment was repeated as described above and the  e l u t i o n p r o f i l e i s shown i n F i g . 9C.  A l l the cytochromes bound very  115.  Fig. 9  Chromatography of the detergent-solubilized ATPase complex on Phenyl-Sepharose CL-4B.  Membrane v e s i c l e s of E^. c o l i ML308-225 were s o l u b i l i z e d with N-lauroyl sarcosine at a detergent:protein r a t i o of 0.5. The s o l u b i l i z e d f r a c t i o n (100-125 mg protein) was applied to a column of Phenyl-Sepharose CL-4B (1.8 x 25 cm) e q u i l i b r a t e d with 50 mM Tris-H S04 buffer, pH 8.0 containing 10 mM MgCl , 10% (v/v) g l y c e r o l and the following: PANEL A, 0.25 M N a S 0 4 and 0.5% (w/v) sodium cholate; PANEL B, 0.25 M Na S04; PANELS C and D, 20% (NH-4) S04 (by saturation). The columns were then washed with 4-5 column volumes of e q u i l i b r a t i o n buffer but with the i n c l u s i o n of 0.5% (w/v) sodium cholate i n PANELS B and C. The columns were then developed with a decreasing l i n e a r gradient (8-10 column volumes) of e i t h e r 0.25 M to 0 M Na S04 (PANELS A and B) or 20% to 0% (NH ) S04 (by saturation) (PANELS C and D) i n 50 mM T r i s - H S 0 buffer, pH 8.0 containing 10 mM MgCl , 10% (v/v) g l y c e r o l and 0.5% (w/v) sodium cholate. Following e l u t i o n with the gradient, the columns were washed with 50 mM Tris-H S04 buffer, pH 8.0, containing 2% (w/v) T r i t o n X-100. The arrows indicate the p o s i t i o n of the buffer changes. Fractions (10 ml) were c o l l e c t e d and assayed f o r ATPase a c t i v i t y (•-•), protein (O-O), t o t a l cytochromes (a^, b^ and d) ( ), and conductivity ( A - A ) described i n MATERIALS AND METHODS. 2  2  2  2  2  2  4  2  2  4  2  2  A _ A  a  s  116.  Fraction  Number  117.  t i g h t l y to the r e s i n and were eluted with 2% (w/v)  T r i t o n X-100.  a c t i v i t y was again detected only i n the fractions from the wash.  ATPase However,  the a c t i v i t y eluted as a single peak during e l u t i o n with the l a s t column volumes of buffer.  two  It appeared that the presence of sodium cholate  i n the buffer was causing the enzyme to be released from the r e s i n .  To  test f o r t h i s p o s s i b i l i t y , sodium cholate was omitted i n the wash buffer and the experiment performed as described for F i g . 9C. i s shown i n F i g . 9D.  Again, a l l the cytochromes were completely  from the ATPase a c t i v i t y . gradient e l u t i o n .  The e l u t i o n p r o f i l e separated  The a c t i v i t y eluted as a single peak during the  The f r a c t i o n with the highest a c t i v i t y eluted at an  ammonium sulphate concentration of 15-16% (34-36 mmho). Thus, the presence of sodium cholate decreased  the i n t e r a c t i o n of the  ATPase enzyme with the resin, whilst ions with "salting-out" properties caused greater binding of the protein to the r e s i n .  The l a t t e r was also  suggested by the e l u t i o n p r o f i l e of the cytochromes.  When bound i n the  presence of Na2S0^, the cytochromes were subsequently  eluted as a broad  peak (Figs. 9A and 9B).  In contrast, they were eluted as a sharp peak  (Figs. 9C and 9D) when bound i n the presence of ammonium sulphate. As I t was possible to bind the s o l u b i l i z e d enzyme to the Phenyl Sepharose CL-4B resin, i t was under i d e n t i c a l conditions.  of interest to determine how  ECF.^ behaved  ECF^ of E_. c o l i ML308-225 obtained a f t e r  p u r i f i c a t i o n on a sucrose gradient or a f t e r d i a l y s i s of membranes i n low ionic strength buffer, as described i n MATERIALS AND METHODS, was chromatographed onto a Phenyl-Sepharose CL-4B column under conditions i d e n t i c a l to those i n F i g . 9D.  In both cases, the ATPase a c t i v i t y eluted  as a single peak during the e l u t i o n with the gradient. p r o f i l e s were similar to F i g . 9D.  The e l u t i o n  However, the fractions containing the  118.  highest a c t i v i t i e s eluted at an ammonium sulphate concentration d i f f e r e n t to that required to elute the detergent-solubilized enzyme. P u r i f i e d E C F ^ or E C F ^ obtained  a f t e r d i a l y s i s of the membranes were eluted at  ammonium sulphate concentrations (24-26 mmho), respectively.  of 9.5-10% (22-24 mmho) and 10.5-11.5%  Therefore,  s o l u b i l i z e d ATPase enzyme was  i t appeared that the detergent-  of a d i f f e r e n t composition than soluble  The e l u t i o n of the s o l u b i l i z e d ATPase at a higher i o n i c  ECF-j^.  than p u r i f i e d ECF^ conformation.  concentration  suggested that the former must have a d i f f e r e n t  This might be expected of an i n t a c t ECF^FQ complex.  The presence of an i n t a c t ECF-^FQ complex was  confirmed when f r a c t i o n s  from the Phenyl-Sepharose CL-4B column were found to be sensitive to DCCD. The a c t i v i t y was  i n h i b i t e d by 50% at a DCCD concentration of 600 yM  (not  shown).  Other Hydrophobic-Interaction Resins In addition to Phenyl-Sepharose CL-4B, a number of other hydrophobic resins were tested for t h e i r p o t e n t i a l use i n the p u r i f i c a t i o n of the ECF^FQ  complex.  The  resins, w-Amino Butyl Agarose, w-Amino Hexyl agarose  (AH-Sepharose CL-4B), Butyl Agarose, Octyl Sepharose CL-4B and Decyl Agarose were selected f o r t h i s purpose. Membrane v e s i c l e s of E. c o l i ML308-225 i n 0.5 M T r i s - H S 0 2  pH 8.0  containing 0.25  M Na S0 2  4  and 10%  4  (v/v) g l y c e r o l were s o l u b i l i z e d  with N-lauroyl sarcosine at a detergent to protein r a t i o of 0.5. s o l u b i l i z e d f r a c t i o n was  buffer,  applied to a column of  The  hydrophobic-interaction  r e s i n , under conditions i d e n t i c a l to those described i n the legend to F i g . 9D. Under these conditions, only a small amount (15%) of the protein  was  119.  bound to either w-Amino Butyl Agarose or w-Amino Hexyl Agarose CL-4B).  (AH-Sepharose  In both cases, the ATPase a c t i v i t y co-eluted as a single peak with  the cytochromes (a^, b^ and d) during the i n i t i a l column wash step. Therefore, both of these resins were considered to be unsuitable f o r the p u r i f i c a t i o n of the ATPase complex.  I t i s probable that both functioned  predominantly as ion-exchange r e s i n s . The i d e n t i c a l experiment was repeated with Butyl Agarose, Octyl Sepharose CL-4B and Decyl Agarose.  In a l l three cases, the ATPase enzyme  bound very t i g h t l y to the resin and was only released during e l u t i o n with the gradient. 9D.  The e l u t i o n p r o f i l e s were almost i d e n t i c a l to that i n F i g .  The ATPase a c t i v i t y eluted as a single peak at an ammonium sulphate  concentration of 15-16%.  With Butyl Agarose and Octyl Sepharose CL-4B, the  cytochromes (a^, b^ and d) could be eluted from the resin with 2% T r i t o n X-100.  (w/v)  However, cytochromes were not detected i n the fractions when  Decyl Agarose was eluted with buffer containing either 2% (w/v) or 5% T r i t o n X-100.  (w/v)  I t was not known I f the cytochromes were eluted i n a  denatured form or were s t i l l t i g h t l y bound to the r e s i n . The s p e c i f i c a c t i v i t y of the eluted ATPase enzyme was determined i n each case.  The highest s p e c i f i c a c t i v i t i e s i n the fractions from Butyl  Agarose, Decyl Agarose and Octyl Sepharose CL-4B were 2.25, 4.7 and  5.2  units per mg protein, and represented 4.7-, 6.1- and and 8.7-fold p u r i f i c a t i o n s , respectively.  Although some p u r i f i c a t i o n was observed i n  each case, i t was not as high as that obtained i n the f r a c t i o n eluted from Phenyl-Sepharose CL-4B (24.8-fold p u r i f i c a t i o n ) .  When the most active  ( fractions from these columns were analyzed on SDS-polyacrylamide gels, the fewest number of polypeptide bands was observed i n the f r a c t i o n from the Phenyl-Sepharose CL-4B column.  Therefore, Phenyl-Sepharose CL-4B was  120.  chosen as the r e s i n f o r the p u r i f i c a t i o n of the  Further P u r i f i c a t i o n of the  ECF^FQ  ECF^FQ  complex.  Complex  The ATPase complex was p a r t i a l l y p u r i f i e d on Phenyl-Sepharose CL-4B as described e a r l i e r .  Analysis of the f r a c t i o n with the highest s p e c i f i c  a c t i v i t y on SDS-polyacrylamide g e l revealed the presence of  approximately  14 major protein-staining bands (including the subunits of ECF^) and many minor bands (the gel was  similar to F i g . 11A,  lane c ) .  The ATPase complex  of the thermophile, PS3, was p u r i f i e d by Sone et a l . (102) and shown to consist of eight d i f f e r e n t polypeptides. t i o n of the  ECF^FQ  It was  complex should be s i m i l a r .  thought that the composi-  Therefore, an attempt  was made to remove some of the polypeptides (contaminants) of the p a r t i a l l y purified  complex by sucrose gradient centrifugation.  ECF^FQ  Membrane v e s i c l e s of E_. c o l i ML308-225 were s o l u b i l i z e d with N-lauroyl sarcosine at a detergent AND METHODS.  to protein r a t i o of 0.5 as described i n MATERIALS  The s o l u b i l i z e d f r a c t i o n was applied to a column of Phenyl-  Sepharose CL-4B and chromatography carried out as described i n the legend to F i g . 9D.  The active f r a c t i o n s eluted during the gradient were pooled  and concentrated  by u l t r a f i l t r a t i o n using an Amicon PM-10  concentrated material was applied on a 15 to 25% (w/v) gradient and centrifuged at 280 000 xg for 23 h. separation are shown i n F i g . 10.  membrane.  The  sucrose density  The results of the  The ATPase a c t i v i t y peak was well  J  separated from the majority of the protein, which remained at the top of the gradient.  Approximately 10% of the t o t a l a c t i v i t y applied to the  sucrose gradient was  recovered.  Attempts to stimulate the ATPase enzyme by  addition of L - a - l y s o l e c i t h i n , p r i o r to assaying for a c t i v i t y , were not successful.  As shown i n F i g . 10, the added lyso-phospholipid i n h i b i t e d the  121.  0  5  (Bottom)  F i g . 10  10  15  20  Fraction  25  (Top)  P u r i f i c a t i o n of the E C F ^ F Q complex by sucrose gradient centrifugation.  The E C F ^ F Q complex obtained a f t e r chromatography on PhenylSepharose CL-4B was concentrated by u l t r a f i l t r a t i o n using an Amicon PM-10 filter. The concentrated material (13 mg protein i n a volume of 1.2 ml) was applied to a l i n e a r gradient of 15% - 25% (w/v) sucrose i n 50 mM Tris-H S04 buffer, pH 8.0 containing 0.5% (w/v) sodium cholate, 5 mM MgCl and 0.25 mM DTT. The gradient was centrifuged at 280 000 xg f o r 23 h and ten drop f r a c t i o n s c o l l e c t e d . Protein ( A - A ) , nd ATPase a c t i v i t y determined i n the absence (p-a) or presence (•-•) of 0.02% (w/v) L-a-lysophosphatidylcholine were measured as described i n MATERIALS AND METHODS. 2  2  a  122.  enzyme a c t i v i t y .  S i m i l a r l y , the i n c l u s i o n of 0.1%  (w/v)  soybean phospha-  t i d y l c h o l i n e i n the sucrose gradients did not prevent the loss of a c t i v i t y . When the concentrated f r a c t i o n , which was applied to the sucrose gradient, was  stored at 4°C f o r the same length of time spanned by the sucrose  gradient centrifugation step (24-28 h), about 70% of the ATPase a c t i v i t y was l o s t .  These results indicated that the enzyme was unstable at 4°C.  The highest s p e c i f i c a c t i v i t y i n the sucrose gradient fractions was  7.7  units per mg protein and t h i s represented a 22-fold p u r i f i c a t i o n over the intact membranes. Because of the d i f f e r e n t activating properties of the detergents as well as the unstable nature of the ATPase enzyme during p u r i f i c a t i o n , a comparison of the p u r i f i c a t i o n with respect to s p e c i f i c a c t i v i t i e s i s meaningless.  A better method f o r comparing the p u r i f i c a t i o n procedures i s  the analysis of the fractions on SDS-polyacrylamide  gels.  The active fractions from the Phenyl-Sepharose CL-4B column, when analyzed on SDS-polyacrylamide  gels, revealed that the fractions from the  leading edge (the g e l was again s i m i l a r to F i g . 11A, lanes b to d) of the a c t i v i t y peak appeared to contain many more minor bands than those from the t r a i l i n g edge.  Many of these contaminants were subsequently removed by  sucrose gradient centrifugation.  In addition to the subunits of ECF^  (ct-e), polypeptides with molecular weights of 48 000, 35 000, 24  000,  18 000, 14 000 and 9000 were also present i n invariant stoichiometry. Although the sucrose gradient removed many of the minor bands with molecular weight less than 30 000 as well as a major contaminant of 70  000  daltons, many minor bands of less than 50 000 daltons were s t i l l present. /Many more polypeptides than those shown i n F i g . 11, were present i n this preparation.  In order to remove some of these minor bands, an ammonium  123.  sulphate p r e c i p i t a t i o n step was  included p r i o r to chromatography on  Phenyl-Sepharose CL-4B. Membrane v e s i c l e s of _E. c o l i ML308-225 were s o l u b i l i z e d at a detergent to protein r a t i o of 0.25  rather than 0.5,  f r a c t i o n p r e c i p i t a t i n g between 0.35 (0.35-0.5 P f r a c t i o n ) was  as described e a r l i e r .  The  and 0.5 saturation of ammonium sulphate  suspended i n the buffer used f o r s o l u b i l i z a t i o n  at a protein concentration of 5-7 mg/ml.  N-lauroyl sarcosine was added to  give a detergent to protein r a t i o of 0.8 to 1.0.  This f r a c t i o n  was  chromatographed on a column of Phenyl-Sepharose CL-4B under conditions i d e n t i c a l to those described i n the legend to F i g . 9D except that the column was eluted with a l i n e a r decreasing gradient of 20% to  12.5%  ammonium sulphate. On occasion, the 0.35-0.5 P f r a c t i o n did not p e l l e t following centrifugation at 30 000 xg f o r 20 min, but floated to the surface.  This  problem was a l l e v i a t e d by using 10% (v/v) methanol rather than g l y c e r o l i n the s o l u b i l i z a t i o n buffer.  The presence of 10% (v/v) methanol did not  r  appear to a f f e c t the p u r i f i c a t i o n and was  subsequently  used i n the other  experiments. Applying a shallower gradient (20-12.5%) did not result i n a change i n the e l u t i o n p r o f i l e of the enzyme from that seen i n F i g . 9D.  The  activity  eluted as a single peak at an ammonium sulphate concentration of 15-16%. The active f r a c t i o n s were concentrated and applied to a l i n e a r 17.5 (w/v)  sucrose gradient as described i n MATERIALS AND METHODS.  approximately  15% of the a c t i v i t y was  centrifugation. to F i g . 10.  to 25%  As before,  recovered following sucrose gradient  The separation on the sucrose density gradient was  similar  The majority of the protein (contaminants) remained at the top  of the gradient, whereas the ATPase a c t i v i t y was associated with a smaller  124.  i protein peak close to the bottom of the tube. i  Inclusion of the ammonium sulphate f r a c t i o n a t i o n step reduced  the  number of minor bands of less than 50 000 daltons i n the fractions from the Phenyl-Sepharose CL-4B column and subsequently i n the fractions from the sucrose gradient ( F i g . 11).  In the fractions from the sucrose gradient,  the subunits of ECF^ (a - e) as well as major-protein staining bands with molecular weights of 48 000, 35 000, 24 000, 18 000, 14 000 and 9 000 were present i n invariant stoichiometry.  Relative to the other subunits of  ECF^, the <5 subunit (molecular weight, 21 000) was present i n f a i r l y  low  amounts i n the active fractions ( F i g . 11B) from the sucrose gradient.  It  has been reported that t h i s subunit i s very susceptible to protease digestion (6,24). Foster and Fillingame (94) have also reported the p u r i f i c a t i o n of the ECF^FQ  complex.  The  FQ  1  complex consisted of three subunits of molecular  weight 24 000, 18 000 and 8 500.  They reported that d i a l y s i s of the  detergent-solubilized f r a c t i o n followed by r e s o l u b i l i z a t i o n with detergent removed many of the minor protein-staining bands. Membrane v e s i c l e s of E. c o l i ML308-225 were s o l u b i l i z e d at a detergent to protein r a t i o of 0.25 a described e a r l i e r . dialyzed against buffer f o r 24-30 h.  The s o l u b i l i z e d f r a c t i o n  was  The reaggregated material i n the  dialysate was collected by centrifugation and r e s o l u b i l i z e d with N-lauroyl sarcosine at a detergent to protein r a t i o of 0.8 to 1.0. a c t i v i t y was recovered following d i a l y s i s .  Only 60% of the  The r e s o l u b i l i z e d material was  subjected to ammonium sulphate p r e c i p i t a t i o n , chromatography on PhenylSepharose CL-4B and sucrose gradient centrifugation as described i n the previous experiment.  Again, only 15% of the t o t a l a c t i v i t y applied to the  sucrose gradient was recovered.  However, the a c t i v i t y peak was well  r  125.  F i g . 11  SDS-polyacrylamide gel electrophoresis of the E C F ] F Q complex p u r i f i e d by chromatography on Phenyl-Sepharose CL-4B and sucrose gradient centrifugation.  Membrane v e s i c l e s of E_. c o l i ML308-225 were s o l u b i l i z e d with N-lauroyl sarcosine at a detergent to,protein r a t i o of 0.25. The s o l u b i l i z e d f r a c t i o n was subjected to ammonium sulphate p r e c i p i t a t i o n and the f r a c t i o n p r e c i p i tating between 35 and 50% of saturation was suspended i n the s o l u b i l i z i n g buffer at a protein concentration of 5-7 mg/ml. N-lauroyl sarcosine was added to give a detergent to protein r a t i o of 0.8-1.0, as described i n MATERIALS AND METHODS. The r e s o l u b i l i z e d material was applied to a column of Phenyl-Sepharose CL-4B (1.8 x 25 cm) under conditions i d e n t i c a l to those described i n the legend to F i g . 9D. The active fractions from the PhenylSepharose CL-4B column were concentrated and applied to a l i n e a r 17.5 to 25% (w/v) sucrose gradient and centrifuged at 280 000 xg f o r 23 h as described i n the MATERIALS AND METHODS section. The active f r a c t i o n s from the Phenyl-Sepharose CL-4B column and the sucrose gradient were analyzed by SDS-gel electrophoresis. The separating g e l (Tris-buffered system) consisted of a l i n e a r 7.5 - 16.5% (w/v) acrylamide gradient. The stacking gel consisted of 4% (w/v) acrylamide. Following electrophoresis, the gels were stained with 0.1% (w/v) Coomassie Blue. PANEL A: Fractions from the Phenyl-Sepharose CL-4B column. Lane a represents the material applied to , the column. Lanes b-f represent the f r a c t i o n s eluted from the column, with the f r a c t i o n highest i n ATPase a c t i v i t y i n lane c. Lane g i s p u r i f i e d ECF^. The p o s i t i o n of migration of the subunits of ECF^ (a-e) are i n d i cated. PANEL B: Fractions from the sucrose gradient. Lane a represents the material loaded on the sucrose gradient. Lanes b - i represent the fractions from the sucrose gradient. The f r a c t i o n containing the highest ATPase a c t i v i t y i s i n lane e. Lane j i s p u r i f i e d ECF^. The p o s i t i o n of migration of the subunits of ECF^ ( -e) are also indicated. a  126.  a b c d e  f g  127.  separated from the majority of the protein (as i n F i g . 10). Inclusion of the d i a l y s i s step appeared to decrease the number and i n t e n s i t y of the minor protein-staining bands with molecular weights greater than 20 000, i n the fractions from the Phenyl-Sepharose CL-4B J  column (Fig. 12A), when compared to those i n F i g . 11A.  However, those  minor bands with molecular weights less than 20 000 were increased. Nevertheless, the i n c l u s i o n of the d i a l y s i s step did improve the o v e r a l l p u r i f i c a t i o n as seen i n the active f r a c t i o n s from the sucrose gradient (Fig. 12B).  The subunits of ECF  1  (ct - e), as well as major p r o t e i n -  staining bands with molecular weights of 35 000,  24 000, 18 000, 14 000  and  9 000, appeared to be present i n invariant stoichiometry.  In contrast to  the results i n F i g . 11B, the 48 000 dalton polypeptide was  almost  completely absent i n this preparation.  In addition, the s t a i n i n g - i n t e n s i t y  of the 18 000 and 14 000 dalton polypeptides were greater i n t h i s preparation.  Furthermore, the 6 subunit was more distinguishable, suggesting  that t h i s preparation was perhaps less contaminated with proteases. i n c l u s i o n of the protease i n h i b i t o r s PMSF (0.1 mM) (6 mM)  The  and p-aminobenzamidine  during the concentration step, as well as i n the sucrose gradient,  did not result i n any change i n the pattern of bands seen i n F i g . 12B.  It  was not known i f these protease i n h i b i t o r s were capable of i n h i b i t i n g the a c t i v i t y of the protease(s) which may  have been present i n the preparation.  The f r a c t i o n from the sucrose gradient which was was  found to be sensitive to DCCD.  30% at a DCCD concentration of 200  richest i n ATPase a c t i v i t y  The ATPase a c t i v i t y was  inhibited by  uM.  More recently, Schneider and Altendorf (107) have also p u r i f i e d the ECF^FQ  complex by chromatography on DEAE-Sepharose CL-6B i n the presence  of Aminoxid WS-35, followed by centrifugation of the active f r a c t i o n s at  128. F i g . 12  SDS-polyacrylamide g e l electrophoresis of the ECF^FQ complex obtained after, chromatography on Phenyl-Sepharose CL-4B and sucrose gradient centrifugation.  Membrane v e s i c l e s from E_. c o l i ML308-225 (25-30 g wet weight) were s o l u b i l i z e d with N-lauroyl sarcosine at a detergent to protein r a t i o of 0.25. The s o l u b i l i z e d material was dialyzed (1:100) against 50 mM T r i s H 2 S O 4 buffer, pH 8.0 containing 1 mM DTT, 0.1 mM EGTA, 25 mM Na S0 and 10% (v/v) methanol f o r 24-30 h at 4°C, with changes of the external buffer at 6-8 h i n t e r v a l s . The reaggregated material was c o l l e c t e d by centrifugation at 250 000 xg f o r 3 h and r e s o l u b i l i z e d with N-lauroyl sarcosine at a detergent to protein r a t i o of 0.8-1.0. The r e s o l u b i l i z e d f r a c t i o n was then subjected to ammonium sulphate p r e c i p i t a t i o n , as described i n MATERIALS AND METHODS. The f r a c t i o n p r e c i p i t a t i n g between 35 and 50% of saturation was applied to a column of Phenyl-Sepharose CL-4B (1.8 x 25 cm) under conditions i d e n t i c a l to those described i n the legend to F i g . 9D, except that the enzyme was eluted with a l i n e a r decreasing gradient of 20% - 12.5% ammonium sulphate (by saturation). The active f r a c t i o n s from the column were concentrated and applied to a l i n e a r 17.5 to 25% (w/v) sucrose gradient and centrifuged at 280 000 xg f o r 23 h as described previously. The active fractions were analyzed by SDS-gel electrophoresis. The separating g e l (Tris-buffered system) consisted of a l i n e a r 7.5-16.5% (w/v) acrylamide while the stacking g e l was 4% (w/v) acrylamide. The gels were fixed i n a solution containing 5% (w/v) TCA, 5% (w/v) s u l f o s a l i c y l i c acid and 10% (v/v) methanol, and then stained with 0.1% (w/v) Coomassie Blue. PANEL A: Lane a represents the material applied to the column of Phenyl-Sepharose CL-4B. Lanes b - i represent the active f r a c t i o n s eluted from the column, with the material i n lane e containing the highest a c t i v i t y . Lane j i s p u r i f i e d ECF^. The migration positions of the subunits of ECF^ (a-e) are indicated. PANEL B: Lane a represents the material loaded onto the sucrose gradient. Lanes b-j represent the active fractions from the sucrose gradient, with the material i n lane d containing the highest a c t i v i t y . Lane k i s p u r i f i e d ECF^. The migration positions of the subunits of ECF^ (a-e) are also shown. 2  4  ® a b c d e f g h i j  Iffissts-  k  130.  220 000 xg f o r 15 h. Therefore, centrifugation of the active fractions from the Phenyl-Sepharose CL-4B column was used to overcome two of the problems associated with the p u r i f i c a t i o n on a sucrose gradient:  the poor  recovery of a c t i v i t y and the small quantity of the p u r i f i e d enzyme which could be obtained with a sucrose gradient. The experiment described previously (legend to F i g . 12) was repeated except that the sucrose gradient step was omitted.  Instead, the active  f r a c t i o n s from the Phenyl-Sepharose CL-4B column were made up to 0.1 mM PMSF and 6 mM p-aminobenzamidine p r i o r to centrifugation at 250 000 xg f o r 16-17  h. The p e l l e t was taken up i n 50 mM MOPS-KOH buffer, pH 7.5  containing 10 mM MgCl , 20% (v/v) g l y c e r o l and 0.2% (w/v) Triton X-100. 2  Approximately  40% and 10% of the t o t a l a c t i v i t y centrifuged was recovered  i n the p e l l e t and supernatant f r a c t i o n s , respectively.  A recovery of 40%  was a substantial improvement over.the 8-10% y i e l d usually obtained following sucrose gradient centrifugation. The preparation of the samples f o r SDS-polyacrylamide phoresis was modified at this stage.  gel e l e c t r o -  Detergent and s a l t s , which interfered  with the migration of the polypeptides i n the gels were removed by the column chromatography-method of Penefsky AND METHODS.  (170) as described i n MATERIALS  This i s i n contrast to the previous preparations, i n which  the samples were dialyzed against water f o r 1-2 days and then l y o p h i l i z e d . The f r a c t i o n from Phenyl-Sepharose CL-4B richest i n ATPase a c t i v i t y revealed the presence of at least 15 major protein-staining bands and several minor ones on SDS-polyacrylamide  gels.  The gel resembled that i n  F i g . 12A. Analysis of the sedimented ATPase enzyme on gels i s shown i n F i g . 13 (lane a).  SDS-polyacrylamide  Centrifugation of the active fractions  resulted i n the enrichment of 11 polypeptides.  Of these, f i v e polypeptides  131.  F i g . 13  SDS-polyacrylamide gel electrophoresis of the E C F ^ F Q complex obtained by chromatography on Phenyl Sepharose CL-4B and sedimentation at 250 000 xg for 16-17 h.  The experiment described i n the legend to F i g . 12 was repeated except that p u r i f i c a t i o n of the E C F ^ F Q complex by sucrose gradient centrifugation was omitted. Instead, the active f r a c t i o n s from the Phenyl-Sepharose CL-4B column were pooled and made up to 0.1 mM PMSF and 6 mM-pr-aminobenzamidine, and centrifuged at 250 000 xg f o r 16-17 h. The sedimented enzyme (lane a) was subjected to further p u r i f i c a t i o n on a column of DEAE-Sepharose CL-6B, as described i n MATERIALS AND METHODS. It was taken up i n 50 mM Tris-HCl buffer, pH 8.0, containing 1 mM MgCl , 0.2 mM DTT, 0.2 mM EGTA, 0.1 mM PMSF, 100 mM KC1, 20% (v/v) methanol, 50 mg/ml soybean phospholipid and 0.9% (w/v) Aminoxid WS-35 p r i o r to loading onto the column. The f r a c t i o n containing the highest a c t i v i t y was analyzed by SDS-gel electrophoresis (lane b). The composition of the SDS-gel was the same as that described i n the legend to F i g . 12 except that the concentration of SDS i n the gel was 0.5% (w/v). Lane c represents p u r i f i e d ECF^. The migration positions of the subunits of ECF^ (<*-e) as well as of the DCCD-binding protein ( i d e n t i f i e d using [ " c p C C D , see F i g . 15), the 28 000 and 18 000 dalton polypeptides of F Q , are also indicated. 2  132.  were the subunits of ECF^ and the remaining had molecular weights of 30 000, 28 000, 18 000, 14 000, 9 000 and 7 500. Minor bands of 85 000, 71 000 and 24 000 daltons were also present: thought to be contaminants.  Some of these polypeptides were  Attempts were made to remove these  contaminat  ing polypeptides by subjecting the sedimented ATPase enzyme to further fractionation. The f i r s t method involved the treatment of the enzyme with PEG 6 000 and 400, as described by F r i e d l et a l . (108).  This was not successful.  P r e c i p i t a t i o n of the ATPase complex with PEG 6000 and 400 resulted i n almost complete (98%) i n a c t i v a t i o n of the enzyme a c t i v i t y .  In addition to  being -y-deficient, the precipitated enzyme also contained very low amounts of the a and B subunits of E C F ^ The second method involved ion-exchange chromatography on DEAEs  Sepharose CL-6B i n the presence of Aminoxid WS-35, as described by F r i e d l and Schairer (106).  The sedimented ATPase complex dissociated during  ion-exchange chromatography.  The active fractions from the DEAE-Sepharose  CL-6B column were analyzed by SDS-polyacrylamide  gel electrophoresis and  found to contain only 6-def i c i e n t ECF^ ( F i g . 13", lane b). of intact  ECF^FQ  The absence  complex was confirmed, by determining the s e n s i t i v i t y  of the ATPase a c t i v i t y of t h i s preparation to DCCD ( F i g . 14).  INTACTNESS OF THE  ECF^^FQ C O M P L E X  In t h i s study, centrifugation of the active fractions from the Phenyl-Sepharose CL-4B column, at 250 000 xg f o r 16-17 h resulted i n optimal p u r i f i c a t i o n of the ATPase enzyme. ECF^FQ  complex was determined  F i g . 14.  The intactness of the p u r i f i e d  by using DCCD.  The results are shown i n  133.  DCCD — LI M  Fig.  14  E f f e c t of DCCD on the ATPase a c t i v i t y of the E C F J F Q complex.  The ATPase enzymes obtained a f t e r chromatography on Phenyl-Sepharose CL-4B and sedimentation at 250 000 xg f o r 16-17 h ( F i g . 13, lane a ) , or a f t e r further p u r i f i c a t i o n of the sedimented enzyme on DEAE-Sepharose CL-6B ( F i g . 13, lane b), were incubated with various l e v e l s of DCCD i n 0.1 M Tris-HCl buffer, pH 8.0, containing 5 mM MgCl ( f i n a l volume, 0.1 ml) at 37°C f o r 45 min. ECF^ obtained a f t e r AH-Sepharose 4B chromatography was also treated with DCCD. Following incubation with DCCD, a sample of the mixture was removed and the ATPase a c t i v i t y measured as described i n MATERIALS AND METHODS. The s p e c i f i c a c t i v i t i e s of the sedimented enzyme (O-O), DEAE-Sepharose CL-6B-purified enzyme (•-•) and ECF]_ (*-*) were 18, 31.4 and 26.3 units per mg protein and the amount of protein per assay was 1.75, 0.85 and 0.8 ng, respectively. 2  134.  Over a DCCD concentration range of 500 uM, the a c t i v i t y of the p u r i f i e d ECF^ was i n h i b i t e d by only 5-10%.  Similar results were obtained  with the DEAE-Sepharose CL-6B-purified enzyme.  This was expected  since  t h i s preparation appeared to be 6-defIcient ECF^ and not the ECF^F^ complex. By contrast, the enzyme sedimented by centrifugation at 250 000 xg f o r 16-17  h, was sensitive to DCCD. The ATPase a c t i v i t y was inhibited by 30%  and 45% at DCCD concentrations of 100 uM and 500 uM, respectively. Treatment of a l l three enzyme preparations with [ C]DCCD (136 UM) llf  at pH 8.0 i n the presence of 5 mM MgC^, under conditions i d e n t i c a l to those described i n the legend to F i g . 14, resulted i n the l a b e l l i n g of the B subunit (52 000 daltons).  In addition, a polypeptide of about 9 000  daltons was labelled i n the enzyme sedimented by centrifugation at 250 000 xg f o r 16-17 h ( F i g . 15). Other evidence that the sedimented ATPase enzyme was present i n intact form was otained by using [l *CJiodoacetamide -1  5-iodoacetamidofluorescein.  and the fluorescent l a b e l ,  Recently, Paradies et a l . (35) have shown that  the compounds TAMM andjNEM-Hg labelled only the fl subunit of p u r i f i e d ECF^. However, the labels were associated only with the a subunit when the F^FQ complex was used. [ *C]- iodoacetamide was found to l a b e l both the a and B subunits ll  of the sedimented ATPase complex but only the a of the p u r i f i e d ECF^. With the fluorescent probe 5-iodoacetamidofluorescein, both the a and B subunits of the i n t a c t ATPase complex were l a b e l l e d , with a higher amount of the label on the a subunit.  But with p u r i f i e d E C F ^ only the B  subunit was labelled (these experiments were done i n collaboration with Drs. Helga Stan-Lotter and P.D. Bragg). These results suggested  that the ECF remained associated with F 1  n  135.  F i g . 15  SDS-polyacrylamide g e l electrophoresis of ECF^ and ECFJFQ complex l a b e l l e d with ['"CJDCCD.  ECF^ (0.34 mg), obtained a f t e r chromatography on AH-Sepharose 4B, and the p u r i f i e d ECF-^FQ complex (0.35 mg, F i g . 13, lane a) were suspended i n 0.25 ml of 50 mM Tris-HCl buffer, pH 8.0 containing 5 mM MgCl . To each was added 7 H i of 5 mM ['"CJDCCD (Specific a c t i v i t y , 50 mCi/nmol) and the mixtures incubated at 37°C f o r 45 min. The unreacted l a b e l was removed by the centrifugation column-chromatography method described by Penefsky (170), The eluents were depolymerized and 82.5 ug of the ECF^ (PANEL A) and 70 yg of the E C F ^ Q complex (PANEL B) were applied to the SDS-gel. The slab g e l (Tris-buffered system) consisted of a separating gel of 13% (w/v) acrylamide and a stacking gel of 4% (w/v) acrylamide with the SDS concentration i n the g e l being 0.5% (w/v). After electrophoresis, the g e l was stained with 0.1% (w/v) Coomassie Blue, the separating g e l s l i c e d into 1 mm segments, and the r a d i o a c t i v i t y of the s l i c e s determined as described i n MATERIALS AND METHODS, a - e r e f e r to the migration positions of the subunits of ECF^ i n the g e l . The position of the tracking dye, bromophenol blue (BP) i s also indicated. 2  136.  0  40  80 Slice  137.  following centrifugation at 250 000 xg f o r 16-17 h, but dissociated during subsequent ion-exchange  chromatography. >  REPRODUCIBILITY OF PURIFICATION ON PHENYL SEPHAROSE CL-4B Problems were encountered when attempts were made to use PhenylSepharose more than once.  Although the position of e l u t i o n of the a c t i v i t y  (15-16% ammonium sulphate concentration) was almost i d e n t i c a l when the r e s i n was re-used, analysis of the active fractions from the column on SDS-polyacrylamide gels revealed that a d i f f e r e n t pattern of polypeptide bands was obtained each time. The experiment was repeated as described f o r F i g . 12 except that the f r a c t i o n was applied to a column of Phenyl-Sepharose CL-4B which had been used and regenerated under d i f f e r e n t conditions.  The fractions richest i n  ATPase a c t i v i t y were analyzed on SDS-polyacrylamide gels. About 14-16 protein straining bands were found when the ATPase enzyme was chromatographed a).  on a column of freshly-prepared resin ( F i g . 16, lane  When the enzyme was chromatographed  on a column of resin which had  been used several times and regenerated as recommended by the manufacturer (Pharmacia), more than 22 d i f f e r e n t polypeptides were found to be present. These extra protein bands ranged i n molecular weight from 10 000 to 100 000 ( F i g . 16, lane b ) . A more complete regeneration of the r e s i n was obtained when the previously used r e s i n was washed with 2% (w/v) SDS and then regenerated as recommended.  The most active f r a c t i o n contained  (Fig. 16, lane c ) .  13-15 major polypeptides  However, the resin continued to deteriorate with  repeated use, even with i n c l u s i o n of a 2% (w/v) SDS washing step ( F i g . 16, lane d). \  138.  Fig.  16  SDS-polyacrylamide g e l electrophoresis of the E C F ^ F Q complex obtained a f t e r chromatography on Phenyl-Sepharose CL-4B: Reproducibility of the p u r i f i c a t i o n .  The experiment described i n the legend to F i g . 12 (PANEL A) was repeated except that the f r a c t i o n was applied to a column of PhenylSepharose CL-4B (1.8 x 25 cm) which had been regenerated under several d i f f e r e n t conditions. The most active f r a c t i o n from the column was analyzed on SDS-gels. The gel consisted of 13% (w/v) acrylamide with a 4% (w/v) acrylamide stacking g e l , as described by Laemmli (171). Protein bands were visualized with 0.1% (w/v) Coomassie Blue as described i n MATERIALS AND METHODS. Lane a, freshly-prepared Phenyl-Sepharose CL-4B; lane b, resin was used and regenerated according to the manufacturer's (Pharmacia) recommendations; lane c, r e s i n used f o r experiment i n lane b was regenerated as before but an extra wash with 2% (w/v) SDS was included; lane d, the r e s i n used f o r the experiment i n lane c was regenerated as f o r the experiment i n lane c; lane e consists of molecular weight marker proteins (M , 94 000 - 14 400). r  139.  F a i r l y reproducible p u r i f i c a t i o n s could be obtained column of freshly-prepared  each time with a  resiri.  COMPARISON OF THE' GEL ELECTROPHORESIS AND  PROTEIN-DETECTION SYSTEMS  The main determinant i n assessing the purity of the ATPase complex was by SDS-polyacrylamide g e l electrophoresis.  The number of polypeptide bands  observed has been reported to depend on the resolving power of the gel electrophoresis system (5,95).  In t h i s study, a comparison of the resolving  power of the phosphate-buffered (174) and the Tris-buffered (171,173) gel electrophoresis systems as well as the protein-detection systems was made. A preparation of the ATPase complex p u r i f i e d on a sucrose gradient subjected to the d i f f e r e n t electrophoresis systems. i n F i g . 17.  was  The results are shown  In each case, the gels were stained with one of the staining  systems described i n MATERIALS AND METHODS. The Tris-buffered system ( F i g . 17A) was more resolving than the phosphate-buffered system (Fig; 17B). ECF^  In the former, the subunits of  (q - e) were highly resolved, whereas i n the l a t t e r , the a and fl  subunits overlapped. phosphate system.  The e subunit could barely be i d e n t i f i e d with the  In addition, many of the minor protein bands seen i n the  T r i s system were not resolved i n the phosphate system. polypeptide  In general,  the  bands were more d i s t i n c t i n the Tris-buffered system.  The number of protein-staining bands seen on the gel also depended on the staining properties of the dye used. staining systems i s shown i n F i g . 17.  A comparison of four d i f f e r e n t  The most intensely-stained prep-  a r a t i o n and the greatest number of bands was  obtained with 0.05%  (w/v)  Coomassie Blue i n 25% (v/v) isopropanol and 10% (v/v) acetic acid ( F i g . 17A,  I).  A s l i g h t l y weaker s t a i n i n g - i n t e n s i t y , but equally as resolving,  140. i  F i g . 17  Comparison of the SDS-gel electrophoresis and protein-detection systems. *  P a r t i a l l y p u r i f i e d ECF-^FQ complex obtained a f t e r chromatography on Phenyl-Sepharose CL-4B and sucrose gradient centrifugation, and sucrose gradient-purified ECF^, were subjected to SDS-gel electrophoresis on a l i n e a r 7.5% - 16.5% (w/v) acrylamide gradient g e l i n the Trisrbuffered system (PANEL A), or i n the phosphate-buffered system (PANEL B), as described i n MATERIALS AND METHODS. Following electrophoresis, the gels were fixed with a solution containing 5% (w/v) TCA, 5% (w/v) s u l f o s a l i c y l i c acid and 10% (v/v) methanol f o r 30 min at 60°C. The gels were then stained for 9 h at 20°C with ( I ) , 0.05% (w/v) Coomassie Blue i n isopropanol:acetic acid:water (25:10:65); ( I I ) , 0.25% (w/v) Coomassie Blue i n methanol:acetic acid:water (45:10:45); ( I I I ) , 0.12% (w/v) Coomassie Blue i n ethanol:acetic acid:water (25:8:67); (IV), 1% (w/v) Amido Black i n 7% (v/v) acetic acid. Lanes a and b are p a r t i a l l y p u r i f i e d E C F ^ F Q complex, with lane b containing twice the amount of E C F ^ F Q complex of that i n lane a. Lane c i s p u r i f i e d ECF-^. The migration p o s i t i o n of the subunits of ECF^ (a - e) are indicated.  i  141,  ® 1 "II a b c a  WW  '. w  III  II  1  1 b c  i  IV  ; i * —i a b c a b c  ST'*"} IHj .  - •m  St 11  n: •  ii  «- £  ® _M_  1  b  MM  •  IN  IV  a b a b a b  M  ffi  r  i  142.  was obtained with 0.25% (w/v) Coomassie Blue i n 45% (v/v) methanol and 10% (v/v) Acetic acid ( F i g . 17A, I I ) . The number of minor bands observed decreased considerably when the g e l was stained with either 0.12% (w/v) Coomassie Blue i n 25% (v/v) ethanol and 8% (v/v) acetic acid or 1% (w/v) Amido Black i n 7% (v/v) acetic acid. least number of polypeptide bands as well as the weakest staining was observed with Amido Black.  The  143.  PART II  STUDIES ON MUTANTS OF E. c o l l DEFECTIVE IN PROTON-TRANSLOCATING ACTIVITY  The  ECF^FQ  complex p u r i f i e d i n the preceding section consisted of  eleven major polypeptides.  Other workers have shown that the complex  consists of eight subunits (Tables 2 and 3).  Five of these polypeptides  were subunits of ECF^ (a - e ) . The remaining polypeptides of 24 000, 18 000 and 8 400 daltons were considered to be subunits of F Q .  However,  the mechanism of proton conduction through F Q and the relationship of these polypeptides to each other are not known. One approach to t h i s problem has been through the i s o l a t i o n of mutants of E_. c o l i which are defective i n oxidative phosphorylation (unc mutants). It i s conceivable that a defect i n any of the three subunits of F Q would a l t e r i t s proton-translocating properties. Several of these mutants have been isolated and the properties of those studied i n t h i s thesis are l i s t e d i n Table 12. wild-type s t r a i n .  Both of the unc mutants, E_. c o l i N  not exhibit ATPase a c t i v i t y .  E_. c o l i WS1 i s a I 4 4  and CBT-302 did  Analysis of membrane preparation from these  two mutants on O'Farrell gels (176) revealed the absence of any subunits of ECF  1#  ATPase a c t i v i t y was detected i n the mutant, E_. c o l i AN382.  In  contrast to the wild-type s t r a i n , the a c t i v i t y of E_. c o l i AN382 was r e l a t i v e l y Insensitive to DCCD.  At a DCCD concentration of 63 uM, the  ATPase a c t i v i t i e s of E. c o l i WS1 and AN382 were inhibited by 60% and 8%, respectively.  The ECF^ from both strains were analyzed on O'Farrell gels  and found to be i d e n t i c a l .  Thus, i t i s u n l i k e l y that the difference i n the  s e n s i t i v i t y of the ATPase a c t i v i t y to DCCD was due to differences i n the ECF  portion of the E C F F n  n  complex.  144.  Table  12  Some Properties of the unc Mutants of E. c o l i used i n t h i s Thesis  3  Strain  ATPase a c t i v i t y -DCCD +DCCD  WSI  0.294  0.116  60  +  0  0  -  0  CBT-302  0  0  -  0  AN382  0.199  0.183  8  +  N  b  I44  % Inhibition  Presence of ECF^  a.  umol/min/mg protein.  b.  63 yM DCCD  c.  Determined by two-dimensional g e l electrophoresis of the membranes +, present; -, absent.  Membrane v e s i c l e s of _E. c o l i WSI, N ,,, CBT-302 and AN382 were suspended i n 50 mM Tris-HCl buffer, pH 8.0, containing 10 mM MgCI and 10% (v/v) g l y c e r o l at a p r o t e i n concentration of 4 mg/ml. The membrane suspensions were incubated with DCCD f o r 30 min. at 37 C a f t e r which the ATPase a c t i v i t y was determined as described i n MATERIALS AND METHODS. 2  145.  SENSITIVITY OF THE MEMBRANE-BOUND ATPase ACTIVITY TO INHIBITION BY DCCD IN THE PRESENCE OF CATIONS 2+ 2+ The e f f e c t of Ca and Mg on the s e n s i t i v i t y of ATPase a c t i v i t y to DCCD i n membrane v e s i c l e s of E. c o l i ML308-225, AN180, AN382 and WS1 was also examined.  In a l l four strains, maximum i n h i b i t i o n of a c t i v i t y by DCCD 2+  was observed i n the presence of Mg  ( F i g . 18). In the wild-type strains,  E. c o l i WS1, AN180 and ML308-225, the enzyme a c t i v i t y was i n h i b i t e d h a l f maximally at 3-6 nmol DCCD per mg protein.  By contrast, i n E. c o l i AN382,  the enzyme was i n h i b i t e d by only 35% at 100 nmol DCCD per mg protein. Therefore, the resistance of the a c t i v i t y to DCCD appeared to be due to defect(s) i n the  FQ  portion of the E C F ^ F Q complex.  Other methods  were then used to characterize t h i s mutant as well as the unc mutants E_. coli N  I 4 4  and CBT-302.  MEASUREMENT OF THE PROTON GRADIENT USING 9-AMINOACRIDINE Everted membrane v e s i c l e s of E. c o l i have the ECF^ portion of the ATPase complex exposed to the external medium such that during ATP hydrolysis or substrate oxidation a proton gradient i s generated i n these vesicles. The proton gradient within these everted v e s i c l e s can be detected by using the fluorescent, l i p o p h i l i c weak base, 9-aminoacridine. Fig.  As shown i n  19 (trace 1), the fluorescent dye equilibrated across the membrane of  these everted v e s i c l e s i n response to a proton gradient generated by o x i dation of substrate (NADH). This resulted i n a decrease i n fluorescence. When the system went anaerobic, the respiratory chain became non-functional and the fluorescence returned to i t s o r i g i n a l l e v e l as a result of r e - e q u i l i b r a t i o n of the proton gradient across the membrane.  The addition  146.  Fig.  18  S e n s i t i v i t y of the membrane-bound ATPase a c t i v i t y to I n h i b i t i o n by DCCD i n the presence of cations.  Membrane v e s i c l e s from E_. c o l i AN382, AN180, ML308-225, and WS1 were prepared i n 50 mM Tris-H2S04 buffer, pH 8.0, containing 1 mM MgCl as described i n MATERIALS AND METHODS. The membrane v e s i c l e s were suspended at a protein concentration of 4 mg/ml In 50 mM Tris-H S04 buffer, pH 8.0, containing 10% (v/v) g l y c e r o l (*-*) and either 10 mM C a C l ( O - O ) or 10 mM MgCl ( • - • ) . Various concentrations of DCCD i n ethanol (10 u l ) were added to 1 ml of membrane v e s i c l e s . After 45 min at 3 7 ° C , the ATPase a c t i v i t y of the membrane v e s i c l e s was determined as previously described. The s p e c i f i c a c t i v i t i e s of the membrane-bound ATPase of IS. c o l i AN382, AN180, ML308-225 and WS1 i n the absence of cations were 0.09, 0.30, 0.67 2  2  2  2  2+  and 0.30 units per mg p r o t e i n ; i n the presence of Ca were 0.11, 0.28, 0.67 and 0.26 u n i t s per mg protein and i n the presence of Mg^ were 0.16, 0.36, 0.66 and 0.38 units per mg p r o t e i n , respectively. +  D C C D -- n m o l / m g p r o t e i n  148.  NADH 1 1 1 | _ CONTROL  STRIPPED  USTRIPPED DCCD  s _STRIPPED CONTROL ATPase  UJ  o ? o <n t o <  tu  LU CC OC O  O q  2 MINUTES  F i g . 19  Measurement of the proton gradient i n everted membrane v e s i c l e s using the fluorescent dye, 9-aminoacridine.  Everted membrane v e s i c l e s of E_. c o l i WSI were dialyzed against a low i o n i c strength buffer to remove ECF^ from the membranes, as described i n MATERIALS AND METHODS. The ECF^-stripped membrane v e s i c l e s were suspended i n 50 mM HEPES-KOH buffer, pH 7.5, containing 5 mM MgCl at a protein concentration of 10 mg/ml. Samples (0.1 ml) were removed and assayed f o r the a b i l i t y to quench the fluorescence of 9-aminoacridine during substrate (NADH) oxidation, or during hydrolysis of ATP, as described i n the MATERIALS AND METHODS section. Trace 1, untreated everted membrane v e s i c l e s ; Trace 2, ECF^-stripped membrane v e s i c l e s ; Trace 3, ECF^-stripped everted membrane v e s i c l e s incubated with (375 uM)DCCD f o r 5 min at 20°C p r i o r to assay; Trace 4, ECF^-stripped everted membrane v e s i c l e s incubated with ECF^ (90 ug protein) f o r 5 min at 20°C p r i o r to assay. 2  149.  of ATP at t h i s point again energized fluorescence was observed.  the membrane and a decrease i n  Quenching of fluorescence was not observed i n  everted v e s i c l e s which had been stripped of ECF^ ( F i g . 19, trace 2). However, addition of DCCD or p u r i f i e d ECF^ to these stripped membranes restored i t s a b i l i t y to be energized  during substrate oxidation.  membranes which had been reconstituted with ECF^  Stripped  could also be energized  by ATP hydrolysis ( F i g . 19, trace 4). These r e s u l t s suggested that the FQ portion of the ATPase complex i s a proton pore and that ECF^ and DCCD could block the passive proton leakage through FQ. The mutants, E. c o l i wild-type  CBT-302 and AN382, as well as the  s t r a i n , E^. c o l i WS1, were investigated i n a s i m i l a r manner.  Normal and mutant membrane v e s i c l e s were treated with 2M urea, 2M guanidine hydrochloride or 2% (w/v) s i l i c o t u n g s t i c acid to remove any subunits of ECF^ which might be retained by the membrane.  In this respect,  the mutant membrane v e s i c l e s were also treated with trypsin. shown i n F i g . 20 are t y p i c a l of a l l these treatments.  The results  Oxidation of  ascorbate ( i n the presence of PMS to introduce electrons into the cytochrome region of the respiratory chain) by untreated  ("washed")  membranes resulted i n the quenching of fluorescence of 9-aminoacridine indicating that a proton gradient had been established across the membrane v e s i c l e ( F i g . 20, traces 1 and 3). addition of ATP.resulted  When the system became anaerobic,  i n the restoration of the proton gradient only i n  the wild-type membranes ( F i g . 20, trace 1). Treatment of the membranes of the wild-type  s t r a i n with urea  ("stripping") resulted i n the removal of ECF^ with the loss of the a b i l i t y to set-up and maintain a proton gradient ( F i g . 20, trace 2).  By  150.  1 MIN  Fig.  20  E f f e c t of s t r i p p i n g everted membrane vesicles from the parent QL* i WS1) and mutant (IS. c o l i ^ 4 4 ) on the energization of the membrane. c  o  l  Membrane v e s i c l e s of E. c o l i WS1 and N 1 4 4 were treated with 2 M urea to remove ECF^ from the membrane. The membrane v e s i c l e s were suspended at a p r o t e i n concentration of 10 mg/ml i n 50 mM HEPES-KOH buffer, pH 7.5, containing 5 mM MgCl2« Samples (0.1 ml) were removed and assayed f o r the a b i l i t y to quench the fluorescence of 9-aminoacridine during oxidation of ascorbate (Asc) i n the presence of phenazine methosulphate (PMS) or during ATP h y d r o l y s i s , as described i n MATERIALS AND METHODS. 1 and 3 are traces of the quenching observed with the untreated everted v e s i c l e s of the parent (WS1) and the mutant ( N J 4 4 ) , r e s p e c t i v e l y . 2 and 4 are traces of the quenching observed with urea-treated ("ECF^-stripped") everted v e s i c l e s of the parent (WS1) and mutant ( N J 4 4 ) , respectively. S i m i l a r r e s u l t s (trace 4 ) were obtained i f the membrane v e s i c l e s from the mutant ( N J 4 4 ) were treated with 2 M guanidine hydrochloride, 2% (w/v) s i l i c o t u n g s t i c acid or TPCK-trypsin.  151 )  contrast, none of the stripping procedures described e a r l i e r destroyed the capacity of the mutant membranes to generate a proton gradient during substrate oxidation (Fig-. 20, trace 4).  Thus, i t i s unlikely that reten-  t i o n of individual subunits on the membrane was responsible f o r the r e l a t i v e impermeability of these membranes to protons. Although ATPase a c t i v i t y was detected i n the membranes of the mutant, E_. c o l i AN382 (Table 12), and the ECT^ was i d e n t i c a l to that of the w i l d type s t r a i n , ATP-dependent fluorescence quenching was not observed i n t h i s mutant. These results indicated that the l e s i o n ( s ) responsible f o r the r e l a t i v e proton-impermeability N  I 4 4  of the membranes of the unc mutants, E_. c o l  , CBT-302 and AN382 resided i n the F  Q  portion of the E C F ^ complex.  This was then investigated.  LABELLING OF MEMBRANES OF E. c o l i WITH [ C]DCCD ll>  It has been shown that the i n h i b i t i o n of ATPase a c t i v i t y i n E. c o l i vesicles by DCCD was associated with the l a b e l l i n g of a 8-9 000 dalton polypeptide (141,144). S i m i l a r l y , the addition of DCCD to "stripped" everted membranes of E_. c o l i blocked proton-translocation, as shown i n F i g 19 (trace 3). Therefore, i t was postulated that the 9 000 dalton p o l y peptide (DCCD-binding protein) must be involved i n proton (144,145).  conduction  The r e l a t i v e impermeability of the stripped membranes of the  mutant c e l l s suggested that the l e s i o n responsible f o r t h i s might reside i t h i s polypeptide.  The presence of t h i s polypeptide i n the mutant membrane  was investigated by l a b e l l i n g the membranes of the normal and mutant E_. c o l i with [ c p C C D followed by separation of the polypeptides by SDS1%  polyacrylamide g e l electrophoresis.  A t y p i c a l result of the l a b e l l i n g  152.  experiment (E.  of the membranes of the wild-type (E_. c o l i WS1)  and the mutant  c o l i Nj^^) strains i s shown i n F i g . 21 (traces 1 and 2).  Besides  material at the top of the g e l , two major peaks of r a d i o a c t i v i t y were observed both with the normal and mutant membranes.  The larger of these  peaks coincided with a Coomassie Blue staining polypeptide of 9 000 daltons (data not shown).  The smaller peak was not associated with a Coomassie  Blue staining polypeptide. A c h a r a c t e r i s t i c property of the DCCD-binding protein of F a b i l i t y to be extracted into chloroform-methanol  Q  is its  (2:1) (144,145,150).  [' "* C] -labelled membrane v e s i c l e s were extracted with chlorof orm-methanol and the proteins precipitated with ether.  Gel electrophoresis of this  material from both normal and mutant membranes gave mainly a single polypeptide band of molecular weight 9 000 (data not shown) coincident with the main peak of r a d i o a c t i v i t y ( F i g . 21, traces 3 and 4).  Similar r e s u l t s were  obtained with the unc mutants, E. c o l i CBT-302 and AN382 as well as with the wild-type s t r a i n E. c o l i ML308-225.  These results indicated that the  DCCD-binding protein occurs i n the mutants and that the aspartyl residue, which reacts with DCCD and i s involved i n proton translocation, i s present (159,160).  PURIFICATION OF THE DCCD-BINDING PROTEIN Although the DCCD-binding protein was found to be present i n the mutants, E_. c o l i AN382 and CBT-302 and N-j-^^, there remained the p o s s i b i l i t y that the amino acid composition of the protein was altered i n these mutants.  Therefore, attempts were made to p u r i f y this polypeptide.  ['"CJDCCD labelled membrane vesicles of E. c o l i WS1 were extracted with chloroform-methanol  or ML308-225  (2:1) and the proteins precipitated  153.  Slice  F i g . 21  number  SDS-polyacrylamide g e l electrophoresis of [ "C]DCCD-labelled membranes and of ether-precipitated proteins of chlorof orm-methanol extracts of the l a b e l l e d membranes of E_. c o l i . 1  Membrane v e s i c l e s of the parent (WSI) and mutant (N-j-^) strains of E. c o l i were l a b e l l e d with [^CJDCCD. The DCCD-binding proteins were i s o l a t e d from the l a b e l l e d membrane v e s i c l e s by extraction with chloroform: methanol (2:1) followed by p r e c i p i t a t i o n with ether, as described i n MATERIALS AND METHODS. The l a b e l l e d membrane v e s i c l e s (150-200 Mg protein) and the i s o l a t e d DCCD-binding proteins (12-15 yg protein) were subjected to electrophoresis on SDS-urea (8 M) gels. Following e l e c t r o phoresis, the gels were fixed with 50% (w/v) TCA, stained with 0.25% (w/v) Coomassie Blue, cut into 2 mm s l i c e s and the r a d i o a c t i v i t y of each s l i c e determined as described previously. Traces 1 and 2 show the d i s t r i b u t i o n of the r a d i o a c t i v i t y i n the [ " CJDCCD-labelled membranes of the parent (WSI) and the mutant ( N J 4 4 ) s t r a i n s , respectively. Traces 3 and 4 show the d i s t r i b u t i o n of the r a d i o a c t i v i t y of the i s o l a t e d DCCD-binding protein from the parent (WSI) and mutant ( N J 4 4 ) s t r a i n s , respectively. The migration positions of the molecular weight marker proteins (M , 94 000 14 400) and the bromophenol blue (BP) tracking dye are shown. 1  r  154. \  *  \  with ether.  •  The DCCD-binding protein was  chromatography. 22A).  further p u r i f i e d by thin-layer  Several bands were v i s i b l e on the chromatogram ( F i g .  About 80% of the t o t a l r a d i o a c t i v i t y applied was  band 4 (R  = 0.9) and 10% with band 1 (R  f  f  = 0.05).  r a d i o a c t i v i t y was d i s t r i b u t e d between bands 2 (R 0.68).  f  The remaining  = 0.45)  The material eluted from the chromatogram was  SDS-polyacrylamide g e l electrophoresis.  associated with  and 3 (R  analyzed  by  A peak of r a d i o a c t i v i t y was  observed with both bands 1 and 4 ( F i g . 22B),  but only the r a d i o a c t i v i t y  peak of band 1 coincided with a Coomassie Blue staining polypeptide 9 000 daltons (not shown).  =  f  The peak of band 4 was  of  associated with an  opaque region on the g e l , which migrated with a molecular weight of less than 9 000  daltons.  The amino acid composition of the material eluted from band 1 revealed the presence of the amino acids, serine, h i s t i d i n e and cysteine.  This  was  diagnostic of a contaminated preparation of DCCD-binding protein since these amino acids are not present i n t h i s polypeptide  (146).  another procedure f o r p u r i f y i n g the DCCD-binding protein was The  [ "C]DCCD-binding protein i s o l a t e d from E. c o l i WS1 l  of the membranes with chloroform-methanol (2:1) was column of Whatman CM-32 or BioRad Cellex CM, solvents, as described i n MATERIALS AND quite d i f f e r e n t ( F i g . 23).  Therefore, needed. by extraction  chromatographed on a  i n the presence of organic  METHODS.  The e l u t i o n p r o f i l e s were  Most of the radioactive material applied to the  column of BioRad Cellex CM did not bind to the r e s i n ( F i g . 23A). unbound f r a c t i o n e s s e n t i a l l y consisted of phospholipids. detected i n t h i s f r a c t i o n .  The  Protein was  not  By contrast, most of the r a d i o a c t i v i t y applied  to the column of Whatman CM-32 was  bound to the r e s i n and eluted i n the  subsequent washing steps ( F i g . 23B).  Fractions A and B ( F i g . 23B)  155. Fig.  22  Thin layer chromatography of the DCCD-binding protein.  The DCCD-binding protein was i s o l a t e d from [**C]DCCD-labelled membranes of IS. c o l i WSI by extraction with chlorof orm methanol (2:1) and p r e c i p i t a t i o n with ether. The ether-precipitated protein(s) i n chloroform: methanol (2:1) was applied to a s i l i c a gel G plate. The chromatogram was developed by ascending chromatography i n a mixture of chlorof ormmethanol: water (65:25:4) containing 20 mM HC1, as described i n MATERIALS AND METHODS. The positions of the bands were v i s u a l i z e d with iodine (PANEL A). The stained spots were scraped off the s i l i c a plate and extracted with chloroformmethanol (2:1) containing 20 mM HC1. The extracts were dried, p r e c i p i tated with ether and the p r e c i p i t a t e subjected to electrophoresis on SDSurea (8 M) gels. The gels were fixed with 50% (w/v) TCA, stained with ' 0.25% (w/v) Coomassie Blue, cut Into 2 mm s l i c e s and the r a d i o a c t i v i t y of each s l i c e determined (PANEL B). The migration positions of the bromophenol blue (BP) tracking dye and of the molecular weight marker proteins (M , 94 000 - 14 400) are also shown. r  \  ®  Solvent  Front  •  Band 4  R = 0.9  •  \\\\\\\  Band 3  R, =0.68  •  WWW  Band 2  R, =0.45——*•  Band 1  R =0.05 Origin  f  f  • *"  I  WWW  I  157.  F i g . 23  Chromatography of the DCCD-binding protein on CM-cellulose.  DCCD-binding protein was extracted from [ C]DCCD-labelled membranes of IS. c o l i WSI with chlorof ormmethanol (2:1) and p r e c i p i t a t e d with ether. The ether-precipitated proteins were taken up i n chloroformmethanol (2:1) and applied to a column of CM-cellulose, as described i n MATERIALS AND METHODS. PANEL A: The i s o l a t e d [ "C]DCCD-binding protein (4-5 mg protein) was applied to a column of BioRad Cellex CM ( 0 . 9 x 14 cm) e q u i l i brated with chloroform:ethanol (2:1). The column was washed sequentially with 10 column volumes of chlorof ormmethanol (2:1) and 5 column volumes each of chlorof ormmethanol (1:1), chloroform methanol :water (10:10:1) and chlorof ormmethanol :water (5:5:1). 10 ml fractions were c o l l e c t e d and the absorbance at 280 nm (A280) O" ) d the r a d i o a c t i v i t y (O-O) determined. 1-4 r e f e r to the f r a c t i o n s which were pooled and subjected to SDS-gel electrophoresis (see t e x t ) . PANEL B: The experiment described f o r Panel A was repeated, except that the r e s i n was Whatman CM-32 c e l l u l o s e . &-280* (•-•); r a d i o a c t i v i t y , (O-O). 1-4 and A, B and C r e f e r to the f r a c t i o n s which were pooled and subjected to SDS-gel electrophoresis (see F i g . 24). C, chloroform; M, methanol; H, water. l11  1  --  a n  158.  C/M I  0  (2:1)  C/M '  (1:1) 1  100  C/M/H '  (10:10:1 ) 1  200 Volume - -  C/M/H '  (5:5:1) 1  300 ml  1  400  159.  contained high amounts of phospholipids. Analysis of the pooled fractions (A, B and C) obtained from the column of Whatman CM-32 on SDS-polyacrylamide gels revealed that the r a d i o a c t i v i t y was distributed throughout the length of the gel i n fractions A and B ( F i g . 24, panels A and B).  However, only the r a d i o a c t i v i t y at 9 000 daltons  coincident with a Coomassie Blue staining polypeptide (not shown).  was  The  other broad r a d i o a c t i v i t y peaks were associated with an opaque region extending throughout most of the g e l .  By contrast, f r a c t i o n C contained  e s s e n t i a l l y one peak of r a d i o a c t i v i t y ( F i g . 24, panel C), which coincided with a single protein-staining band of 9 000 daltons. The fractions eluted from the column of BioRad Cellex CM were also analyzed i n the same manner.  Fractions 2 and 3 ( F i g . 23A) contained large  amounts of phospholipids, but a protein-staining band of 9 000 daltons present only i n f r a c t i o n 3. SDS-polyacrylamide g e l was  was  The' r a d i o a c t i v i t y p r o f i l e of f r a c t i o n 3 on similar to that i n F i g . 24 (panel B).  However,  the fractions ( f r a c t i o n 4) eluted with chloroform-methanol-water (5:5:1), contained very l i t t l e phospholipid.  On SDS-gels, e s s e n t i a l l y one radio-  a c t i v i t y peak coincident with a Coomassie Blue staining polypeptide  was  observed, as i n F i g . 24 (panel C). Therefore, BioRad Cellex CM was of the DCCD-binding protein.  subsequently used for the p u r i f i c a t i o n  E l u t i o n with chloroform-methanol-water  (10:10:1) was omitted from the p u r i f i c a t i o n procedure (150).  The amino  acid composition of the material eluted with chloroform-methanol-water (5:5:1) revealed that the preparation of DCCD-binding protein was  still  contaminated with a polypeptide(s) containing the amino acids serine, h i s t i d i n e and cysteine.  The contaminant(s) could be removed by adsorption  chromatography on Sephadex LH-60 i n the presence of chloroform-methanol  F i g . 24  SDS-polyacrylamide g e l electrophoresis of DCCD-binding protein obtained by chromatography on CM-cellulose.  The f r a c t i o n s eluted from the column of Whatman CM-32 ( F i g . 23, PANEL B) were pooled (A, B and C), dried and subjected to electrophoresis on SDS-urea (8 M) gels as described i n MATERIALS AND METHODS. The gels were f i x e d with 50% (w/v) TCA, stained with 0.25% (w/v) Coomassie Blue, cut Into 2 mm s l i c e s and the r a d i o a c t i v i t y of each s l i c e determined as described previously. BP indicates the p o s i t i o n of the bromophenol blue tracking dye. The migration positions of the molecular weight marker proteins (M , 94 000 - 14 400) are also shown. PANELS A, B and C correspond to the pooled f r a c t i o n s i n F i g . 23, PANEL B. r  161.  (2:1) containing 20 mM ammonium acetate (149). were obtained (Fig. the  25).  Two major protein peaks  The DCCD-binding protein eluted as the larger of  two peaks immediately a f t e r the void volume.  Judicious pooling of the  f r a c t i o n s from t h i s peak was required f o r obtaining pure DCCD-binding protein (149).  AMINO ACID COMPOSITION OF THE DCCD-BINDING PROTEIN The DCCD-binding protein from normal and mutant c e l l s were extracted with chloroform-methanol (2:1) and p u r i f i e d to homogeneity as described i n MATERIALS AND METHODS.  Their amino acid compositions were similar,  suggesting that the polypeptides from the wild-type and mutant strains were i d e n t i c a l (Table 13).  Other evidence f o r i d e n t i t y was obtained by peptide-  mapping of the cyanogen-bromide cleaved fragments of the DCCD-binding protein.  Separation of the cleaved fragments by only one-dimensional  thin-layer chromatography was not adequate to resolve a l l the fragments. Better resolution of the fragments was obtained with two-dimensional t h i n layer chromatography.  The pattern of migration of the peptides of the  DCCD-binding protein from the normal and mutant c e l l s were i d e n t i c a l ( F i g . 26).  Therefore, the l e s i o n responsible f o r the r e l a t i v e impermeability of  the mutant membranes to protons did not appear to be present on the DCCDbinding protein.  ANALYSIS OF THE MEMBRANES OF E. c o l i BY TWO-DIMENSIONAL ISOELECTRIC FOCUSING GEL ELECTROPHORESIS The previous r e s u l t s showed that the DCCD-binding protein from the wild-type (WSI and ML308-225) and mutant strains of E. c o l i ( N CBT-302) were i d e n t i c a l .  I 4 4  , AN382  In order to determine i f the defect responsible  162.  F i g . 25  Chromatography of the CM-cellulose-purified DCCD-binding p r o t e i n on Sephadex LH-60.  The DCCD-binding protein (6 mg protein) p u r i f i e d by chromatography on CM-cellulose was applied to a column of Sephadex LH-60 (1.2 x 40 cm) which was e q u i l i b r a t e d with chloroformmethanol ((2:1) containing 20 mM ammonium acetate. The column was eluted with t h i s solvent and 1 ml f r a c t i o n s c o l l e c t e d as described i n MATERIALS AND METHODS. The f r a c t i o n s between the arrows were pooled and stored at -20°C.  163. Table 13  Amino Acids  Amino Acid Composition of the DCCD-Binding Protein from D i f f e r e n t Strains of E. c o l i  ML 308-225  WS1  N  144  AN382  CBT-302  DNA b sequence  Asp  5.0  4.80  4.67  4.95  5.0  5  Thr  1.06  1.03  1.20  1.04  1.02  1  Ser  0.09  0  0.10  0.04  0.03  0  Glu  4.71  4.47  4.53  4.48  4.34  4  Pro  3.30  2.13  2.64  2.77  2.2  3  Gly  10.29  10.23  10.40  10.23  10.26  10  Ala  12.99  13.20  13.24  13.40  13.30  13  Cys  0  0  0  0  0  0  Val  5.83  5.90  5.84  5.95  5.84  6  Met  6.95(8.0)*  7.09(8.0)  6.61(8.2)  6.90(8.1 7.05(8.3)  Ile  7.81  7.82  7.88  7.78  7.88  8  Leu  12.41  12,39  12.50  12.21  12.37  12  Tyr  1.14  1.30  1.23  1.32  1.33  2  Phe  4.19  4.41  4.68  4.45  4.24  4  His  0  0  0  0  0  0  Lys  1.39  1.26  1.23  1.31  1.44  1  Arg  2.01  2.14  2.15  2.13  2.09  2  *  8  values i n parentheses were obtained a f t e r performic acid oxidation of the DCCD-binding protein and are r e l a t i v e to alanine = 13 and g l y c i n e = 10.  a.  Mean of four determinations; polypeptide.  b.  from (128, 131, 132).  values are i n mol amino acid per mol  164.  F i g . 26  Two-dimensional thin-layer chromatography of cyanogen bromide cleaved fragments of the DCCD-binding protein of E. c o l i CBT-302.  The DCCD-binding protein was treated with cyanogen bromide and the fragments (30-50 ug protein) separated by two-dimensional t h i n layer chromatography on c e l l u l o s e . Separation of the cyanogen bromide fragments i n the f i r s t dimension was by electrophoresis and i n the second dimension by ascending chromatography, as described i n MATERIALS AND METHODS. The p o s i t i o n of migration of the peptides were v i s u a l i z e d with ninhydrin.  165.  for proton-impermeability was present on the other subunits of the FQ complex, the mutant membranes were analyzed by two-dimensional  isoelectric  focusing g e l electrophoresis. F i g . 27 i l l u s t r a t e s the pattern of polypeptides obtained with the membranes from the parent (WSI) and the mutant (N^.^^) s t r a i n s .  None of  the subunits of ECF^ were found to be present i n the membranes of E_. c o l i N^ . 44  The Y subunit of ECF^ i s not seen on these gels since i t migrates  o f f because of i t s very basic nature ( p i - 8.9). Careful examination of the gels shown i n F i g . 27 revealed that a major polypeptide of molecular weight 18 000 (shown i n the square) was absent i n the mutant, _E. c o l i N^.^.  (An adjacent minor polypeptide of i d e n t i c a l  molecular weight was also absent i n the mutant.  This adjacent spot  may  have been caused by carbamylation of the major polypeptide by the cyanate formed from the urea present i n the sample.  A l t e r n a t i v e l y , this doublet  could be electrophoretic variants of the same subunit.  Fillingame et a l .  (204) have attempted to explain t h i s occurrence as due to spontaneous deamination of the polypeptide since i t occurred most frequently i n aged samples).  I t i s l i k e l y that t h i s polypeptide i s polypeptide b of FQ since  i t migrates i n t h i s p o s i t i o n on gels of t h i s type (126).  Unfortunately,  polypeptide a of FQ (24 000 daltons) does not enter these polyacrylamide gels (126). be determined  Thus, i t s presence i n the membranes of the mutants could not by using this gel system.  Comparison of the pattern of polypeptides obtained with the membranes of the mutants E_. c o l i CBT-302 and i t s parent, E_. c o l i CBT-1 absence of the subunits of ECF^ i n the mutant membranes.  revealed the  However, no  differences were observed i n the 18 000 molecular weight region. By contrast, the subunits of ECF, were present i n the membranes of  166.  F i g . 27  Two-dimensional I s o e l e c t r i c focusing g e l electrophoresis of membranes of parent (WSI) and mutant ( N J 4 4 ) strains of 15. c o l i .  The first-dimension g e l (horizontal direction) contained 0.4% (w/v) pH 3.5-10 and 1.6% (w/v) pH 5-7 ampholytes. The second dimension ( v e r t i c a l direction) was a discontinuous polyacrylamide g e l (Tris-buffered system) consisting of a layer of 15% (w/v) acrylamide below a layer of 11% (w/v) acrylamide. The samples f o r electrophoresis were prepared by the method of M e r r i l et a l . (175), as described i n MATERIALS AND METHODS. The a, A, 6" and e subunits of the ECF^-ATPase, which are present only i n the membranes of the parent s t r a i n , are indicated. The polypeptide outlined by the rectangle i s absent i n the mutant s t r a i n .  167.  the mutant E_. c o l i AN382. 18 000 dalton region.  But again, no differences were observed i n the  However, minor changes which do not involve a change  i n the molecular weight or the net charge of the 18 000 dalton polypeptide of FQ could not be excluded.  Furthermore, since polypeptide a (24  000  daltons) does not enter these gels, any changes i n t h i s polypeptide could not be ascertained.  168.  PART I I I  STUDIES ON. THE DCCD-BINDING PROTEIN OF THE F  Q  COMPLEX OF .  E. c o l i  r EFFECT OF ANTISERUM TO THE DCCD-BINDING PROTEIN ON THE ENERGIZATION OF THE MEMBRANE OF UREA-STRIPPED EVERTED MEMBRANE VESICLES In my study, attempts to demonstrate proton-translocating a c t i v i t y by reconstituting the DCCD-binding protein into phospholipids, by various methods (206,207), were not successful.  But evidence from reconstitution  experiments (7) involving ECF^ and ECF^-depleted membranes or by the use of mutants (25,121) suggested that the DCCD-binding protein i s d i r e c t l y involved i n proton translocation i n E_. c o l i .  This polypeptide should be  transmembranous i f i t i s the sole component of the proton translocation pathway through FQ.  The orientation of the DCCD-binding protein i n the  membrane of E_. c o l i was investigated by using antiserum to the DCCD-binding protein. As was shown i n the previous section, removal of ECF^ from the membrane vesicles of normal strains of E_. c o l i resulted i n the leakage of protons through FQ.  Consequently, reactions such as the quenching of the  fluorescence of the dye 9-aminoacridine, which required the presence of a transmembrane pH gradient, could not occur.  Fluorescence quenching could  be observed i f the leakage of protons through FQ was blocked by the readdition of ECF or by the reaction with DCCD. 1  F i g . 28A shows the  e f f e c t on fluorescence quenching of various additions to urea-stripped everted vesicles of E_. c o l i .  Bovine serum albumin at a concentration  normally present i n serum (approximately 37 mg per ml) had no e f f e c t on the residual fluorescence quenching of the ECF^-depleted v e s i c l e s .  Both  antiserum to ECF- and preimmune serum had only a s l i g h t effect on the  169.  Fig.  28  E f f e c t of the antiserum to the DCCD-binding p r o t e i n and of ECF^ on the ascorbate-oxidatipn-dependent quenching of fluorescence of 9-aminoacridine by urea-stripped everted membrane v e s i c l e s .  PANEL A: 2M urea-stripped everted v e s i c l e s of E_. c o l i WS1 (10 mg p r o t e i n ) i n 50 mM HEPES-KOH buffer, pH 7.5, containing 5 mM MgCl were Incubated f o r 5 h at A°C with d i f f e r e n t amounts of antiserum to the DCCD-binding p r o t e i n (•-•), antiserum to ECF^ (•-•), preimmune serum (O-O) or bovine serum albumin (37 mg/ml) ( A - A ) , i n a f i n a l volume of 1 ml. Samples (0.1 ml) were assayed f o r the a b i l i t y to quench the fluroescence of 9-aminoacridine with ascorbate as substrate ( i n the presence of phenazine methosulphate) as described i n MATERIALS AND METHODS. PANEL B: Ureastripped everted v e s i c l e s of E_. c o l i WS1 (1.0 mg p r o t e i n ) i n 50 mM HEPESKOH b u f f e r , pH 7.5, containing 5 mM MgCl were incubated at 20°C f o r 5 min with various l e v e l s of ECF^ i n a f i n a l volume of 0.1 ml. Ascorbateoxidation-dependent quenching of fluorescence of 9-aminoacridine was measured as described i n MATERIALS AND METHODS. 2  2  170.  quenching.  This small e f f e c t was  l i k e l y due to non-specific i n t e r a c t i o n of  serum proteins with the subunits of  FQ.  By contrast, antiserum to the DCCD-binding protein markedly stimulated fluorescence quenching indicating that the reaction of the antiserum with t h i s polypeptide  blocked  the leakage of protons through FQ.  If the data  of F i g . 28A were examined as a Lineweaver-Burk p l o t , a value could be calculated f o r the maximum quenching to be expected at saturating l e v e l s of antiserum.  This value (62% quenching) was  i n good agreement with that  observed following the addition of saturating levels of ECF^ Fig.  (69% quenching,  28B).  EFFECT OF ANTISERUM TO THE DCCD-BINDING PROTEIN ON THE BINDING OF ECF-^  TO  UREA-STRIPPED EVERTED MEMBRANE VESICLES The addition of antiserum to the DCCD-binding protein or ECF ECF^-depleted membrane v e s i c l e s resulted i n the stimulation of quenching during substrate oxidation.  1  to  fluorescence  This suggested that both ECF.^  and  antiserum to the DCCD-binding protein, were reacting at the same binding s i t e ( s ) to prevent the leakage of protons through FQ.  The e f f e c t of  antiserum to the DCCD-binding protein on the binding of ECF depleted membranes, was IE. c o l i WSI  examined as follows.  1  to  ECF^  Stripped everted v e s i c l e s of  were incubated with d i f f e r e n t amounts of antiserum.  The  vesicles were sedimented by centrifugation and then washed to remove unbound antiserum.  Various amounts of ECF^ were added to the suspension  of the treated v e s i c l e s and the extent of binding of ECF^ measured by the increase i n the ATPase a c t i v i t y of the v e s i c l e s . ATPase a c t i v i t y . amount of ECF  n  The extent of binding of ECF^  Stripped vesicles had  as a function of the  added to the treated and untreated  vesicles i s shown as a  no  ,  171.  Lineweaver-Burk plot i n F i g . 29.  The l i n e s intersecting close to the  ordinate suggested that antiserum to the DCCD-binding protein i n t e r f e r r e d with the binding of ECF^ i n a near competitive manner.  Preimmune serum  at a concentration of 110 u l serum per mg membrane protein also s l i g h t l y i n h i b i t e d the binding of ECF^ to the stripped v e s i c l e s .  However, the  extent of i n h i b i t i o n was much lower than that seen with antiserum to the DCCD-binding protein. Similar binding studies were c a r r i e d out on the unc mutants E_. c o l i N  I 4 4  and CBT-302.  Stripped everted v e s i c l e s of E. c o l i N  l 4 4  and CBT-302  were treated with preimmune serum or antiserum to the DCCD-binding protein at a concentration of 120 u l serum per mg membrane protein.  Reconstitution  with ECF^ was performed as described e a r l i e r and the r e s u l t s shown as Lineweaver-Burk p l o t s i n F i g . 29.  In both mutants, the results were  similar to those obtained with the wild-type  s t r a i n , E. c o l i WS1, i n that  the antiserum to the DCCD-binding protein interfered with the' binding of ECF^ i n a near competitive manner. However, i n contrast to the wild-type  s t r a i n , preimmune serum d i d not  i n h i b i t the binding of ECF^ to the mutant membranes.  In addition, the  extent of i n h i b i t i o n of the binding of ECF^ by antiserum to the DCCDbinding protein was lower i n the mutants than i n the wild-type  membranes.  This was further evidence that the FQ complex i n E_. c o l i Nj^^ and CBT-302 was d i f f e r e n t from that of the wild-type  s t r a i n (E_. c o l i WS1) .  EFFECT OF ANTISERUM TO THE DCCD-BINDING PROTEIN ON THE PROTON PERMEABIITY OF THE RIGHT-SIDE OUT VESICLES OF E. c o l i The experiments described e a r l i e r had detected the exposure of the DCCD-binding protein at the cytoplasmic  surface of ECF -depleted membrane 1  172. Fig.  29  E f f e c t of antiserum to the DCCD-binding protein on the binding of ECF^ to urea-stripped everted membrane v e s i c l e s .  Urea-stripped everted v e s i c l e s (25 mg protein) from 15. c o l i WSI, N and CBT-302 i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM MgCl and 10% (v/v) g l y c e r o l were incubated at 4°C f o r 20 h with d i f f e r e n t amounts of antiserum i n a f i n a l volume of 4.4 - 4.5 ml ml. After a 4-fold d i l u t i o n i n buffer, the mixture was centrifuged at 250 000 xg f o r 2.5 h. The sedimented v e s i c l e s were washed once by suspension i n buffer followed by resedimentation as before. The washed v e s i c l e s were resuspended i n buffer at 6.5 mg protein/ml. D i f f e r e n t amounts of ECF^ were incubated at 4°C f o r 45 min with 2.5 mg of v e s i c l e s protein i n buffer. The mixtures were d i l u t e d 8 to 10-fold i n buffer and the v e s i c l e s sedimented as before. The ATPase a c t i v i t y of the v e s i c l e s was assayed as described i n MATERIALS AND METHODS. A c t i v i t y i s expressed as units per mg protein. PANEL A: 0 y l ( A — A ) 50 y l (•-•), 75 y l (a-n) and 110 u l (O-O) of antiserum to the DCCD-binding p r o t e i n per mg of membrane protein. PANELS B and C: 0 u l (A-A), and 120 u l ( ~ ) of antiserum to the DCCD-binding protein per mg membrane p r o t e i n . 120 u l ( O - O ) of preimmune serum per mg membrane protein. I 4 4  2  t  A  A  174.  vesicles.  The e x t e n t of exposure  of t h i s p o l y p e p t i d e a t the e x t e r n a l  ( p e r i p l a s m i c ) s u r f a c e of the c e l l membrane was a n t i s e r u m to the DCCD-binding p r o t e i n . was  a l s o examined by u s i n g  In t h i s experiment,  the a n t i s e r u m  p a r t i a l l y p u r i f i e d by ammonium s u l p h a t e p r e c i p i t a t i o n to reduce  b u f f e r i n g e f f e c t of the serum. determined  The p a r t i a l l y p u r i f i e d a n t i s e r u m  by c r o s s e d Immunoelectrophoresis  from i t s b i n d i n g s i t e s on F Q .  l e a k protons through FQ (26,205).  was  to be s t i l l a c t i v e ( F i g . 3 0 ) .  The ECF-pATPase-defective mutant, E_. c o l i DL-54, r e a d i l y ECF^  the  Consequently,  loses  v e s i c l e s of DL-54  A d d i t i o n of v a l i n o m y c i n to K+-loaded  " r i g h t - s i d e o u t " v e s i c l e s r e s u l t s i n an e f f l u x of K+ c o n c o m i t a n t l y w i t h a compensatory i n f l u x of p r o t o n s , p r o v i d e d t h a t a pathway f o r p r o t o n s i s available. proteins  T h i s pathway i s p r o v i d e d i n DL-54 by the ECF^-depleted  FQ  (26,205).  The b a s i s of t h i s assay i s i l l u s t r a t e d i n F i g . 31.  The i n f l u x of  protons i s d e t e c t e d as a r i s e i n the pH of the medium e x t e r n a l t o the vesicles.  A t y p i c a l r e s u l t i s shown i n F i g . 32.  A d d i t i o n of DCCD to the  v e s i c l e s b l o c k e d the movement of p r o t o n s through FQ ( F i g . 33B) . mum  of about  A maxi-  60-65% of the t o t a l p r o t o n s e n t e r i n g the v e s i c l e s c o u l d be  i n h i b i t e d by DCCD (880 P M ) .  The r e s i d u a l uptake of protons was  due t o p a s s i v e movement of the protons a c r o s s the membrane.  likely  In c o n t r a s t to  the e f f e c t of DCCD, the a n t i s e r u m to the DCCD-binding p r o t e i n had no  effect  on t h e - t o t a l amount of protons taken up ( F i g . 33A). However, i f the d a t a were a n a l y z e d w i t h r e s p e c t to the r a t e , not e x t e n t , of p r o t o n uptake, an e f f e c t was DCCD-binding p r o t e i n . D).  The  observed w i t h a n t i s e r u m to the  These r e s u l t s a r e shown i n F i g . 33 ( p a n e l s C and  r a t e of p r o t o n uptake was  c o n c e n t r a t i o n of 440-880 P M .  i n h i b i t e d to a maximum of 65% a t a DCCD  Antiserum to the DCCD-binding p r o t e i n a l s o  175.  Fig. 30  Crossed Immunoelectrophoresis o f antiserum to the DCCD-binding protein.  Crossed Immunoelectrophoresis of the DCCD-binding protein was carried out as described i n MATERIALS AND METHODS. UPPER SLIDE: Antigen i n the first-dimension was 4 vg DCCD-binding protein i n Bjerrum buffer, pH 8.8, containing 100 mM glycine, 38 mM T r i s base and 1% (w/v) T r i t o n X-100. 150 u l of antiserum to the DCCD-binding protein was present i n the seconddimension g e l . LOWER SLIDE: Antigen i n the first-dimension was 8 Ug DCCD-binding protein i n Bjerrum buffer, pH 8.8. 175 u l of ammonium sulphate-purified antiserum to the DCCD-binding protein was present i n the second-dimension g e l .  176.  Fig.  31  Schematic representation of the proton-pathway provided by the " r i g h t - s i d e out" v e s i c l e s of _E. c o l i DL-54.  The figure on the l e f t shows that the addition of valinomycin to K -loaded " r i g h t - s i d e out" v e s i c l e s of IS. c o l i r e s u l t s i n the e f f l u x of K with a concomitant i n f l u x of protons through FQ. The i n f l u x of proton i s blocked by addition of antibody to polypeptide(s) of FQ (figure on the r i g h t ) . Details of t h i s type of experiment are described i n the legend to F i g . 32. +  +  V a l , valinomycin;  Ab, antibody;  RSO, "right-side out" v e s i c l e s .  (Drawings courtesy of Dr. P.D. Bragg.)  177.  r"  Valinomycin  F i g . 32  E f f e c t of DCCD and of antiserum to the DCCD-binding protein on the proton permeability of " r i g h t - s i d e out" membrane v e s i c l e s .  E_. c o l i D L - 5 4 was grown to the l a t e exponential phase and converted to spheroplasts by treatment with lysozyme i n the presence of EDTA. The spheroplasts were then loaded with K as described i n MATERIALS AND METHODS. The pH changes occurring following the addition of 3 u l of valinomycin ( 5 mg/ml) to 0.85 ml K -loaded v e s i c l e s ( 1 . 5 mg protein) i n 0 . 4 M sucrose - 1 0 mM MgCl were measured with a glass pH electrode connected to a Fisher Accumet Model 325 expanded scale pH meter as described i n the MATERIALS AND METHODS section. In some experiments, the v e s i c l e s were pre-incubated with DCCD (15 u l i n ethanol) or with ammonium sulphate-purified antiserum to the DCCD-binding protein f o r 4 5 min at 2 0 ° C . Each assay was i n t e r n a l l y c a l i b r a t e d by addition of a known concentration of a c i d ( H C 1 or H 2 S O 4 ) . 1 , no addition; 2 , 50 u l antiserum to the DCCD-binding protein; 3, 880 uM DCCD. The amounts and rates of proton i n f l u x are summarized i n F i g . 33. +  2  178.  -  240  ®  Q.  3  •  C 0) (0  +  120  o E c  1  I  50  100  1  1  120  c E a a  3 C  a> ro  O  E c  Serum-ul  F i g . 33  400  800  DCCD-uM  E f f e c t of DCCD and of antiserum to the DCCD-binding protein on the proton-permeability of "right-side out" membrane v e s i c l e s of l i . c o l i DL-54.  The experiment was carried out as described i n the legend to F i g . 32. PANELS A and B show the amounts of protons taken up by the "right-side out" v e s i c l e s , whereas PANELS C and D show the rates of proton-uptake, i n the presence of DCCD or antiserum to the DCCD-binding protein.  179.  decreased  the rate of proton uptake, but only to a maximum of  30%.  Thus, there appeared to be some c o r r e l a t i o n between the e f f e c t of DCCD oh the rate of proton uptake and on the t o t a l amount of protons taken up by these v e s i c l e s .  This r e l a t i o n s h i p was not shown when the antiserum to the  DCCD-binding protein was used, suggesting that DCCD and antiserum to the DCCD-binding protein affected proton movement through FQ by d i f f e r e n t mechanisms.  EFFECT OF ANTISERUM TO THE DCCD-BINDING PROTEIN ON THE ENERGIZATION OF  THE  MEMBRANE OF NATIVE EVERTED MEMBRANE VESICLES The e f f e c t of antiserum on the quenching of fluorescence during substrate oxidation and ATP hydrolysis by native everted v e s i c l e s was also investigated. Everted v e s i c l e s of E_. c o l i WS1  were incubated i n the presence of  d i f f e r e n t amounts of antiserum and then assayed f o r fluorescence quenching as previously described.  Bovine serum albumin and preimmune serum did not  a f f e c t fluorescence quenching during oxidation of ascorbate presence of PMS)  ( F i g . 34, panel A).  ( i n the  By contrast, antiserum to the  DCCD-binding protein increased the l e v e l of quenching from 50% to Addition of saturating l e v e l s of ECF^ quenching to  60%.  to these v e s i c l e s stimulated the  70%.  Similar r e s u l t s were obtained with quenching during ATP hydrolysis (Fig. 34, panel B).  Bovine serum albumin and preimmune serum had, at the  most, only a s l i g h t e f f e c t on the fluorescence quenching of the everted vesicles.  Stimulation of fluorescence quenching was observed when a n t i -  serum to the DCCD-binding protein was  present.  These results suggested that there was  loss of some ECF  1  from the  180.  V o l u m e -- u l  Fig.  34  E f f e c t of antiserum to the DCCD-binding protein on the energization of untreated everted membrane v e s i c l e s .  Membrane v e s i c l e s of E. c o l i WSI (10 mg protein) i n 50 mM HEPES-KOH buffer, pH 7.5, containing 5 mM MgCl were incubated f o r 5 h at 4°C with various amounts of antiserum to the DCCD-binding protein (•-•), antiserum to ECFj (•-•), preimmune serum ( A - A ) or bovine serum albumin (37 mg/ml) (•-•), i n a f i n a l volume of 1 ml. Samples (0.1 ml) were assayed f o r the a b i l i t y to quench the fluorescence of a 9-arainoacridine during: PANEL A, oxidation of ascorbate i n the presence of phenazine methosulphate; PANEL B, ATP h y d r o l y s i s . 2  t  181.  membrane during preparation. The most s t r i k i n g e f f e c t was observed when native everted v e s i c l e s were treated with antiserum to ECF^. During substrate oxidation, a n t i serum to ECF^ caused a decrease i n the quenching of fluorescence ( F i g . 34, panel A) indicating that the membranes became leaky to protons. Fluorescence quenching during ATP hydrolysis was completely abolished i n v e s i c l e s treated with antiserum to ECF^ ( F i g . 34, panel B). The cause(s) of the decrease i n fluorescence quenching during substrate oxidation i n the treated v e s i c l e s i s not known.  I t has been  suggested (208,209) that the membrane and/or membrane proteins undergo conformational changes during membrane energization. possible that antiserum  to ECF^ prevented  Therefore, i t i s  the conformational change of  ECF^ during substrate oxidation such that the FQ was not completely blocked.  A l t e r n a t i v e l y , the antiserum to ECF^ could have caused the  detachment of ECF^ from the membrane (65). Quenching during ATP hydrolysis was abolished i n v e s i c l e s treated with antiserum  to ECF^ because of i n h i b i t i o n of the ATPase a c t i v i t y rather  than because of membranes becoming leaky to protons. to ECF  1  Addition of antiserum  to p u r i f i e d ECF inhibited the ATPase a c t i v i t y . 1  ENERGIZATION OF THE MEMBRANE OF TRYPSIN-TREATED UREA-STRIPPED EVERTED VESICLES ATP and respiration-dependent  fluorescence quenching could be restored  to urea-stripped everted v e s i c l e s reconstituted with ECF^. By contrast, ECF^ could not restore these a c t i v i t i e s i n stripped v e s i c l e s which had been treated with t r y p s i n (Table 14).  Since the energized membrane was  detected i n d i r e c t l y by the quenching of the fluorescence of  9-aminoacridine,  182. Table  14  Energization of the membrane of trypsin-treated everted membrane v e s i c l e s of E. c o l i  Percent Quenching System  Urea-treated  vesicles  NADH  7.5  + DCCD  35.1  + ECF  40.5  1  Trypsin -treated stripped vesicles + DCCD + ECF  1  5.0 15.0 7.1  Ascorbate/PMS  ATP  5.9  0-2  7.3  0-2  28.3  37.5  5.2  3.5  6.4  0-2  4.8  4.3  Urea-treated everted v e s i c l e s of E. c o l i WSI were treated with TPCKt r y p s i n . The treated v e s i c l e s were suspended i n 50mM HEPES-KOH buffer, pH 7.5, containing 5mM MgCI^ and 10% (v/v) g l y c e r o l at a protein concentration of 10 mg/ml. Samples (0.1 ml.) were removed and assayed for the a b i l i t y to quench the fluorescence of 9-aminoacridine during subs t r a t e (NADH; ascorbate, i n the presence of PMS) oxidation or during hydrolysis of ATP, as described i n MATERIALS AND METHODS. In experiments i n which DCCD or ECF, was used, each was incubated with the membrane 1 o suspension f o r 5 min. at 20 C, p r i o r to assay. The f i n a l concentration of DCCD i n the assay mixture was 375 uM and the amount of ECF used was 80 - 100 yg p r o t e i n . 1  183.  i t was not known i f the absence of quenching i n trypsin-treated v e s i c l e s was due to the complete destruction of the respiratory chain or to the vesicles becoming leaky to protons as a result of protease treatment. It was u n l i k e l y that the absence of fluorescence quenching a c t i v i t y was due to the destruction of the respiratory chain since NADH-oxidationdependent fluorescence quenching i n the presence of DCCD was observed (Table 14). Quenching was not observed during ascorbate oxidation because DCCD i n h i b i t e d i t s a c t i v i t y .  This was supported  by the observation that  trypsin-treatment of the membranes from the unc mutants, E_. c o l i CBT-302 d i d not destroy NADH- or ascorbate-oxidation-dependent  AN382 and  fluorescence  quenching ( F i g . 20). Since ECF^ d i d not i n h i b i t the proton-translocating properties of FQ i n trypsin-treated v e s i c l e s , i t was of interest to determine i f antiserum to DCCD-binding protein could restore  respiration-dependent  fluorescence quenching. Urea-stripped everted v e s i c l e s of E. c o l i WS1 were treated with trypsin and then incubated with d i f f e r e n t amounts of antiserum.  F i g . 35  shows the e f f e c t of various additions to trypsin-treated v e s i c l e s on the fluorescence quenching.  These r e s u l t s appeared to be similar to those i n  F i g . 28, panel A, f o r urea-treated v e s i c l e s . effect on the residual fluorescence quenching.  Bovine serum albumin had no Preimmune serum and  antiserum to ECF^ only s l i g h t l y stimulated the quenching of fluorescence. By contrast, antiserum to the DCCD-binding protein markedly stimulated fluorescence quenching indicating that reaction of the antiserum with t h i s polypeptide blocked the leakage of protons through F Q . These results were d i f f e r e n t from those obtained with urea-treated everted v e s i c l e s ( F i g . 28A) i n two ways.  F i r s t l y , the maximum quenching  184.  V o l u m e -- u l  Fig.  35  E f f e c t of antiserum to the DCCD-binding protein on the ascorbate-oxidation^dependent quenching of fluorescence of 9-aminoacridine by trypsin-treated everted membrane vesicles,  Urea-stripped everted v e s i c l e s of _E. c o l i WS1 were treated with TPCKt r y p s i n at 20°C f o r 30 min as described i n MATERIALS AND METHODS. The trypsin-treated v e s i c l e s (10 mg) i n 50 mM HEPES-KOH buffer, pH 7.5, containing 5 mM MgCl were incubated f o r 5 h at 4°C with various l e v e l s of antiserum to the DCCD-binding protein (•-•), antiserum to E C F 2 ( • - • ) , preimmune serum (O-O) or bovine serum albumin (37 mg/ml) ( A - A ) , i n a f i n a l volume of 1 ml. Samples (0.1 ml) were assayed f o r the a b i l i t y to quench the fluorescence of 9-aminoacridine with ascorbate as substrate ( i n the presence of phenazine methosulphate) as described i n the MATERIALS AND METHODS section. 2  185.  expected at saturating levels of antiserum was lower (42% quenching) than with urea-treated everted v e s i c l e s (62% quenching).  Secondly, the addition  of ECF^ to trypsin-treated v e s i c l e s (at levels which caused maximum fluorescence quenching i n urea-treated v e s i c l e s ) , did not stimulate fluorescence quenching.  The lower extent of quenching may  be due to damage  of the membrane by trypsin, causing an increase i n proton leakage other than through FQ.  BINDING OF ECF  1  TO PROTEASE-TREATED MEMBRANE VESICLES  Treatment of stripped everted v e s i c l e s with trypsin did not destroy the respiratory chain.  However, the absence of fluorescence  quenching i n trypsin-treated v e s i c l e s reconstituted with ECF^ that the coupling- and/or ECF^-binding by protease treatment.  completely  suggested  s i t e s on the membrane were affected  This was examined as follows.  Urea-stripped everted v e s i c l e s of E. c o l i WSI were treated with trypsin and reconstituted with ECF^ as described e a r l i e r . The extent of v  binding of ECF^ was measured by the increase i n the ATPase a c t i v i t y of the v e s i c l e s since the t r y p s i n - or urea-treated v e s i c l e s had no ATPase activity.  ATP-  and respiration-dependent  fluorescence quenching was  not  observed i n these trypsin-treated v e s i c l e s which had been reconstituted with  ECF^ The results i n F i g . 36 (panel A) shows that the digestion of the  stripped v e s i c l e s with t r y p s i n f o r up to 4 h at 37°C did not affect the a b i l i t y of the membranes to bind Trypsin-treatment N  I 4 4  ECF^.  of stripped membranes of the unc mutants, E. c o l i  and CBT-302 also did not a l t e r the capacity of these membranes to  bind ECF  ( F i g . 36, panels B and C).  186.  F i g . 36  B i n d i n g o f ECF^ t o t r y p s i n - t r e a t e d e v e r t e d membrane v e s i c l e s .  U r e a - s t r i p p e d e v e r t e d v e s i c l e s o f 15. c o l i WSI, N 1 4 4 and C B T - 3 0 2 were t r e a t e d w i t h T P C K - t r y p s i n and t h e n r e c o n s t i t u t e d w i t h v a r i o u s l e v e l s o f ECF! a s d e s c r i b e d i n MATERIALS AND METHODS. Treatment w i t h T P C K - t r y p s i n was f o r 4 h a t 37°C. The amount o f membrane v e s i c l e p r o t e i n r e c o n s t i t u t e d w i t h ECF;L was 2 mg. The e x t e n t o f b i n d i n g o f ECF^ t o u r e a - s t r i p p e d o r t r y p s i n - t r e a t e d ( A - A ) v e s i c l e s was measured by t h e i n c r e a s e I n ATPase a c t i v i t y . A c t i v i t y i s e x p r e s s e d i n u n i t s p e r mg p r o t e i n .  187.  ECF  1  --  H  g  188.  Double-reciprocal  plots of the data i n F i g . 36 (not shown) revealed  that the maximum ATPase a c t i v i t y to be expected i n urea-treated vesicles of E_. c o l i WS1, N ECF^,  I 4 4  membrane  and CBT-302, at saturating l e v e l s of  was s i m i l a r (8.33 units per mg protein).  The half-saturation  values f o r E. c o l i WS1, N .. and CBT-302 were determined to be 0.55, 1.11 — 144 T/  and 1.11 mg ECF^, respectively.  This suggested that the urea-treated  v e s i c l e s of E_. c o l i WS1 (wild-type) had a higher a f f i n i t y f o r ECF , as 1  would be expected.  Similar analysis of the data on the binding of ECF  to "trypsin-treated vesicles resulted i n more complex k i n e t i c s (not shown). Attempts to abolish the a b i l i t y of urea-treated  vesicles to bind  ECF^ were made also by treating the stripped vesicles with Staphylococcus aureus Vg protease.  Stripped v e s i c l e s which were treated with Vg protease  were s t i l l able to bind ECF  1  ( F i g . 37). Analysis of the data on  Lineweaver-Burk p l o t s , revealed that the maximum a c t i v i t y expected at saturating l e v e l s of ECF^ was 8.33 units per mg protein f o r E_. c o l i WS1 only.  Similar analysis of the data on the binding of ECF^ to Vg  protease-treated  vesicles of E_. c o l i  and CBT-302 were also very  complex (not shown).  EFFECT OF DCCD ON THE ATPase ACTIVITY OF THE ECF^^ BOUND TO TRYPSIN-TREATED VESICLES The a c t i v i t y of the i n t a c t membrane-bound ATPase enzyme can be inhibited by DCCD.  Since trypsin-treated v e s i c l e s were s t i l l capable of  binding ECF^, i t was of interest to determine i f the a c t i v i t y of the rebound ATPase enzyme was s t i l l sensitive to DCCD. Urea-stripped  everted v e s i c l e s of E_. c o l i WS1 were treated with  trypsin and then reconstituted with ECF.. as previously described.  The  Fig.  37  Binding of everted  ECF^ to  membrane  S t a p h y l o c o c c u s a u r e u s VR  protease-treated  vesicles.  U r e a - s t r i p p e d v e s i c l e s o f _E. c o l i WSI, ^ 4 4 and CBT-302 were t r e a t e d w i t h S t a p h y l o c o c c u s a u r e u s VR p r o t e a s e a n d t h e n r e c o n s t i t u t e d w i t h v a r i o u s l e v e l s o f ECF-L a s d e s c r i b e d i n M A T E R I A L S AND METHODS. Treatment w i t h V g p r o t e a s e was f o r 4 h a t 3 7 ° C . 2 mg o f membrane v e s i c l e p r o t e i n was r e c o n s t i t u t e d w i t h E C F ^ . The e x t e n t o f b i n d i n g o f E C F ^ t o u r e a - s t r i p p e d (•-•) o r Vg p r o t e a s e - t r e a t e d (•-•) v e s i c l e s was m e a s u r e d b y t h e i n c r e a s e i n ATPase a c t i v i t y . A c t i v i t y i s e x p r e s s e d i n u n i t s p e r mg p r o t e i n .  ECF  1  H  g  191.  reconstituted v e s i c l e s were incubated with d i f f e r e n t amounts of DCCD and the ATPase a c t i v i t y measured.  The r e s u l t s are i l l u s t r a t e d i n F i g . 38.  After a short period of incubation (45 min at 37°C), the enzyme a c t i v i t i e s present i n the native membrane v e s i c l e s and i n the trypsin-treated v e s i c l e s were i n h i b i t e d by about 40% at a concentration of 120 nmol DCCD per mg protein.  With a longer period of incubation ( i 2 h at 4°C), the i n h i b i t i o n  of the ATPase a c i t i v t y i n both cases increased to 60%. Under i d e n t i c a l conditions, the ATPase a c t i v i t y of the p u r i f i e d ECF^ was i n h i b i t e d by 5-10%. Therefore, DCCD i n h i b i t e d the ATPase a c t i v i t y of the rebound ECF^ to an extent similar to that found i n native everted membrane v e s i c l e s .  IMMUNOPRECIPITATION OF THE ECF-^Q COMPLEX WITH ANTISERUM Since ECF^ could s t i l l bind to trypsin-treated v e s i c l e s , i t was conceivable that the reconstituted v e s i c l e s could be s o l u b i l i z e d with detergents and the ECF^FQ complex immunoprecipitated  with antiserum to  determine which polypeptide of FQ was l i k e l y to be affected by t r y p s i n treatment. I n i t i a l l y , attempts were made to determine which polypeptides of the ECFJFQ complex could be immunoprecipitated. " Native everted vesicles of c  °li WS1 were washed with a low i o n i c strength buffer i n the presence of  protease i n h i b i t o r s and then s o l u b i l i z e d with Aminoxid WS-35 as described i n the legend to F i g . 39. The s o l u b i l i z e d f r a c t i o n was treated with the test  antiserum. The immunoprecipitate  obtained with antiserum to the DCCD-binding  protein contained many bands when analyzed by SDS-polyacrylamide g e l electrophoresis ( F i g . 39).  The major protein-staining polypeptides had  192.  F i g . 38  E f f e c t of DCCD on the ATPase a c t i v i t y of the ECF± bound to trypsin-treated everted membrane v e s i c l e s .  Urea-stripped everted v e s i c l e s of E_. c o l i WSI were treated with TPCK-trypsin f o r 45 min at 20°C as described i n MATERIALS AND METHODS. Reconstituti on of the v e s i c l e s with ECF-i was carried out as follows! 40 mg of membrane v e s i c l e protein i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM MgCl and 10% (v/v) g l y c e r o l was reconstituted with 2.4 mg of ECF^ i n a f i n a l volume of 4 ml. The suspension was incubated at 4°C f o r 45 min, d i l u t e d 8-10 f o l d with buffer, and the v e s i c l e s sedimented by centrifugation at 250 000 xg f o r 2.5 h. The sedimented v e s i c l e s were resuspended i n buffer and samples containing 4 mg membrane protein incubated with various l e v e l s of DCCD i n a f i n a l volume of 1 ml either f o r 45 min at 37°C or f o r 45 min at 37°C followed by 12 h at 4°C before the ATPase a c t i v i t y was determined. Reconstituted urea-treated (•-•) or trypsin-treated ( D - D ) v e s i c l e s incubated f o r 45 min at 37°C; reconstituted urea-treated (•-•) or trypsin-treated (O-O) v e s i c l e s incubated f o r 45 min at 37°C followed by 12 h at 4°C. ECFj^ ( A - A ) treated with DCCD f o r 45 min at 37°C followed by 12 h at 4°C. The s p e c i f i c a c t i v i t i e s of ECF and of the reconstituted urea-stripped and trypsin-treated v e s i c l e s were 18.5, 1.34 and 1.08 units/mg protein, r e s p e c t i v e l y . 2  X  193.  F i g . 39  SDS-polyacrylamide g e l electrophoresis of the ECF^FQ complex immunoprecipitated with antiserum.  Everted membrane vesicles of E_. c o l i ML 308-225 were suspended i n 50 mM Tris-HCl buffer, pH 7.4 containing 100 mM KC1, 40 mM e-aminocaproic acid, 24 mM p-aminobenzamidine, 0.8 mM PMSF, 0.4% (w/v) Merthiolate and 2% (v/v) methanol at a protein concentration of 10 mg/ml. The membrane v e s i c l e s were s o l u b i l i z e d with Aminoxid WS-35 at a detergent to protein r a t i o of 6, as described i n MATERIALS AND METHODS. To 10 ml of s o l u b i l i z e d material was added 0.5 ml of antiserum to the DCCD-binding protein or antiserum to ECF^ and the mixture incubated at 4°C f o r 24-30 h. The p r e c i p i t a t e was collected by centrifugation at 12 000 xg f o r 30 min and washed once with 0.9% (w/v) NaCl containing 1% (w/v) Aminoxid WS-35, and twice with 0.9% (w/v) NaCl. The immunoprecipitate was subjected to SDS-electrophoresis, as described by Laemmli (171). The separating g e l consisted of 13% (w/v) acrylamide and the stacking g e l was 4% (w/v) acrylamide. The gel was stained with 0.1% (w/v) Coomassie Blue as described previously. Lanes a and c, p u r i f i e d ECF^; lane b, immunoprecipitate obtained with antiserum to the DCCD-binding protein; lanes d and e, immunoprecipitate obtained with antiserum to ECF-^ with lane e containing twice the amount of material as that i n lane d. The migration positions of the subunits of ECF-^, of the DCCD-binding protein (DBP), and of the 18 000 dalton polypeptide of FQ are Indicated.  194.  molecular weights of 60 000, 55 000, 32 000, 30 000, 28 000, 22-25 000  1  (stained d i f f u s e l y ) , 18 000, 11 000 and 9 000. daltons  The polypeptide of 9 000  migrated as a diffuse Coomassie Blue staining region and was  i d e n t i f i e d as the DCCD-binding protein since i t co-migrated with p u r i f i e d DCCD-binding protein.  However, the 56 000 (a), 52 000 (B), 22 000 (6)  and 13 000 (e) dalton subunits of ECF^ were absent.  S i m i l a r l y , the  subunits of ECF^ could not be detected i n the immunoprecipitate when the ATPase complex, p u r i f i e d by Phenyl-Sepharose  CL-4B and centrifugation at  250 000 xg f o r 16-17 h, was treated with antiserum to the DCCD-binding protein.  I t i s possible that the i n t e r a c t i o n of the antibody to the DCCD-  binding protein with the  ECF^FQ  complex resulted i n the displacement of  ECF^ since i t has been shown that the antibody binds close to the ECF^binding s i t e on F  Q  ( F i g . 29A). By contrast, treatment of the s o l u b i l i z e d  f r a c t i o n with antiserum to ECF^ precipitated polypeptides with molecular weights of 94 000, 90 000, 56 000 ( a ) , 52 000 (fi), 48 000, 35 000, 32 000, 30 000, 28 000, 22-25 000 (stained d i f f u s e l y ) , 18 000, 13 000 ( e ) , 11 000 and 9 000 (stained d i f f u s e l y ) .  The presence of the a, B and e  subunits suggested that the 32 000 dalton polypeptide was l i k e l y to be the y subunit of ECF^.  The 9 000 dalton polypeptide was i d e n t i f i e d as the  DCCD-binding protein as before.  These results indicated that the immuno-  p r e c i p i t a t e obtained with antiserum to ECF^ contained a more " i n t a c t " ECF-JFQ  complex.  Urea-stripped v e s i c l e s of E_. c o l l WS1 also were treated with trypsin and reconstituted with ECF^.  The reconstituted vesicles were s o l u b i l i z e d  with Aminoxid WS-35 and the s o l u b i l i z e d f r a c t i o n immunoprecipitated with antiserum to ECF^.  The immunoprecipitates were analyzed on the two-  dimensional g e l electrophoresis system of O'Farrell (176).  This technique  195.  was used to analyze the contents of the immunoprecipitates  because of the  c h a r a c t e r i s t i c migration patterns of the a, J3, <S and e subunits of ECF^ and the 18 000 dalton subunit (polypeptide b) of F preliminary results are shown i n F i g . 40.  Q  ( F i g . 27).  These  Many polypeptides were present  i n the p r e c i p i t a t e from the urea-stripped vesicles which had been reconstituted with ECF  1  ( F i g . 40A).  presumably i n t a c t . distinguishable.  The  ECF^FQ  complex i n t h i s preparation was  The <*, J3, e subunits of ECF^ were e a s i l y The 6 subunit of ECF^ was missing i n t h i s preparation.  This was probably due to the s u s c e p t i b i l i t y of t h i s subunit to proteases (Dr. Helga Stan-Lotter, personal communication) and the problem was further enhanced by the prolonged incubation period (30-36 h) involved i n the experiment.  In addition to these three subunits of ECF^,  a polypeptide  of 18 000 daltons was present (shown i n square) and was l i k e l y to be polypeptide b of F Q . S i m i l a r l y , the <*, J3 and e subunits of ECF^ were present i n the p r e c i p i t a t e obtained from the trypsin-treated v e s i c l e s which had been reconstituted with ECF^  ( F i g . 40 B).  In contrast to the urea-treated  v e s i c l e s , trypsin-treatment resulted i n the cleavage of the 18 000 dalton subunit.  This polypeptide was absent, even though the g e l was loaded with  more protein than i n F i g . 40A. the  B and e subunits of  (Note the r e l a t i v e staining i n t e n s i t y of  ECF^)  Analysis of the precipitates from the urea- and trypsin-treated vesicles on one dimensional SDS-polyacrylamide gels revealed that the 9 000 dalton polypeptide (DCCD-binding protein) did not appear to be affected by trypsin-treatment.  However, the p o s s i b i l i t y that trypsin digested a  f r a c t i o n of the DCCD-binding protein as well as the 24 000 dalton subunit (polypeptide a) of F  n  could not be  excluded.  196. F i g . 40  Two-dimensional gel electrophoresis of the ECF^FQ complex obtained by immunoprecipitation with antiserum to ECF-^.  Urea-stripped everted v e s i c l e s of E_. c o l i ML308-225 were treated with TPCK-trypsin and suspended i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM MgCl and 10% (v/v) g l y c e r o l at a protein concentration of 10 mg/ml. Twenty ml of the treated v e s i c l e s were reconstituted with 0.3 ml of ECF^ (9.5 mg/ml) as described i n MATERIALS AND METHODS. The reconstituted v e s i c l e s were suspended i n 50 mM Tris-HCl buffer, pH 7.4 containing 100 mM KC1, 40 mM e-aminocaproic acid, 24 mM p-aminobenzamidine, 0.8 mM PMSF, 0.4% (w/v) Merthiolate and 2%(v/v) methanol at a protein concentration of 10 mg/ml and s o l u b i l i z e d with Aminoxid WS-35 at a detergent to protein r a t i o of 6, as described i n the legend to F i g . 39. To 25 ml of the s o l u b i l i z e d f r a c t i o n was added 1.5 ml of antiserum to ECF]_ and the ( mixture incubated at 4°C f o r 30-36 h. The immunoprecipitate was collected by centrifugation at 12 000 xg for 30 min and washed three times with 50 mM Tris-H2S04 buffer, pH 8.0, containing 10 mM MgCl2- The immunoprecipitate was subjected to two-dimensional gel electrophoresis. The first-dimension g e l (horizontal d i r e c t i o n ) consisted of 0.8% (w/v) pH 3.5-10 and 1.6% (w/v) pH 5-7 ampholytes. The second-dimension gel ( v e r t i c a l direction) was a l i n e a r gradient of 7.5-16.5% (w/v) acrylamide i n the Tris-buffered system. The gels were stained with 0.1% (w/v) Coomassie Blue, as described previously. PANEL A: Immunoprecipitate obtained from urea-stripped everted v e s i c l e s reconstituted with ECF^. PANEL B: Immunoprecipitate obtained from trypsin-treated everted v e s i c l e s reconstituted with ECF^. The a, Q and e subunits of ECF^ are indicated. The polypeptide outlined by the rectangle i s absent i n the immunoprecipitate from t r y p s i n treated everted v e s i c l e s . 2  (A)  Urea - treated  198.  DETECTION BY SOLID PHASE RADIOIMMUNE ASSAY OF THE REACTION OF ANTIBODY WITH MEMBRANE VESICLES The experiments described e a r l i e r , i n which the decrease i n proton leakage through FQ i n the presence of antiserum to the DCCD-binding protein was measured, were attempts to determine i f the DCCD-binding protein was transmembranous.  The a b i l i t y of the antibody to the DCCD-binding  protein to react with t h i s polypeptide i n "right-side out" and everted membrane vesicles was also examined by the competitive i n h i b i t i o n assay (195).  In t h i s assay, the DCCD-binding protein of the membrane v e s i c l e s  (urea-stripped everted vesicles or right-side out v e s i c l e s ) competed f o r the antibody with the DCCD-binding protein immobilized i n microtitre wells. Poly-L-lysine was required to immobilize the antigen.  The t i t r a t i o n  curve i n F i g . 41 could not be reproduced If the antigen was passively dried (55-60°C) onto the m i c r o t i t r e wells.  The results of the competitive  i n h i b i t i o n assay are shown i n F i g . 42 (panel A). vesicles of E_. c o l i WS1  The right-side out  were approximtely one-third as e f f e c t i v e i n binding  the antibody compared with urea-stripped everted v e s i c l e s .  Almost  i d e n t i c a l results were obtained with the vesicles from E_. c o l i ML380-225. In both of these s t r a i n s , 50% i n h i b i t i o n of binding was obtained with a concentration of 10 ug everted v e s i c l e protein per ml, while that by the right-side out v e s i c l e s of E_. c o l i WS1  and ML308-225 was 26.6 yg protein  per ml. The a b i l i t y of both types of v e s i c l e s to bind the antibody, but with d i f f e r e n t effectiveness, could be due to the presence of a mixed population of antibodies ( i . e . antibodies to determinants exposed on the inside (cytoplasmic side) and outside of the c e l l ) .  A l t e r n a t i v e l y , the binding of  antibody to the right-side out vesicles was l i k e l y due to reaction with  199.  F i g . 41  T i t r a t i o n of the DCCD-binding protein with antiserum to t h i s polypeptide.  DCCD-binding protein (3.3 yg) i n 100 mM sodium borate buffer, pH 8.8, containing 2% (w/v) T r i t o n X-100 was immobilized onto p o l y l y s i n e coated m i c r o t i t r e plate w e l l s . Non-specific binding s i t e s were quenched with RIA buffer and the immobilized DCCD-binding protein was t i t r a t e d with s e r i a l d i l u t i o n s of antiserum to the DCCD-binding protein (•-•) or preimmune serum (O-O). The extent of binding of the rabbit antiserum was measured with I - l a b e l l e d goat a n t i - r a b b i t immunoglobulin as described i n MATERIALS AND METHODS. 1 2 5  200. Fig.  42  I n h i b i t i o n of antibody binding to immobilized DCCD-binding p r o t e i n by membrane v e s i c l e s of 15. c o l i , PS3 and rat l i v e r mitochondria, and by phospholipid v e s i c l e s .  Urea-stripped everted v e s i c l e s were suspended i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM MgCl and 10% (v/v) g l y c e r o l . "Rightside out" v e s i c l e s were suspended i n 0.4 M sucrose containing 10 mM MgCl . The phospholipids were taken up i n 100 mM sodium borate b u f f e r , pH 8.8 containing 2% (w/v) T r i t o n X-100. DCCD-binding p r o t e i n (3.3 yg protein) was immobilized to polylysine-coated m i c r o t i t r e plate w e l l s . I n h i b i t i o n of antibody binding to the immobilized polypeptide by various amounts of membrane v e s i c l e s or phospholipid v e s i c l e s was measured by the "competitive i n h i b i t i o n assay" as described i n MATERIALS AND METHODS. PANEL A: (•-•), urea-stripped everted v e s i c l e s of E. c o l i WSI; (O-O), r i g h t - s i d e out v e s i c l e s of E_. c o l i WSI. PANEL B: (•-•), urea-stripped everted v e s i c l e s of 15. c o l i WSI; (O-O), urea-stripped everted v e s i c l e s of PS3; (•-•), phosphate-washed mitochondrial inner membranes; ( ), sonicated phosphate-washed mitochondrial inner membranes; (•-•), ureastripped phosphate-washed mitochondrial inner membanes. PANEL C: (•-•) urea-stripped everted v e s i c l e s of E. c o l i WSI; ( A - A ) , soybean phosphatidylcholine vesicles; (O-O), egg yolk phosphatidylcholine; (•-•), synthetic phosphatidylcholine ( l - p a l m i t y l - 2 - o l e o y l phosphatidylcholine). Concentration of the everted membrane v e s i c l e s i s expressed i n yg protein/ml whereas the phospholipid concentration i s expressed as yg/ml (w/v). 2  2  A - A  202.  re-oriented ATPase complexes (210-212) or to a contamination  of the r i g h t -  side out v e s i c l e s with a population of inside-out v e s i c l e s . In contrast to the results obtained with E. c o l i WSI and ML308-225, 50% i n h i b i t i o n of binding by the urea-stripped v e s i c l e s of the unc mutants E_. c o l i DL-54, N  l 4 4  and AN382 occurred at a concentration of 19 yg  everted v e s i c l e protein per ml.  In addition, 50% i n h i b i t i o n of binding by  right-side out v e s i c l e s of E_. c o l i DL-54 and  were at 24 and 19 yg  membrane protein per ml, respectively. The cause(s) of this difference i n the value of the 50% i n h i b i t i o n of binding by everted and r i g h t - s i d e out vesicles of the wild-type and mutant strains of E. c o l i i s not known. However, Cox et a l . (140) have proposed, on the basis of genetic studies, that the a and /or B subunits of ECF.. were needed f o r the proper / 1 assembly of a functional F Q .  In i t s (their) absence, the ( F Q polypeptide)  DCCD-binding protein was suggested to insert randomly into the membrane. Therefore, i f the DCCD-binding protein does exist as a "loop" i n the membrane, then i n a random i n s e r t i o n , one would expect that 50% of the antigenic determinant normally exposed on the cytoplasmic surface to be also exposed on the external surface of the c e l l .  In this event, d i f f e r e n -  t i a t i o n between "right-side" out and "inside-out" v e s i c l e s would not be possible and the 50% i n h i b i t i o n of antibody-binding by both types of vesicles should be i d e n t i c a l . The ATPase complex of the thermophilic bacterium PS3 has been well characterized (4).  In spite of considerable homology between the DCCD-  binding proteins of IS. c o l i and PS3 (146), the antibody reacted only weakly with everted membrane v e s i c l e s of PS3 ( F i g . 42, panel B).  Everted inner  mitochondrial membrane v e s i c l e s (submitochondrial p a r t i c l e s ) from rat l i v e r , or p u r i f i e d mitochondrial membranes from which MF, had been stripped, d i d  203.  not react with the antibody to the E_. c o l i DCCD-binding protein, at least at the concentration of antigen used ( F i g . 42, panel B). I t would appear that a very high concentration of membrane vesicles (E_. c o l i , PS3 or mitochondrial membranes) could result i n the trapping or non-specific i n t e r action of antibody with the v e s i c l e s .  As shown i n F i g . 42 (panel C), at  extremely high concentrations (greater than 1.8 mg per ml) of phospholipids, a s i g n i f i c a n t amount of antibody was bound by the phospholipid v e s i c l e s .  REACTION SITE(S) FOR THE ANTIBODY ON THE DCCD-BINDING PROTEIN The antiserum to the DCCD-binding protein could bind to the p u r i f i e d DCCD-binding protein.  Therefore, attempts were made to determine the  reaction s i t e ( s ) f o r the antibody on this polypeptide.  The DCCD-binding  protein was modified with several group-specific reagents (213).  The  modified polypeptide was then used i n the competitive i n h i b i t i o n assay. The immobilized antigen i n the m i c r o t i t r e wells was unmodified DCCD-binding protein. Cleavage of the polypeptide at methionyl residues with cyanogen bromide, or oxidation of methionyl residues with performic acid to greater than 95% (Table 13) (184,213), resulted i n a reduction i n the a f f i n i t y of the modified polypeptide f o r the antibody by almost two orders of magnitude ( F i g . 43). The polypeptides modified by these two reagents were soluble i n borate-Triton buffer only up to a l e v e l of 90-100 ug protein per ml. Although the extent of cleavage of the DCCD-binding protein by TPCK-trypsin or Staphylococcus  aureus Vg protease was not known, the a f f i n i t y of the  protease-treated polypeptide was also reduced  3-fold.  Modifiction of the  arginyl residue(s) of the DCCD-binding protein with phenylglyoxal or 2,3-butanedione had no e f f e c t on i t s reaction with the antibody.  Similarly,  20A.  6]  log  F i g . 43  Protein  I n h i b i t i o n of antibody binding to immobilized DCCD-binding p r o t e i n by protease-treated or chemically-modified DCCD-binding p r o t e i n .  The protease-treated or chemically-modified DCCD-binding p r o t e i n was suspended i n 100 mM sodium borate buffer, pH 8.8, containing 2% (w/v) T r i t o n X-100. DCCD-binding protein (3.3 yg) was immobilized to p o l y l y s i n e coated m i c r o t i t r e plate wells. I n h i b i t i o n of antibody binding to the immobilized polypeptide by various amounts of protease-treated or chemicallymodified DCCD-binding protein was measured by the "competitive i n h i b i t i o n assay", as described i n MATERIALS AND METHODS. PANEL A: ( X - X ) , untreated DCCD-binding p r o t e i n ; (•-•), 2,3-butanedipne-treated DCCD-binding p r o t e i n ; (O-O), phenylglyoxal-treated DCCD-binding protein; (•-•), performic acid-treated DCCD-binding protein; ( ) , antibody replaced i n (control) experiments by preimmune serum. PANEL B: (•-•), untreated DCCD-binding p r o t e i n ; (O-O), TPCK-trypsin-treated DCCD-binding protein; (*-*), VQprotease-treated DCCD-binding protein; (•-•), cyanogen bromide-treated DCCD-binding p r o t e i n . A _ A  205.  treatment  o f t h e DCCD-binding p r o t e i n w i t h c h l o r a m i n e  T o r hydrogen  peroxide a l s o d i d not a f f e c t i t s a b i l i t y t o bind the antibody  ( n o t shown).  But the e x t e n t of m o d i f i c a t i o n of t h e DCCD-binding p r o t e i n by  these  r e a g e n t s was not known. I n c o n t r a s t t o t h e e f f e c t of t r y p s i n and Vg p r o t e a s e on the a b i l i t y v  v  o f t h e p u r i f i e d DCCD-binding p r o t e i n t o r e a c t w i t h t h e a n t i b o d y ,  urea-  s t r i p p e d v e s i c l e s t r e a t e d w i t h t r y p s i n o r Vg p r o t e a s e d i d not cause a r e d u c t i o n i n the a f f i n i t y f o r the antibody  ( F i g . .44). S i m i l a r r e s u l t s were  o b t a i n e d w i t h u r e a - s t r i p p e d v e s i c l e s w h i c h had been t r e a t e d w i t h p h e n y l g l y o x a l o r Chloramine T (not shown).  BINDING OF ECFj^ BY PURIFIED DCCD-BINDING PROTEIN The a b i l i t y o f u r e a - s t r i p p e d v e s i c l e s o f the unc mutant E_. c o l i ^I44»  w n  * h presumably c o n t a i n s o n l y t h e DCCD-binding p r o t e i n i n t h e c  FQ  complex, suggested t h a t t h i s p o l y p e p t i d e may be i n v o l v e d i n t h e b i n d i n g of ECF^  Indeed, ECF.^ c o u l d be bound by t h e p u r i f i e d DCCD-binding p r o t e i n . T h i s was shown i n two ways.  immobilized  I n t h e f i r s t method, DCCD-binding p r o t e i n  i n m i c r o t i t r e p l a t e w e l l s ( " f i x e d a n t i g e n " ) was r e a c t e d w i t h  ECF^ ("free a n t i g e n " ) . of anti-ECF^ measured w i t h  serum. 1 2 s  Bound ECF^ was t i t r a t e d w i t h v a r i o u s d i l u t i o n s  The e x t e n t of- b i n d i n g of the a n t i b o d y was t h e n  I - l a b e l l e d goat a n t i - r a b b i t i m m u n o g l o b u l i n .  i s schematically represented  i n Fig. 45.)  (This assay  N o n s p e c i f i c b i n d i n g of  antiserum  t o the f i x e d a n t i g e n (and t o t h e w e l l s ) and n o n s p e c i f i c b i n d i n g o f ECF^ t o the w e l l s were c o r r e c t e d f o r by o m i t t i n g t h e ECF^ and t h e DCCD-binding p r o t e i n , r e s p e c t i v e l y . As shown i n F i g . 4 6 , p a n e l A, n o n s p e c i f i c b i n d i n g o f a n t i - E C F ^ serum was n e g l i g i b l e . ECF  S i g n i f i c a n t nonspecific binding of  c o u l d be d e t e c t e d but t h i s was much l e s s t h a n the b i n d i n g o f ECF..  206.  log  Fig.  44  Protein  I n h i b i t i o n of antibody binding to immobilized DCCD-binding p r o t e i n by protease-treated or chemically-modified everted membrane vesicles.  Urea-stripped everted membrane v e s i c l e s of E. c o l i WS1 were treated with phenylglyoxal, Staphylococcus aureus Vs protease, or TPCK-trypsin as described i n MATERIALS AND METHODS. The treated v e s i c l e s were then suspended i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM MgCl and 10% (v/v) g l y c e r o l . DCCD-binding p r o t e i n (3.3 ug) was immobilized to polylysine-coated m i c r o t i t r e p l a t e w e l l s . I n h i b i t i o n of antibody binding to the immobilized polypeptide by various amounts of protease-treated or chemically-modified v e s i c l e s was measured by the "competitive i n h i b i t i o n assay" as described i n the MATERIALS AND METHODS section. (•-•), ureastripped everted v e s i c l e s ; ( ) , phenylglyoxal-treated everted v e s i c l e s ; (A-A), V 3 protease-treated everted v e s i c l e s ; (O-O), TPCK-trypsin-treated everted v e s i c l e s . 2  A _ A  207.  • DCCD-BP  Fig.  45  12^  a  Rabbit anti-Rj  1  ? i 5  >  Goat antirabbit lg  Schematic representation of the radioimmune binding assay.  This figure shows the binding of the free antigen (F^) to the fixed antigen (DCCD-binding p r o t e i n ) . The amount of F^ bound was measured by treating the contents of the wells with antiserum to F^ and f i n a l l y with I - l a b e l l e d goat a n t i - r a b b i t immunoglobulin. See MATERIALS AND METHODS for d e t a i l s . 1 2 5  (Drawings courtesy of Dr. P . D . Bragg.)  208.  F i g . 46  Binding of ECF^ to the DCCD-binding protein.  See MATERIALS AND METHODS f o r d e t a i l s of the binding assay (Solid Phase Radioimmune Assay) PANEL A; Immobilized DCCD-binding protein (3.3 yg protein) i n the presence (•-•) or absence ( ) of ECF^ (19 yg protein) was t i t r a t e d with various d i l u t i o n s of anti-ECF^ serum. (O-O), ECF^ (19 yg protein) added but the immobilized DCCD-binding protein was omitted. PANEL B: Immobilized ECF^ (19 yg protein) i n the presence (•-•) or absence (O-O) of DCCD-binding p r o t e i n (3.3 yg protein) was t i t r a t e d with various d i l u t i o n s of anti-DCCD-binding p r o t e i n serum. ( A - A ) , DCCD-binding protein was present but immobilized ECFj_-was omitted. A _ A  209.  found i n the presence of the DCCD-binding protein.  The extent of  nonspecific binding could be decreased by lowering the concentration of ECF^ used i n the experiment.  In the second method ( F i g . 46, panel B),  the fixed antigen was p u r i f i e d ECF  1  and the binding of DCCD-binding  protein to t h i s was measured using antiserum to the polypeptide.  Again,  s i g n i f i c a n t l y more DCCD-binding protein was bound to ECF^ than i n the control. P r i o r reaction of immobilized DCCD-binding protein with ECF^ did not a f f e c t i t s subsequent reaction with antibody ( F i g . 47, panel A).  (A  s i m i l a r result was obtained f o r the reaction of immobilized ECF^ with i t s antibody ( F i g . 47, panel B).) this.  There are two possible explanations of  Either the reaction of the DCCD-binding protein with i t s antibody i s  s u f f i c i e n t l y strong to displace pre-bound ECF-j^ or the ECF^-binding  site  on the polypeptide i s separate from the binding s i t e f o r the antibody.  As  described below, our r e s u l t s favour the l a t t e r explanation.  REACTION SITE(S) ON ECFj^ FOR THE DCCD-BINDING PROTEIN The subunits of ECF protein were explored.  1  responsible f o r binding to the DCCD-binding  Treatment of ECF^ with TPCK-trypsin followed by  r e i s o l a t i o n of the enzyme on a sucrose gradient resulted i n the complete removal of the 6 and e subunits ( F i g . 48) and cleavage of the aminoterminal f i f t e e n residues of the <* subunits (68,214). also appeared to be cleaved i n our preparation.  The y subunit  The trypsin-treated ECF^  was bound by the DCCD-binding protein almost as e f f e c t i v e l y as the native enzyme ( F i g . 49, panel A).  Pronase treatment of ECF^ reduced the extent  of binding dramatically due to extensive cleavage of a l l subunits of the ECF .  Treatment of ECF.. with other proteases also reduced the extent  210.  E a  Reciprocal  F i g . 47  D i l u t i o n x 10  E f f e c t of ECF^ on the binding of anti-DCCD-binding protein serum to the DCCD-binding protein and the e f f e c t of DCCD-binding p r o t e i n on the binding of anti-ECF! serum to ECF^.  See MATERIALS AND METHODS f o r d e t a i l s of the radioimmune binding assay. PANEL A: Immobilized DCCD-binding protein (3.3 Pg protein) was t i t r a t e d with s e r i a l d i l u t i o n s of anti-DCCD-binding protein serum i n the absence (•-•) and presence (n-o) of ECF^ (19 Pg p r o t e i n ) . (*-*), ECF]^ (19 Pg protein) was added but the immobilized DCCD-binding protein was omitted. PANEL B: Immobilized ECF^ (19 Pg protein) was t i t r a t e d with s e r i a l d i l u t i o n s of anti-ECF-^ serum i n the absence (•-•) and presence (O-O) of DCCD-binding protein (3.3 pg). ( A - A ) , DCCD-binding protein (3.3 pg) was added but the immobilized ECF^ was omitted.  211.  F i g . 48  SDS-polyacrylamide  g e l electrophoresis of subunits of ECF^.  The subunits of ECF^ were prepared from E. c o l i ML308-225 as described i n MATERIALS AND METHODS. The SDS-gel consisted of 13% (w/v) acrylamide with a 4% (w/v) stacking g e l , i n the Tris-buffered system. The gel was stained with 0.1% Coomassie Blue as previously described. Lane a, purified ECFj; lane b, TPCK-trypsin treated ECFj; lane c, p u r i f i e d a subunit of ECF-^; lane d, p u r i f i e d J3 subunit of ECFj^. The migration positions of the subunits of ECF^ (a - e) are indicated.  F i g . 49  Binding of ECF^ to the DCCD-binding protein: treatment.  E f f e c t of protease  ECF^ and DCCD-binding protein were treated with various proteases and the binding assay c a r r i e d out as described i n MATERIALS AND METHODS. PANEL A: Binding of DCCD-binding protein to TPCK-trypsin treated ECF;L. Immobilized ECF ( • - • ) ( 1 9 yg protein) and TPCK-trypsin treated ECF-L (•-•) ( 1 4 yg protein) were t i t r a t e d with DCCD-binding protein. PANEL B: Binding of DCCD-binding protein to protease-treated ECF^. ECFj^ ( 1 9 yg protein) was treated with Vg protease (•-•), a-chymotrypsin (A~ ) or pronase (•-•), or not treated (•-•), and immobilized on p o l y l y s i n e coated m i c r o t i t r e plate wells and then t i t r a t e d with DCCD-binding p r o t e i n . PANEL C: Binding of ECF^ to protease-treated DCCD-binding p r o t e i n . T i t r a t i o n by ECF-j^ of immobilized DCCD-binding protein ( 3 . 3 yg) which had been treated with Vg protease ( D - D ) , a-chymotrypsin ( A - A ) , TPCKt r y p s i n (O-O), or pronase (•-•), or not treated (•-•). X  A  214.  of i t s binding of the DCCD-binding protein but the effect was not as large as that seen with pronase ( F i g . 49, panel B).  Similarly, the binding of  ECF^ to the immobilized DCCD-binding protein was almost  completely  abolished when the polypeptide was treated with pronase or TPCK-trypsin p r i o r to immobilization ( F i g . 49, panel C). The r o l e of the major subunits of ECF^ i n binding the DCCD-binding protein was investigated following the p u r i f i c a t i o n of the a and B subunits from the salt-dissociated enzyme by the procedure of Dunn and Futai (43).  The purity of these subunits i s shown i n F i g . 48.  While the  i s o l a t e d a subunit was about 100% pure, the isolated Q subunit was s t i l l contaminated  (5-10%) with some low molecular weight polypeptides.  These  isolated subunits were immobilized i n polylysine-treated m i c r o t i t r e plate wells and the extent of binding of the DCCD-binding protein measured using i t s antibody.  Both subunits bound the DCCD-binding protein e f f e c t i v e l y  (Fig., 50).  EFFECT OF CHEMICAL MODIFICATION OF THE DCCD-BINDING PROTEIN ON ITS REACTION WITH ECFj^ By contrast with i t s i n h i b i t o r y e f f e c t on the binding of antibody, performic oxidation of methionyl residues of the DCCD-binding protein had l i t t l e e f f e c t on the binding of ECF^ ( F i g . 51, panel A). However, modification of i t s a r g i n y l residues with phenylglyoxal and 2,3-butanedione reduced the a f f i n i t y of the DCCD-binding protein f o r ECF with a 1  r e l a t i v e l y small effect on the t o t a l ECF^-binding capacity of the polypeptide ( F i g . 52, panel A).  Double-reciprocal plots of binding versus  concentration gave half-saturation values (under the conditions of this experiment) of 0.26, 0.59 and 0.95 wg f o r untreated, phenylglyoxal-, and \  215.  F i g . 50  Binding of DCCD-binding p r o t e i n to subunits of ECF^.  See MATERIALS AND METHODS f o r d e t a i l s of the radioimmune binding assay. T i t r a t i o n of the immobilized ECF^ (•-•) (19 ug p r o t e i n ) , a subunit ( A — A ) (6 ug protein) and J3 subunit (O-O) (9 ug protein) by DCCD-binding p r o t e i n .  216.  F i g . 51  E f f e c t of chemical modification of the DCCD-binding protein on the binding of ECF^.  The experimental d e t a i l s are described i n the MATERIALS AND METHODS section. The following modification was made. The chemically-modified and the mock-treated DCCD-binding protein (controls) were suspended i n 90% formic acid at a p r o t e i n concentration of 0.13 mg/ml. 25 u l of t h i s mixture (3.3 Ug DCCD-binding protein) was immobilized on p o l y l y s i n e coated m i c r o t i t r e plate wells and the binding experiment c a r r i e d out as described previously. PANEL A: T i t r a t i o n of the immobilized mock-treated (•-•) or performic acid-treated ( ~ ) DCCD-binding protein (3.3. ug protein) with ECF^. PANEL B: T i t r a t i o n of the immobilized mock-treated (•-•) or cyanogen bromide-treated (O-O) DCCD-binding protein (3.3 ug protein) with ECFj. A  A  Fig.  52  Effect of modification of the a r g i n y l residue(s) of the DCCD-binding p r o t e i n on the binding of ECFj.  D e t a i l s of the experiment are described i n MATERIALS AND METHODS. PANEL A: T i t r a t i o n of immobilized ECFj (19 yg protein) with mocktreated ( • - • ) , phenylglyoxal-treated (O-O) and 2,3-butanedione-treated ( A - A ) DCCD-binding p r o t e i n . PANEL B: The above data analyzed as a Lineweaver-Burk p l o t .  218.  219.  2,3-butanedione-treated B).  DCCD-binding protein, respectively ( F i g . 52, panel  This suggests that ECF  1  binds to the polypeptide i n the region of  i t s a r g i n y l residues and i s supported by the finding that the binding of ECF^ to trypsin-treated DCCD-binding protein was s i g n i f i c a n t l y (Fig.  49C).  reduced  These a r g i n y l residues are located i n the central polar region  (residues 41 to 50) ( F i g . 55) of the DCCD-binding polypeptide molecule. This region should remain i n t a c t when the polypeptide i s cleaved by cyanogen bromide at methionyl residues 17 and 57.  As shown i n F i g . 51 (panel B),  the DCCD-binding protein had increased a f f i n i t y f o r ECF following 1  cleavage by cyanogen bromide.  Half-saturation of the binding s i t e s on the  untreated and cleaved polypeptide under the conditions of the experiments were given by 0.55 and 0.2 ug ECF.^, respectively. The  ECF^binding  capacities of the polypeptides were s i m i l a r .  EFFECT OF PHENYLGLYOXAL ON THE BINDING OF ECF^ TO UREA-STRIPPED EVERTED VESICLES Since i n v i t r o binding of ECF^ to the DCCD-binding protein was reduced by treatment of the polypeptide with phenylglyoxal, i t was of i n t e r e s t to determine the e f f e c t on intact v e s i c l e s .  The e f f e c t of  phenylglyoxal on the binding of ECF^ to urea-stripped vesicles was examined as follows.  Stripped everted v e s i c l e s of E_. c o l i WS1 and N-j-^  were treated with phenylglyoxal and then washed to remove any unreacted phenylglyoxal.  Various amounts of ECF^ were added to the treated v e s i c l e s  and the extent of binding of ECF^ was measured by the increase i n ATPase a c t i v i t y of the v e s i c l e s .  Phenylglyoxal inhibited the binding of ECF.^ to  the stripped membranes of the wild-type (E_. c o l i WS1) and the mutant (E_. coli N  ) strains ( F i g . 53).  220.  ECF., - -  Fig.  53  ug  B i n d i n g o f ECF^ t o p h e n y l g l y o x a l - t r e a t e d v e s i c l e s .  U r e a - s t r i p p e d v e s i c l e s o f E_. c o l i WSI and were t r e a t e d w i t h p h e n y l g l y o x a l a s d e s c r i b e d i n MATERIALS AND METHODS. R e c o n s t i t u t i o n w i t h ECF-L was c a r r i e d o u t as f o l l o w s : 2 mg samples o f t h e t r e a t e d membrane v e s i c l e s a t 10 mg p r o t e i n p e r m l i n 50 mM HEPES-KOH b u f f e r , pH 7.5, c o n t a i n i n g 10 mM M g C l and 10% ( v / v ) g l y c e r o l were i n c u b a t e d w i t h v a r i o u s l e v e l s o f ECF-L a t 4°C f o r 45 m i n i n a f i n a l volume o f 0.5 m l . The m i x t u r e was t h e n d i l u t e d 8-10 f o l d w i t h b u f f e r and t h e membranes sedimented a t 250 000 xg f o r 2.5 h. The membranes were resuspended i n b u f f e r and t h e ATPase a c t i v i t y i n t h e r e c o n s t i t u t e d u r e a - s t r i p p e d (•-•) and p h e n y l g l y o x a l - t r e a t e d (O-O) v e s i c l e s d e t e r m i n e d as d e s c r i b e d p r e v i o u s l y . Enzyme a c t i v i t y i s e x p r e s s e d as u n i t s p e r mg p r o t e i n . 2  221.  It was possible that the effect of phenylglyoxal on the binding of ECF^ was not due to the modification of the arginyl residue(s) of the DCCD-binding protein.  In order to determine whether the arginyl residues  of the DCCD-binding protein were modified, the urea-stripped v e s i c l e s of E_. c o l i WSI were treated with [7-  111  C]phenylglyoxal.  The DCCD-binding protein  was extracted from the treated v e s i c l e s with chloroform-methanol (2:1) and precipitated with ether.  Gel electrophoresis of t h i s material gave three  polypeptide bands of molecular weights 96 000, 19 000 and 9 000. dalton polypeptide was panel A).  The 19  000  stained very intensely with Coomassie Blue ( F i g . 54,  However, more than 95% of the t o t a l r a d i o a c t i v i t y recovered from  the g e l was associated with the protein-staining peak of 9 000 daltons (DCCD-binding p r o t e i n ) . The remaining  r a d i o a c t i v i t y was coincident with  the peak of 96 000 daltons ( F i g . 54, panel B).  The product(s) of the  reaction,of phenylglyoxal with the a r g i n y l residues has been reported to be quite unstable at a l k a l i n e pHs (213).  This may  l i k e l y be the cause of the  decreased amount of r a d i o a c t i v i t y recovered from the gel following staining with Coomassie Blue ( F i g . 54, panel A).  These results suggested  that the  a r g i n y l residue(s) of the DCCD-binding protein were modified i n the stripped vesicles. Treatment of the untreated v e s i c l e s with chloroform-methanol  (2:1)  does not normally result i n the extraction of the 19 000 dalton polypeptide. Only the DCCD-binding protein i s extracted from the membrane.  Therefore,  the extraction of a 19. 000 dalton polypeptide from the phenylglyoxal-treated v e s i c l e s suggested that the reaction of the arginyl residue(s) of t h i s polypeptide with phenylglyoxal caused the polypeptide to become quite non-polar.  This resulted i n i t s extraction from the membrane with  chloroform-methanol (2:1) ( F i g . 54B).  222.  F i g . 54  SDS-polyacrylamide g e l electrophoresis of the DCCD-binding protein of E. c o l i l a b e l l e d with [7- "C]phenylglyoxal. l  1.25 ml of urea-stripped everted v e s i c l e s of 15. c o l i WS1 (13.5 mg protein per ml) i n 50 mM HEPES-KOH buffer, pH 7.5, containing 10 mM MgCl and 10% (v/v) g l y c e r o l was mixed with 15 u l of 16.45 mM [7- "C]phenylglyoxal ( s p e c i f i c a c t i v i t y , 15.1 mCi/mmol) ( f i n a l concentration, 195 uM). The reaction mixture was incubated at 20°C f o r 2 h a f t e r which the phenylglyoxal concentration was increased to 34.1 mM with the non-radioactive reagent. The mixture was incubated f o r another 60 min. The reaction was stopped by a d d i t i o n of 1 volume of 100 mM arginine hydrochloride i n buffer. After incubation f o r another 30 min at 20°C, the reaction mixture was d i l u t e d 10-15 f o l d with buffer and then centrifuged at 250 000 xg f o r 2.5 h. The sedimented v e s i c l e s were resuspended i n d i s t i l l e d water (10 ml) and the DCCD-binding p r o t e i n extracted with chloroform-methanol (2:1) as described i n MATERIALS AND METHODS. The ether p r e c i p i t a t e containing the DCCD-binding p r o t e i n was subjected to electrophoresis on an SDS-urea (8M) g e l . The g e l was fixed with 50% (w/v) TCA f o r 4-6 h at 20°C, stained with 0.1% (w/v) Coomassie Blue, s l i c e d into 1 mm segments and the r a d i o a c t i v i t y of each s l i c e determined. PANEL A: Coomassie Blue-stained g e l of the [7- C]phenylglyoxal l a b e l l e d proteins. The migration positions of the molecular weight marker proteins (M , 94 000 - 14 400), the 19 000 dalton polypeptide, the DCCD-binding protein (DBP) and the bromophenol blue (BP) tracking dye are indicated. PANEL B: D i s t r i b u t i o n of the r a d i o a c t i v i t y of i d e n t i c a l gels which had been cut into 1 mm s l i c e s immediately a f t e r f i x i n g i n 50% (w/v) TCA ( A - A ) or a f t e r s t a i n i n g with Coomassie Blue (•-•). BP indicates the p o s i t i o n of the bromophenol blue tracking dye. The migration positions of the molecular weight marker proteins (M , 94 000 - 14 400) are also shown. 2  1  1H  r  r  223.  67  K—•  43  K—•  30K—• 20.1  K—•  ll «-19  K  14.4K—• - «-DBP « • BP  224.  Furthermore, the absence of r a d i o a c t i v i t y associated with the 19 000 dalton polypeptide indicated the reactive a r g i n y l residue(s) on this polypeptide had a low a f f i n i t y f o r phenylglyoxal such that reaction occurred only at a high concentration of phenylglyoxal (34 mM cold phenylglyoxal). Labelling with phenylglyoxal was also suggested  by the brownish-yellow  polypeptide band (19 000 dalton) observed when the gel was fixed with 50% TCA p r i o r to staining with Coomassie Blue.  I t has been reported (215) that  the reaction of the a r g i n y l residue(s) of a polypeptide with phenylglyoxal results i n an orange-brown product.  Therefore, the reactive a r g i n y l  residue(s) of the DCCD-binding protein must have a higher a f f i n i t y f o r phenylglyoxal.  225.  DISCUSSION .  PURIFICATION OF THE  ECFJFQCOMPLEX  Attempts to purify the  ECF^FQ  graphy only were not successful. with the ATPase a c t i v i t y .  complex by g e l f i l t r a t i o n chromatoThe majority of the proteins coeluted  In addition, the a c t i v i t y was eluted as a broad  peak suggesting that the ATPase complexes were of d i f f e r e n t molecular weights (Table 11).  The enzyme eluted as larger aggregates (M , 680  000  - 890 000) i n the presence of non-ionic detergents than i n the presence of i o n i c detergents (M  r>  450 000 - 580 000).  This difference i n the  molecular weight of the s o l u b i l i z e d enzyme was l i k e l y due to the assoc i a t i o n of the detergent molecules with the membrane proteins to form lipoprotein-detergent complexes as suggested by Tanford and Reynolds (216).  Therefore, the size of the eluted enzyme and the broadness of the  a c t i v i t y peak w i l l depend on the extent of association of the detergent molecules with the  ECF^FQ  complex and on the CMC of the detergent.  In the present study, the best p u r i f i c a t i o n of the  complex  ECF^FQ  was achieved by chromatography on Phenyl-Sepharose CL-4B followed by sedimentation of the enzyme at 250 000 xg f o r 16-17 h. was judged to be an intact  ECF^FQ  complex.  The p u r i f i e d enzyme  This was determined by the  s e n s i t i v i t y of i t s a c t i v i t y to i n h i b i t i o n by DCCD ( F i g . 14) and l a b e l l i n g studies involving [ "C]-iodoacetic acid and x  fluorescein.  The p u r i f i e d  ECF^FQ  through  5-iodoacetamido-  complex consisted of eleven major  polypeptides of molecular weight 56 000, 52 000, 32 000, 30 000, 28 22 000, 18 000, 14 000, 12 000, 9 000 and 7 500. 000 and 24 000 were also present.  000,  Minor bands of 85 000,  71  The polypeptides with molecular weights  of 56 000 («), 52 000 (B), 32 000 ( Y ) , 22 000 (6), and 12 000  (e)  226.  were subunits of ECF^  Labelling studies with ['"CJDCCD revealed that  the polypeptide of 9 000 daltons was the DCCD-binding protein.  The  polypeptides of molecular weights 28 000, 18 000 and 14 000 may  also be  subunits of F Q . The i d e n t i f i c a t i o n of some of these polypeptides as subunits of FQ was made by comparison with the p u r i f i c a t i o n of others. The subunit composition of FQ obtained i n the present study and those obtained by other workers i s summarized i n Table 15.  The DCCD-binding  protein ( 8 - 9 000 daltons) and the 18-19 000 dalton polypeptides are c l e a r l y subunits of F Q . However, i t was not clear whether the 28 000, 24 000 and 14 000 dalton subunits were polypeptides of F Q . Genetic and DNA ECF^FQ  (a),  50  sequencing evidence  (42,128-133)  indicates that the  complex consists of eight subunits of molecular weights 157  (J3), 34  100  (y),  30  258  (a),  19  310  ( 6 ) , 17  233  55  (b),  264 14  and 8 365 ( c ) , and are coded for by the unc genes l i s t e d i n Table 4.  (e)  194  The  polypeptides of molecular weight 30 258 (a), 17 233 (b) and 8 365 (c) are the subunits of F Q , although polypeptide a migrates with an apparent molecular weight of  24  000  on SDS-polyacrylamide  gels  Foster and  (138).  Fillingame ( 9 4 ) reported the i d e n t i c a l subunit composition to the above i n a preparation of the  ECF^FQ  complex.  This subunit composition  subsequently confirmed by F r i e d l et a l .  was  (108).  Schneider and Altendorf ( 1 0 7 ) have also p u r i f i e d the ECF.F. complex J  1  and showed that the F Q also consisted of three subunits.  0  But, i n contrast  to that found by Foster and Fillingme ( 9 4 ) , the F Q subunits had molecular weights of 28 000, 19 000 and 8 300. present i n only a minor amount.  The 24 000 dalton polypeptide was  In this respect, the  appears to be similar to that p u r i f i e d i n this thesis. DNA  ECF^FQ  complex  However, from the  sequence of the unc operon and the known anomalous migration of poly-  227.  Table 15 Subunit Composition of F Q from Various ECF^F and FQ Purifications.  Subunit composition Reference  Source  Foster and F i l l i n g a m e ( 9 4 )  F  Negrin et a l ( 9 )  i o F  F  F  o  i o F  (M  of F Q  x 10" ) 3  r  24;  19; 8 . 4  a  24;  19;  a  24;  1 9 ; 8.3  8.4  F r i e d l and Schairer ( 1 0 8 ) F  F  o  i o F  2 4 ; 1 9 ; 8.3  28;  19; 8 . 3  a  Schneiders. Altendorf ( 1 0 7 ) F  This Thesis  a. This subunit [ C] 14  DCCD  F  o  i o F  19; 1 4 ; 8.3  28; 24; 18; 14;  i d e n t i f i e d as the DCCD - binding protein using  9  a  228. peptide a on SDS gels, i t i s unlikely that the 28 000 dalton polypeptide i s a subunit of FQ.  Therefore, the question remains as to the identity of  the 28 000 dalton polypeptide. Foster and Fillingame (94) also reported that a 14 000 dalton polypepT  tide copurified with the preparation of ECF^FQ complex only from c e l l s grown i n a medium containing succinate, acetate and malate. dalton polypeptide could be the Gene  The 14 000  1 product of the unc operon (128).  The presence of a 28 000 and a 14 000 dalton subunit i n the E C F ^ complex p u r i f i e d i n the present study suggest that the former might be a dimer of the l a t t e r . and Altendorf  (107).  This p o s s i b i l i t y was f i r s t postulated by Schneider They found that urea-treatment of t h e i r p u r i f i e d  ECF^FQ complex consisting of the 28 000, 19 000 and 8 300 dalton polypeptides as the major subunits of FQ resulted i n the disappearance of the 28 000 dalton polypeptide, with a concomitant appearance of a 14 000 dalton polypeptide.  The resulting FQ preparation consisted of the 19 000, 14 000  and 8 300 dalton polypeptides. The function,of the 14 000 dalton subunit i s not known, although i t has been postulated (128) that i t might function i n regulating the assembly of the FQ polypeptides.  Schneider and Altendorf (107) also reported that  the absence of the 14 000 dalton polypeptide i n FQ preparations resulted i n a non-functional FQ complex.  However, recent genetic studies have  shown that the 14 000 dalton subunit (Gene 1 product) i s not essential f o r the biosynthesis or the a c t i v i t y of a functional ECF^FQ complex  (217).  C r i t i c a l examination of the densitometric scans of the gels of the ECF^FQ complex prepared  by other workers  (105-108) c l e a r l y revealed the  presence of variable amounts of 28 000 and 14 000 dalton polypeptides. This contrasts with the apparent stoichiometric amounts of these polypep-  - 229.  t i d e s found i n t h e p r e p a r a t i o n o f  ECF^FQ  complex d e s c r i b e d i n t h i s  thesis. The causes o f t h i s i n c o n s i s t e n c y i n t h e r e l a t i v e amounts and t y p e ( s ) of s u b u n i t s o f F Q i n t h e v a r i o u s p r e p a r a t i o n s a r e n o t known.  It is  p o s s i b l e t h a t these d i f f e r e n c e s a r e a f u n c t i o n o f the d e t a i l s of the p u r i f i c a t i o n p r o c e d u r e s (Tables 2 and 3 ) , o f t h e c e l l s t r a i n , o f t h e growth c o n d i t i o n s (94), of the oligomeric nature of the FQ polypeptides (149,155) and o f p r o t e o l y t i c d i g e s t i o n ( 9 5 ) . A l t e r n a t i v e l y , t h e s e d i f f e r e n c e s c o u l d a l s o be due t o t h e p o s s i b i l i t y that there a r e polypeptides which a r e c l o s e l y associated w i t h the  ECF^FQ  complex i n v i v o .  F o r example, t h e p o l y p e p t i d e s r e q u i r e d by t h e l o c a l i z e d  proton hypothesis  (218).  extruded  According t o t h i s hypothesis, protons a r e not  i n t o t h e e x t e r n a l medium d u r i n g membrane e n e r g i z a t i o n .  the p r o t o n s a r e l o c a l i z e d w i t h i n t h e b i l a y e r and a r e t r a n s p o r t e d s p e c i f i c channels  through  c o n s i s t i n g o f a s e r i e s o f c o n n e c t i n g p o l y p e p t i d e s between  the r e s p i r a t o r y c h a i n and t h e ATPase complex. copurify with the  Instead,  ECF^FQ  Such p o l y p e p t i d e s c o u l d  complex.  In c o n c l u s i o n , i t i s l i k e l y t h a t t h e p o l y p e p t i d e s o f m o l e c u l a r weight 28 000, 24 000, 18 000, 14 000 and 9 000 a r e s u b u n i t s o f F  Q  or are  c l o s e l y a s s o c i a t e d w i t h i t i n t h e membrane.  SOME MUTANTS OF E. c o l l DEFECTIVE I N PROTON TRANSLOCATION Removal o f ECF^ from membranes o f normal s t r a i n s o f 15. c o l i i n the leakage of protons through F Q .  As a r e s u l t , r e a c t i o n s such as t h e  quenching of f l u o r e s c e n c e o f t h e dye 9-aminoacridine energy-dependent t r a n s h y d r o g e n a t i o n  results  o f NADP  +  ( F i g . 19),or the  by NADH ( 2 3 ) , w h i c h r e q u i r e  the presence o f a transmembrane p r o t o n g r a d i e n t cannot o c c u r .  The unc  230.  mutants of E_. c o l i N-j.^ and CBT-302 were isolated as ATPase-deficient mutants (Table 7).  Although the membranes of these mutants were found to  be lacking ECF^ (Table 12), they were s t i l l capable of maintaining a transmembrane proton gradient ( F i g . 20). Several explanations f o r this are possible.  ( i ) Although intact ECF^ i s absent i n these mutants, a subunit  of the ECF^ could s t i l l be present and prevent the leakage of protons through F Q .  Mutants i n which only the J3 or 6 subunits of ECF^ are retained  by the membrane (following removal of ECF^) without i t becoming leaky have been described (219-221).  (ii) F  be absent from the mutant membranes,  Q  or the DCCD-binding protein could  ( i i i ) The aspartyl residue of the  DCCD-binding protein with which DCCD reacts s p e c i f i c a l l y to block proton translocation through F Q has been replaced (159-161).  Mutants i n which  t h i s aspartyl residue has been replaced by a g l y c y l or glutamyl residue are defective i n proton translocation (Table 6).  ( i v ) A polypeptide of F Q  other than the DCCD-binding protein and which i s involved i n the proton channel could be absent from or modified i n the mutant membranes.  These  p o s s i b i l i t i e s were investigated i n the unc mutants, E_. c o l i N^.^, CBT-302 and AN 382. Although the membranes of E_. c o l i AN 382 contain an active ECF^,  the membrane cannot be energized through the hydrolysis of ATP,  suggesting that there i s a defect i n the F Q portion of the ATPase complex. The extensive stripping procedures used to remove ECF^ and subunits of ECF^ resulted i n the loss of the a b i l i t y of the normal membranes (E_. c o l i WS1) to set up and maintain a proton gradient.  By contrast, none of  these treatments destroyed the capacity of the mutant membranes to generate and maintain a proton gradient.  Therefore, i t was unlikely that retention  of i n d i v i d u a l subunits of ECF^ on the membranes of these mutants was responsible f o r the r e l a t i v e impermeability of these membranes to protons.  /  231.  The absence of the a, B, 6 and e subunits of ECF^ on the membranes of E_. c o l i N j  4 4  and CBT-302 was confirmed by comparing t h e i r polypeptide  composition with that of the parent s t r a i n s , 15. c o l i WSI and CBT-1. The presence of a [ "C]DCCD-binding protein of 8 000 daltons i n the 1  membranes of these mutants, and the s i m i l a r i t y of the amino acid compositions to the parent strains (Table 13), indicated that the DCCD-binding protein occurred i n these three mutants and that the DCCD-reacting aspartyl Therefore, the defect(s) i n F Q responsible f o r the  residue was present.  r e l a t i v e impermeability of these three mutant membranes to protons was l i k e l y to be on the other subunits(s) of F Q (polypeptides a and b ) . Analysis of the membranes of these mutants by two-dimensional i s o e l e c t r i c focusing g e l electrophoresis revealed that the 18 000 dalton polypeptide b /  of F Q was missing i n the mutant, E. c o l i N . ^ ( F i g . 27). This d i f f e r ence was not observed  i n the membranes of the unc mutants, E_. c o l i , AN382  and CBT-302. However, changes i n t h i s polypeptide which did not a f f e c t i t s molecular weight or i t s net charge could not be excluded. Recently, F r i e d l et a l . (222) have reported the i s o l a t i o n of an unc mutant of E_. c o l i which contained only the 24 000 dalton (polypeptide a) and the DCCD-binding protein (polypeptide c) of the  ECF^FQ  complex.  The  absence of the 18 000 dalton subunit (polyeptide b) of F Q i n this mutant d r a s t i c a l l y reduced the permeabiity of the membranes to protons.  Thus, the  mutant, 15. c o l i N^^^ appears to be s i m i l a r to that isolated by F r i e d l et a l . (222).  From the arrangement of the genes of the unc operon, i t would t  appear either that E_. c o l i N-j-^ i s a deletion mutant with only the proximal unc B and E genes (coding f o r polypeptide a and c of F Q ) of the whole operon being retained or there i s a polar effect of the mutation on the expression of the d i s t a l genes of the operon.  232.  Therefore, these results suggest that the DCCD-binding protein and polypeptide a are i n s u f f i c i e n t to form a functional proton channel through the membrane.  However, i t i s not known i f polypeptide b forms part of the  proton channel or acts i n d i r e c t l y by influencing the conformation of the DCCD-binding protein and/or of polypeptide a of F Q . Fillingame et a l . (204) have recently characterized the defect i n the unc mutant E_. c o l i AN382 by genetic techniques. lesion responsible f o r r e l a t i v e impermeabiity  I t was shown that the  of the membranes to protons  i n t h i s mutant was due to an amber-suppressible,  chain-terminating mutation  i n the unc B gene that resulted i n the loss of the 24 000 dalton subunit (polypeptide a) from the FQ complex. Although the membranes of E_. c o l i CBT-302 did not contain any subunits of ECFp no change(s) i n the composition of F Q could be detected".  Thus,  the nature of the l e s i o n responsible f o r proton impermeability i n E. c o l i CBT-302 could not be characterized. Therefore, the picture which emerges from the studies on the unc mutants, E_. c o l i N^.^ and AN.382 i s that a l l three subunits of F Q (polypeptides a, b and c) are required f o r a functional proton channel and this has recently been confirmed by the results of F r i e d l et a l . (232).  This i s  also supported by the finding that an unc mutant containing only the 24 000 dalton subunit (polypeptide a) of FQ was r e l a t i v e l y impermeable to protons (140).  This picture i s complicated by the observation that another unc  mutant of E_. c o l i containing a l l three subunits of F Q (a, b and c) was also r e l a t i v e l y impermeable to protons (140).  Apparently, the presence of  a l l three subunits of FQ i s not adequate to form a functional proton channel through the membrane.  Cox et a l . (140) have suggested  that only  the proper assembly of a l l three subunits i n the membrane w i l l result i n  233.  functional proton channel and that proper assembly of F Q requires the involvement of the a and/or Q subunit of ECF^. But the presence of the a and 13 subunits i n the membranes of E_. c o l i AN382 suggests that mutations i n the-polypeptide(s) of FQ can also a f f e c t the assembly of a functional proton  channel.  The a b i l i t y of the F  of _E. c o l i AN382 to bind ECT^, although more  Q  weakly than i n the wild-type s t r a i n , i n the absence of the 24 000 dalton subunit of F Q suggests that t h i s polypeptide i s not necessary f o r the binding of ECF^ to the membrane.  This i s supported  by the finding that  the membranes of another unc mutant containing only the 24 000 dalton subunit of F  Q  was incapable of binding ECF^ (140).  Therefore, the a b i l i t y  of the isolated membranes of E_. c o l i N^.^^, containing only the 24 000 and 9 000 dalton subunits of F , to bind p u r i f i e d ECF suggests that the Q  1  DCCD-binding polypeptide i s responsible f o r binding ECF^ to the membrane. The absence of the 24 000 or the 18 000 dalton polypeptide i n the mutant affected only the a f f i n i t y , but not the capacity of the membrane for ECF^. The decreased  affinity  of the mutant membranes (half saturation of binding  for E. c o l i WSI, 0.55 mg ECF^; E. c o l i  1.10 mg ECF^) could be  explained on the basis that the other FQ subunits most l i k e l y influence the conformation  of the DCCD-binding protein.  binding protein could affect the conformation  A l t e r n a t i v e l y , the DCCDof the missing polypeptide,  thus causing i t to interact with ECF^. Therefore, the membranes of the unc mutants, E_. c o l i N^.^, AN382 and CBT-302 could s t i l l bind ECF^ but not with as high an a f f i n i t y as i n the wild-type s t r a i n (E. c o l i WSI). and N ^  44  Studies on the unc mutants,  c o l i AN382  suggest that a l l three subunits of F Q may be needed f o r optimal  ECF^-binding  a f f i n i t y and are absolutely required f o r a functional proton  234.  channel.  ORIENTATION OF THE DCCD-BINDING PROTEIN IN THE MEMBRANE An understanding of the arrangement of the polypeptides of F Q i n the membrane i s obviously an important prerequisite for, determining the mechanism of proton translocation through F Q .  The orientation of the  DCCD-binding protein i n the membrane of E_. c o l i was studied by using antiserum against t h i s polypeptide. The antiserum to the DCCD-binding protein blocked the leakage of protons through F Q i n urea-stripped everted vesicles suggesting that the DCCD-binding protein was exposed on the cytoplasmic surface of the c e l l membrane ( F i g . 28A). Competitive i n h i b i t i o n assays confirmed that the antibodies to the DCCD-binding protein reacted p r e f e r e n t i a l l y with the cytoplasmic surface of the c e l l membrane. Although the antibody was raised against the p u r i f i e d DCCD-binding protein, i t could s t i l l recognize and bind to t h i s polypeptide when i t was assembled as an oligomer i n the membrane.  The antibody i s presumably  recognizing a limited amino acid sequence i n the molecule since i t i s u n l i k e l y that the isolated polypeptide would retain i t s native oligomeric structure following extraction by chloroformmethanol  (2:1).  The amino  acid sequence of the DCCD-binding polypeptide exposed on the cytoplasmic , surface of the c e l l membrane and which i s recognized by the^antibody i s not known. However, rn v i t r o studies with p u r i f i e d DCCD-binding protein have shown that the oxidation of methionyl residues i n the isolated polypeptide almost completely abolished i t s a b i l i t y to react with the antibody. Cleavage of the polypeptide at the methionyl residues with cyanogen bromide  235.  had a s i m i l a r e f f e c t .  These results are consistent with the  binding s i t e being close to one or more methionyl residues.  antibodyIf the binding-  s i t e on the isolated polypeptide i s the same as that of the membrane-bound polypeptide, then the most l i k e l y methionyl residue i s that at p o s i t i o n 57 (Fig.  55).  This assignment depends on the v a l i d i t y of the evidence that  t y r o s y l residues 10 and 73 are on the periplasmic surface of the membrane (223).  Modification of a r g i n y l residues of the DCCD-binding protein ( i n  the isolated or membrane-bound form) with phenylglyoxal and 2,3-butanedione had l i t t l e e f f e c t on the binding of the antibody  ( F i g . 43).  Thus i t i s  u n l i k e l y that the antibody reacts with residues 41 to 50 of the polar segment of the polypeptide molecule.  On this basis, the DCCD-binding  protein molecule must have a looped arrangment i n the membrane and  the  other methionyl residues at p o s i t i o n 6, 11, 16, 17, 65 and 75 could not be close to the cytoplasmic surface of the membrane. _ The exposure of the DCCD-binding protein^on the external (periplasmic) surface of the c e l l membrane could not be unambiguously determined. Addition of valinomycin to K -loaded "right-side out" v e s i c l e s of E_. c o l i +  DL-54 resulted i n an e f f l u x of K  +  concomitantly  with a compensatory  i n f l u x of protons primarily through the ECF^-depleted FQ proteins. i n f l u x of protons was  inhibited by DCCD.  well as the rate of proton uptake was 65%.  This  The t o t a l amount of protons as  inhibited by DCCD to a maximum of  By contrast, antiserum to the DCCD-binding protein did not reduce the  t o t a l amount of protons taken up by these v e s i c l e s , but did reduce the rate of proton uptake to a maximum of 30% ( F i g . 33). The absence of an e f f e c t on the movement of the t o t a l amount of protons could be due to several causes.  ( i ) The DCCD-binding protein may  not be exposed on the external surface of the c e l l membrane.  However, the  236.  results of Schneider et a l . (223) indicate that the amino- and carboxyterminals of this polypeptide are exposed on the external surface, ( i i ) The determinants of the DCCD-binding protein on the external surface are inaccessible to the antibodies,  ( i i i ) The portion(s) of the DCCD-binding  protein exposed on the external surface may  be so weakly antigenic that the  antiserum does not,contain antibodies to them.  ( i v ) The antibodies did  bind to the portion of the molecule exposed on the external surface without a f f e c t i n g proton translocation. The e f f e c t of the antiserum to the DCCD-binding protein on the rate of proton movement could also be due to several causes.  ( i ) Antibodies to the  determinants on the DCCD-binding protein exposed on the external surface of the membrane are present.  ( i i ) There may  be a re-orientation of the ATPase  complex to the external surface during preparation of the membranes. ( i i i ) The  "right-side out" v e s i c l e s may  "inside-out" v e s i c l e s . than ( i i ) .  be contaminated with a population of  Energetically, t h i s appears to be more feasible  However, Wickner (210), Adler and Rosen (211) and Owen and  Kaback (212), have suggested that the re-orientation event occurs r e a d i l y . The extent to which t h i s occurs seems to vary between workers and perhaps i s a function of the c e l l s t r a i n and of the d e t a i l s of the procedures used to prepare the v e s i c l e s .  Thus one explanation of the above results i s that  the antibody i s reacting preferentialy with the DCCD-binding protein exposed on the cytoplasmic  surface of the c e l l membrane of E_. c o l i and that the  binding to r i g h t - s i d e out vesicles i s due to the reaction with re-orientated ATPase complexes.  Recently, van der Plas et a l . (224) reported that 31% of  the ATPase enzyme re-orientated to the external surface i n right-side out vesicles.  This would account f o r the r e s u l t s that "right-side out" vesicles  of E. c o l i WS1  and ML308-225 ( F i g . 42 A) were only one-third as e f f e c t i v e  237.  i n competing f o r the antibody to the DCCD-binding protein compared with everted v e s i c l e s . It i s l i k e l y that the problems associated with the determination  of  the antigenic s i t e s of the DCCD-binding protein which are exposed and possible re-orientation of the ATPase enzyme across the membrane may  the only  be solved by using monoclonal antibodies.  INTERACTION OF THE DCCD-BINDING PROTEIN WITH ECFj^ The amino acid sequence of the DCCD-binding protein i s suggestive of structure.  Four regions of the molecule can be recognized  (146,150).  Seven amino-terminal, predominantly polar amino acids are  followed by a non-polar region (residues 8 to 32). are predominantly polar. polar.  ( F i g . 55)  Amino acids 34 to 52  The fourth region (residues 53 to 79) i s non-  I t has been suggested by Altendorf et a l . (150) that the  two  non-polar regions of the molecule are transmembranous, forming a looped structure i n which the middle polar region i s exposed at the surface of the membrane. findings that  cytoplasmic  The looped arrangement i s supported by the  ( i ) replacement of amino a c y l residue 28 (with isoleucine or  valine, Table 6) i n the f i r s t non-polar segment influenced the reaction of DCCD with aspartyl-61 i n the other non-polar segment (162),  ( i i ) tyrosyl  residues 10 and 73 were accessible on the periplasmic (outer) surface of the c e l l membrane (223), and  ( i i i ) antiserum against the DCCD-binding  protein blocked the leakage of protons through FQ i n ECF^-stripped everted membrane v e s i c l e s ( F i g . 28A).  The a c c e s s i b i l i t y of the DCCD-binding  protein at the cytoplasmic surface suggested that i t could react with  ECF^.  In contrast to the effect of modification of amino acyl residues of the DCCD-binding protein on i t s reaction with antibodies, the e f f e c t on the  238.  F i g . 55  Amino acid sequence of the DCCD-binding protein of E_. c o l i  (146)  The amino acid sequence of the DCCD-binding protein i s drawn as a "hairpin" loop. The two nonpolar regions of the molecule are shown. DCCD i s bound to the c i r c l e d a s p a r t y l residue. The methionyl (Q) and a r g i n y l (-¥") residues of the polypeptide are also Indicated.  239.  binding of ECF^ was d i f f e r e n t .  Modification of the arginyl residues of  the DCCD-binding protein with phenylglyoxal 2,3-butanedione ( F i g . 52), or treatment  of the DCCD-binding protein with TPCK-trypsin  the binding of ECF^ to the polypeptide.  ( F i g . 49C),  reduced  However, oxidation of the  methionyl residues of the"DCCD-binding protein, or cleavage of the chain at these residues with cyanogen bromide, did not affect the reaction of with the polypeptide (Fig. 51).  Therefore, the ECF^binding  ECF^  s i t e must be  near residues 41 to 50 i n the polar segment of the polypeptide.  This s i t e  i s consistent with the "looped model" i n which this region i s proposed to be at the cytoplasmic surface of the c e l l membrane (223).  The independence  of the ECF^-binding and the antibody-binding s i t e s was confirmed  directly.  Binding of ECF^ to the DCCD-binding protein did not prevent the binding of the antibody.  The reverse was also true (Fig. 47).  However, i t was  shown ( F i g . 29) with everted membrane vesicles that the binding of antibody interfered with the rebinding of ECF^ to ECF^-stripped membranes.  The  most l i k e l y reason i s that the antibody-binding s i t e i s closer to the ECF -binding 1  s i t e i n the oligomeric form of the DCCD-binding protein  present i n the membrane (5,146).  Such conformational differences of the  DCCD-binding protein i n vivo and i n v i t r o i s also suggested  by the lower  l e v e l of i n h i b i t i o n by antiserum to the DCCD-binding protein i n the binding of ECF^ to the membranes of the unc mutants, E_. coli_ N-j-^ and CBT-302 (Fig.  29).  As discussed e a r l i e r , the polypeptides of FQ may  not be  correctly assembled i n these two mutants. The significance of the binding of ECF protein i s unclear;  1  by the isolated DCCD-binding-  Evidence for the i n vivo involvement  of the DCCD-  binding protein i n binding F^-ATPase to the membrane comes from the p u r i f i c a t i o n of the F F 1  \  n  complex from Clostridium pasteurianum (16) and  240.  from the chloroplast (225).  In both cases, the DCCD-binding protein was  the only subunit of the FQ complex.  It was mentioned previously that  a r g i n y l residues are involved i n the i n t e r a c t i o n of ECF^ with the isolated DCCD-binding protein, and they also appear to be involved i n the i n t e r a c t i o n of ECF^ with F Q i n everted v e s i c l e s .  Treatment of ECF^-stripped  everted v e s i c l e s with phenylglyoxal, an arginyl-modifying reagent, almost completely abolished the rebinding of ECF^ i n both the wild-type (E_. c o l i WSI)  and the mutant s t r a i n (E. c o l i N ^ )  ( F i g . 53).  Labelling of  stripped everted vesicles with [7- *C]phenylglyoxal modified the a r g i n y l 1  residue(s) of the DCCD-binding protein ( F i g . 54).  This suggests that the  binding of ECF^ to the isolated DCCD-binding protein may significant.  be physiologically '  Binding to the polypeptide involved the a and/or fl subunits  of ECF^ since both of these subunits could bind independently. not surprising i n view of t h e i r sequence homology (226).  This i s  However, the 6  and e subunits of ECF^ have also been implicated i n the binding of ECF^ to the membranes of E_. c o l i (6,63).  These subunits were not required f o r the  Interaction with the isolated DCCD-binding protein. Mutants have been i s o l a t e d i n which the & (unc D) or 6" (unc H) subunits are retained by the membranes i n the absence of other subunits (219-221).  The polypeptide(s)  with which the B or 6 subunits interact has not been i d e n t i f i e d , but c l e a r l y ECF^ i s able to form linkages with F Q not involving the 6 and e subunits.  Recently, Andreo et a l . (227) have concluded also that the <5  subunit of chloroplast F^ i s not absolutely required f o r binding to the membrane, but i s required to block the leakage of protons through F Q . Therefore, i t i s becoming clearer that ECF^ may not be attached to the membrane solely by a "stalk" consisting of the 6 and e subunits  (19,20).  It has been suggested instead (231), that a large portion of the ECF,  241.  molecule i s embedded i n the membrane and that the extent to which i t i s embedded i s regulated by the energy state of the c e l l . Walker et a l . (228) have proposed that ECF  1  i s linked v i a i t s <S and e  subunits to polypeptide b of F Q . The amino acid sequence of polypeptide i  b suggests that the molecule i s composed of two a - h e l i c a l regions which are anchored by the amino-terminal sequence of non-polar amino acids.  The  helices are suggested to provide a pathway f o r conduction of protons to ECF^  (132,228).  Walker et a l . (228) also quoted unpublished work of  Hoppe et a l . (229) which suggests that t h i s polypeptide of F Q i s p a r t i c u l a r l y sensitive to degradation by proteases. 40) have been obtained i n my studies.  Similar results ( F i g .  The immunoprecipitate  obtained from  the ECF^-reconstituted trypsin-treated vesicles did not contain a 18  000  dalton polypeptide indicating that i t had been removed by proteolysis. Hoppe et a l . (233) have recently confirmed that trypsin-treatment of stripped-everted vesicles of E_. c o l i results i n the proteolytic degradation of the 18 000 dalton (polypeptide b) of F . Q  In the present study, trypsin-treatment of ECF^-stripped everted vesicles of E. c o l i did not prevent the rebinding of ECF^. ECF^ was s t i l l sensitive to DCCD.  The rebound  However, even following rebinding of  ECF^, F Q was s t i l l leaky to protons.  (A somewhat similar behaviour  observed with trypsin-treated submitochondrial p a r t i c l e s (230).)  was  One  possible explanation of these data i s that i n the absence of polypeptide b, destroyed by protease treatment, the proton pathway through F Q and the 6 and e subunits of ECF^ i s disrupted.  In t h i s case, binding of ECF^ to F  could be explained by the interaction of the a and/or J3 subunits with the DCCD-binding protein.  The involvement of the DCCD-binding protein i n the  proton pathway i s supported by the fact that the antibody to this polypep-  Q  242.  tide w i l l also block proton leakage even i n the trypsin-treated v e s i c l e s (Fig. 35).  ,  In summary, the results presented i n this thesis are consistent with the lopped arrangement of the DCCD-binding protein i n the membrane, as proposed by Altendorf et a l . (150), i h which the polar central region of this molecule i s at the cytoplasmic surface of the c e l l membrane.  This  region may interact with the a and/or Q subunits of ECF^. Additional linkage(s) to the FQ may involve the i n t e r a c t i o n of the <S and e subunits of ECF  with polypeptide b of F  n  as proposed by Walker et a l . (228).  243. REFERENCES  1. Simoni, R.D. and Postma, P.W. 2. Harold, F.M. (1977)  (1975)  Ann. Rev. Biochem. 43, 523-554.  Curr. Top. Bioenerg. 6_, 83-149.  3. Haddock, B.A. and Jones, C.W.  (1977)  B a c t e r i o l . Rev. 4l_, 47-99.  4. Kagawa, Y., Sone, N., Hirata, H. and Yoshida, M. (1979) Biomemb. 11_, 39-78.  J . Bioenerg.  5. Fillingame, R.H. (1980) Ann. Rev. Biochem. _49, 1079-1113. 6. Dunn, S.D. and Heppel, L.A. (1981) 421-436.  Arch. Biochem. Biophys. 210, /  7. Bragg, P.D. (1979) i n Microbiology (Schlessinger, D., ed.), pp. 54-57, Amer. Soc. Microbiol., Washington, D.C. 8. Futai, M. and Kanazawa, H. (1980)  Curr. Top. Bioenerg. l£, 181-215.  9. Negrin, R.S., Foster, D.L., and Fillingame, R.H. (1980) Chem. 255, 5643-5648. 10. F r i e d l , P. and Schairer, H.U. (1981) 11. M i t c h e l l , P. (1961)  J. Biol.  FEBS L e t t . 128, 261-264.  Nature (London) 191, 144-148.  (i  12. Boyer, P.D., Chance, B., Ernster, L., M i t c h e l l , P., Racker, E. and Slater, E.C. (1977) Ann. Rev. Biochem. 46, 955-1026. 13. M i t c h e l l , P. (1979)  Eur. J . Biochem. 95, 1-20.  14. M i t c h e l l , P. (1976)  Biochem. Soc. Trans. 4_, 399-430.  15. Kashket, E.R. (1982)  Biochemistry 21, 5534-5538.  16. Clarke, D.J., F u l l e r , F.M. and Morris, J.G. (1979) ^8, 597-612. 17. Abrams, A., McNamara, P. and Johnson, F.B. (1960) 3659-3662. 18. Voelz, H. (1964)  Eur. J . Biochem. J . B i o l . Chem. 253,  J . B a c t e r i o l . J56, 1196-1198.  19. Openheim, J.D. and Salton, M.J.R. (1973) 297-322. 20. Hinkle, P.C. and McCarty, R.E. (1978)  Biochim. Biophys. Acta 298,  S c i . Amer. 238, 104-123.  244.  21.  Kanner, B.I., Nelson; N. and Gutnick, D.L. (1975) Acta 396, 347-359.  22.  Kobayashi, H. and Anraku, Y. (1972)  23.  Bragg, P.D. and Hou, C. (1972)  24.  Bragg, P.D. (1979) i n Membrane Proteins i n Energy Transduction (Capaldi, R.A., ed.) pp. 341-449, Marcel Decker, Inc., New York,  J . Biochem. 71. 387-399.  FEBS Lett.^28, 309-312.  25.  Downie, J.A., Gibson, F. and Cox, G.B. (1979) 103-131.  26.  Bragg, P.D. and Hou, C. (1973) 729-736.  27.  Rosen, B.P. (1973)  Biochim. Biophys.  N.Y.  Ann. Rev. Biochem. 4£,  Biochem. Biophys. Res. Commun. 50,  J . B a c t e r i o l . 116,  1124-1129.  28.  Altendorf, K., Harold, F.M. and Simoni, R.D. (1974) 249, 4587-4593.  J . B i o l . Chem.  29.  Boonstra, J . , Gutnick, D.L. and Kaback, H.R. 124, 1248-1255.  J. Bacteriol.  30.  Hasan, S.M. and Rosen, B.P. (1977) 225-240.  31.  Kanazawa, H. and Futai, M. (1979)  32.  Fillingame, R.H. and Wopat, A.F. (1978)  33.  Davies, P.L. and Bragg, P.D. (1972) 273-284.  34.  Laget, P.P. (1978)  35.  Paradies, H.H., Mertens, G., Schmid, R., Schneider, E. and Altendorf, K. (.1982) Biophys. J . 37, 195-197.  36.  Paradies, H.H. and Schmidt, V.D. (1979) 5257-5263. ,  37.  Satre, M. and Zaccal, G. (1979)  38.  Yoshida, M., Sone, N., Hirata, H., Kagawa, Y. and U i , N. (1979) J . B i o l . Chem. 254, 9525-9533.  39.  Biketov, S.F., Kasho, V.N., Kozlov, I.A., Mileykovskaya, Y.I., Ostrovsky, D.N., Skulachev, V.P., Tikhonora, G.V. and Tsuprun, V.L. (1982) Eur. J . Biochem. 129, 241-250.  40.  Kagawa, Y. (1982) i n Membranes and Transport (Martonosi, A.N., ed.) Vol. 1, pp. 439-446, Plenum Press, New York, N.Y.  (1975)  Biochim. Biophys. Acta 459, FEBS Lett. 105, 275-277. J . Bacteriol. 134, 687-689.  Biochim. Biophys. Acta 266,  Arch. Biochem. Biophys. 189, 122-131.  J . B i o l . Chem. 254,  FEBS Lett. 102, 244-248.  245. 41.  Bragg, P.D. and Hou, C. (1975)  Arch. Biochem. Biophys. 167, 311-321.  42.  Saraste, M., Gay, N.J., Eberle, A., Runswick, M.J. and Walker, J.E. (1981) Nucleic Acids Res. 9, 5287-5296.  43.  Bragg, P.D. (1975)  44.  Yoshida, M., Sone, N., Hirata, H. and Kagawa, Y. (1978) Biophys. Res. Commun. j?4, 117-122.  45.  Pedersen, P.L. (1975)  46.  Shavit, N. (1980)  47.  Baird, B.A. and Hammes, G.G. (1979) 31-53.  48.  Amzel, L.M. and Pedersen, P.L. (1978)  49.  Merchant, S., Shaner, S.L. and Selman, B.R. (1983). 258, 1026-1031.  50.  Harris, D.A. (1978)  51.  Maeda, M., Kobayashi, H., Futai, M. and Anraku, Y. (1976) Biophys. Res. Commun. _70, 228-234.  52.  Maeda, M., Kobayashi, H., Futai, M. and Anraku, Y. (1977) J . Biochem. 82, 311-314.  53.  Nelson, N., Kanner, B.I. and Gutnick, D.L. (1974) S c i . , U.S.A. 71_, 2720-2724.  54.  Futai, M., Sternweiss, P.C. and Heppel, L.A. (1974) Acad. S c i . , U.S.A. 71_, 2725-2729.  55.  Hanson, R.L. and Kennedy, E.P. (1973)  56.  Bragg, P.D., Davies, P.L. and Hou, C. (1973) 159, 664-670.  57.  Smith, J.B. and Sternweiss, P.C. (1975) Commun. 62, 764-771.  Biochem. Biophys. Res.  58.  Smith, J.B. and Sternweiss, P.C. (1977)  Biochemistry 16_, 306-311.  59.  Smith, J.B. and Sternweiss, P.C. (1977).  60.  Younis, H.M., Winget, G.D. and Racker, E. (1977) 1814-1818.  61.  Yoshida, M., Okamoto, H., Sone, N., Hirata, H. and Kagawa, Y. (1977) Proc. Natl. Acad. S c i . , U.S.A. 74, 936-940.'  J . Supramol. Struct. _3, 297-303. Biochem.  J . Bioenerg. 6_, 243-275.  Ann. Rev. Biochem. 49^, 111-138. Biochim. Biophys. Acta 549, J . B i o l . Chem. 252, 4743-4748. J . B i o l . Chem.  Biochim. Biophys. Acta 463, 245-273. Biochem.  Proc. Natl. Acad. Proc. Natl.  J . B a c t e r i o l . 114, 772-781. Arch. Biochem. Biophys.  Biochemistry 16_, 4020-4025. J . B i o l . Chem. 252,  246. 62.  Abrams, A., J e n s e n , C. and M o r r i s , D.H. (1976) Biochem. B i o p h y s . Res. Commun. j>9, 804-811.  63.  S t e r n w e i s s , P.C. ( 1 9 7 8 ) .  64.  L a g e t , P.P. and S m i t h , J.B. (1979) A r c h . Biochem. B i o p h y s . 197, 83-89. i S m i t h , J.B. and S t e r n w e i s s , P.C. (1982) A r c h . Biochem. B i o p h y s . 217, 376-387.  65.  J . B i o l . Chem. 253, 3123-3128.  66.  Van de S t a d t , R . J . , de B o e r , B.L. and Van Dam, K. (1973) B i o p h y s . A c t a . 292, 338-349.  67.  Asami, K., J u n t t i , K. and E r n s t e r , L. (1970) B i o c h i m . B i o p h y s . A c t a 205, 307-311.  68.  Dunn, S.D., H e p p e l , L.A. and F u l l m e r , C.S. (1980) 255, 6891-6896.  69.  L a r s o n , R. and S m i t h , J.B. (1977)  70.  Dunn, S.D. (1982)  71.  V o g e l , G. and S t e i n h a r t , R. (1976)  72.  Dunn, S.D. and F u t a i , M. (1980)  73.  Bragg, P.D. and Hou, C. (1977)  74.  Biochim.  J . B i o l . Chem.  B i o c h e m i s t r y .16, 4266-4270.  J . B i o l . Chem. 257, 7354-7359. B i o c h e m i s t r y 15_, 208-216.  J . B i o l . Chem. 255, 113-118. A r c h . Biochem. B i o p h y s . 178, 486-494.  V o g e l , G., S c h a i r e r , H.U. and S t e i n h a r t , R. (1978)  E u r . J . Biochem.  JB7, 155-160.  75.  Dunn, S.D. (1978)  Biochem. B i o p h y s . Res. Commun. J32, 596-602.  76.  Kanazawa, H., S a i t o , S. and F u t a i , M. (1978)  J . Biochem. 84,  1513-1517.  77. 78.  S a t r e , M., L u n a r d i , J . , P o u g e o i s , R. and V i g n a i s , P.V. (1979) B i o c h e m i s t r y 18_, 3134-3140. P o u g e o i s , R., S a t r e , M. and V i g n a i s , P.V. (1980)  FEBS L e t t . 117,  344-348.  79.  80.  V e r h e i j e n , J.H., Postma, P.W. and Van Dam, K. (1978) B i o p h y s . A c t a 502, 345-353. L u n a r d i , J . , S a t r e , M. and V i g n a i s , P.V. (1981)  Biochim.  B i o c h e m i s t r y 20,  473-480.  81.  Bragg, P.D., S t a n - L o t t e r , H. and Hou, C. (1981) B i o p h y s . 207, 290-299.  A r c h . Biochem.  247. 82.  Bragg, P.D., Stan-Lotter, H. and Hou, C. (1982) Biophys. 213, 669-679.  Arch. Biochem.  83.  Schatz, G., Penefsky, H.S. and Racker, E. (1967) 2552-2560.  84.  L i u , S.S., T s a i , H.L., Tsou, T.M. and Gao, F.H. (1976) Biophys. S i n . 8_, 307-317.  85.  L i u , S.S., Gao, F.H., T s a i , H.L. and Ding, Y.Z. (1982) ' Biophys. J . 37, 88-91.  86.  Futai, M., Kanazawa, H. and Takeda, K. (1980) Commun. 96, 227-234.  87.  Takeda, K., Hirano, M., Kanazawa, H., Nukiwa, N., Kagawa, Y. and Futai, M. (1982) J . Biochem. 91, 695-701.  88.  Bragg, P.D. and Hou, C. (1976) 1042-1048.  Biochem. Biophys. Res. Commun. 72,  89.  Bragg, P.D. and Hou, C. (1980)  Eur. J . Biochem. 106, 495-503.  90.  Helenius, A. and Simons, K. (1975)  91.  Helenius, A., McCaslin, D.R., F r i e s , E. and Tanford, C. (1979) i n Methods i n Enzymology (Fleischer, S. and Packer, L., eds.) V o l . 56, pp. 735-749, Academic Press, Inc., New York, N.Y.  92.  Gonenne, A. and Ernst, R. (1978)  93.  Ryrie, I . J . (1977)  94.  Foster, D.L. and Fillingame, R.H. (1979) 8230-8236.  95.  Ryrie, I . J . and Gallagher, A. (1979) 1-14.  96.  Ludwig, B., Prochaska, L. and Capaldi, R.A. (1980) 1516-1523.  Biochemistry 19,  97.  Soper, J.W., Decker, G.L. and Pedersen, P.L. (1979) ' 254, 11170-11176.  J . B i o l . Chem.  J . B i o l . Chem. 242,  Acta Biochem.  Biochem. Biophys. Res.  Biochim. Biophys. Acta 415, 29-79.  Anal. Biochem. 87, 28-38.  Arch. Biochem. Biophys. 184, 464-475.  98.  Sebald, W. (1977)  99*.  Pick, U. and Racker, E. (1979)  J . B i o l . Chem. 254,  Biochim. Biophys. Acta 545,  Biochim. Biophys. Acta 463, 1-27. J . B i o l . Chem. 254,' 2793-2799.  100.  Nelson, N., Nelson, H. and Schatz, G. (1980) U.S.A. 77_, 1361-1364.  101.  Zakharov, S.D. and Mal'yan, A.N. (1981)  Proc. Natl. Acad. S c i . ,  Biokhimiya 46, 1236-1243.  248.  102.  Sone, N., Yoshida, M., Hirata, H. and Kagawa, Y. (1975) Chem. 250, 7917-7923.  J. Biol.  10a.  Sone, N., Yoshida, M., Hirata, H. and Kagawa, Y. (1978) Acad. S c i . , U.S.A. 75_, 4219-4223.  Proc. Natl.  104.  Hare, J.F. (1975)  105.  Rosen, B.P. and Hasan, S.M. (1979)  106.  F r i e d l , P., F r i e d l , C. and Schairer, H.U. (1979) 100, 175-180.  107.  Schneider, E. and Altendorf, K. (1980)  108.  F r i e d l , P. and Schairer, U. (1981)  109.  Okamoto, H., Sone, N., Hirata, H., Yoshida, M. and Kagawa, Y. (1977) J. B i o l . Chem. 252, 6125-6131.  110.  Cohen, N.S., Lee, S.-H. and Brodie, A.F. (1978) 8_, 111-117.  111.  Leimgruber, R.M., Jensen, C. and Abrams, A. (1981) 147, 363-372.  112.  Sone, N., Yoshida, M., Hirata,- H. and Kagawa, Y. (1977) Chem. 252, 2956-2960.  J. Biol.  113.  Sone, N., Takeuchi, Y., Yoshida, M. and Ohno, K. (1977) 82_, 1751-1758.  J . Biochem.  114.  Foster, D.L., Mosher, M.E., F u t a i , M. and Fillingame, R.H. (1980) J . B i o l . Chem. 255, 12037-12041.  115.  Kanazawa, H., Miki, T., Tamura, F., Yura, T. and Futai, M. (1979) Proc. Natl. Acad. S c i . , U.S.A. 76_, 1126-1130.  116.  B u t l i n , J.D., Cox, G.B. and Gibson, F. (1971)  117.  Schairer, H.U. and Haddock, B.A. (1972) Commun. _48, 544-551.  118.  B u t l i n , J.D., Cox, G.B. and Gibson, F. (1973) 292, 366-375.  119.  Gutnick, D.L., Kanner, B.I. and Postma, P.W. (1972) Biophys. Acta 283, 217-222.  120.  Cox, G.B.', Gibson, F. and McCann, L. (1973) 1015-1021.  121.  Gibson, F. (1982)  Biochem. Biophys. Res. Commun. 66, 1329-1337. FEBS L e t t . 104, 339-342. Eur. J . Biochem.  FEBS L e t t . 116, 173-176.  FEBS L e t t . 128, 261-264.  J . Supramol. Struct. J . Bacteriol.  Biochem. J . 124, 75-81.  Biochem. Biophys. Res. Biochim. Biophys. Acta  Biochim.  Biochem. J . 134,  Proc. R. Soc. (London) 215, 1-18.  249. 122.  Ames, B.N. (1966) i n Methods i n Enzymology (Estabrook, R.W. and Pullman, M.E., ed.) V o l . 8^, pp. 115-118, Academic Press, Inc., New York, N.Y.  123.  Gibson, F., Cox, G.B., Downie, J.A. and Radik, J . (1977) 164, 193-198.  Biochem. J .  124.  Gibson, F., Cox, G.B., Downie, J.A. and Radik, J . (1977) 162, 665-670.  Biochem. J .  125.  Gibson, F., Downie, J.A., Cox, G.B. and Radik, J . (1978) J . B a c t e r i o l . 134, 728-736.  126.  Downie, J.A., Langman, L., Cox, G.B., Yanofsky, C. and Gibson, F. (1980) J . B a c t e r i o l . 143, 8-17.  127.  Downie', J.A., Cox, G.B., Langman, L., Ash, G., Becker, M. and Gibson, F. (1981) J . B a c t e r i o l . 145, 200-210.  128.  Gay, N.J. and Walker, J.E. (1981)  129.  Mabuchi, K., Kanazawa, H., Kayano, T. and Futai, M. (1981) Biophys. Res. Commn. 102, 172-179.  Biochem.  130.  Kanazawa, H., Kayano, T., Mabuchi, K. and Futai, M. (1981) Biophys. Res. Commun. 103, 604-612.  Biochem.  131.  Kanazawa, H., Mabuchi, K., Kayano, T., Noumi, T., Sekiya, T. and Futai, M. (1981) , Biochem. Biophys. Res. Commun. 103, 613-620.  132.  Nielsen, J . , Hansen, F.G., Hoppe, J . , F r i e d l , P. and von Meyenburg, K. (1981) Mol. Gen. Genet. 184, 33-39.  133.  Kanazawa, H., Kayano, T., Kiyasu, T. and Futai, M. (1982) Biophys. Res. Commun. 105, 1257-1264.  134.  Brusilow, W.S.A., Klionsky, D.J. and Simoni, R.D. (1982) J . B a c t e r i o l . 151, 1363-1371.  135.  Brusilow, W.S.A., Gunsalus, R.P., Hardeman, E . C , Decker, K.P. and Simoni, R.D. (1981) J . B i o l . Chem. 256, 3141-3144.  136.  Bridgen, J . and Walker, I.D. (1976)  137.  Anderson, S., Bankier, A., Barrel, B.G., de Bruijn, M.H.L., Coulson, A.R., Drouin, J . , Eperori^ I.C., N i e r l i c h , D.P., Roe, B.A., Sanger, F., Schreier, P.H., Smith, A.J., Staden, R. and Young, I.G. (1981) Nature (London) 290, 457-465.  138.  Steffens, K., K i l t z , H.H., Schneider, E., Schmid, R. and Altendorf, K. (1982) FEBS L e t t . 142, 151-154."  Nucleic Acids Res. 9_, 3919-3926.  Biochem.  Biochemistry 15, 792-797.  /  250. 139.  Decker, K.P., Brusilow, W.S.A., Gunsalus, R.P. and Simoni, R.D. (1982) J . B a c t e r i o l . 152, 815-821.  140.  Cox, G.B., Downie, J.A., Langman, L., Senior, A.E., Ash, G., Fayle, D.R.H. and Gibson, F. (1981) J . B a c t e r i o l . 148, 30-42.  141.  Altendorf, K. and Zitzman, W. (1975)  142.  Sebald, W., Hoppe, J . and Wachter, E. (1979) i n Function and Molecular Aspects of Biomembrane Transport ( Q u a g l i a r i e l l o , E., ed.) pp. 63-74, Elsevier/North-Holland, New York, N.Y.  143.  Beechey, R.B., Roberton, A.M., Holloway, C.T. and Knight, I.G. (1967) Biochemistry 3867-3879.  144.  Fillingame, R.H. (1975)  145.  C a t t e l l , K.J., Lindop, C.R., Knight, I.G. and Beechey, R.B. (1971) Biochem. J . 125, 169-177.  146.  Sebald, W. and Hoppe, J . (1981)  147.  Sierra, M.F. and Tzagoloff, A. (1973) 70, 3155-3159.  148.  Blondin, G.A. (1979)  J . B a c t e r i o l . 124, 870-883.  Curr. Top. Bioenerg. JL2, 1-64. Proc. Natl. Acad. S c i . , U.S.A.  Biochem. Biophys. Res. Commun. 87_, 1087-1094.  149. - Fillingame, R.H. (1976) 150.  FEBS L e t t . 59, 268-272.  J . B i o l . Chem. 251, 6630-6637.  Altendorf, K., Hammel, U., Deckers, G., K i l t z , H.-H. and Schmid, R. (1979) i n Function and Molecular Aspects of Biomembrane Transport ( Q u a g l i a r i e l l o , E., Ed.) pp. 53-61, Elsevier/North-Holland, New York, N.Y. v  151.  Nelson, N., Eytan, E., Notsani, B.-E., S i g r i s t , H., Sigrist-Nelson, K. and G i t l e r , C. (1977) Proc. Natl. Acad. S c i . , U.S.A. 74, 2375-2378.  152.  C e l i s , H. (1980)  153.  Criddle, R.S., Packer, L. and Shieh, P. (1977) S c i . , U.S.A. _74, 4306-4310.  154.  Criddle, R.S., Johnston, R., Packer, L., Shieh, P. and Konishi, T. (1979) i n Cation Flux Across Biomembranes (Mukohata, Y. and Packer, L., eds.) pp. 399-407,., Academic Press, Inc., New York, N.Y.  155.  Foster, D.L. and Fillingame, R.H. (1982) 2009-2015.  156.  Kagawa, Y., Sone, Ni, Yoshida, M., Hirata, H. and Okamoto, Hv (1976) J . Biochem. 80, 141-151.  Biochem. Biophys. Res. Commun. 92., 26-31. Proc. Natl. Acad.  J . B i o l . Chem. 257,  251.  157. ,158.  Turner, G., Imam, G. and Kuntzel, H. (1979) 565-571.  Eur. J . Biochem. 97,  Schmid, R., K i l t z , H.H., Schneider, E. and Altendorf, K. (1981) Lett. 125, 97-100.  FEBS  159.  Hoppe, J . , Schairer, H.U. and Sebald, W. (1980) 107-111.  FEBS L e t t . 109,  160.  Wachter, E., Schmid, R., Deckers, G. and Altendorf, K. (1980) L e t t . 113, 265-270.  161.  Hoppe, J . , Schairer, H.U., F r i e d l , P. and Sebald, W. (1982) Lett. 145, 21-24.  162.  Hoppe, J . , Schairer, H.U. and Sebald, W. (1980) 112, 17-24.  163.  Engelman, D.M. and Zaccai, G. (1980) 77_, 5894-5898.  164.  Bragg, P.D., Davies, P.L. and Hou, C. (1972) Commun. 47_, 1248-1255.  165.  Yoshida, M., Sone, N., Hirata, H. and Kagawa, Y. (1975) Chem. 250, 7910-7916.  166.  Singh, A.P. and Bragg, P.D. (1976) 450-461.  167.  Hirata, H., Altendorf, K. and Harold, F.M. (1974) 249, 2939-2945.  168.  Soper, J.W. and Pedersen, P.L. (1979) i n Methods i n Enzymology (Fleischer, S. and Packer, L., eds.) Vol 55, pp. 328-333, Academic Press, Inc., New York, N.Y.  169.  Rouser, G., Kritchevsky, G., Yamamoto, A., Simon, G., G a l l i , C. and Bauman, A.J. (1969) i n Methods i n Enzymology (Lowenstein, J.M., ed.) Vol 1_4, pp. 272-317, Academic Press, Inc., New York, N.Y.  170.  Penefsky, H.S. (1974)  171.  Laemmli, U.K. (1970)  172.  Ames, G.F. (1974)  173.  Cox, G.B., Downie, J.A., Fayle, D.R.H., Gibson, F. and Radik, J . (1978) J . B a c t e r i o l . 133, 287-292.  174.  Weber, K. and Osborn, M. (1969)  FEBS  FEBS  Eur. J . Biochem.  Proc. Natl. Acad. S c i . , U.S.A. Biochem. Biophys. Res. J. Biol.  Biochim. Biophys. Acta 423, J . B i o l . Chem.  J . B i o l . Chem. 249, 3579-3585. Nature (London) 227, 680-685.  J . B i o l . Chem. 249, 634-644.  J . B i o l . Chem. 244, 4406-4412.  252. 175.  M e r r l l , C.R., Switzer, R.C. and Van Keuren, M.L. (1979) Acad. S c i . , U.S.A. 76., 4335-4339.  Proc. Natl.  176.  O'Farrell, P;H. (1975)  177.  Nowotny, A. (1979) Basic Exercises i n Immunochemistry, pp. 1-3, Springer-Verlag, New York, N.Y.  178.  Bjerrum, O.J. and Lundhal, P. (1974) 69-80.  179.  Mayer, R.J. and Walker, J.H. (1980) Immunochemical Methods i n B i o l o g i c a l Sciences: Enzymes and Proteins, pp. 131-132, Academic Press, Inc., New York, N.Y.  180.  Fairbanks, G., Steck, T.L. and Wallach, D.F.H. (1971) 10, 2606-2617.  181.  Cuatrecasas, P.J. and Anfinsen, C.B. (1971) i n Methods i n Enzymology (Jakoby, W.B., ed.) V o l . 22, pp. 345-378, Acadmic Press, Inc., New York, N.Y.  182.  Moore, S. and Stein, W.H. (1963) i n Methods i n Enzymology (Colowick, S.P. and Kaplan, N.O., eds.) V o l . 6, pp. 819-831, Academic Press, Inc., New York, N.Y.  183.  Sebald, W., Machleidt, W. and Wachter, E. (1980) S c i . , U.S.A. 77, 785-789.  184.  Hirs, C.H.W. (1967) i n Methods i n Enzymology (Hirs, C.H.W., ed.) Vol 11, pp. 59-62, Academic Pres, Inc., New York, N.Y.  185.  Guide to the Care and Use of Experimental Animals (1980) (Canadian Council on Animal Care, ed.) V o l . 1_, Ottawa, Ont.  186.  Herbert, W.J. (19.78) i n Handbook of Experimental Immunology, (Weir, H., ed.), 3rd ed. , V o l . _3_, Appen. 4.16-4.17, Blackwell S c i e n t i f i c Publications, Oxford, Great B r i t a i n .  187.  Garvey, J.S., Cremer, N.E. and Sussdorf, D.H. (1977) Methods i n Immunology, 3rd ed., pp. 36-38, W.A. Benjamin, Inc., Reading, M.A.  188.  Weeke, B. (1973)  189.  Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J . B i o l . Chem. 193, 265-275.  190.  Bradford, M.M. (1976)  191.  Singh, A.P. and Bragg, P.D. (1976)  192.  Rottenberg, H. and Lee, C P . (1975)  J . B i o l . Chem. 250, 4007-4021.  Biochim. Biophys. Acta 342,  Biochemistry  Proc. Natl. Acad.  Scand. J . Immunol. V o l . 2_, Suppl. 1, 15-35.  Anal. Biochem. 72., 248-254. Eur. J . Biochem. 67, 177-186. Biochemistry 14, 2675-2680.  253.  193.  Pudek, M.R. (1976)  Ph.D. Thesis, University of B r i t i s h Columbia.  194.  Beers, R.F. and Sizer, I.W. (1952)  195.  MacKenzie, D. and Molday, R.S. (1982)  196.  Roisin, M-P. and Kepes, A. (1973) 249-259.  Biochim. Biophys. Acta 305,  197.  Carreira, J . and Munoz, E. (1975)  Mol. C e l l . Biochem. 9_, 85-95.  198.  Bragg, P.D. and Hou, C. (1976)  199.  Ritz-Gold, C.J. and Brodie, A.F. (1979) 18-26.  200.  Huberman, M. and Salton, M.R.J. (1979) 230-240. -  201.  K i t a , K., Yamato, I. and Anraku, Y. (1978) 8910-8915^.  202.  Yon, R.J. (1978)  203.  Dickie, P. and Weiner, J.H. (1979)  204.  Fillingame, R.H., Mosher, M.E., Negrin, R.S. and Peters, L.K. (1983) J. B i o l . Chem. 258, 604-609.  205.  Altendorf, K., Harold, F.M., and Simoni, R.D. (1974) 249, 4587-4593.  206.  Kagawa, Y. and Racker, E. (1971)  207.  Racker, E. (1973)  208.  Ohta, S., Tsuboi, M., Yoshida, M., and Kagawa, Y. (1980) Biochemistry 19_, 2160-2165.  209.  Dunn, S.D. (1980)  210.  Wickner, W. (1976)  211.  Adler, L.W. and Rosen, B.P. (1977)  212.  Owen, P. and Kaback, H.R. (1979)  213.  Means, G.E. and Feeney, R.E. (1971) Chemical Modification of Proteins, pp. 195-197, Holden-Day, San Francisco, C.A.  214.  Bragg, P.D. and Hou, C. (1978)  215.  Takahashi, K. (1968)  J . B i o l . Chem. 195, 133-150. J . B i o l . Chem. 257, 7100-7105.  Arch. Biochem. Biophys. 174, 553-561. Biochim. Biophys. Acta 547,  Biochim. Biophys. Acta 547,  J . B i o l . Chem. 253,  Int. J . Biochem. j), 373-379. Can. J . Biochem. 57, 813-821.  J." B i o l . Chem.  J . B i o l . Chem. 246, 5477-5487.  Biochem. Biophys. Res. Commun. 55_, 224-230.  J . B i o l . Chem. 255, 11857-11860. J . B a c t e r i o l . 127, 16,2-167. J . B a c t e r i o l . 129, 959-966.  Biochemistry 18, 1422-1426.  Can. J . Biochem. _56, 559-564.  J . B i o l . Chem. 243, 6171-6179.  254. 216.  Tanford, C. and Reynolds, J.A. (1976) 133-170.  Biochim. Biophys. Acta 457,  217.  von Meyenburg, K., Jorgensen, B.B., Nielsen, J . and Hansen, F.G. (1982) Mol. Gen. Genet. 188, 240-248.  218.  Srere, P.A. (1982)  219.  Senior, A.E., Fayle, D.R.H., Downie, J.A., Gibson, F. and Cox, G.B. (1979) Biochem. J . 180, 111-118.  220.  Humbert, R., Brusilow, W..A., Gunsalus, R.P., Klionsky, D.J. and Simoni, R.D. (1983) J . B a c t e r i o l . 153, 416-422.  221.  Noumi, T. and Kanazawa,, H. (1983) I l l , 143-149.  222.  F r i e d l , P., Bienhaus, G., Hoppe, J . and Schairer, U. (1981) Natl. Acad. S c i . , U.S.A. 78, 6643-6646.  223.  Schneider, E., Schmid, R., Deckers, G., Steffens, K., K i l t z , H.H. and Altendorf, K. (1981) i n V e c t o r i a l Reactions i n Electron and Ion Transport i n Mitochondria and Bacteria (Palmieri, F., ed.) pp. 231-234, Elsevier/North-Holland, New York, N.Y.  224.  van der Plas, J . , Hellingwerf, K.J., Seijen, H.G., Guest, J.R.,' Weiner, J.H. and Konings, W.N. (1983) J . B a c t e r i o l . 153, 1027-1037.  225.  Suss, K.-H. and Manteuffel, R. (1983)  226.  Walker, J.E., Eberle, A., Gay, N.J., Runswick, M.J. and Saraste, M. (1982) Biochem. Soc. Trans. jLO, 203-206.  227.  Andreo, C.S., Patrie, W.J. and McCarty, R.E. (1982) 257, 9968-9975.  228.  Walker, J.E., Saraste, M. and Gay, N.J. (1982)  229.  Hoppe, J . et a l .  230.  Pedersen, P.L., Hullihen, J . and Wehrle, J.P. (1981) 256, 1362-1369.  Trends Biochem. S c i . _7, 375-378.  Biochem. Biophys. Res. Commun.  Proc.  FEBS L e t t . 153, 134-140.  J . B i o l . Chem.  Nature 298, 867-869.  Eur. Bioenerget. Conf. Rep. (1982) 2_, 85-86. J . B i o l . Chem.  231.  Kozlov, I.A. and Skulachev, V.P. (1977) >29-89.  Biochim. Biophys. Acta 463,  232.  F r i e d l , P., Hoppe, J . , Gunsalus, R.P., Michelsen, 0., von Meyenburg, K. and Schairer, H.U. (1983) EMBO. J . 2, 99-103.  233.  Hoppe, J . , F r i e d l , P., Schairer, H.U., Sebald, W., von Meyenburg, K. and Jorgensen, B.B. (1983) EMBO. J . 2_, 105-110.  

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