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Studies on the pyridine nucleotide transhydrogenase of Escherichia coli Homyk, Mona 1981

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STUDIES ON THE PYRIDINE NUCLEOTIDE TRANSHYDROGENASE OF ESCHERICHIA CQLI by MONA HOMYK M.Sc, A i n Shams U n i v e r s i t y , 1971 THESIS SUBMITTED IN PARTIAL FULFILMENT THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY -i n ( THE DEPARTMENT OF BIOCHEMISTRY) THE FACULTY OF GRADUATE STUDIES We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA January, 1981 <gj Mona Homyk, 1981 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e a t t h e U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and stu d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f ~cS\ ooft-<TctsyST f e - s T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date ^ ^ Q ^ \ ^ i. ABSTRACT P y r i d i n e n u c l e o t i d e transhydrogenase c a t a l y z e s the r e v e r s i b l e t r a n s f e r of hydride i o n e q u i v a l e n t s between NADP(H) and NAD(H). In t h i s study, the a c t i v i t y of the enzyme was measured by f o l l o w i n g the r a t e of r e d u c t i o n of + + + an analogue of NAD , 3-acetylpyridxne-NAD (APNAD ) by NADPH. The enzyme was s o l u b i l i z e d by d e t e r g e n t s such as l y s o l e c i t h i n , sodium c h o l a t e ( i n the presence of ammonium sulphate) or T r i t o n X-100. The molecular s i z e of the s o l u b i l i z e d enzyme was examined u s i n g sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n the presence of B r i j 58. These de t e r g e n t s gave s o l u b l e fragments of d i f f e r e n t s i z e s . That s o l u b i l i z e d by T r i t o n X-100 or sodium c h o l a t e ( i n the presence of ammonium sulphate) e x i s t e d as l a r g e aggregates w i t h sedimentation c o e f f i c i e n t s of 24.5 t o 25.4S, whereas t h a t obtained w i t h l y s o l e c i t h i n c o n s i s t e d mainly of a s p e c i e s w i t h a sedimentation c o e f f i c i e n t of 7.3 t o 16.5S. The fragment r e s u l t i n g from the s o l u b i l i z a -t i o n w i t h T r i t o n X-100 c o u l d be c l e a v e d i n t o a s m a l l e r s p e c i e s (8.4S) by l y s o l e c i t h i n . A n a l y s i s by chromatography on Sepharose 6B of the enzyme p r e p a r a t i o n s o l u b i l i z e d by sodium c h o l a t e ( i n the presence of ammonium sulphate), r e v e a l e d the presence of other c o n s t i t u e n t s of the mem-brane, such as s u c c i n a t e dehydrogenase, ATPase and cytochrome b^. The molecular weight of the aggregate was estimated t o be between 0.25 x 10 6 and 4 x 10 6. The enzyme i n t h i s p r e p a r a t i o n c o u l d not be f u r t h e r d i s a g g r e g a t e d by Tween 80, B r i j 3 5 or T r i t o n X-100. Chromatography of t h i s p r e p a r a t i o n on DEAE-Sepharose CL-6B y i e l d e d a maximum p u r i f i c a t i o n of 37 t o 6 8 - f o l d over t h a t o f the membrane p a r t i c l e suspension. The s p e c i f i c a c t i v i t y of the enzyme was 8.8 t o 15.7 ymol per min per mg p r o t e i n . A n a l y s i s of the p a r t i a l l y p u r i f i e d enzyme on p o l y -acrylamide g e l s i n the presence of sodium dodecy l sulphate r e v e a l e d enrichment of s e v e r a l major p o l y p e p t i d e bands of molecular weights 90 000, 57 000, 50 000 and 40 000, c o i n -c i d i n g with the transhydrogenase a c t i v i t y . The p a r t i a l l y p u r i f i e d enzyme c o u l d be a c t i v a t e d by deter g e n t s of the Tween or B r i j s e r i e s and by l y s o l e c i t h i n , p a l m i t i c a c i d and p h o s p h o l i p i d e x t r a c t s from E. c o l i . Measurements of the s t e a d y - s t a t e k i n e t i c s of the membrane-bound enzyme gave v a l u e s of 45.6 and 106.7 yM f o r the s u b s t r a t e s APNAD + and NADPH, and d i s s o c i a t i o n . : constants of 3.6 and 16.2 yM, r e s p e c t i v e l y . Lineweaver-Burk p l o t s f o r each s u b s t r a t e a t d i f f e r e n t f i x e d c o n c e n t r a -t i o n s of the oth e r s u b s t r a t e r e v e a l e d a unique p a t t e r n of l i n e s t h a t i s c h a r a c t e r i s t i c of r a p i d e q u i l i b r i u m random b i r e a c t a n t mechanisms w i t h two dead-end products. In t h i s type of mechanism each substrate i s able to interac t at the binding s i t e of theother substrate to cause i n h i b i t i o n of enzyme a c t i v i t y . This mechanism was confirmed by k i n e t i c studies using the alternate substrates deamino-NADPH and + . . . NAD , as well as by product xnhibitxon studies. The adenine nucleotides 51-AMP and ADP were competitive i n h i b i t o r s of the APNAD +-binding s i t e , while 2'-AMP was a competitive i n h i b i t o r of the NADPH-binding s i t e on the enzyme. Studies on the active s i t e using 2,3-butanedione or phenyl glyoxal revealed the presence of one modifiable a r g i n y l residue per active s i t e on the enzyme. Protection against modification by 2,3-butanedione was afforded by 2'-AMP, 5'-AMP, NAD+ and NADP+. In h i b i t i o n by 2,3-butanedione was enhanced i n the presence of low concentrations of NADH or NADPH suggesting that binding of the reduced pyridine nucleotides, possibly at an a l l o s t e r i c s i t e , causes a conformational change i n the enzyme. Enhancement of i n -a c t i v a t i o n of the enzyme by TPCK-trypsin was also observed in the presence of reduced pyridine nucleotides. NAD(P)H was oxidized by 2,3-butanedione i n the presence of l i g h t . The rate of photooxidation was greatest at pH 7 and when the wavelength of incident l i g h t was 410 nm. This indicates that absorption of l i g h t by the diketone was necessary for the occurrence of the photooxidation iv. r e a c t i o n . The stochiometry of the r e a c t i o n between NADH and 2,3-butanedione was 1:1. The p o s s i b l e nature of the r e a c t i o n product i s d i s c u s s e d i n the t h e s i s . V . TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS v LIST OF TABLES xi LIST OF FIGURES x&ii ABBREVIATIONS x v i i i ACKNOWLEDGEMENTS xx PART I INTRODUCTION 2 B B - S p e c i f i c transhydrogenases 3 A B - S p e c i f i c transhydrogenases 5 Occurrence 5 L o c a l i z a t i o n i n the membrane 5 R e l a t i o n s h i p t o the ener g y - c o u p l i n g system . 6 P h y s i o l o g i c a l importance 7 I d e n t i t y of energy-dependent and energy-independent transhydrogenase 13 Assay of the energy-independent t r a n s h y -drogenase 13 P u r i f i c a t i o n 14 L i p i d dependence « 20 R e c o n s t i t u t i o n of the transhydrogenase system , 21 S i t e - s p e c i f i c i n h i b i t o r s 24 Stead y - s t a t e k i n e t i c s of tr a n s h y d r o g e n a t i o n . 27 C a t a l y t i c mechanism 2 9 vi. Page O b j e c t i v e o f t h i s study ,.. 34 MATERIALS AND METHODS 3 6 Reagents 36 B u f f e r s 37 B a c t e r i a l s t r a i n s and t h e i r maintenance . 38 Growth c o n d i t i o n s 3 9 P r e p a r a t i o n of membrane p a r t i c l e s 4 0 P r e p a r a t i o n of EDTA-lysozyme s p h e r o p l a s t s 41 Determination o f p r o t e i n 41 Determination o f Cytochrome b^ 42 Enzyme assay procedures 42 Energy-independent transhydrogenase 42 Energy-dependent transhydrogenase 43 Suc c i n a t e dehydrogenase 45 C a t a l a s e 45 ATPase 4 6 Treatment of membrane p a r t i c l e s w i t h phenyl g l y o x a l 47 Treatment of membrane p a r t i c l e s w i t h 2,3-butanedione 47 Treatment o f E. c o l i W6 w i t h TPCK-trypsin 49 S o l u b i l i z a t i o n o f membrane p a r t i c l e s with. d e t e r g e n t s , , 49 Sodium c h o l a t e , 4 9 Sodium deoxycholate 51 Ammonyx LO, T r i t o n X-100 or l y s o l e c i t h i n ... 52 v i i . Page Chromatographic procedures 52 A d s o r p t i o n chromatography on h y d r o x y l -a p a t i t e 52 Ion-exchange chromatography on DEAE-c e l l u l o s e 52 Ion-exchange chromatography on DEAE-Sepharose CL-6B . . . . 53 Gel f i l t r a t i o n chromatography on Sepharose 6B 54 Gel f i l t r a t i o n chromatography on Sepharose 6B/4B 54 P r e p a r a t i o n of sucrose d e n s i t y g r a d i e n t s 55 Pol 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 55 Depolymerization of samples 55 Depol y m e r i z a t i o n of molecular weight marker p r o t e i n s 56 P r e p a r a t i o n of g e l s 56 Sepa r a t i n g gel.. 56 St a c k i n g g e l 57 E l e c t r o p h o r e s i s 58 S t a i n i n g and d r y i n g 58 P r e p a r a t i o n of l i p i d s and f a t t y a c i d s 59 T o t a l l i p i d e x t r a c t from E. c o l i K-12 ...... 59 Soybean p h o s p h o l i p i d s ( a s o l e c t i n ) 59 P a l m i t i c a c i d 60 D e l i p i d a t i o n of p a r t i a l l y p u r i f i e d (0.33-0.6P) f r a c t i o n 60 v i i i . Page Ph o t o o x i d a t i o n of NADH by 2,3-butanedione 60 Determination of the stochiometry of photooxida-t i o n of NADH by 2 ,3-butanedione 61 RESULTS 62 The energy-dependent p y r i d i n e n u c l e o t i d e t r a n s -hydrogenase 62 The energy-independent p y r i d i n e n u c l e o t i d e transhydrogenase 72 O r i e n t a t i o n of the enzyme i n the membrane .. 72 Co n d i t i o n s of assay 7 4 pH optimum of the enzyme 7 4 E f f e c t of b a c t e r i a l s t r a i n type on the s p e c i f i c a c t i v i t y of the membrane-bound enzyme 7 4 E f f e c t o f b u f f e r s 77 E f f e c t of s u b s t r a t e s 80 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 p y r i d i n e n u c l e o t i d e transhydrogenase 83 S o l u b i l i z a t i o n of the energy-dependent transhydrogenase 83 S o l u b i l i z a t i o n of the energy-independent transhydrogenase 86 Choice of deter g e n t 8 6 C o n d i t i o n s of s o l u b i l i z a t i o n 88 Mole c u l a r s i z e of the s o l u b i l i z e d enzyme 95 Di s a g g r e g a t i o n by l y s o l e c i t h i n 118 P u r i f i c a t i o n of energy-independent transhydrogenase 126 ix. Page The e f f e c t of l i p i d s and d e t e r g e n t s on s o l u b i -l i z e d energy-independent transhydrogenase 141 E f f e c t o f l i p i d s and dete r g e n t s on the enzyme a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex 143 E f f e c t of p h o s p h o l i p i d s on l i p i d - d e p l e t e d p r e p a r a t i o n s of the enzyme 157 K i n e t i c c o n s t a n t s of p a r t i a l l y p u r i f i e d energy-independent transhydrogenase 161 St e a d y - s t a t e k i n e t i c s of the membrane-bound energy-independent transhydrogenase 165 E f f e c t of s i t e - s p e c i f i c i n h i b i t o r s on the a c t i v i t y of the enzyme 165 Low c o n c e n t r a t i o n s of adenosine phosphates 166 High c o n c e n t r a t i o n of adenosine phosphates 171 E f f e c t of s a l t on the a c t i v i t y of the enzyme 17 9 Mechanism of a c t i o n of the enzyme 187 I n i t i a l v e l o c i t y p a t t e r n s 187 Product i n h i b i t i o n 18 9 A l t e r n a t e s u b s t r a t e s 215 St u d i e s on the a c t i v e s i t e of the enzyme by chemical m o d i f i c a t i o n , 220 I n a c t i v a t i o n by 2,3-butanedione and phenyl g l y o x a l 220 P r o t e c t i o n a g a i n s t i n a c t i v a t i o n by 2,3-butanedione , 221 P r o t e c t i o n a g a i n s t i n a c t i v a t i o n by TPCK-t r y p s i n 235 X . Page DISCUSSION , 24 0 S o l u b i l i z a t i o n of the energy-independent t r a n s -hydrogenase of E. c o l d 240 P u r i f i c a t i o n o f energy-independent t r a n s h y -drogenase 244 E f f e c t of detergents and p h o s p h o l i p i d s on the energy-independent transhydrogenase 247 St e a d y - s t a t e k i n e t i c s of the energy-independent transhydrogenase r e a c t i o n 254 The a c t i v e s i t e of the energy-independent t r a n s -hydrogenase 260 C o n c l u s i o n 265 PART I I P h o t o o x i d a t i o n of reduced p y r i d i n e n u c l e o t i d e s by 2,3-butanedione and phenyl g l y o x a l 2 67 The product of the 2,3-butanedione-dependent p h o t o o x i d a t i o n of NADH 280 D i s c u s s i o n 288 REFERENCES 296 LIST OF TABLES Table Page 1. K i n e t i c Constants f o r S u b s t r a t e s of the Energy-Independent Transhydrogenase from D i f f e r e n t Sources 3 0 2. E f f e c t o f Breakage of EDTA-lysozyme Spheroplasts on Energy-Independent Transhydrogenase A c t i v i t y . 73 3. The E f f e c t of B a c t e r i a l S t r a i n Type on the S p e c i f i c A c t i v i t y o f Energy-Independent Trans-hydrogenase i n Membrane P a r t i c l e s 7 8 4. The E f f e c t of B u f f e r s on Energy-Independent Transhydrogenase A c t i v i t y 7 9 5. D i s t r i b u t i o n of Transhydrogenase A c t i v i t y F o l l o w i n g Treatment of Membrane P a r t i c l e s w i t h T r i t o n X-100 85 6. S o l u b i l i z a t i o n of Energy-Independent Transhy-drogenase of E. c o l i by V a r i o u s Detergents 87 7. E f f e c t of V a r y i n g Detergent and P r o t e i n Con-c e n t r a t i o n s on the S o l u b i l i z a t i o n of Energy-Independent Transhydrogenase 8 9 8. Recovery of Energy-Independent Transhydrog-enase A c t i v i t y F o l l o w i n g S o l u b i l i z a t i o n w i t h V a r i o u s Detergents and S e p a r a t i o n on Sucrose G r a d i e n t s 100 9. P u r i f i c a t i o n of Energy-Independent Transhy-drogenase i n the (0.33-0.6P) F r a c t i o n , by Chromatography on Sepharose 6B i n the Presence of T r i t o n X-100 117 10. E f f e c t of L y s o l e c i t h i n on T r i t o n X-100-S o l u b i l i z e d Membrane P a r t i c l e s a f t e r Chrom-atography on DEAE-Cellulose 122 11. P u r i f i c a t i o n of Energy-Independent Transhy-drogenase from F r a c t i o n (0.33-0.6P) by Chrom-atography on DEAE-Sepharose CL-6B i n the Presence of T r i t o n X-10 0 131 Table Page 12. P u r i f i c a t i o n of Energy-Independent Transhydrog-enase from F r a c t i o n (0.33-0.6P) by Treatment w i t h 0.4% (w/v) Sodium Deoxycholate and Chromatography on DEAE-Sepharose CL-6B i n the Presence of B r i j 35 142 13. E f f e c t of P h o s p h o l i p i d s and Detergents on Trans-hydrogenase A c t i v i t y i n S o l u b l e R e s p i r a t o r y Complex 147 14. The R e l a t i o n s h i p Between P h y s i c a l P r o p e r t i e s of Some Detergents and t h e i r E f f e c t on Energy-Independent Transhydrogenase A c t i v i t y i n S o l u b l e R e s p i r a t o r y Complex 249 15. E f f e c t of Membrane P a r t i c l e s and 2,3-Butanedione on the O x i d a t i o n of NADPH 268 16. E f f e c t of Yeast A l c o h o l Dehydrogenase on the O x i d a t i o n of NADH by Acetaldehyde or 2,3-Butanedione i n the Presence or Absence of L i g h t 28 4 LIST OF FIGURES F i g u r e Page 1. E f f e c t of pH on the ATP-driven energy-dependent transhydrogenase r e a c t i o n 64 2. E f f e c t of ATP c o n c e n t r a t i o n on the ATP-driven energy-dependent transhydrogenase r e a c t i o n 66 3. Hanes-Woolf p l o t f o r the d e t e r m i n a t i o n of V and K m f o r ATP i n the energy-dependent t r a n s -hydrogenase r e a c t i o n 68 4. E f f e c t of ADP on the ATP-driven energy-dependent transhydrogenase a c t i v i t y 71 5. E f f e c t o f pH on the a c t i v i t y of the energy-independent transhydrogenase 7 6 6. E f f e c t of c o n c e n t r a t i o n s of APNAD + and NADPH on the a c t i v i t y of energy-independent t r a n s h y d r o g -enase 82 7. S t a b i l i t y of energy-independent transhydrogenase i n the presence of dete r g e n t 94 8. S e p a r a t i o n o f membrane p a r t i c l e s o f E. c o l i s o l u b i l i z e d w i t h 3% (w/v) l y s o l e c i t h T n on 10-50% sucrose d e n s i t y g r a d i e n t s 98 9. A n a l y s i s by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a -t i o n o f membrane p a r t i c l e s from E. c o l i s o l u b i -l i z e d with 3% (w/v) T r i t o n X^lOO or 2% (w/v) sodium c h o l a t e ( i n the presence of ammonium sulphate) 104 10. Chromatography of the (0.33-0.6P) f r a c t i o n s on Sepharose 6B i n the presence of 0.2% (w/v) B r i j 35 110 11. Chromatography of the (0.33-0.6P) f r a c t i o n on Sepharose 6B i n the presence of 1% (w/v) Tween 80 113 xiv. F i g u r e Page 12. Chromatography of the (0.33-0.6P) f r a c t i o n on Sepharose 6B i n the presence of 0.5% (w/v) T r i t o n X-100 115 13. Chromatography of T r i t o n X - 1 0 0 - s o l u b i l i z e d membrane p a r t i c l e s of E. c o l i on D E A E - c e l l u l o s e . 121 14. S e p a r a t i o n of f r a c t i o n s F and L on 10-50% sucrose d e n s i t y g r a d i e n t s 125 15. Chromatography of the (0.33-0.6P) f r a c t i o n on DEAE-Sepharose CL-6B i n the presence of 0.2% (w/v) T r i t o n X-100 13 0 16. Chromatography of p a r t i a l l y p u r i f i e d energy-independent transhydrogenase on h y d r o x y l a p a t i t e . 134 17. SDS-polyacrylamide g e l e l e c t r o p h o r e s i s of p a r t -i a l l y p u r i f i e d energy-independent transhydrog-enase 136 18. Chromatography of the (0.33-0.6P) f r a c t i o n , t r e a t e d w i t h 0.4% (w/v) sodium deoxycholate, on DEAE-Sepharose CL-6B i n the presence of 0.1% (w/v) B r i j 35 140 19. P r e p a r a t i o n of s o l u b l e r e s p i r a t o r y complex 146 20. E f f e c t of B r i j on energy-independent t r a n s h y -drogenase a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex f r a c t i o n s 150 21. E f f e c t o f Tween on energy-independent t r a n s h y -drogenase a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex f r a c t i o n s 153 22. E f f e c t of p a l m i t i c a c i d and p h o s p h o l i p i d s on energy-independent transhydrogenase a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex f r a c t i o n s 156 23. E f f e c t o f p h o s p h o l i p i d s on energy-independent transhydrogenase a c t i v i t y i n p a r t i a l l y p u r i f i e d and d e l i p i d a t e d (0.33-0.6P) f r a c t i o n 160 24. E f f e c t of APNAD+ and NADPH c o n c e n t r a t i o n s on the a c t i v i t y of energy-independent transhydrogenase i n s o l u b l e r e s p i r a t o r y complex 164 XV . F i g u r e Page 25. E f f e c t of low c o n c e n t r a t i o n s of 2'-AMP on energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s 168 26. E f f e c t of low c o n c e n t r a t i o n s of 5'-AMP on energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s 17 0 27. E f f e c t of hig h c o n c e n t r a t i o n s of 2'-AMP on the energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s 173 28. E f f e c t of hig h c o n c e n t r a t i o n s of 51-AMP on energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s 17 6 29. E f f e c t of ADP on energy-independent t r a n s h y -drogenase a c t i v i t y i n membrane p a r t i c l e s 17 8 30. E f f e c t of 2'-AMP and KCl on energy-independent transhydrogenase a c t i v i t y 181 31. E f f e c t of 5'-AMP and KCl on energy-independent transhydrogenase a c t i v i t y 183 32. E f f e c t o f 200 mM KCl on the APNAD+- and NADPH-b i n d i n g s i t e s of the energy-independent t r a n s -hydrogenase 18 6 33. E f f e c t o f the c o n c e n t r a t i o n of APNAD"1" on the a c t i v i t y of the energy-independent t r a n s h y -drogenase a t constant c o n c e n t r a t i o n s of NADPH .. 18 9 34. E f f e c t of the c o n c e n t r a t i o n of NADPH on the a c t i v i t y of the energy-independent t r a n s h y -drogenase a t constant c o n c e n t r a t i o n s of APNAD"1" . 191 35. Determination of k i n e t i c c o n s t a n t s from r e p l o t s of 1 versus 1 a t d i f f e r e n t f i x e d con-v [APNAD+] c e n t r a t i o n s of NADPH. 197 36. Determination of k i n e t i c constants..from r e p l o t s of 1 versus 1 at d i f f e r e n t f i x e d con-v [NADPH] c e n t r a t i o n s of APNAD + 2 00 xvi. Figure Page 3 7 . Determination of the d i s s o c i a t i o n constant for NADPH by a H'anes-Woolf p l o t 2 0 1 3 8 . Determination of the d i s s o c i a t i o n constant for APNAD+ by a Hanes-Woolf p l o t 2 0 4 3 9 . E f f e c t of NADP+ on the energy-independent trans-hydrogenase a c t i v i t y i n membrane p a r t i c l e s 2 0 6 4 0 . E f f e c t of APNADH on the energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s 2 0 9 4 1 . E f f e c t of NADH on the energy-independent trans-hydrogenase a c t i v i t y i n membrane p a r t i c l e s 2 1 2 4 2 . E f f e c t of NADH on the energy-independent trans-hydrogenase a c t i v i t y at various concentrations of substrate 2 1 4 4 3 . I n h i b i t i o n of energy-independent transhydrog-enase a c t i v i t y by alternate substrates NAD+ and deamino-NADPH 2 1 8 4 4 . Kinetics of i n a c t i v a t i o n of the energy-independent transhydrogenase by 2,3-butanedione and phenyl glyoxal 2 2 3 4 5 . E f f e c t of APNAD"1" on the in a c t i v a t i o n of the energy-independent transhydrogenase by 2 , 3 -butanedione 2 2 6 4 6 . E f f e c t of substrates and substrate analogues on the i n a c t i v a t i o n of the energy-independent transhydrogenase by 2,3-butanedione 2 2 8 4 7 . E f f e c t of NAD+ and NADP+ on the i n h i b i t i o n of energy-independent transhydrogenase by 2,3--butanedione , 2 3 1 4 8 . E f f e c t of NADPH and NADH on the i n h i b i t i o n of the energy-independent transhydrogenase by 2 ,3-butanedione 2 3 3 4 9 . E f f e c t of substrates on the in a c t i v a t i o n of the energy-independent transhydrogenase by TPCK-trypsin 238 x v i i . Figure Page 50. Photooxidation of NADH by 2,3-butanedione 271 51. Photooxidation of NADH by phenyl glyoxal 273 52. Absorption spectrum of a mixture of 2,3-butanedione and NADH before and after i r r a d i -ation 277 53. E f f e c t of pH and concentration of 2,3-butanedione on the rate of photooxidation of NADH 27 9 54. E f f e c t of the wavelength of the i r r a d i a t i n g l i g h t on the rate of photooxidation of NADH by 2 ,3-butanedione 282 55. Stochiometry of photooxidation of NADH by 2,3-butanedione 286 56. Stochiometry of oxidation of NADH by 2,3-butanedione catalyzed by yeast alcohol dehy-drogenase 290 x v i i i . ABBREVIATIONS ADP Adenosine-5'-diphosphate Ammonyx LO Dimethyl l a u r y l amine oxide 21-AMP Adenosine-2'-monophosphate 3'-AMP Adenosine-3 1-monophosphate 5'-AMP Adenosine-5'-monophosphate APNAD + 3-Acetylpyridine-NAD + ATP Adenosine-5'-triphosphate ATPase ATP phosphohydrolase B r i j 3 5 Polyoxyethylene (23) l a u r y l ether B r i j 36T Polyoxyethylene (10) l a u r y l ether B r i j 58 Polyoxyethylene (20) c e t y l ether CMC C r i t i c a l m i c e l l a r c o n c e n t r a t i o n DCIP 2,6-Dichlorophenolindophenol DNase Deoxyribonuclease DTT D i t h i o t h r e i t o l EDTA Ethylenediamine t e t r a a c e t a t e FAD F l a v i n adenine d i n u c l e o t i d e FCCP para-Trifluoromethoxyphenyl hydrazone HLB H y d r o p h i l e - L i p o p h i l e balance NAD +(H) Nicotinamide adenine d i n u c l e o t i d e NADP +(K) Nicotinamide adenine d i n u c l e o t i d e phosphate p s i Pounds per square i n c h RNase Ribonuclease CC Is CC • SDS Sodium dodecyl sulphate TMED N,N,N',N' tetramet h y l e t h y l e n e d i a m i n e TPCK-trypsin T r y p s i n t r e a t e d with L - ( t o s y l a m i d o 2-phenyl) e t h y l chloromethyl ketone to i n h i b i t contam-i n a t i n g chymotryptic a c t i v i t y T r i s Tris-(hydroxymethyl)-aminoethane T r i t o n X-100 P o l y o x y e t h y l e n e g l y c o l (9-10) p - t - o c t y l p h e n o l Tween 20 Polyoxyethylene (20) s o r b i t a n monolaurate Tween 4 0 Polyoxyethylene (20) s o r b i t a n monopalmitate Tween 60 Polyoxyethylene (20) s o r b i t a n monostearate Tween 8 0 Polyoxyethylene (20) s o r b i t a n monooleate ACKNOWLEDGEMENTS I wish to thank Dr. Bragg f o r h i s p a t i e n t guidance and advice throughout my graduate s t u d i e s and d u r i n g the w r i t i n g of t h i s t h e s i s . I am g r a t e f u l to the M e d i c a l Research C o u n c i l of Canada f o r s u p p o r t i n g t h i s work, to Dr. Burton f o r h i s generous g i f t of r e c r y s t a l l i z e d p a l m i t i c a c i d , to Dr. Zbarsky f o r use of some l a b o r a t o r y equipment and to Mrs. Cyn t h i s Hou f o r k i n d l y p r e p a r i n g the p u r i f i e d ATPase used i n t h i s study. I a l s o a p p r e c i a t e the h e l p f u l d i s c u s s i o n s and p l e a s a n t working atmosphere provided by my c o l l e a g u e s i n the l a b o r a -t o r y . I would l i k e to thank Ms. Diane Wilson f o r k i n d l y t y p i n g t h i s manuscript. L a s t , but not l e a s t , I am g r a t e f u l to my husband, Ted, f o r constant encouragement and moral support throughout my work on t h i s p r o j e c t . 1. PART I 2. INTRODUCTION Pyridine nucleotide transhydrogenases (E.C. 1.6.1.1) are enzymes which catalyze the rev e r s i b l e transfer of a hydride ion equivalent from the reduced pyridine nucleotide to the oxidized form (1) according to the reaction. NADH + NADP+ -—- NAD+ + NADPH These enzymes may be divided into two classes. One class , which i s present i n certain bacteria and plants, contains flavoproteins which are e a s i l y s o l u b i l i z e d . This class i s not functionally linked to the energy-transfer system of the membrane. It i s not known whether the enzyme i s loosely bound to the membrane or whether i t exists as a high molecular weight complex, not associated with the membrane structure (2) . . w i t h such enzymes, transfer of the hydride ion equivalent occurs between the 4B-hydrogen atom of both NADH and NADPH (3,4). The other class of pyridine nucleotide transhydrogenases, i s present i n certain bacteria and i n mitochondria. . These are membrane-bound enzymes that are not flavoproteins (5,6,7) and are functionally linked to the energy-transfer system of the membrane. They are s p e c i f i c for transfer between the 4A-hydrogen atom of NADH and the 4B-hydrogen atom of NADPH. The transfer of the hydride ion equivalent occurs without exchange with the hydrogen atoms of the surrounding medium (8,9). The two classes of enzymes are referred to as BB-specific and AB-specific transhydrogen-ases respectively (10). 3 . BB-Spec i f i c transhydrogenases These occur i n bacteria such as Pseudomonas fluorescens (11-13), Pseudomonas aeruginosa (3), Azotobacter v i n e l a n d i i (14),' Azotobacter chroococcum and Azotobacter a g i l e (15). Transhydrogenases have also been reported to occur i n plants (16) and algae (17), although they have not d e f i n i t e l y been established as being BB- or AB-specific enzymes (10). The transhydrogenase enzymes of Pseudomonas aeruginosa (18) and Azotobacter v i n e l a n d i i (19,20) have been p u r i f i e d to homo-geneity. The enzymes were isola t e d as large filamentous aggregates. In the presence of 21-AMP or NADP+ the enzyme from P. aeruginosa dissociated into smaller fragments composed of 20 polypeptides, each polypeptide of molecular weight 40 000 to 45 000 (21). The enzyme from A. v i n e l a n d i i also disaggregated in the presence of NADP+ into fragments with a minimum molecular weight of 58 000 (19). That both of these enzymes have FAD as a prosthetic group was demonstrated by the fact that heat i n a c t i v a t i o n at pH 7.5, which resulted i n the loss of the prosthetic group from the protein, could be reversed by the addition of FAD. S e n s i t i v i t y of the enzyme to heat i n a c t i v a t i o n was i n -creased i n the presence of NADH or NADPH. Oxidized pyridine nucleotides or FAD, on the other hand, s t a b i l i z e d the enzyme against heat i n a c t i v a t i o n (18,19). Sulphydryl reagents had opposite e f f e c t s on the enzyme from P. aeruginosa i n 4. the presence of reduced or oxidized pyridine nucleotides, respectively (18), suggesting that sulphydryl groups at or near the active s i t e are exposed to d i f f e r e n t extents upon binding of oxidized or reduced substrates, respectively. The enzyme from P. aeruginosa was not inh i b i t e d by treatment with trypsin or palmityl-CoA but was activated by 21-AMP or C a 2 + (2) by a l l o s t e r i c regulation at separate binding s i t e s (22). The presence of multiple binding s i t e s , including a possible regulatory s i t e , was also evident from experiments based on the reduction of thio-NAD + by NADPH. Double r e c i p r o c a l plots revealed a second-order dependence on NADPH but a f i r s t - o r d e r dependence on thio-NAD + (23). In the presence of 21-AMP the dependence with respect to NADPH was f i r s t - o r d e r , suggesting that the second binding s i t e for NADPH (presumably a regu-latory site) was now occupied by 2'-AMP (10). The enzyme from A. v i n e l a n d i i , which appeared to be similar to that of P. aeruginosa, was reported to have separate hydrogen-donor and hydrogen-acceptor s i t e s (14). I n i t i a l v e l o c i t y patterns indicated that transhydrogenation i n t h i s organism proceeded by way of a rapid equilibrium random bireactant mechanism (14,24) . 5. AB-Specific transhydrogenases  Occurrence These occur i n animal tissues such as beef heart, kidney, l i v e r , muscle and a r t e r i a l tissue (.25-28), i n f a c u l t a t i v e anaerobic bacteria such as Escherichia c o l i (29-31) and Salmonella typhimurium (32) , i n the photosynthetic bacteria, •Rhodospirilium rubrum (33,34), Rhodopseudomonas spheroides (35), Rhodopseudomonas p a l u s t r i s and Rhodospirilium mol-ischianum (36). L o c a l i z a t i o n i n the membrane Subcellular d i s t r i b u t i o n studies on the transhydrogenase from animal tissues have shown the enzyme to be l o c a l i z e d i n the mitochondria, where i t i s t i g h t l y bound to the inner membrane (26,37-40). Sweetman et a l . (41) studied the e f f e c t s of 2-phenylisatogen (an i n h i b i t o r of oxidative phosphorylation) (42) on the transhydrogenase reaction i n rat l i v e r mitochon-d r i a . Transhydrogenase was i n h i b i t e d by 2-phenylisatogen. Inhibition of the enzyme was increased when the 2-phenyl-isatogen was added to the mitochondria p r i o r to sonication rather than to the submitochondrial p a r t i c l e s . They i n t e r -preted t h i s to mean that the i n h i b i t o r could reach i t s s i t e of action i n mitochondria but not i n submitochondrial p a r t i c l e s , i ndicating that the enzyme i s located towards the i n t e r -membrane space i n mitochondria. Ernster and coworkers (10,43), 6. however, pointed out that, although part of the transhydrog-enase molecule may be exposed to the intermembrane space, the nicotinamide nucleotide binding site(s) are more l i k e l y exposed to the matrix side. This assumption was based on the impermeability of the mitochondrial inner membrane to nicotinamide nucleotides (4 4) and that i n t a c t mitochondria (27) did not exhibit transhydrogenase a c t i v i t y with externally added nicotinamide nucleotides, whereas submitochondrial p a r t i c l e s d id. The transhydrogenase of E. c o l i , which i s similar i n properties to that of mitochondria (2), i s located on the inner or cytoplasmic side of the inner membrane. This was demonstrated by studies on membrane ve s i c l e s of E. c o l i (45) which have shown that disruption of the vesicl e s by a French pressure c e l l , or treatment with detergent, caused an i n -crease i n the transhydrogenase a c t i v i t y over that of the in t a c t membrane by exposing the previously inaccessible active s i t e s of the enzyme to the external medium. Relationship to the energy-coupling system The pyridine nucleotide transhydrogenase i s an i n t e g r a l membrane protein which i s fun c t i o n a l l y coupled to the energy transfer system of the membrane i n which i t occurs (46). This i s manifested by an energy-dependent increase i n both the rate (10) and the extent (4 6,47) of reduction of NADP+ by NADH. In mitochondria, the apparent equilibrium constant 7 . f o r the r e a c t i o n i s i n c r e a s e d from 1 t o 5 00 i n the presence of an energy source (46). Energy, d r i v i n g the r e a c t i o n may be generated by e l e c t r o n t r a n s p o r t , through c o u p l i n g s i t e s of the r e s p i r a t o r y c h a i n , or by.ATE h y d r o l y s i s (30,40). S i m i -l a r l y , i n E. c o l i , the enzyme may be ene r g i z e d through the r e s p i r a t o r y c h a i n or by ATP h y d r o l y s i s . In p h o t o s y n t h e t i c b a c t e r i a , an a d d i t i o n a l source of energy capable of d r i v i n g the transhydrogenase r e a c t i o n i s the h y d r o l y s i s of i n o r g a n i c pyrophosphate (33,35,36). Transhydrogenation between NADPH and NAD + i s coupled to the uptake of protons by su b m i t o c h o n d r i a l p a r t i c l e s (48). The membrane p o t e n t i a l generated by such a process (49-51) i s s u f f i c i e n t f o r the s y n t h e s i s of ATP from ADP and i n o r g a n i c phosphate (52). Based upon these o b s e r v a t i o n s i t was pro-posed t h a t the transhydrogenase f u n c t i o n s as a proton pump (50,53). T h i s r o l e w i l l be d i s c u s s e d i n a l a t e r s e c t i o n . P h y s i o l o g i c a l importance The p h y s i o l o g i c a l f u n c t i o n of the transhydrogenase i s s t i l l a matter of c o n t r o v e r s y . The energy-dependent t r a n s -hydrogenase of a d r e n o c o r t i c a l m i t o c h o n d r i a has been i m p l i -cated i n the supply of NADPH f o r l l g - h y d r o x y l a t i o n i n s t e r o i d metabolism (54-59). T h i s has been d i s p u t e d by other l a b o r a -t o r i e s who claimed t h a t m a l i c enzyme p r o v i d e s the main source of r e d u c i n g e q u i v a l e n t s f o r t h i s h y d r o x y l a t i o n (.60,61). In 8 . i s o l a t e d a d r e n o c o r t i c a l m i t o c h o n d r i a e i t h e r of these enzymes appear t o be able t o pr o v i d e the redu c i n g e q u i v a l e n t s f o r 1 1 3-hydroxylation under s u i t a b l e c o n d i t i o n s . However, l i t t l e i s known about the f a c t o r s which determine the predominating pathway in. v i v o . The transhydrogenase a l s o appears t o be i n d i r e c t l y i n -v o l v e d i n the o x i d a t i o n of i s o c i t r a t e (62). Mammalian mito-chon d r i a c o n t a i n an NAD +-dependent and an NADP +-dependent. i s o c i t r a t e dehydrogenase (63,64). Although the NADP +-dependent enzyme has a g r e a t e r a f f i n i t y f o r i s o c i t r a t e , the NAD +-dependent enzyme may p r e v a i l i n f u l l y coupled m i t o c h o n d r i a (65) . T h i s i s because, under these c o n d i t i o n s , the t r a n s -hydrogenase i s under energy c o n t r o l and the e q u i l i b r i u m i s d r i v e n towards NADPH formation. Excess NADPH would cause the i s o c i t r a t e to p r e f e r e n t i a l l y b i n d t o the NAD +-dependent i s o c i t r a t e dehydrogenase (62). Thus, the energy-dependent transhydrogenase, which i s r e g u l a t e d by the energy s t a t e of the m i t o c h o n d r i a , i n t u r n r e g u l a t e s the route by which i s o -c i t r a t e i s o x i d i z e d . Based upon the o b s e r v a t i o n t h a t there i s a s o l u b l e and a m i t o c h o n d r i a l NADP +-dependent i s o c i t r a t e dehydrogenase (66) and th a t the l a t t e r can c a r b o x y l a t e a - k e t o g l u t a r a t e t o i s o c i t r a t e , Kaplan (62) proposed the e x i s t e n c e of an a-k e t o g l u t a r a t e - i s o c i t r a t e s h u t t l e whose o p e r a t i o n would provide a f u n c t i o n a l l i n k between the energy-dependent 9. transhydrogenase and e x t r a m i t o c h o n d r i a l NADPH-dependent r e a c -t i o n s . I n t r a m i t o c h o n d r i a l NADPH would be o x i d i z e d d u r i n g the formation of i s o c i t r a t e from a - k e t o g l u t a r a t e and carbon d i o x i d e . I s o c i t r a t e would then pass i n t o the cytoplasm where i t would be o x i d i z e d by the NADP +-dependent enzyme. The e x t r a m i t o c h o n d r i a l NADPH so formed c o u l d be used f o r syn-t h e t i c r e a c t i o n s , w h i l e the a - k e t o g l u t a r a t e would r e - e n t e r the m i t o c h o n d r i a and the c y c l e of t r a n s f e r of reducing equiva-l e n t s across the mitoc h o n d r i a would be repeated. Although a - k e t o g l u t a r a t e - i s o c i t r a t e s h u t t l e systems have been recon-s t i t u t e d , the p h y s i o l o g i c a l r o l e of transhydrogenase i n such systems has not y e t been e s t a b l i s h e d . Bragg e t a l . (67) suggested t h a t the energy-dependent transhydrogenase of E. c o l i i s i n v o l v e d i n g e n e r a t i n g NADPH f o r the b i o s y n t h e s i s of amino a c i d s . T h e i r s p e c u l a t i o n was based upon the f a c t t h a t energy-dependent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s from c e l l s grown on t r y p t i c a s e -soy medium or c a s e i n h y d r o l y s a t e was undetectable, or markedly decreased, from t h a t of c e l l s grown on minimal medium - i n the absence of amino a c i d s . T h i s e f f e c t was a l s o observed i n c e l l s grown on glucose, i n the presence of a broad range o f s i n g l e amino a c i d s or combinations t h e r e o f . The r e p r e s s i o n of the enzyme by growth i n the presence of amino a c i d s sug-gested t h a t the energy-independent transhydrogenase p l a y s a r o l e i n p r o v i d i n g NADPH f o r the b i o s y n t h e s i s of amino a c i d s , and t h a t the formation of t h i s enzyme was rep r e s s e d when the 10. preformed end-product was s u p p l i e d . Repression of t r a n s -hydrogenase i n c e l l s grown i n the presence of amino a c i d s was subsequently shown by other workers (68-71). The enzyme was derepressed by the removal of the amino a c i d ( s ) from the growth medium but the recovery was blocked by r i f a m p i c i n or chloramphenicol (68,69)' i n d i c a t i n g t h a t de novo s y n t h e s i s was necessary f o r i t s a c t i v i t y , r a t h e r than the a c t i v a t i o n of p r e e x i s t i n g p r o t e i n . T h i s phenomenon was made use of to l a b e l newly s y n t h e s i z e d p o l y p e p t i d e s of the enzyme. P a r t i a l p u r i f i c a t i o n of the l a b e l l e d enzyme and a n a l y s i s on p o l y -acrylamide g e l s made p o s s i b l e the de t e r m i n a t i o n o f i t s molecular weight (70). The h y p o t h e s i s o f Bragg e t a l . (67) was c h a l l e n g e d by Gerolimatos and Hanson (69), who argued t h a t transhydrogenase p r o v i d e s o n l y a smal l f r a c t i o n of the NADPH r e q u i r e d f o r amino a c i d b i o s y n t h e s i s (72). They d i d not exclude the pos-s i b i l i t y , however, t h a t i n s t r a i n s of E. c o l i l a c k i n g the hexose monophosphate shunt, and i n which the transhydrogenase i s i n c r e a s e d , NADPH may be pro v i d e d by the energy-dependent transhydrogenase under these c o n d i t i o n s (69). Of the amino a c i d s added t o the growth medium, l e u c i n e , and to a l e s s e r e x t e n t methione and a l a n i n e , were e f f e c t i v e i n r e p r e s s i n g the transhydrogenase enzyme. The l e u c i n e b i o s y n t h e t i c operon and l e u c i n e t r a n s p o r t systems are both r e p r e s s i b l e by l e u c i n e and are r e g u l a t e d s e p a r a t e l y (73) . In. an attempt to study whether the r e p r e s -s i o n of transhydrogenase by l e u c i n e was connected t o the regu-l a t i o n of the t r a n s p o r t or b i o s y n t h e s i s of t h i s amino a c i d , Gerolimatos and Hanson (69) showed t h a t mutant s t r a i n s of E. c o l i t h a t were c o n s t i t u t i v e l y derepressed f o r l e u c i n e , i s o l e u c i n e and v a l i n e b i o s y ntheses were r e p r e s s e d by l e u c i n e w i t h r e s p e c t to l e u c i n e t r a n s p o r t and transhydrogenase ac-t i v i t y . The r e g u l a t i o n of the enzyme wit h the l e u c i n e t r a n s -p o r t systems suggested to these authors t h a t the transhydrog-enase p r o t e i n may have a r o l e i n the uptake of branched-chain amino a c i d s . A l a n i n e and methionine were e f f e c t i v e r e p r e s s o r s of transhydrogenase. These amino a c i d s a l s o r e p r e s s l e u c i n e t r a n s p o r t by i n c r e a s i n g i n t r a c e l l u l a r l e u c i n e l e v e l s (74). In a s t r a i n of E. c o l i w i t h a l t e r e d leucyl-tRNA, t r a n s h y -drogenase was a t a r e p r e s s e d l e v e l without the a d d i t i o n of l e u c i n e (6 9). The data l e d Gerolimatos and Hanson (69) to conclude t h a t the r e g u l a t o r y molecule f o r the r e p r e s s i o n of transhydrogenase i s the leucyl-tRNA. Recently, a s t r a i n of E. c o l i l a c k i n g p y r i d i n e n u c l e o -t i d e transhydrogenase a c t i v i t y has been i s o l a t e d (75). The mutation pnt-1, causing l o s s of the enzyme a c t i v i t y was found to map near min 35, on the E. c o l i genome (7 6). The growth p r o p e r t i e s of the mutant on glucose, s u c c i n a t e or. g l y c e r o l were found to resemble those of the parent s t r a i n , i n d i c a t i n g t h a t transhydrogenase i s a p p a r e n t l y not an e s s e n t i a l source of NADPH f o r the c e l l . However, the n o n s p e c i f i c r e p r e s s i o n 12. of the enzyme by amino a c i d s other than l e u c i n e , i s o l e u c i n e and methionine (67) throw some doubt as to the v a l i d i t y of the suggested f u n c t i o n of transhydrogenase i n the t r a n s p o r t of branched-chain amino a c i d s or i n i t s r e g u l a t i o n by l e u c y l -tRNA. A p o s s i b l e involvement f o r transhydrogenase i n the a s s i m i l a t i o n of ammonia by E. c o l i and S. typhimurium has been proposed by Houghton (71). C e l l s of E. c o l i K-12 grown on glucose i n the presence of 0.5 to 20 mM ammonium'chloride showed an i n c r e a s e i n energy-independent transhydrogenase a c t i v i t y concomitant ' w i t h an i n c r e a s e i n N A D ( P ) + - l i n k e d glutamate dehydrogenase a c t i v i t y . Higher l e v e l s of ammonium c h l o r i d e (60 to 150 mM) caused both a c t i v i t i e s to decrease. The r a t i o of both of these enzymatic a c t i v i t i e s was c o n s t a n t over the range of ammonium c h l o r i d e t e s t e d . Under these con-d i t i o n s , both enzymes were r e p r e s s e d by glutamate. In S. typhimurium both enzymes were i n s e n s i t i v e to ammonium c h l o r i d e or glutamate, but were r e p r e s s e d by a s p a r t a t e . Under growth c o n d i t i o n s where glutamine synthetase was v a r i e d , transhydrog-enase a c t i v i t y c o r r e l a t e d w i t h t h a t of glutamate dehydrogenase when the l a t t e r was i n v o l v e d i n the a s s i m i l a t i o n of ammonia. Thus the p h y s i o l o g i c a l r o l e of p y r i d i n e n u c l e o t i d e t r a n s -hydrogenase i s s t i l l an open q u e s t i o n . F u r t h e r work i n t h i s area i s r e q u i r e d to s o l v e t h i s c o n t r o v e r s y . 13. I d e n t i t y of energy-dependent and energy-independent t r a n s - hydrogenase S o l u b i l i z a t i o n of the transhydrogenase from m i t o c h o n d r i a l membranes by detergents leads to uncoupling of the enzyme from the energy c o n s e r v a t i o n s i t e due to breakage of the membrane s t r u c t u r e . Thus the s o l u b i l i z e d transhydrogenase i s not energy-dependent u n l e s s i t i s r e c o n s t i t u t e d i n t o p h o s p h o l i p i d v e s i c l e s (22,53,77,78). S t u d i e s have shown t h a t the same enzyme p a r t i c i p a t e s i n both the energy-dependent and energy-independent r e a c t i o n s . Thus, both r e a c t i o n s have the same s t e r e o s p e c i f i c i t y w i t h r e s p e c t t o the t r a n s f e r of h y d r i d e i o n e q u i v a l e n t s (8,79,80) and are i n h i b i t e d by the same reagents (46,81-83). R e c o n s t i t u t i o n of the p u r i f i e d enzyme i n t o liposomes r e s u l t e d i n the r e s t o r a t i o n of both energy-dependent and energy-independent a c t i v i t i e s (77,78). The i s o l a t i o n of a mutant of E. c o l i l a c k i n g both the energy dependent and energy-independent transhydrogenase a c t i v i t i e s (76) i s f u r t h e r evidence t h a t both of these r e a c t i o n s are c a t a l y z e d by the same enzyme. Assay of the energy-independent transhydrogenase The pH maximum f o r the energy-independent transhydrogen-a t i o n of NADP + by NADH i s 5.5, w h i l e t h a t f o r the r e v e r s e r e a c t i o n occurs a t pH 7.0 (25). Since a t n e u t r a l pH the maximal i n i t i a l v e l o c i t y of the r e v e r s e r e a c t i o n i s about f i v e times g r e a t e r than t h a t of the forward r e a c t i o n (84), energy-independent 14. transhydrogenation i s measured as the reduction of NAD+ by NADPH at pH 7.0. This cannot be assayed d i r e c t l y , however, since the spectral differences between NADH and NADPH are ne g l i g i b l e . Thus, analogues of NAD+ such as thio-NAD + or acetylpyridme-NAD (APNAD ) whose absorption maximum for the reduced form i s above 340 nm are used. For these substrates the increase in- absorbance at 400 nm or 375 nm,respectively, i s measured, since there i s p r a c t i c a l l y no contribution from NADPH at these wavelengths (5,18). A l t e r n a t i v e l y , the con-centration ' of one of the substrates may be kept constant by means of a suitable enzymic regenerating system, while the change i n concentration of the other substrate i s measured at 340 nm (11). P u r i f i c a t i o n Attempts to s o l u b i l i z e the transhydrogenase from mito-chondrial membranes by extraction with d i g i t o n i n and p u r i f i c a -t i o n by adsorption on calcium phosphate gel was f i r s t i n t r o -duced by Kaplan et a l . (25) . A ten-fold p u r i f i c a t i o n was achieved by t h i s method. This was subsequently improved by the use of sucrose density gradient centrifugation to a 25-fold p u r i f i c a t i o n (5). However, the preparation was not homogeneous and the s e n s i t i v i t y of the enzyme to organic solvents, b i l e s a l t s and phospholipases, rendered p u r i f i c a t i o n by these methods d i f f i c u l t . 15. Rydstrom e t a l . (85) demonstrated t h a t the p h o s p h o l i p i d , l y s o l e c i t h i n , c o u l d be e f f e c t i v e l y used t o s o l u b i l i z e the transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s . D i f f e r e n t i a l c e n t r i f u g a t i o n of the e x t r a c t e d enzyme y i e l d e d a p r e p a r a t i o n w i t h a t w e l v e - f o l d p u r i f i c a t i o n over the submito-c h o n d r i a l p a r t i c l e s (86). L y s o l e c i t h i n was s u c c e s s f u l l y used by Anderson and F i s h e r (7) to o b t a i n a pure p r e p a r a t i o n o f the enzyme from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s . S o l -u b i l i z a t i o n with l y s o l e c i t h i n f o l l o w e d e x t r a c t i o n o f the membrane p r e p a r a t i o n w i t h sodium p e r c h l o r a t e . Treatment of the membrane p a r t i c l e s w i t h sodium p e r c h l o r a t e p a r t i a l l y s o l u b i l i z e d other membrane-bound.enzymes (87) and rendered the remainder of the contaminating enzymes n o n e x t r a c t a b l e d u r i n g the subsequent s o l u b i l i z a t i o n w i t h l y s o l e c i t h i n . P u r i f i c a t i o n was then achieved by f r a c t i o n a t i o n o f the s o l u -b i l i z e d enzyme on alumina g e l , c a l c i u m phosphate g e l and by chromatography on NAD + a f f i n i t y columns, i n the presence of de t e r g e n t s . The minimal molecular weight of the enzyme ob-t a i n e d from t h i s p r e p a r a t i o n was 120 000. The enzyme probably e x i s t s as a dimer (7,53). No p r o s t h e t i c group c o u l d be de-t e c t e d . The transhydrogenase can a l s o be e x t r a c t e d from beef-h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s by sodium c h o l a t e i n the presence of ammonium sulphate (88) . P u r i f i c a t i o n of such an e x t r a c t by chromatography on DEAE-Sepharose CL-6B and h y d r o x y l -a p a t i t e y i e l d e d a homogeneous p r e p a r a t i o n of the t r a n s -hydrogenase, the molecular weight of which was r e p o r t e d to be 97 000. No p r o s t h e t i c group was d e t e c t e d (6). 16. There have been many attempts to s o l u b i l i z e and p u r i f y AB-transhydrogenases from b a c t e r i a l membranes (33,70,80,89-92), but these have o n l y met wi t h p a r t i a l success. P u r i f i c a t i o n to homogeneity has not, as y e t , been achieved. The d i f f i c u l t y i n s o l u b i l i z i n g and p u r i f y i n g the transhydrogenase from E. c o l i and other b a c t e r i a l membranes l i e s p a r t l y i n the f a c t t h a t membranes from s i n g l e c e l l organisms are r e s p o n s i b l e f o r many d i v e r s e f u n c t i o n s . Thus they comprise a much l a r g e r number of p r o t e i n s than membranes of a more s e l e c t i v e nature such as m i t o c h o n d r i a l membranes. The transhydrogenase from a b a c t e r i a l membrane, t h e r e f o r e , c o n s t i t u t e s a s m a l l e r f r a c t i o n of the t o t a l membrane p r o t e i n than i t s m i t o c h o n d r i a l counter-p a r t . An a d d i t i o n a l f a c t o r c o n t r i b u t i n g to the d i f f i c u l t y i n o b t a i n i n g a p u r i f i e d and e n z y m a t i c a l l y a c t i v e t r a nshy-drogenase enzyme from b a c t e r i a l membranes i s the presence of pr o t e a s e s , l o c a t e d mainly i n the outer membrane of these organisms (93-96) . Such proteases are l i k e l y contaminants i n p r e p a r a t i o n s of the transhydrogenase and thus c o u l d con-t r i b u t e to the de g r a d a t i o n of the transhydrogenase and l o s s of i t s enzymatic a c t i v i t y . Of the b a c t e r i a l transhydrogenases, o n l y two s p e c i e s have been e x t e n s i v e l y i n v e s t i g a t e d , t h a t from R. rubrum chromatophores and t h a t from E. c o l i . The chromatophores from R. rubrum c o n t a i n a transhydrogenase complex, c o n s i s t i n g of a membrane-bound component, and a l o o s e l y bound p r o t e i n c a l l e d the s o l u b l e f a c t o r (34,80,91,97,98). N e i t h e r the 17. i s o l a t e d soluble factor nor the remaining membrane component independently catalyze transhydrogenation. However, recon-s t i t u t i o n of both energy-dependent and energy-independent transhydrogenase a c t i v i t i e s i s possible by mixing the membrane component with p a r t i a l l y p u r i f i e d soluble factor (91,97). The formation of a stable complex between the soluble factor and the membrane component was found to require low concen-trations of NADP+ or NADPH (97). The concentration of NADP+ or NADPH required for half-maximal binding was 0.4 yM and 0.2 yM, respectively. Concentrations of NADPH greater than 10 yM, however, in h i b i t e d binding of the soluble factor to the membrane component. Excess NADP+, on the other hand, was not i n h i b i t o r y i n t h i s respect (97). The membrane component was successfully extracted from chromatophores or from membranes depleted of soluble factor. L y s o l e c i t h i n proved to be the most e f f e c t i v e detergent for this extraction (91). The p u r i f i c a t i o n of the membrane com-ponent to homogeneity has not yet been achieved. The soluble factor could be detached from the chromato-phore membranes by extensive washing with buffer or by sub-jecting the chromatophores to chromatography on Sephadex G-200 or sucrose density gradient centrifugation (99). Treat-ment of the chromatophores with 40 to 90% saturated ammonium sulphate, followed by chromatography on DEAE-Sephadex, yielded a 2000-fold p u r i f i c a t i o n of the soluble factor, as compared to the crude c e l l - f r e e extract. The molecular weight of the 18. s o l u b l e f a c t o r , as determined u s i n g chromatography on Sepha-dex G-200, was approximately 70 000 (99). A n a l y s i s of p u r i -f i e d s o l u b l e f a c t o r on p o l y a c r y l a m i d e g e l s r e v e a l e d two (99) t o f o u r (97) p r o t e i n bands. I t i s not c l e a r whether the enzyme has been obtained i n homogeneous form. The transhydrogenase of E. c o l i has r e c e i v e d l i t t l e a t t e n t i o n w i t h regards to i t s 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 . Homyk and Bragg (90) r e p o r t e d i t s s o l u b i l i z a t i o n by the use of d e t e r g e n t s such as T r i t o n X-100, l y s o l e c i t h i n or sodium c h o l a t e i n the presence of ammonium su l p h a t e . A s i d e from these workers, however, on l y two other l a b o r a t o r i e s have been i n v o l v e d i n attempts to p u r i f y the enzyme (70,89,92). Hanson (92) s o l u b i l i z e d membrane p a r t i c l e s of E. c o l i w i t h 15% T r i t o n X-100. The e x t r a c t e d enzyme was subjected to chromatography on D E A E - c e l l u l o s e i n the presence of 1% T r i t o n X-100 and e l u t e d with 0.25 M NaCl i n potassium phosphate b u f f e r . The a c t i v e f r a c t i o n s were con c e n t r a t e d and subjected to chromatography on Sepharose 4B i n the presence of 0.1% B r i j 35. The s p e c i f i c a c t i v i t y of the pooled a c t i v e f r a c -t i o n s thus obtained was 10 u n i t s per mg p r o t e i n . T h i s represented a 7 1 - f o l d p u r i f i c a t i o n over the membrane p a r t i c l e suspension. The l a t t e r f i g u r e , however, may be an over-estimate, s i n c e the a c t i v i t y of the membrane p a r t i c l e sus-pension was measured i n the presence of T r i t o n X-100, which may i n t u r n have c o n t r i b u t e d t o the i n i t i a l l y low a c t i v i t y (0.14 u n i t s per mg p r o t e i n ) . The enzyme was not p u r i f i e d t o homogeneity by t h i s procedure. Membrane p a r t i c l e s from E. c o l i were a l s o s o l u b i l i z e d and the enzyme p a r t i a l l y p u r i f i e d u s i n g 0.5% (w/v) potassium deoxycholate i n the presence of potassium c h l o r i d e . T r i t o n X-100 (0.5%) was added t o the d i a l y z e d s o l u b i l i z e d f r a c t i o n and the s o l u t i o n a p p l i e d t o a column of D E A E - c e l l u l o s e . E l u t i o n was achieved by a l i n e a r s a l t g r a d i e n t i n the presence of 0.5% T r i t o n X-100. The f r a c t i o n s a c t i v e i n enzyme ac-t i v i t y were d e l i p i d a t e d by f u r t h e r treatment w i t h potassium c h o l a t e and ammonium su l p h a t e . The s p e c i f i c a c t i v i t y of t h i s p r e p a r a t i o n was 13.6 u n i t s per mg p r o t e i n when assayed i n the presence of p h o s p h o l i p i d s . T h i s r e p r e s e n t e d a 1 0 - f o l d p u r i f i c a t i o n over the membrane p a r t i c l e suspension (89). F u r t h e r p u r i f i c a t i o n , but not to homogeneity, c o u l d be achieved by an e x t e n s i o n of t h i s procedure. The l i p i d - d e p l e t e d enzyme was f u r t h e r f r a c t i o n a t e d on agarose A50M i n the presence of 0.1% potassium deoxycholate. The a c t i v i t y o f such a prep-a r a t i o n i n the presence of p h o s p h o l i p i d s was 20.2 u n i t s per mg p r o t e i n and r e p r e s e n t e d a 1 6 . 8 - f o l d p u r i f i c a t i o n over t h a t of the membrane p a r t i c l e suspension (70). A l k y l a t i o n of t h i s p r e p a r a t i o n and a n a l y s i s on p o l y a c r y l a m i d e g e l s i n the presence of SDS r e v e a l e d two major and f o u r minor p o l y p e p t i d e bands. The above s o l u b i l i z a t i o n procedure,''but stopped p r i o r t o the d e l i p i d a t i o n step, was used to p a r t i a l l y p u r i f y the t r a n s -hydrogenase from E. c o l i grown on [ 3H] casamino a c i d s and induced i n the presence of n o n - r e p r e s s i v e l e v e l s of [ l l*C]-l e u c i n e . The p o l y p e p t i d e s w i t h h i g h 1 1*C/ 3H r a t i o s i n the 20. p a r t i a l l y p u r i f i e d p r e p a r a t i o n of the enzyme represented newly s y n t h e s i z e d transhydrogenase. A n a l y s i s of t h i s p r e p a r a t i o n on p o l y a c r y l a m i d e g e l s i n the presence of SDS r e v e a l e d a major component of molecular weight 94 000 and one, or pos-s i b l y two, components of molecular weight 50 000 (70). I t i s not known whether the 50 000 m o l e c u l a r weight p r o t e i n i s a cleavage product of the 94 000 component. The presence of proteases (94-96) i n s i m i l a r p r e p a r a t i o n s c o u l d account f o r such a cleavage. The enzyme from E. c o l i i s more s e n s i t i v e t o t r y p s i n than t h a t from m i t o c h o n d r i a (2), render-i n g more l i k e l y the presence of s m a l l e r subunits i n p r e p a r a -t i o n s of the former enzyme than i n the l a t t e r (7 0). I t i s a l s o p o s s i b l e t h a t the enzyme from E. c o l i resembles t h a t of R. rubrum i n c o n s i s t i n g of two p r o t e i n components o r , c o n v e r s e l y , t h a t p r o t e o l y t i c cleavage i s r e s p o n s i b l e f o r the two p o r t i o n s of the transhydrogenase, the membrane-bound and the s o l u b l e f a c t o r s , of t h i s p h o t o s y n t h e t i c organism. L i p i d dependence The i n a c t i v a t i o n of m i t o c h o n d r i a l transhydrogenase by de t e r g e n t s (85,100), o r g a n i c s o l v e n t s (25,100) and phospho-l i p a s e s (25,101) suggests t h a t the enzyme r e q u i r e s l i p i d f o r i t s a c t i v i t y . Transhydrogenase e x t r a c t e d from b e e f - h e a r t mi t o c h o n d r i a w i t h t e r t i a r y amyl•alcohol was a c t i v a t e d by l e c i t h i n (102). Other evidence f o r the l i p i d dependency of the energy-independent transhydrogenase from b e e f - h e a r t mitochondria was subsequently p r o v i d e d by Rydstrom e t a l . (101) and by Anderson and F i s h e r (7) u s i n g d e l i p i d a t e d prep-a r a t i o n s of the enzyme. Mixed m i t o c h o n d r i a l p h o s p h o l i p i d s (7), p h o s p h a t i d y l c h o l i n e and phosphatidylethanolamine from b e e f - h e a r t m i t o c h o n d r i a were s t i m u l a t o r y as was l y s o l e c i t h i n (101) and soybean p h o s p h a t i d y l c h o l i n e (7). C a r d i o l i p i n stimu-l a t e d transhydrogenase a c t i v i t y but became i n h i b i t o r y a t h i g h c o n c e n t r a t i o n s . Between 0.2 and 0.5 umol per mg p r o t e i n of the v a r i o u s p h o s p h o l i p i d s were r e q u i r e d f o r half-maximal s t i m u l a t i o n . T h i s value i s c l o s e to the c o n c e n t r a t i o n of p h o s p h o l i p i d , 0.57 umol per mg p r o t e i n i n s u b m i t o c h o n d r i a l p a r t i c l e s (101). The l i p i d dependence of the transhydrogenase e x t r a c t e d from E. c o l i membranes has a l s o been shown (70,89, 90). E. c o l i p h o s p h o l i p i d s and soybean p h o s p h o l i p i d , c a r d i o -l i p i n , and to a l e s s e r extent, l e c i t h i n , s t i m u l a t e d the enzyme a c t i v i t y (89,90). The s t i m u l a t i o n of transhydrogenase a c t i v i t y by phospho-l i p i d s appears to be n o n - s p e c i f i c . I t i s l i k e l y t h a t the r o l e of p h o s p h o l i p i d s i n t h i s r e s p e c t i s to s t a b i l i z e an a c t i v e conformation of the enzyme by occupying hydrophobic s u r f a c e s of the transhydrogenase, which are normally exposed to the hydrophobic i n t e r i o r of the i n t a c t membrane. R e c o n s t i t u t i o n of the transhydrogenase system The i n c o r p o r a t i o n of p a r t i a l l y p u r i f i e d energy-independent transhydrogenase from b e e f - h e a r t m i t o c h o n d r i a i n t o p h o s p h o l i p i d v e s i c l e s i n the presence of p a r t i a l l y p u r i f i e d F^F ATPase complex, l e d t o the r e s t o r a t i o n of ATP-driven energy-dependent transhydrogenase a c t i v i t y (103). The r e c o n s t i t u t e d ATP-driven energy-dependent transhydrogenase a c t i v i t y was i n h i b i t e d by oli g o m y c i n and FCCP, while the energy-independent r e a c t i o n was not e f f e c t e d by these reagents. T h i s was c o n s i s t e n t w i t h the involvement of the ATPase complex and a proton g r a d i e n t i n e n e r g i z i n g the transhydrogenase r e a c t i o n t h a t had been p o s t u l a t e d e a r l i e r . The r e v e r s e transhydrogenase r e a c t i o n ( r e d u c t i o n of NAD + by NADPH) i n p h o s p h o l i p i d v e s i c l e s c o n t a i n -i n g the p a r t i a l l y p u r i f i e d enzyme showed an u n c o u p l e r - s e n s i t i v e uptake of te t r a p h e n y l b o r o n (TPB ). The tra n s h y d r o g e n a t i o n was s t i m u l a t e d by uncouplers and ionophores, i n d i c a t i n g the forma-t i o n of a membrane p o t e n t i a l i n the r e c o n s t i t u t e d system (88). T h i s i s c o n s i s t e n t w i t h the e a r l i e r f i n d i n g s of Skulachev' and coworkers (49,50) and Van de Stadt e t a l . (52) who showed t h a t i n s u b m i t o c h o n d r i a l p a r t i c l e s and R. rubrum chromatophores (104), the o x i d a t i o n of NADPH by NAD + generates a membrane p o t e n t i a l . S i m i l a r r e s u l t s have been obtained u s i n g t r a n s -hydrogenase p u r i f i e d to homogeneity (6,53) and i n c o r p o r a t e d i n t o d i o l e o y l - L - a - p h o s p h a t i d y l c h o l i n e liposomes. R e c o n s t i -t u t i o n r e s u l t e d i n a decreased enzyme a c t i v i t y . The s t i m u l a -t i o n of the r a t e of tra n s h y d r o g e n a t i o n i n both the forward and rev e r s e d i r e c t i o n by the uncoupler FCCP (53) or by v a l i n o m y c i n p l u s n i g e r i c i n i n the presence of potassium c h l o r i d e (6,77,78) l e d E a r l e e t a_l. (53) t o suggest t h a t r e c o n s t i t u t e d t r a n s -hydrogenase from b e e f - h e a r t m i t o c h o n d r i a a c t s as a r e v e r s i b l e proton pump. The i n h i b i t i o n of t r a n s h y d r o g e n a t i o n d e r i v e s from the pH g r a d i e n t generated a c r o s s the membrane. T h i s would be c o l l a p s e d upon a d d i t i o n of uncouplers t o a l l o w a continuous pumping of protons from the v e s i c l e i n t e r i o r t o the e x t e r i o r d u r i n g t r a n s h y d r o g e n a t i o n i n the forward d i r e c t i o n . More d i r e c t evidence f o r the a c i d i f i c a t i o n of the v e s i c l e i n t e r i o r d u r i n g the r e v e r s e t r a n s h y d r o g e n a t i o n r e a c t i o n was demonstrated by the f l u o r e s c e n c e quenching of the pH probe, 9-aminoacridine (77,78) and by e l e c t r o m e t r i c measurements i n the presence of v a l i n o m y c i n (105). R e c o n s t i t u t i o n of transhydrogenase a c t i v i t y w i t h d i o l e o y l p h o s p h a t i d y l c h o l i n e r e v e a l e d t h a t a p h o s p h o l i p i d t o p r o t e i n r a t i o of a t l e a s t 200 was r e q u i r e d t o achieve maximal i n c o r -p o r a t i o n of the enzyme i n t o the liposomes (77) . Uncoupler-s t i m u l a t e d v e s i c l e s d i d not show any f u r t h e r i n c r e a s e i n enzyme a c t i v i t y i n the presence of detergents (77,78) suggest-i n g t h a t the a c t i v e s i t e s of the enzyme molecule which normally face the m i t o c h o n d r i a l matrix, were l o c a t e d on the o u t s i d e of the v e s i c l e s . T h i s i n d i c a t e s t h a t the i n c o r p o r a t i o n of the transhydrogenase i n t o the s y n t h e t i c membrane i s asymmetric (77) . S i t e - s p e c i f i c i n h i b i t o r s Both energy-dependent and energy-independent pyridine nucleotide transhydrogenase a c t i v i t i e s from mitochondria are inh i b i t e d by a variety of cations such as C a 2 + and Mn 2 +, (81,106). The energy-independent reaction i s in h i b i t e d p r e f e r e n t i a l l y by Mg 2 + (7,46,47,81,106,107). Deuterium oxide, espe c i a l l y at low pH, has also been found to i n h i b i t the mito-chondrial * transhydrogenase reaction (108--110) . This e f f e c t i s not due to breakage of a C-H bond i n which hydrogen i s exchanged for deuterium, since deuterium does not i n h i b i t transhydrogenation when substituted for the 4A-hydrogen of NADH (109) and the hydrogen transferred from NADH to NADP+ does not exchange with the medium (79). Deuterium could i n h i b i t the reaction by i t s interference with the formation of an active form of the enzyme (109,110) or the coupling of trans-port of protons across the membrane to that of hydride ion equivalent transfer between the two substrates (50). Adenine nucleotides substituted at the 2'- or 3'-position (2'-AMP, 3'-AMP and 3':5'-AMP) were found to s p e c i f i c a l l y i n h i b i t mitochondrial transhydrogenase a c t i v i t y by competing with the substrate NADP(H), while adenine nucleotides without such a substituent (Adenosine, 51-AMP and ADP) were competitive i n h i b i t o r s with respect to NAD(H) (83,111). This led to the concept that there exists two d i s t i n c t s i t e s on the enzyme molecule, one for NAD(H) and one for NADP(H). The NAD(H) s i t e 25. was c o m p e t i t i v e l y i n h i b i t e d to the same extent by a c e t y l -dephospho-CoA and palmityl-dephospho-CoA (83,111). On the other hand, c o m p e t i t i v e i n h i b i t i o n a t the NADP(H) s i t e was g r e a t e r f o r pa l m i t y l - C o A than f o r acetyl-CoA or CoA, suggest-ing t h a t the NADP(H)-binding s i t e of the enzyme i s i n a hydro-phobic environment (83). The p a t t e r n of i n h i b i t i o n of the energy-independent t r a n s -hydrogenase of E. c o l i by adenine n u c l e o t i d e s i s s i m i l a r to t h a t of the enzyme f o r b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s (68,92,112). In a d d i t i o n , n o n - s p e c i f i c i n h i b i t i o n by p a l -m i t y l c a r n i t i n e and s t e a r o y l c a r n i t i n e was observed w i t h the enzyme from E. c o l i (68). The i n h i b i t i o n of transhydrogenases from beef h e a r t (113) , E. c o l i (89,112) and R. rubrum (98) by 2,3-butanedione, and the p r o t e c t i o n a g a i n s t t h i s by NAD+, NADP + and s u b s t r a t e analogues i m p l i e s the presence o f an a r g i n y l r e s i d u e a t or near the c a t a l y t i c s i t e of the enzyme. A mixture of NAD + and NADP + had a g r e a t e r e f f e c t than e i t h e r alone i n p r o t e c t i n g the transhydrogenase a g a i n s t m o d i f i c a t i o n by 2,3-butanedione, suggesting t h a t the b i n d i n g of these p y r i d i n e n u c l e o t i d e s to the transhydrogenase promotes a c o n f o r m a t i o n a l change i n the enzyme (98,112,113). I n h i b i t i o n s t u d i e s u s i n g 2,3-butanedione have, i n a d d i t i o n , been employed to l o c a l i z e NAD +- and NADP +-binding s i t e s on the R. rubrum transhydrogenase complex. The s o l u b l e f a c t o r ^ was p a r t i a l l y protected against modification by 2,3-butanedione by NAD or NADP at optimal concentrations. A 1:1 mixture of the two substrates afforded nearly complete protection against modification, implying the presence of separate binding s i t e s for NAD+ and NADP+ on th i s component of the transhydrogenase complex (9 8). That NADP+ does not bind to the NAD+ s i t e i n th i s factor was evidenced by the fact that soluble factor, bound to agarose-NAD+ gel, could not be eluted by NADP+. The membrane component, depleted of soluble factor, was completely protected against modification by 2,3-butanedione i n the presence of NADP+ or NADPH, but not NAD+. This indicates the presence of an NADP(H) s i t e on the membrane component (98) and was supported by thermal and t r y p t i c i n a c t i v a t i o n studies (114) i n which conformational changes were demonstrated upon binding of NADP+ or NADPH. Thus there are' at least three binding s i t e s for pyridine nucleotides on the R. rubrum transhydrogenase complex. The two NADP +-binding s i t e s have d i f f e r i n g a f f i n i t i e s for the substrate (97). I t was proposed that the high a f f i n i t y NADP +-binding s i t e regulates the forma-tion of the active transhydrogenase complex, while the lower a f f i n i t y NADP +-binding s i t e may be part of the active s i t e (98). In h i b i t i o n of the E. c o l i transhydrogenase a c t i v i t y by 2,3-butanedione or trypsin i s enhanced i n the presence of low concentrations of NADH or NADPH. This suggests that binding of the reduced pyridine nucleotides i s also able to cause a 27. c o n f o r m a t i o n a l change i n the enzyme (112). T r y p t i c and thermal i n a c t i v a t i o n of the enzyme from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s (107,115) r e v e a l e d t h a t b i n d i n g of e i t h e r NADP + or NADPH may promote a c o n f o r m a t i o n a l change i n t h i s t r a n s h y -drogenase, although the r e s u l t i n g conformers are d i f f e r e n t . M o d i f i c a t i o n by N-ethylmaleimide of s u l p h y d r y l groups of the enzymes from R. rubrum (116) and E. c o l i (89) was enhanced i n the presence of NADPH. T h i s p r o v i d e s f u r t h e r evidence t h a t the conformation of the enzyme i s a l t e r e d by the b i n d i n g of low c o n c e n t r a t i o n s of reduced p y r i d i n e n u c l e o t i d e . The s u b s t r a t e - i n d u c e d changes i n the conformation of the membrane-bound transhydrogenase support the p r o p o s a l t h a t proton t r a n s l o c a t i o n may be a f f e c t e d by a co n f o r m a t i o n a l change i n the transhydrogenase (115) d u r i n g the f u n c t i o n of the enzyme as a r e v e r s i b l e protonpump. Ste a d y - s t a t e k i n e t i c s o f transhydrogenation The mechanism of s t e a d y - s t a t e k i n e t i c s of the energy-independent and energy-dependent transhydrogenase from beef-h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s was s t u d i e d by Rydstrom e t a l . (117) and T e i x e i r a da Cruz e t a l . (118). They r e p o r t e d t h a t the tr a n s h y d r o g e n a t i o n r e a c t i o n proceeds by way of a t e r n a r y complex of a very s h o r t l i f e t i m e , w i t h the a d d i t i o n of s u b s t r a t e s and r e l e a s e of products o c c u r r i n g i n a d e f i n i t e o rder, t h a t i s , by a Theorell-Chance mechanism. T h i s con-c l u s i o n was based on l i n e a r and convergent double r e c i p r o c a l 28. plots of i n i t i a l v e l o c i t i e s versus substrate concentrations, as well as on product i n h i b i t i o n patterns. These revealed competitive relationships between the oxidized and reduced forms of the same pyridine nucleotide, and noncompetitive relationships between the substrates NAD+ and NADP+ and NADH and NADPH. They suggested that the transhydrogenase has sep-arate binding s i t e s for NAD(H) and NADP(H), and that NAD(H) i s the f i r s t substrate bound by the enzyme i n the reaction sequence (83). Similar r e s u l t s and conclusions were reported by Houghton et a l . (68) with the enzyme from E. c o l i . Cleland (119) pointed out that steady-state k i n e t i c s of a Theorell-Chance mechanism can also apply to a rapid e q u i l i b -rium random bireactant mechanism with two dead-end complexes. In such a mechanism the binding and release of substrates occurs i n no fixed order. The dead-end complexes are formed by the binding of one substrate by the enzyme at the binding s i t e of the second substrate. I n i t i a l v e l o c i t y patterns (83) revealed converging l i n e s i n the s i t e - s p e c i f i c i n h i b i t i o n by 2'-AMP at the NAD +-binding s i t e . These l i n e s , however, were interpreted as being p a r a l l e l by the author (83) who concluded that the rel a t i o n s h i p between 2'-AMP and NAD+ was uncompetititve. Such a conclusion led him to eliminate the rapid equilibrium random bireactant mechanism i n favour of a Theorell-Chance mechanism. The misinterpretation of Rydstrom's data led Homyk and Bragg (112) and Hanson (92) to re-evaluate the steady-state k i n e t i c s of the energy-independent transhydrogenase o f E. c o l i . Hanson (92)!, u s i n g a p a r t i a l l y p u r i f i e d , s o l u b i l i z e d enzyme, and Homyk and Bragg (112) w i t h the membrane-bound enzyme have found t h a t the energy-independent transhydrogenation i n E. c o l i proceeds by way of a r a p i d - e q u i l i b r i u m random b i r e a c t a n t mechanism and not by the Theorell-Chance mechanism as p r e v i o u s l y p o s t u l a t e d (68,117,118). These c o n c l u s i o n s were based on s i t e -s p e c i f i c i n h i b i t o r s t u d i e s by 21-AMP and 5'-AMP (92,112). I n i t i a l v e l o c i t y data taken over a wide range of s u b s t r a t e c o n c e n t r a t i o n s r e v e a l e d a unique p a t t e r n of l i n e s t h a t i s c h a r a c t e r i s t i c o f a r a p i d e q u i l i b r i u m random b i r e a c t a n t mech-anism w i t h two dead-end complexes (120). Furthermore, s t u d i e s with a l t e r n a t e s u b s t r a t e s (112) gave a p a t t e r n of i n h i b i t i o n s u p p o r t i n g such a mechanism and completely e l i m i n a t i n g the p o s s i b i l i t y of a Theorell-Chance mechanism. A summary of k i n e t i c constants determined f o r the energy-independent t r a n s -hydrogenase from d i f f e r e n t sources i s g i v e n i n Tabl e 1. C a t a l y t i c mechanism The mechanism of hyd r i d e i o n t r a n s f e r i n p y r i d i n e n u c l e o -t i d e transhydrogenase i s unknown. Experimental r e s u l t s i n -d i c a t e the d i r e c t t r a n s f e r of hy d r i d e i o n e q u i v a l e n t s between the p y r i d i n e n u c l e o t i d e r i n g s , t h a t i s , without exchange wi t h water i n the medium (8,9,79). T h i s r a i s e s the q u e s t i o n as t o the e x i s t e n c e of a c a r r i e r group oh the enzyme, capable of being reduced i n an in t e r m e d i a t e step, d u r i n g the exchange of Table 1. K i n e t i c Constants f o r S u b s t r a t e s o f the Energy-Independent Transhydrogenase from D i f f e r e n t Sources Source M i c h a e l i s c o n s t a n t , MxlO 5 Reference RAPNAD+ KNAD + NADPH m m m Submitochondrial 1.5 - 7.5 (27) p a r t i c l e s Submitochondrial - 2.8 2 (118) p a r t i c l e s R. rubrum - - 0.59 (97) chromatophores E. c o l i membrane 14.3 - 6.2 (68) p a r t i c l e s E. c o l i membrane 4.7 - 10.7 (112) p a r t i c l e s E. c o l i ( s o l u b i l i z e d 2.8 - 1.4 (92) pr e p a r a t i o n s ) h y d r i d e i o n e q u i v a l e n t s between the two p y r i d i n e n u c l e o t i d e s . Examination of homogeneous p r e p a r a t i o n s of transhydrogenase from b e e f - h e a r t submitochondrial p a r t i c l e s r e v e a l e d the absence of d e t e c t a b l e p r o s t h e t i c groups (6,7). T h i s might suggest t h a t the p a r t i c i p a t i o n of an acceptor f o r the hyd r i d e i o n e q u i v a l e n t s on the enzyme would be u n l i k e l y (80). However, Jacobs and F i s h e r (116), working w i t h the transhydrogenase o f R. rubrum, have pr o v i d e d evidence t h a t the enzyme appears t o be reduced i n an in t e r m e d i a r y step d u r i n g t r a n s h y d r o g e n a t i o n . The path of h y d r i d e i o n e q u i v a l e n t t r a n s f e r i n the t r a n s -hydrogenation r e a c t i o n o f R. rubrum has been proposed t o proceed a c c o r d i n g t o the r e a c t i o n : NADH + E NAD + + E-H (1) NADP + + E-H s?=^NADPH + E (2) A P N A D + + E-H ^ i A P N A D H + E (3) The reduced enzyme i n t e r m e d i a t e (E-H) c o u l d subsequently reduce NADP + (Equation 2) or APNAD4" (Equation 3). The s e q u e n t i a l occurrence of r e a c t i o n s (1) and (2) cou l d c a t a l y z e a r e a c t i o n termed "TD transh y d r o g e n a t i o n " by F i s h e r and G u i l l o r y (80), whereas t h a t of r e a c t i o n s (1) and (3) would c a t a l y z e "DD transhyd r o g e n a t i o n " . Since the transhydrogenase of R. rubrum resembles t h a t of mitochondria and E. c o l i i n many ways, i t i s of i n t e r e s t t o s p e c u l a t e upon a mechanism by which the enzyme can be r e -duced. The presence of s u l p h y d r y l groups has been demonstrated i n the transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t -i c l e s , R. rubrum chromatophores and E. c o l i (89,99,116,121,122). D i s u l p h i d e r e d u c t i o n by p y r i d i n e n u c l e o t i d e s i s c a t a l y z e d by s e v e r a l f l a v o p r o t e i n enzymes, f o r example, d i h y d r o l i p o y l de-hydrogenase, t h i o r e d o x i n reductase and g l u t a t h i o n e r e d u c t a s e . Although the transhydrogenase i s not a f l a v o p r o t e i n (6,7), r e d u c t i o n of a s u l p h y d r y l group on the enzyme by NADH or NADPH might be i n v o l v e d i n an analogous manner d u r i n g transhydrogen-a t i o n , however, i n v e s t i g a t i o n of e s s e n t i a l s u l p h y d r y l groups of transhydrogenase have shown t h i s t o be u n l i k e l y . In t h i s enzyme, two types of s u l p h y d r y l groups w i t h d i f f e r i n g s e n s i -t i v i t i e s t o modifying reagents e x i s t (122,123). In E. c o l i , the e s s e n t i a l s u l p h y d r y l group (s) are presen t i n both the NADP - and NAD - b i n d i n g s i t e of the enzyme (89). In the mito-c h o n d r i a l transhydrogenase the presence of one type of s u l p -h y d r y l group was demonstrated a t the NADP +-binding s i t e of the enzyme, w h i l e the other type was p e r i p h e r a l t o the a c t i v e s i t e . The p e r i p h e r a l s u l p h y d r y l group was proposed t o f u n c t i o n i n the maintenance o f enzyme conformation through i o n i c of hydrogen-bonding i n t e r a c t i o n s . N e i t h e r group was ap p a r e n t l y e s s e n t i a l f o r c a t a l y s i s s i n c e m o d i f i c a t i o n w i t h methyl methane-t h i o s u l p h o n a t e (MMTS) d i d not completely i n h i b i t .the enzyme a c t i v i t y (122). M o d i f i c a t i o n of s u l p h y d r y l groups i h the transhydrogenase of R. rubrum chromatophores by N-ethylmaleimide i n d i c a t e d t h a t the r e a c t i v e groups are not l i k e l y t o occupy the NADP^-binding s i t e s of the transhydrogenase complex s i n c e NADPH o f f e r e d no p r o t e c t i o n a g a i n s t the m o d i f i c a t i o n (116). Thus the reduced enzyme i n t e r m e d i a t e d u r i n g t r a nshydrogenation i n t h i s system probably does not i n v o l v e s u l p h y d r y l groups. However, f u r t h e r i n v e s t i g a t i o n i s necessary t o determine whether r e d u c t i o n of the transhydrogenase enzymes of mi t o c h o n d r i a and E. c o l i i n v o l v e s s u l p h y d r y l groups. There i s some evidence f o r the involvement of oth e r amino a c y l groups a t the a c t i v e s i t e . The i n a c t i v a t i o n of the t r a n s -hydrogenase complex and membrane component of R. rubrum by 2,4-pentanedione, and the r e v e r s a l of t h i s m o d i f i c a t i o n by hydroxylamine, suggests the presence of l y s y l r e s i d u e ( s ) at the a c t i v e s i t e . That the l y s y l r e s i d u e e x i s t s a t the NADP4" s i t e of the membrane component of the transhydrogenase i n t h i s organism i s supported by thermal i n a c t i v a t i o n s t u d i e s which demonstrated t h a t NADP + or NADPH c o u l d not bin d to the 2,4-pentanedione-modified membrane component. M o d i f i c a t i o n of the membrane component by 2,.4-pentanedione i n h i b i t e d the r e -c o n s t i t u t i o n o f DD-transhydrogenation as w e l l as TD-trans-hydrogenation. P r o t e c t i o n a g a i n s t m o d i f i c a t i o n was a f f o r d e d by NADP4" or NADPH, but not by NAD4". I t was proposed t h a t the r e d u c t i o n of NAD4" by NADH i s a p a r t i a l r e a c t i o n c a t a l y z e d by the transhydrogenase complex and t h a t the same membrane component i s i n v o l v e d i n both TD- and DD-transhydrogenation which i s s e n s i t i v e t o 2,4-pentanedione (116). S t u d i e s w i t h 2,3-butanedione (89,98,112,113) have a l s o r e v e a l e d the involvement of a r g i n y l r e s i d u e s ( s ) a t or near the s u b s t r a t e b i n d i n g s i t e ( s ) of the enzyme from mi t o c h o n d r i a , R. rubrum and E. c o l i . In R. rubrum chromatophores a r g i n y l r e s i d u e s on the mem-brane component of the transhydrogenase r e s i d e o u t s i d e the NADP +-binding domain and have been r e p o r t e d to f u n c t i o n i n the r e c o g n i t i o n and b i n d i n g of the soluble, f a c t o r . These r e s i d u e s would be p r o t e c t e d by 2,.3-butanedione upon b i n d i n g of NADP + to the membrane component, s i n c e t h i s promotes a c o n f o r m a t i o n a l change i n the enzyme (116). I t i s l i k e l y t h a t the a r g i n y l and l y s y l r e s i d u e s i n the transhydrogenase enzyme are i n v o l v e d i n b i n d i n g of the n u c l e o -t i d e coenzyme s u b s t r a t e s , as has been demonstrated i n other enzyme systems (124,125). O b j e c t i v e of t h i s study Although the p y r i d i n e n u c l e o t i d e transhydrogenase enzyme was d i s c o v e r e d i n 1952, l i t t l e was known about the importance of t h i s transmembranous enzyme u n t i l r e c e n t l y . I t s p o t e n t i a l f u n c t i o n as a r e v e r s i b l e proton pump as suggested by M i t c h e l l (126) has spurred renewed i n t e r e s t i n the mechanism of t h i s enzyme, i t s p u r i f i c a t i o n and r e c o n s t i t u t i o n i n t o a r t i f i c i a l membranes. The l a c k of s u c c e s s f u l p u r i f i c a t i o n of the transhydrogen-ase from E. c o l i made i t d e s i r a b l e to seek f o r methods of s o l u b i l i z i n g and p u r i f y i n g t h i s enzyme, and to study some of i t s p r o p e r t i e s . T h i s has been attempted i n the work d e s c r i b e d i n t h i s t h e s i s . Moreover, the mechanism of the s t e a d y - s t a t e k i n e t i c s of the enzyme has been r e - e v a l u a t e d , s i n c e t h a t pro-posed e a r l i e r (83,117,118) appeared t o be i n c o r r e c t . MATERIALS AND METHODS Reagents A l l chemicals used were of reagent-grade p u r i t y . Chemicals were purchased from the f o l l o w i n g s u p p l i e r s : A s s o c i a t e d Concentrates, Inc.: A s o l e c t i n ( P u r i f i e d Soya phosphatides)-A t l a s Chemical I n d u s t r i e s , Canada: B r i j 58 BBL: T r y p t i c a s e - s o y b r o t h powder BDH Chemicals L t d . : Sodium dodecyl sulphate (SDS) Bio-Rad L a b o r a t o r i e s : H y d r o x y l a p a t i t e (Bio-Gel HTP), acrylamide, N,N 1-methylene-bis-acrylamide Calbiochem: Bovine serum albumin, bovine c a t a l a s e , bovine pancreas RNase, y e a s t a l c o h o l dehydrogenase, phenazine metho-sulphate (PMS), P i p e r a z i n e - N , N 1 - b i s - ( 2 - e t h a n e s u l p h o n i c acid) -monosodium monohydrate (PIPES), N - t r i s - ( h y d r o x y m e t h y l ) m e t h y l -2-aminoethanesulphonic a c i d (TES), N - t r i s - ( h y d r o x y m e t h y l ) -methyl g l y c i n e (TRICINE) Canamex Productos Quimicos, Mexico: B r i j 3 6T DIFCO L a b o r a t o r i e s : Agar, y e a s t e x t r a c t E.C. Apparatus C o r p o r a t i o n : Ammonium p e r s u l p h a t e , cyanogen 41 g e l l i n g agent Eastman Organic Chemicals: 2,6-dichlorophenolindophenol (DCIP), N,N,N',N 1-tetramethylethylenediamine (TMED) F i s h e r S c i e n t i f i c Company: B r i j 35, bromophenol bl u e Mann Research L a b o r a t o r i e s , Inc.: Tween 20, Tween 40, Tween 60, Tween 80, thyreoglobulin 37. Matheson, Coleman and B e l l Manufacturing Chemists: Acetaldehyde N u t r i t i o n a l Biochemicals C o r p o r a t i o n : G l y c y l g l y c i n e Onyx Chemical Company: Ammonyx LO Pharmacia F i n e Chemicals, Inc.: Sephadex G-25, Sepharose 4B, Sepharose 6B, DEAE-Sepharose CL-6B Schwartz Mann: Bovine pancreas DNase Sigma Chemical Company: Phenyl g l y o x a l , 2,3-butanedione, l y s o l e c i t h i n (egg y o l k ) , T r i t o n X-100, sodium deoxycholate, Cooma'ssie; b r i l l i a n t b l u e R, (N-2-hydroxyethylpiperazine-N 1-2-ethanesulphonic acid) (HEPES), morpholinopropane s u l p h o n i c a c i d (MOPS), 2(N-morpholino)ethane s u l p h o n i c a c i d (MES), 2'-AMP, 5'-AMP, ADP, NAD(H), APNAD(H), NADP(H), deamino NADPH Whatman Biochemicals L t d . : DEAE-Cellulose (DE52) Worthington B i o c h e m i c a l C o r p o r a t i o n : Phosphorylase a, lysozyme ( p i g ) , T BCK-trypsin, soybean t r y p s i n i n h i b i t o r . Acetaldehyde and 2,3-butanedione were r e d i s t i l l e d p r i o r to use. P a l m i t i c a c i d was r e c r y s t a l l i z e d and was a generous g i f t from Dr. A. Burton. P u r i f i e d ATPase from E. c o l i ML308-225••' was prepared by Mrs. C y n t h i a Hou. B u f f e r s The a b b r e v i a t i o n s f o r the b u f f e r s commonly used i n the experimental s e c t i o n of t h i s t h e s i s are l i s t e d below. The c o n c e n t r a t i o n s of the T r i s or potassium phosphate component of the b u f f e r as w e l l as i t s pH are i n d i c a t e d i n each case i n 38. * the t e x t . TED-1: T r i s - H 2 S O i t / l mM EDTA/1 mM DTT TED-2: Tris-CH 3COOH/3 mM EDTA/2 mM DTT TEGD: Tris-CH3COOH/3 mM EDTA/10% (v/v) g l y c e r o l / 0 . 6 mM DTT TGD-1: T r i s - H C l / 0 . 1 mM DTT/10% (v/v) g l y c e r o l TGD-2: T r i s - H C l / 1 mM DTT/20% (v/v) g l y c e r o l TGDS/KCl/cholate: T r i s - H C l / 1 0 % (v/v) g l y c e r o l / 0 . 6 mM DTT/ 20 mM disodium s u c c i n a t e / 1 M KC1/0.1% (w/v) sodium c h o l a t e TM: T r i s - H 2 S O 4 / 1 0 mM MgSOk TMDG: Tris-HaSO^/S mM MgSO^/1 mM DTT/10% (v/v) g l y c e r o l PDGX: potassium phosphate/1 mM DTT/20% (v/v) g l y c e r o l / 0.5% (w/v) T r i t o n X-100 B a c t e r i a l s t r a i n s and t h e i r maintenance E. c o l i w i l d - t y p e K-12, NRC-482, ML308-225 or the p r o l i n e auxotroph E. c o l i W6 (ATCC 25377) were the s t r a i n s used i n t h i s study. E. c o l i K-12 c e l l s were purchased as harvested c e l l s , commercially grown on e n r i c h e d medium and were s t o r e d at -20°C. They were used i n t h i s study s o l e l y as a source of l i p i d s . The three other s t r a i n s were maintained on s l a n t s c o n t a i n i n g t r y p t i c a s e - s o y , b r o t h powder, y e a s t e x t r a c t powder and agar. The s l a n t s were prepared by d i s s o l v i n g 3 g * For example, 50 mM TED-1 b u f f e r , pH 7.8 i s 50 mM T r i s -E2S0^ b u f f e r , pH 7.8, c o n t a i n i n g 1 mM EDTA and 1 mM DTT. t r y p t i c a s e - s o y b r o t h and 0.3 g y e a s t powders i n 100 ml d i s -t i l l e d water. T h i s was brought to a b o i l , a f t e r which 1.5 g agar was added. The mixture was b r i e f l y reheated to d i s s o l v e the agar and was then dispensed i n 5 ml p o r t i o n s i n t o t e s t tubes. The tubes were s t e r i l i z e d by a u t o c l a v i n g f o r 20 min a t a pressure of 15 p s i . A f t e r t h e i r removal from the auto-c l a v e they were allowed to c o o l i n a s l i g h t l y t i l t e d p o s i t i o n so t h a t the n u t r i e n t b r o t h c o u l d s o l i d i f y as a s l a n t . <The b a c t e r i a l c u l t u r e s were stre a k e d onto the s l a n t s , incubated a t 37°C o v e r n i g h t and then s t o r e d a t 4°C u n t i l needed. C u l t u r e s were maintained by t r a n s f e r to f r e s h s l a n t s every two months. C u l t u r e s to be s t o r e d f o r longer p e r i o d s of time were i n o c u l a t e d i n t o n u t r i e n t medium prepared the same way as s l a n t s , but c o n t a i n i n g 0.7% (w/v) agar i n s t e a d of 1.5% (w/v) and dispensed, as 2 ml a l i q u o t s i n t o v i a l s which were kept i n u p r i g h t p o s i t i o n s a f t e r a u t o c l a v i n g . Growth c o n d i t i o n s E. c o l i s t r a i n s NRC-482 and ML308-225 were grown on minimal s a l t s / g l u c o s e media c o n t a i n i n g : 0.7% (w/v) K 2 H P O i * , 0.3% (w/v) K H 2 P O 4 , 0.05% (w/v), sodium c i t r a t e - 2 H 2 O , 0.02% (w/v) MgSOit • 7 H 2 O , 0.1% (w/v) (NHU) 2 S O 4 and 0.4% (w/v) g l u c o s e . F e r r i c c i t r a t e (12 uM) was added to growth media of volumes exceeding 1 i. The growth medium f o r E. c o l i s t r a i n W6 was supplemented w i t h 50 yg/ml p r o l i n e . 40. C e l l t r a n s f e r s were made from s l a n t s or stabs i n t o -10 ml of growth medium which was then incubated o v e r n i g h t a t 37°C. The growing c e l l s were t r a n s f e r r e d as. 10% (v/v) i n o c u l a i n t o large. , volumes of media. Batches of 2-2.5 I were grown at 37°C w i t h v i g o r o u s a e r a t i o n p r o v i d e d through g l a s s s i n t e r e d s p a r g e r s . C e l l growth was monitored by measuring the absorb-ance of the c u l t u r e a t 420 nm. The c e l l s were harvested a t the l a t e l o g a r i t h m i c phase of growth by c e n t r i f u g a t i o n a t 4500 x g f o r 15 min at 2-4°C. C e l l p e l l e t s were then washed by resuspension i n 50 mM TM b u f f e r , pH 7.8 or 50 mM TED-1 b u f f e r , pH 7.8 and c e n t r i f u g e d at 17 600 x g f o r 10 min. The c e l l p e l l e t s were s t o r e d a t -20°C u n t i l needed. P r e p a r a t i o n of membrane p a r t i c l e s The method f o l l o w e d by Bragg e t a l . (67) was used. C e l l s were suspended i n 50 mM TM b u f f e r , 50 mM TED-1 b u f f e r or 50 mM sodium borate b u f f e r , pH 7.8 to a d e n s i t y of approximately 0.1 g wet weight/ml b u f f e r . Approximately 5-10 ug of DNase was added and the c e l l s were d i s r u p t e d by one, or i n the case of dense suspensions, two passage(s) , through an Aminco French p r e s s u r e c e l l , p r e - c o o l e d t o 0°C, a t a p r e s s u r e of 20 000 p s i . The suspension was c e n t r i f u g e d at 17 000 x g f o r 15 min to remove unbroken c e l l s and c e l l d e b r i s . The supernatant was then c e n t r i f u g e d a t 120 000 - 138 000 x g f o r 1.5-2 h. A l l c e n t r i f u g a t i o n steps were c a r r i e d out a t 0-5°C. In some cases the p e l l e t of membrane p a r t i c l e s was washed by resuspension, i n the p a r t i c u l a r b u f f e r used d u r i n g d i s r u p t i o n by the French p r e s s u r e c e l l , and r e c e n t r i f u g e d as b e f o r e . The p e l l e t was then processed a t 0°C i n a v a r i e t y of ways as d e s c r i b e d i n the RESULTS s e c t i o n . P r e p a r a t i o n of EDTA-lysozyme s p h e r o p l a s t s The method of Weiner and Heppel (127) was used f o r the p r e p a r a t i o n of EDTA-lysozyme s p h e r o p l a s t s . C e l l s of E. c o l i . ML308-225 (8 g) were washed with , and suspended in,10 volumes (w/v) of 0.01 M T r i s - H C l b u f f e r , pH 8, a t 4°C. EDTA and lysozyme were added to c o n c e n t r a t i o n s of 0.01 M and 1 mg/ml, r e s p e c t i v e l y . The suspension was incubated a t 37°C f o r 25 min. I t was then f r o z e n by immersion i n a mixture of e t h a n o l and dry i c e and thawed at 37°C f o r 5 min. The freeze-thawing procedure was repeated twice more, a f t e r which M g C l 2 was added to the suspension to a c o n c e n t r a t i o n of 0.02 M. Follow-i n g the a d d i t i o n of RNase (10 yg per ml) and DNase (10 ug per ml), the mixture was incubated a t 37°C f o r 30 min, then c e n t r i f u g e d a t 48 000 x g and 0°C f o r 20 min. The p e l l e t was then t r e a t e d as d e s c r i b e d i n the RESULTS section'. Determination of p r o t e i n P r o t e i n c o n c e n t r a t i o n was determined by the method of Lowry e t a l . (128) over a c o n c e n t r a t i o n range of 0-500 ug/ml. C r y s t a l l i n e bovine serum albumin was used as a standard. To c o r r e c t f o r i n t e r f e r e n c e by b u f f e r components i n the assay, standard curves were made to i n c o r p o r a t e the a p p r o p r i a t e sample b u f f e r i n volumes e q u i v a l e n t to the volume of sample assayed. Due to t u r b i d i t y , samples- i n which T r i t o n X-100 was a b u f f e r component were c e n t r i f u g e d a t 14 000 x g f o r 10 min p r i o r to measurement of the absorbance. Determination of Cytochrome b-j_ The sample (1.5 ml) a t 22°C was t r a n s f e r r e d i n t o each of two c u v e t t e s having a 1 cm l i g h t path. Approximately 0.05 ml of 0.3% (v/v) hydrogen peroxide was added to the r e f e r e n c e c u v e t t e w h i l e the sample i n the other c u v e t t e was reduced w i t h excess sodium d i t h i o n i t e . The reduced versus the o x i d i z e d spectrum was then scanned between 400 and 600 nm i n a Perkin-Elmer model 3 56 double beam spectrophotometer, equipped w i t h a r e c o r d e r . The h e i g h t of the Soret band i n the d i f f e r e n c e spectrum was taken as the d i f f e r e n c e i n absorbance between 410 nm and 429 nm. B a s e l i n e readings were obtained i n the 400-600 nm range by scanning two c u v e t t e s c o n t a i n i n g u n t r e a t e d samples. The cytochrome b^ content was c a l c u l a t e d u s i n g an e x t i n c t i o n c o e f f i c i e n t of 153 &/mmol/cm (129). Enzyme assay procedures Energy-independent transhydrogenase T h i s was based upon the method of Kaplan (5). The r e a c t i o n was c a r r i e d out a t room temperature (22-25°C). The mixture i n a cuvette with a 1 cm l i g h t path contained approximately 50-2 ug of membrane protein, 0.5 ml of 1 M potassium phosphate buffer, pH 7.0, containing 2 mM KCN (unless otherwise i n d i -cated i n the RESULTS section), and d i s t i l l e d water to give a f i n a l volume of 0.9 ml. 50 ul of 21 mM APNAD+ was then added, followed by 50 u l of 11 mM NADPH and the cuvette was immediately placed i n a Perkin-Elmer model 124 spectrophoto-meter. The reduction of APNAD+ was measured as an increase i n absorbance at 375 nm. The sample was read against a d i s t i l l e d water blank containing 25-50 u l of 11 mM NADPH. This was done i n order to keep the absorbance reading within the operating range of the spectrophotometer. The extinction c o e f f i c i e n t was taken as 5.1 Ji/mmol/cm (5,27). This w i l l subsequently be referred to as the "standard assay". In some experiments the substrate concentrations were varied, or else i n h i b i t o r s , activators or substrate analogues were added. These modifications are i n d i v i d u a l l y discussed i n the RESULTS section. One unit of enzyme a c t i v i t y represents the conversion of 1 umol of APNAD+ to APNADH per min. Energy-dependent transhydrogenase This was measured by a modification of the method of Fisher and Sanadi (31). One ml of 50 mM TM buffer, pH 7.8, containing 0.1% (w/v) bovine serum albumin, 1.3 mM d i t h i o -t h r e i t o l and 0.16 M sucrose was incubated i n a cuvette with a 1 cm l i g h t path a t 37°C f o r 5 min wit h 50 u l of membrane p a r t i c l e s ( c o n t a i n i n g 0.6 to 1.7 mg p r o t e i n ) . The c u v e t t e was then t r a n s f e r r e d to a Perkin-Elmer model 124 s p e c t r o -photometer, maintained a t 37°C by means of. a c i r c u l a t i n g water bath, and equipped w i t h a r e c o r d e r . 10 u l of e t h a n o l , 50 u l of yeast a l c o h o l dehydrogenase (4 mg/ml) and 25 u l of 2.7 mM NAD + was then added. The r e a c t i o n mixture was immed-i a t e l y scanned a t 340 nm f o r 0.66 min whereupon 50 u l of 15.7 mM NADP + was added and the r e d u c t i o n of NADP + measured as an i n c r e a s e i n absorbance a t 34 0 nm ("aer o b i c - d r i v e n t r a n s -hydrogenase") . When the oxygen i n the c u v e t t e was exhausted, the r a t e o f NADP + r e d u c t i o n a b r u p t l y f e l l . (This r a t e c o r -responds t o the energy-independent transhydrogenase.) A f t e r t h i s r a t e had been e s t a b l i s h e d , 10 u l of 65 mM ATP ( d i s -s o l v e d i n 50 mM Tris-HaSCK and adj u s t e d t o pH 7.8 w i t h con-c e n t r a t e d KOH) was added, and the new r a t e of NADP + r e d u c t i o n was then measured ("ATP-driven transhydrogenase"). The r a t e of the ATP-driven transhydrogenase r e a c t i o n was c o r r e c t e d by s u b t r a c t i n g from i t the r a t e of the energy-independent t r a n s -hydrogenase r e a c t i o n . The e x t i n c t i o n c o e f f i c i e n t was taken as 6.22 5,/mmol/cm. One u n i t o f enzyme a c t i v i t y r e p r e s e n t s the c o n v e r s i o n o f one umol of NADP + to NADPH. S u c c i n a t e dehydrogenase The method used was a m o d i f i c a t i o n of t h a t of King (130). The stock assay medium was prepared as f o l l o w s . 7 mg KCN, 3 mg d i c h l o r o i n d o p h e n o l and 270 mg disodium s u c c i n a t e hexa-hydrate were d i s s o l v e d i n 50 ml o f 0.1 M potassium phosphate b u f f e r , pH 7.5. The mixture was s t i r r e d f o r 0.5-1 h and f i l t e r e d i n t o a dark g l a s s b o t t l e . 1.5 ml of the stock assay medium, c o n t a i n i n g 2.15 mM KCN, 1.8 4 mM DCIP and 2 0 mM d i -sodium s u c c i n a t e hexahydrate, was t r a n s f e r r e d i n t o a c u v e t t e w i t h a 1 cm l i g h t path and p l a c e d i n a Perkin-Elmer model 124 spectrophotometer s e t a t 600 nm and equipped with a r e c o r d e r . 0.1-0.2 ml of sample to be assayed was added, f o l l o w e d by 25 u l of 18.3 mM phenazine methosulphate and the decrease i n absorbance a t 6 00 nm was measured. The e x t i n c t i o n c o e f f i c i e n t was taken as 21 £/mmol/cm (131). One u n i t of a c t i v i t y i s the amount of enzyme r e q u i r e d t o o x i d i z e 1 umol of s u c c i n a t e per minute. C a t a l a s e T h i s was based upon the method of Beers and S i z e r (132) and was c a r r i e d out a t 22°-25°C. 1.2 ml of 3% hydrogen peroxide was mixed w i t h 2 0 ml of 50 mM potassium phosphate b u f f e r , pH 7.0. One ml of t h i s mixture was p l a c e d i n t o a c u v e t t e f o l l o w e d by the a d d i t i o n of 0.1-0.4 ml of the enzyme t o be measured. The change i n absorbance was measured a t 240 nm u s i n g a spectrophotometer equipped w i t h a r e c o r d e r . Enzyme a c t i v i t y i s expressed as the rate of change in. absorbance per minute rather than quantitated i n mmol/min since i t was only used for comparative purposes i n gel f i l t r a t i o n or sucrose gradient experiments where catalase was employed as a standard of molecular weight 247 500 (133). ATPase The incubation of ATPase with ATP was done according to Davies and Bragg (134). The inorganic phosphate which was released by the above reaction was determined by a modification of the method described by Ames (135). The enzyme sample was incubated at 37°C with 0.5 ml of 100 mM Tris-HCl buffer, pH 8.5, containing 5 mM ATP and 2.5 mM C a C l 2 - After 60 min of incubation, 2.5 ml of "inorganic phosphate assay mixture" consisting of 1.8 ml ammonium molybdate reagent (0.42% (w/v) ammonium molybdate*4Hz0 i n 2.86% (v/v) H 2SCM, 0.3 ml of 10% (w/v) ascorbic acid, 0.25 ml of 10% (v/v) t r i c h l o r o a c e t i c acid and 0.15 ml water was added. The reaction was allowed to proceed for 15 min at 37°C, a f t e r which i t was stopped by placing the samples at 0°C. The absorbance at 660 nm was measured against a reagent blank. The amount of inorganic phosphate li b e r a t e d was c a l -culated from a standard curve. One unit of ATPase a c t i v i t y i s defined as the amount of enzyme which l i b e r a t e s 1 umol of phosphate per min. 47. Treatment of membrane p a r t i c l e s w i t h phenyl g l y o x a l 100 mM phenyl g l y o x a l s o l u t i o n was prepared by d i s s o l v i n g 13.4 mg i n 0.7 ml 50 mM sodium borate b u f f e r , pH 7.8. The pH was r e a d j u s t e d by the a d d i t i o n of a few m i c r o l i t e r s of c o n c e n t r a t e d NaOH i n sodium bo r a t e b u f f e r and the f i n a l volume made up to 1 ml w i t h sodium borate b u f f e r , pH 7.8. E. c o l i W6 c e l l s (0.65 g) were washed i n 50 mM TED-1 b u f f e r , pH 7.8. Membrane p a r t i c l e s were prepared and r e -suspended i n 50 mM sodium borate b u f f e r , pH 7.8, a t a concen-t r a t i o n o f 3.92 mg/ml p r o t e i n . The i n c u b a t i o n mixtures a t 22°C c o n t a i n e d 0-10 mM phenyl g l y o x a l , 250 u l of enzyme suspension ( f i n a l p r o t e i n c o n c e n t r a -t i o n , 1.96 mg/ml) and 50 mM sodium borate b u f f e r , pH 7.8, t o g i v e a f i n a l volume of 0.5 ml. Immediately upon mixing of the i n c u b a t i o n components, 50 u l was withdrawn i n t o a c u v e t t e and assayed f o r energy-independent transhydrogenase a c t i v i t y . A c o n t r o l v a l u e was obtained f o r each of the p h e n y l g l y o x a l c o n c e n t r a t i o n s . Samples were withdrawn a t timed i n t e r v a l s f o r assay and t h e i r a c t i v i t i e s -expressed as a percentage of the c o n t r o l v a l u e s taken a t the onset of the experiment. Treatment o f membrane p a r t i c l e s w i t h 2,3-butanedione In the experiments s t u d y i n g the e f f e c t i v e n e s s of 2,3-butanedione as a modifying reagent, a 100 mM s o l u t i o n of 2,3-butanedione was made up i n 50 mM sodium borate b u f f e r and the pH a d j u s t e d t o 7.8. E. c o l i W6 c e l l s (0.75 g) were 48. washed i n 50 mM TED-1 b u f f e r , pH 7.8. Membrane p a r t i c l e s were prepared, washed and resuspended i n 50 mM sodium borate b u f f e r , pH 7.8, a t a c o n c e n t r a t i o n of 2.2 mg p r o t e i n / m l . The i n c u b a t i o n s a t 22°C were c a r r i e d out i n the same way as those with phenyl g l y o x a l , but u s i n g 2,3-butanedione a t a co n c e n t r a -t i o n of 0-30 mM and membrane p r o t e i n a t a c o n c e n t r a t i o n of 1.1 mg/ml i n a f i n a l volume of 0.5 ml sodium borate b u f f e r . Energy-independent transhydrogenase a c t i v i t i e s were assayed at timed i n t e r v a l s as s t a t e d above. In the experiments to demonstrate the e f f e c t of n u c l e o -t i d e s upon 2,3-butanedione t r e a t e d membranes 54 mM 2,3-butanedione i n 50 mM sodium borate b u f f e r , pH 7.8, was used i n the i n c u b a t i o n mixtures. The pH of the i n c u b a t i o n mixture was 7.2. N u c l e o t i d e s were d i s s o l v e d i n borate b u f f e r and t h e i r pH a d j u s t e d t o 7.8. The enzyme suspension was incubated w i t h the n u c l e o t i d e and the m o d i f i c a t i o n i n i t i a t e d by the a d d i t i o n o f the 2,3-butanedione. The f i n a l volume of the i n c u b a t i o n mixture was 0.5 ml. 50 y l samples were assayed as above. P r o t e i n and n u c l e o t i d e c o n c e n t r a t i o n s are g i v e n i n the d e s c r i p t i o n s of the i n d i v i d u a l experiments (RESULTS s e c t i o n ) . The experiments i n v o l v i n g the use of reduced p y r i d i n e n u c l e o -t i d e s w i t h butanedione-modified enzymes were c a r r i e d out i n the dark. The i n c u b a t i o n mixtures were p r o t e c t e d from l i g h t by u s i n g aluminum f o i l - c o v e r e d tubes. The samples were p r o t e c t e d from exposure to l i g h t as f a r as p o s s i b l e d u r i n g h a n d l i n g and a s s a y i n g . 49. Treatment of E. c o l i W6 with TPCK-trypsin Membrane p a r t i c l e s from E. c o l i W6 c e l l s were prepared, washed and resuspended i n 50 mM TED-1 buffer, pH 7.8, to a pro^ t e i n concentration of 1.6-3.0 mg/ml. The incubation mixture at 22°C contained the membrane suspension, TPCK-trypsin at a concentration of 0.42-0.9 yg per mg membrane protein, the appropriate pyridine nucleotide (concentrations are given for the i n d i v i d u a l experiments i n the RESULTS section), and 50 mM TED-1 buffer, pH 7.8, to a f i n a l volume of 0.25-0.275 ml. 50 y l samples of the incubation mixture were taken out at timed i n t e r v a l s a f t e r the addition of TPCK-trypsin and transferred to test tubes containing trypsin i n h i b i t o r i n 50 mM TED-1 buffer," pH 7.8 (2-2.2 yg per yg TPCK-trypsin) and kept i n i c e . Transhydrogenase a c t i v i t y was measured as described above. S o l u b i l i z a t i o n of membrane p a r t i c l e s with detergents  Sodium cholate One of two procedures was used. A: The f i r s t i s a modification of that described by Rydstrom et a l . - (88). • Membrane' p a r t i c l e s from E. c o l i ML308-225 were prepared i n 50 mM TM buffer, pH 7.8. They were suspended i n 2 0 mM TED-2, buffer pH 8, at a protein concentration of 62.5 mg/ml. The suspension was s t i r r e d i n an ice bath for 30 min with sodium cholate (0.46 mg/mg protein) and saturated ammonium sulphate solution (adjusted to pH 8 with N H i j O H ) was added to give 10% of saturation. 50. The mixture was centrifuged for 40 min at 0°C and 145 000 x g. So l i d ammonium sulphate was added to the supernatant to give 33% of saturation. The solution was s t i r r e d f or 20 min i n an ice bath followed by recentrifugation as above. The super-natant was brought to 43% saturation with s o l i d ammonium sulphate and s t i r r e d i n ice for 20 min after which the cen-tr i f u g a t i o n step was repeated. The p e l l e t p r e c i p i t a t i n g at 33-43% saturation of ammonium sulphate was taken up i n 20 mM TED-2 buffer, pH 8. The supernatant was brought to 60% saturation with ammonium sulphate, s t i r r e d at 0°C and then centrifuged as above. The p e l l e t from the l a s t step contained the 43-60% ammonium sulphate f r a c t i o n and was taken up i n 2 0 mM TED-2 buffer, pH 7.8 as above. The fractions were then subjected to sucrose density gradient centrifugation. B: The second procedure used for extraction with sodium cholate i s based upon the method of B a i l l i e et a l . (136). Membranes from 8-16 g of E. c o l i ML308-225 or W6 prepared i n 50 mM TM buffer, pH 7.8, were suspended at a protein concen-t r a t i o n of 7-16.7 mg/ml i n 50 mM TGDS/KCl/cholate buffer, pH 7.5. Sodium cholate was added to a concentration of 1.56-3.70 mg/mg protein and the mixture was s t i r r e d i n an ice bath for 15 min. Saturated ammonium sulphate solution, pH 7.2, was then added dropwise to give 3 3% saturation. This was s t i r r e d for 30 min and then centrifuged for 30 min at 0°C and 115 000 x g. The supernatant was taken to 60% saturation with s o l i d ammonium sulphate, s t i r r e d i n an ice bath for 15 min, and then centrifuged for 15 min at 0°C and 10 000 x g. The p e l l e t containing the 33-60% ammonium sulphate f r a c t i o n was taken up i n various buffers and subjected to the tr e a t -ments i n d i v i d u a l l y described i n the RESULTS section. Sodium deoxycholate The method was based on that described by Hare (137). The f r a c t i o n a t i o n procedures were ca r r i e d out at 0°C. C e l l s from 9.9 g of E. c o l i ML308-225 were suspended i n 50 mM Tris-H 2 S O i , . buffer, pH 7.8, containing 5 mM MgSCK , 0.1 mM EDTA and 10 mM 3-mercaptoethanol. They were broken by passage through a French pressure c e l l at 6000 p s i . The sus-pension was centrifuged at 17 600 x g for 15 min and the membranes is o l a t e d by centrifuging the supernatant at 145 000 x g for 2 h. The membranes were resuspended at 20 mg protein/ ml i n 50 mM TMDG buffer, pH 7.5, containing 1 M KCl and 0.2% (w/v) sodium deoxycholate. The suspension was s t i r r e d i n an ice bath for 10 min and centrifuged at 145 000 x g for 1 h. The supernatant was dialyzed against 50 volumes of 50 mM TMDG buffer, pH 7.5, for 10 h at 4°C. It was then centrifuged for 60 min at 145 000 x g and the p e l l e t was resuspended i n 50 mM TMDG buffer, pH 7.5, containing 0.4% (w/v) sodium deoxycholate. 52. Ammonyx LP, T r i t o n X-100 or l y s o l e c i t h i n T h i s i n v o l v e d homogenizing the membrane i n 50 mM TM b u f f e r , pH 7.8, adding the de t e r g e n t to the c o n c e n t r a t i o n s g i v e n i n the RESULTS s e c t i o n , s t i r r i n g the mixture i n i c e f o r 30 min, and then c e n t r i f u g i n g a t 120 000 x g f o r 2 h. The supernatant c o n t a i n i n g the s o l u b i l i z e d membrane was then processed as d e s c r i b e d i n the RESULTS s e c t i o n . Chromatographic procedures A d s o r p t i o n chromatography on h y d r o x y l a p a t i t e H y d r o x y l a p a t i t e (Bio-Gel HTP) i n a column of 1.9 cm diameter and packed t o a h e i g h t of 15 cm, was washed and e q u i l i b r a t e d a t 4°C w i t h 500 ml of 100 mM PDGX b u f f e r , pH 7.5. The sample a p p l i e d c o n t a i n e d 4.1 mg p r o t e i n i n a t o t a l volume of 75 ml of 300 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.5% (w/v) T r i t o n X-100 (see RESULTS s e c t i o n ) . E l u t i o n was e f f e c t e d by a l i n e a r g r a d i e n t of 100-500 mM PDGX b u f f e r , pH 7.5. The flow, r a t e was 23 ml/h. Ion-exchange chromatography on D E A E - c e l l u l o s e 15 g of p r e - s w o l l e n D E A E - c e l l u l o s e (DE52) was s t i r r e d w ith 90 ml d i s t i l l e d water. Concentrated HC1 was added t o a d j u s t the pH of the s o l u t i o n to 4.5. The s l u r r y was de-gassed under reduced p r e s s u r e and a d j u s t e d t o pH 7.8 w i t h T r i s base t o a f i n a l c o n c e n t r a t i o n of 20 mM. The DEAE-c e l l u l o s e was allowed t o s e t t l e and the supernatant then 53. decanted and d i s c a r d e d . The D E A E - c e l l u l o s e was resuspended i n b u f f e r , s t i r r e d and allowed to s e t t l e . T h i s procedure was repeated f i v e times u s i n g a t o t a l volume of 500 ml 20 mM T r i s -HC1 b u f f e r , pH 7.8, u n t i l the pH and c o n d u c t i v i t y of f i l t e r e d supernatant f l u i d (measured u s i n g a Radiometer c o n d u c t i v i t y meter) were the same as those of the f r e s h b u f f e r . The column was then packed w i t h D E A E - c e l l u l o s e , e q u i l i b r a t e d a t 4°C f i r s t w i t h 2 mM T r i s - H C l b u f f e r , pH 7.8, and f i n a l l y w i t h 2 mM TGD-1 b u f f e r , pH 7.8. The dimensions of the packed column were 1.9 x 5.7 cm. The sample c o n t a i n i n g 3-6% (w/v) T r i t o n X-100 was a p p l i e d i n the f i n a l e q u i l i b r i u m b u f f e r . E l u t i o n was e f f e c t e d by s e q u e n t i a l a d d i t i o n of 2-1000 mM TGD-1 b u f f e r , pH 7.8. The column was regenerated between experiments by washing w i t h 500 ml of 1 M NaCl and r e - e q u i l i b r a t e d w i t h 2 mM TGD-1 b u f f e r , pH 7.8. Ion-exchange chromatography on DEAE-Sepharose CL-6B The p r e - s w o l l e n g e l was suspended i n 20 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.1% (w/v) B r i j 35. A 1.9 cm diameter column was packed a t 4°C t o a h e i g h t of 19.5 cm, and e q u i l i -b r a t e d w i t h the same b u f f e r . The flow r a t e was 22 ml/h. When the column was t o be used w i t h another b u f f e r system, i t was washed w i t h 3 bed volumes of 1 M T r i s - H C l b u f f e r , pH 7.8, c o n t a i n i n g 20% (v/v) g l y c e r o l and then e q u i l i b r a t e d w i t h 500 ml of the new b u f f e r t o be used, u n t i l the c o n d u c t i v i t y and pH of the e f f l u e n t were the same as those of the e q u i l i b r a t i n g medium. E l u t i o n of the p r o t e i n s was by l i n e a r g r a d i e n t s of i n c r e a s i n g T r i s c o n c e n t r a t i o n i n the e l u t i n g b u f f e r s . When not i n use the g e l was s t o r e d a t 4°C i n the presence of 0.01% (v/v) H i b i t a n e t o prevent b a c t e r i a l con-t a m i n a t i o n . Gel f i l t r a t i o n chromatography on Sepharose 6B Pr e - s w o l l e n Sepharose 6B was mixed t o a s l u r r y w i t h 3 00 ml of 100 mM potassium phosphate b u f f e r , pH 7.8. I t was packed a t 4°C i n t o a column (2.5 x 38.5 cm) and p r e - e q u i l i b r a t e d u s i n g 3 bed volumes of the b u f f e r c o n t a i n i n g 100 mM potassium phosphate and 10% g l y c e r o l , pH 7.8. The column was then e q u i l i b r a t e d with 3-4 bed volumes of the b u f f e r d e s i r e d f o r a p a r t i c u l a r s e p a r a t i o n . The sample to be e l u t e d was made up i n the same b u f f e r as t h a t e q u i l i b r a t i n g the g e l . Gel f i l t r a t i o n chromatography on Sepharose 6B/4B Pr e - s w o l l e n Sepharose 6B and Sepharose 4B g e l s were pr e -pared as above and each packed a t 4°C i n t o a column. The dimensions of the packed column f o r the Sepharose 6B were 2.5 x 37 cm while those f o r the Sepharose 4B were 2.5 x 39 cm. The two columns were then connected i n s e r i e s w i t h the o u t l e t of the Sepharose 6B column l e a d i n g i n t o the Sepharose 4B column. They were e q u i l i b r a t e d w i t h s e v e r a l bed volumes of 50 mM TGDS/KCl/cholate b u f f e r , pH 7.5. Samples t o be a p p l i e d t o t h i s system were d i s s o l v e d i n the same b u f f e r . 55. The columns were regenerated between experiments by washing w i t h 1000 ml of the same b u f f e r without disodium s u c c i n a t e or u n t i l the absorbance of the e f f l u e n t read a t 280 nm a g a i n s t f r e s h b u f f e r had dropped to zero. P r e p a r a t i o n of sucrose d e n s i t y g r a d i e n t s The sucrose s o l u t i o n s were d i s s o l v e d i n 50 mM T r i s -CH3COOH (or 50 mM T r i s - H C l ) b u f f e r , pH 7.4 c o n t a i n i n g 1.5 mM disodium EDTA, and 2 mM d i t h i o t h r e i t o l . In some e x p e r i -ments 0.2-0.5% (w/v) B r i j 58 or 0.1% (w/v) a s o l e c t i n p l u s 1.5% sodium c h o l a t e was i n c l u d e d i n the d e n s i t y g r a d i e n t b u f f e r , as d e s c r i b e d i n the RESULTS s e c t i o n . The g r a d i e n t s were c e n t r i f u g e d a t 0°C i n a swinging bucket r o t o r a t 138 000 x g f o r 12 h. Twenty drop f r a c t i o n s were c o l l e c t e d by means of a Beckman f r a c t i o n a t i o n system att a c h e d to a f r a c t i o n c o l l e c t o r . Sucrose g r a d i e n t s made to determine the p o s i t i o n of the sedimentation markers, thyreoglobulin and c a t a l a s e , were prepared by d i s s o l v i n g the sucrose i n 50 mM TED-1 b u f f e r , pH 7.8, 2 mg each of c r y s t a l l i n e t h y r o g l o b u l i n and c a t a l a s e d i s s o l v e d i n 1 ml b u f f e r was then a p p l i e d to the g r a d i e n t . The samples were c e n t r i f u g e d a t 0°C f o r 14 h at 138 000 x g. 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  Depolymerization,.' of samples 100 y l p o r t i o n s of f r a c t i o n s (16-204 yg/ml p r o t e i n ) c o l l e c t e d from columns of DEAE-Sepharose CL-6B or: hydroxy 1-a p a t i t e , were t r e a t e d w i t h 10 y l of a s o l u t i o n c o n t a i n i n g 10% (w/v) sodium dodecyl s u l p h a t e , 10% (v/v) mercaptoethanol and one c r y s t a l of bromophenol bl u e i n water. Each mixture was heated f o r 3 minutes i n a b o i l i n g water bath, c o o l e d to.i 22°C and 10 u l o f i t a p p l i e d t o the g e l . Depolymerization of molecular weight marker p r o t e i n s The d e p o l y m e r i z i n g b u f f e r was a m o d i f i c a t i o n of t h a t used by King and Laemmli (13 8). The p r o t e i n s to be used as stand-ards were d i s s o l v e d i n a s o l u t i o n c o n t a i n i n g 62.5 mM T r i s -HC1 b u f f e r , pH 6.8, 1% (w/v) sodium dodecyl s u l p h a t e , 10% (v/v) g l y c e r o l , 1% (v/v) mercaptoethanol and s u f f i c i e n t bromophenol bl u e t o c o l o u r the b u f f e r a l i g h t b l u e . The con-c e n t r a t i o n of standard p r o t e i n s i n t h i s b u f f e r was 0.2-1 mg/ ml. Depolymerization was e f f e c t e d by h e a t i n g a t 100°C f o r 3 min. A f t e r c o o l i n g , 5-25 u l of the depolymerized m a t e r i a l was a p p l i e d to the g e l . P r e p a r a t i o n of g e l s  S e p a r a t i n g g e l 15 ml of s e p a r a t i n g g e l was prepared to c o n t a i n 0.375 M T r i s - H C l b u f f e r , pH 8.8, 0.1% (w/v) sodium dodecyl s u l p h a t e , 9% (w/v) acrylamide and 0.24% (w/v) N,N'-methylenebisacrylamide. (The l a t t e r two components were made up as a stock s o l u t i o n of 30% (w/v) acrylamide and 0.8% (w/v) N, N 1 - m e t h y l e n e b i s a c r y l a -mide i n water.) The s o l u t i o n was then deaerated w i t h a water pump. P o l y m e r i z a t i o n was e f f e c t e d by the a d d i t i o n of 15 u l yg TEMED and 75 y l of f r e s h l y prepared 10% (w/v) ammonium p e r s u l p h a t e i n water. The s o l u t i o n was then q u i c k l y i n t r o -duced by means of a pasteur p i p e t t e between the p l a t e s of a s l a b g e l former (139) c o n s i s t i n g of two 20 x 20 cm g l a s s p l a t e s separated by two narrow l u c i t e s pacers, 0.7 5 mm t h i c k , and f i r m l y clamped t o g e t h e r . The g l a s s p l a t e s were clamped to a support so t h a t the lower edges were s e a l e d by a rubber gasket. A pocket former c o n s i s t i n g o f a l u c i t e "comb", 0.75 mm t h i c k , and with twenty t e e t h was i n s e r t e d between the upper edges of the g l a s s p l a t e s . The s e p a r a t i n g g e l was poured t o w i t h i n 1 cm of the bottom of the comb. .0.3 ml of t e r t i a r y b utanol was then c a r e f u l l y l a y e r e d over the g e l to maintain a s t r a i g h t boundary w h i l e the g e l was s e t t i n g . The s e p a r a t i n g g e l was allowed t o polymerize f o r 0.5-1 h, a f t e r which the comb was removed and the t e r t i a r y b utanol removed by a b s o r p t i o n onto f i l t e r paper. The g e l s u r f a c e was then r i n s e d twice w i t h d i s t i l l e d water. The comb was r e i n s e r t e d and the s t a c k i n g g e l then poured t o the top of the pocket former. S t a c k i n g g e l 5 ml of s t a c k i n g g e l was prepared to c o n t a i n 0.125 M-T r i s - H C l b u f f e r , pH 6.8, 0.1% (w/v) sodium dodecyl sulphate and 4% (w/v) cyanoguiri' 41, g e l l i n g agent. The mixture was de-aerated u s i n g a water pump, a f t e r which p o l y m e r i z a t i o n was s t a r t e d : by the a d d i t i o n o f 10 y l TEMED and 15 y l of f r e s h l y prepared 10% (w/v) ammonium p e r s u l p h a t e i n water. The g e l 58. mixture was l a y e r e d over the s e p a r a t i n g g e l and was allowed to polymerize f o r 0.5 h a f t e r which i t was r i n s e d w i t h a few m i l l i l i t e r s of " e l e c t r o d e b u f f e r " (see below), and the pocket former c a r e f u l l y removed. The pockets were then f i l l e d w i t h e l e c t r o d e b u f f e r and the samples and p r o t e i n standards a p p l i e d to the g e l beneath the b u f f e r by means of a narrow cannula attached to a Lang-Levy p i p e t t e . E l e c t r o p h o r e s i s The two g l a s s p l a t e s c o n t a i n i n g the g e l were attached to a g e l e l e c t r o p h o r e s i s c e l l (Bio-Rad model 220, dual v e r t i c a l s l a b g e l e l e c t r o p h o r e s i s c e l l ) . The " e l e c t r o d e b u f f e r " , pH 8.4, had the f o l l o w i n g composition: 25 mM T r i s base, 192 mM g l y c i n e and 0.1% (w/v) sodium dodecyl s u l p h a t e . E l e c t r o p h o r e s i s was c a r r i e d out a t 22-25°C at a constant c u r r e n t of 30 m i l l i a m p e r e s (139) f o r 1.25-1.75 h. S t a i n i n g and d r y i n g At the end of the e l e c t r o p h o r e s i s run, the g e l was r e -moved from between the g l a s s p l a t e s and covered w i t h the s t a i n i n g s o l u t i o n recommended by F a i r b a n k s e t a l . (140) f o r 1 h. T h i s c o n t a i n e d 0.05% (w/v) Coomassie b r i l l i a n t blue R i n a mixture of i s o p r o p a n o l (25%), a c e t i c a c i d (10%) and water (65%). The s o l u t i o n was f i l t e r e d through a Whatman grade 1 f i l t e r paper be f o r e use. The g e l was d e s t a i n e d w i t h 10% (v/v) a c e t i c a c i d f o r 16 h. Drying of the g e l was e f f e c t e d by t r e a t i n g i t w i t h a s o l u t i o n of 10% (v/v) a c e t i c a c i d and 1% (v/v) g l y c e r o l f o r 1 h, f o l l o w e d by placement i n a Gel Slab Dryer Model 224 (Bio-Rad L a b o r a t o r i e s ) , equipped w i t h a h e a t i n g u n i t and connected to a vacuum pump. P r e p a r a t i o n of l i p i d s and f a t t y a c i d s T o t a l l i p i d e x t r a c t from E. c o l i K-12 T h i s was based on the method of Cunningham and Hager (141). 97.1 g of E. c o l i K-12 c e l l s were suspended i n 57 ml of d i s t i l l e d water. The volume of the suspension was 145 ml. 130 0 ml of acetone was added t o g i v e an acetone to water r a t i o of 9:1. The mixture was s t i r r e d f o r 4 h a t 22°C and then f i l t e r e d through Whatman, grade 41, f i l t e r paper. The r e s i d u e was resuspended i n 200 ml acetone, s t i r r e d f o r another 4 h a t 22°C and f i l t e r e d as b e f o r e . The f i l t r a t e s were combined and the s o l v e n t removed under reduced p r e s s u r e , u s i n g a r o t a r y evaporator. The y i e l d of l i p i d was 1.7 5 g. I t was d i s s o l v e d i n 125 ml of a 4:1 mixture of c h l o r o f o r m : methanol a t a c o n c e n t r a t i o n o f 14 mg/ml, and s t o r e d under n i t r o g e n , a t -20°C, i n g l a s s - s t o p p e r e d tubes. Soybean p h o s p h o l i p i d s ( a s o l e c t i n ) 200 mg a s o l e c t i n was suspended a t a c o n c e n t r a t i o n of 10 mg/ml i n 20 mM Tris-CH 3COOH b u f f e r , pH 8, c o n t a i n i n g 1 mM disodium EDTA, and homogenized u s i n g a P o t t e r - E l v e h j e m homogenizer. The suspension was then passed through a French p r e s s u r e c e l l (Aminco) at 20 000 p s i . 60. P a l m i t i c a c i d An aqueous d i s p e r s i o n of r e c r y s t a l l i z e d p a l m i t i c a c i d was made by suspending 1 mg of p a l m i t i c a c i d i n 1.2 ml of 50 mM TED-1 b u f f e r , pH 7.8. Small g l a s s beads (0.45-0.5 mm diameter) were added and the mixture a g i t a t e d f o r 5 min u s i n g a vo r t e x mixer. D e l i p i d a t i o n of p a r t i a l l y p u r i f i e d (0.33-0.6P) f r a c t i o n The method was based on t h a t of Houghton e t a l . (89). Transhydrogenase-containing f r a c t i o n s from a s e p a r a t i o n of (0.33-0.6P) f r a c t i o n on DEAE-Sepharose CL-6B (see RESULTS) were pooled. The volume was 62 ml wit h a p r o t e i n c o n c e n t r a t i o n 0.04 mg/ml. Sodium c h o l a t e (0.65 g i n 3 ml water), was added to g i v e a f i n a l c o n c e n t r a t i o n of 1% (w/v) and the mixture s t i r r e d f o r 15 min a t 0°C. C r y s t a l l i n e ammonium sulphate was then added t o 50% s a t u r a t i o n and the s t i r r i n g continued f o r another 15 min. The mixture was c e n t r i f u g e d a t 3 0 000 x g f o r 15 min a t 0°C. The r e s u l t i n g p e l l e t was suspended i n 50 mM TED-1 b u f f e r , pH 7.8, and d i a l y z e d f o r 2 h a t 4°C a g a i n s t 100 volumes of the same b u f f e r . The r e s u l t i n g d e l i p i d a t e d m a t e r i a l (0.66 mg/ml p r o t e i n ) was then s u b j e c t e d to r e a c t i v a t i o n by l i p i d s as d e s c r i b e d i n the RESULTS s e c t i o n . P h o t o o x i d a t i o n of NADH by 2,3-butanedione The r e a c t i o n mixture c o n t a i n e d .0.12 mM NADH, 6.7 mM 2,3-butanedione and 50 mM sodium borate b u f f e r , pH 7.8, i n a f i n a l volume of 2 ml.. The s o l u t i o n was con t a i n e d i n a stoppered quartz c u v e t t e with a 1 cm l i g h t path and kept a t 22°C under ambient l a b o r a t o r y l i g h t c o n d i t i o n s . The ab s o r p t i o n spectrum was scanned between 400 and 280 nm i n a Perkin-Elmer model 124 spectrophotometer a t timed i n t e r v a l s . In some experiments the sodium borate b u f f e r was r e p l a c e d by 50 mM potassium phosphate b u f f e r . Determination of the stochiometry of p h o t o o x i d a t i o n of NADH  by 2,3-butanedione The r e a c t i o n mixture a t 22°C contained 0.12 mM NADH, and 0.054 mM 2,3-butanedione i n 50 mM sodium borate b u f f e r , pH 7.8, to a f i n a l volume of 2 ml. The mixture was c o n t a i n e d i n a stoppered quartz c u v e t t e w i t h a 1 cm l i g h t path, and was pl a c e d i n a fumehood 12 inches away from a t h e r m a l l y p r o t e c t e d double f l u o r e s c e n t lamp (General E l e c t r i c , Model F20T12 C.W.). A s i m i l a r r e a c t i o n mixture, but l a c k i n g 2,3-butanedione was a l s o s e t up as a c o n t r o l . I r r a d i a t i o n was allowed t o proceed f o r 140 hours. The absorbance a t 340 nm was measured at timed i n t e r v a l s . RESULTS The energy-dependent p y r i d i n e n u c l e o t i d e transhydrogenase The ATP-driven energy-dependent transhydrogenase r e a c t i o n measured on membrane p a r t i c l e s from E. c o l i s t r a i n NRC-482 e x h i b i t e d maximum a c t i v i t y around pH 8 ( F i g . 1). At pH 7.8, the ATP c o n c e n t r a t i o n r e q u i r e d f o r maximum v e l o c i t y was 0.5 mM. I n c r e a s i n g t h i s c o n c e n t r a t i o n to 2.5 mM i n the assay mixture produced no s i g n i f i c a n t change i n the maximum v e l o c i t y of the r e a c t i o n ( F i g . 2). The r e c i p r o c a l of the slope o b t a i n e d from a Hanes-Woolf p l o t f o r the assay ( F i g . 3) i s e q u i v a l e n t t o the maximum v e l o c i t y . T h i s was c a l c u l a t e d to be 22.5 nmol per min per mg p r o t e i n . S u b s t i t u t i n g t h i s v a lue i n t o the i n t e r c e p t on the o r d i n a t e ( e q u i v a l e n t to K /V) gave a K m of 4 5 uM ATP f o r the ATP-driven energy-dependent transhydrogenase r e a c t i o n . Using membrane p a r t i c l e s from E. c o l i K-12, Sweetman and G r i f f i t h s (142) r e p o r t e d an apparent K^ of 277 UM ATP f o r the ATP-driven energy-dependent transhydrogenase r e a c t i o n . The maximum v e l o c i t y was a p p r o x i -mately 14 nmol per min per mg p r o t e i n . I t i s not c l e a r why these values are so d i f f e r e n t . One e x p l a n a t i o n c o u l d be a t t r i b u t e d t o a d i f f e r e n c e i n the s t r a i n of E. c o l i employed. I t i s of i n t e r e s t , however, t h a t the K f o r the same r e a c t i o n m c a t a l y z e d by chromatophores of R. rubrum was 24 u M ATP (36), which i s c l o s e to t h a t r e p o r t e d i n t h i s t h e s i s . F i g . 1. E f f e c t of pH on the ATP-driven energy-dependent transhydrogenase reaction. Membrane p a r t i c l e s from E. c o l i s t r a i n NRC-482 were suspended in 0.02 M triethanolamine buffer, pH 7.5, con-taining 10% (v/v) g l y c e r o l . Energy-dependent transhydrog-enase a c t i v i t y was measured as described i n MATERIALS AND METHODS. Membrane p a r t i c l e protein was at 0.62 5 mg per assay. The TM buffer usually employed i n thi s assay was replaced by 50 mM PIPES, pH 6.5 (O), MOPS, pH 7.0 ( A ) , HEPES, pH 7.5 (o) / t r i c i n e , pH 8.3 {%) , or g l y c y l g l y c i n e -NaOH, pH 9.0 (••) buffer, respectively. The pH of the ATP solution was adjusted i n each case to that of the correspond-ing buffer employed and the assay determined i n the presence of 10 mM MgCl 2. NAD+, NADP+ and yeast alcohol dehydrogenase were i n d i v i d u a l l y dissolved i n the buffer under investiga-t i o n . Enzyme a c t i v i t y i s expressed as nmol/min/mg protein. F i g . 2. E f f e c t of ATP c o n c e n t r a t i o n on the ATP-driven energy-dependent transhydrogenase r e a c t i o n . Membrane p a r t i c l e s from E. c o l i NRC-482 were suspended i n 50 mM TM b u f f e r , pH 7.8 and the energy-dependent t r a n s -hydrogenase r e a c t i o n c a r r i e d out as o u t l i n e d i n MATERIALS AND METHODS. The assay mixture c o n t a i n e d 1.7 mg of membrane p a r t i c l e p r o t e i n . ATP was a d j u s t e d t o pH 7.8 and used a t the concentrations i n d i c a t e d on the graph. Enzyme a c t i v i t y i s i n nmol/min/mg p r o t e i n . cjn o 67. F i g . 3. Hanes-Woolf plot for the determination of V and K m for ATP i n the energy-dependent transhydrogen-ase reaction. The conditions of the experiment are the same as those described i n F i g . 2. [5] i s i n (min-mg p r o t e i n - m l - 1 . [S] v i s the concentration of ATP expressed as mM. Approximately two minutes aft e r addition of ATP to the reaction mixture, the ATP-driven energy-dependent trans-hydrogenase reaction rate tended to decrease. To test whether th i s e f f e c t was due to competition by the product, ADP, with ATP for the ATPase, the e f f e c t of adding varying amounts of ADP simultaneously with ATP to the reaction mix-ture was examined. F i g . 4 shows that ADP i n h i b i t s the ATP-driven energy-dependent transhydrogenase, a possible ex-planation for the f a l l - o f f of the i n i t i a l v e l o c i t y of the transhydrogenase i n the standard reaction with ATP. The reaction was 50% in h i b i t e d i n the presence of 0.62 mM ADP. Energy d r i v i n g the energy-dependent transhydrogenase reaction may be obtained by electron transport or by ATP hydrolysis. The energy-dependent transhydrogenase reaction and oxidative phosphorylation u t i l i z e a common energy pool (14 3). The generation of energy by the reverse energy-dependent transhydrogenase has been demonstrated. At high NAD+ and NADPH concentrations, inward translocation of protons occurred i n submitochondrial p a r t i c l e s . In the presence of high concentrations of ADP and inorganic phosphate, the membrane poten t i a l generated was s u f f i c i e n t to bring about ATP synthesis (52). It has been suggested (144) that the ATPase and transhydrogenase were organized i n domains whereby the enzymes interacted either d i r e c t l y or i n d i r e c t l y by l o c a l i z e d proton gradients (84). Rydstrom et a l . (88), 70. F i g . 4. Ef f e c t of ADP on the ATP-driven energy-dependent transhydrogenase a c t i v i t y . Energy-dependent transhydrogenase a c t i v i t y was assayed using membrane p a r t i c l e s from E. c o l i NRC-48 2 i n 50 mM TM buffer, pH 7.8. The conditions are described i n MATERIALS AND METHODS. The ATP concentration was 0.5 mM. The ADP solution was adjusted to pH 7.8 and added to the reaction mixture simultaneously with ATP at the indicated concentra-tions. A c t i v i t y i s expressed as a percentage of the control value i n which no ADP was added to the assay mixture. however, r e c o n s t i t u t e d p a r t i a l l y p u r i f i e d transhydrogenase from beef h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s . w i t h l i p i d s and r e p o r t e d t h a t the r e v e r s e energy-dependent transhydrogenase r e a c t i o n took p l a c e i n the absence of ATPase or cytochromes. Thus energy-dependent transhydrogenation i s not dependent upon a d i r e c t i n t e r a c t i o n between the ATPase and the t r a n s -hydrogenase enzymes. The energy-independent p y r i d i n e n u c l e o t i d e transhydrogenase  O r i e n t a t i o n of the enzyme i n the membrane S u b c e l l u l a r d i s t r i b u t i o n s t u d i e s on the m i t o c h o n d r i a l transhydrogenase have shown t h a t the enzyme i s t i g h t l y bound to the i n n e r membrane with i t s n i c o t i n a m i d e n u c l e o t i d e b i n d i n g s i t e ( s ) exposed to the matrix (10). That the energy-independent transhydrogenase of E. c o l i i s on the c y t o p l a s m i c s i d e of the i n n e r membrane was demonstrated u s i n g EDTA-lysozyme s p h e r o p l a s t s prepared by the freeze-thawing method of Weiner and Heppel (127). EDTA-lysozyme s p h e r o p l a s t s from E. c o l i ML308-225 were suspended i n 0.1 M TEGD b u f f e r , pH 7.8. The energy-independent transhydrogenase a c t i v i t y of t h i s suspension was 42.2 nmol per min per mg p r o t e i n . Breakage of the s p h e r o p l a s t s i n a French p r e s s u r e c e l l a t 20 000 p s i i n c r e a s e d the measureable enzyme a c t i v i t y 2.5- f o l d as i n d i c a t e d i n Table 2. The a d d i t i o n of 1% (w/v) T r i t o n X-100 to the i n t a c t s p h e r o p l a s t suspension caused a 1.6- f o l d i n c r e a s e i n the energy-independent transhydrogenase Table 2. E f f e c t of Breakage of EDTA-lysozyme S p h e r o p l a s t s on Energy-Independent Transhydrogenase A c t i v i t y Treatment Energy-independent Transhydrogenase A c t i v i t y (nmol/ min/mg p r o t e i n ) F o l d A c t i v i t y None French p r e s s u r e c e l l 1% (w/v) T r i t o n X-100 French p r e s s u r e c e l l + 1%(w/v) T r i t o n X-100 42.2 104.6 67.4 60.3 1 2.5 1.6 1.4 EDTA-lysozyme s p h e r o p l a s t s from E. c o l i ML308-225 were prepared as d e s c r i b e d i n MATERIALS AND METHODS. They were suspended i n 0.1 M TEGD b u f f e r , pH 7.8 a t a c o n c e n t r a t i o n of 19.5 mg p r o t e i n / m l and t r e a t e d as d e s c r i b e d i n the t e x t . a c t i v i t y , but did not cause any further increase i n the enzyme a c t i v i t y of the spheroplasts that had been subjected to the French pressure c e l l . Treatment of the spheroplasts (representing a 'rightside i n 1 membrane orientation) with detergent, or breakage i n a French pressure c e l l r e s u l t s i n 'inside out' membrane p a r t i c l e s i n which the cytoplasmic side of the membrane i s exposed to the medium. The increase i n energy-independent transhydrogenase a c t i v i t y of the sphero-plasts by either of these treatments indicates that the active s i t e of t h i s enzyme i s most probably located on the cytoplasmic side of the inner membrane and therefore became more accessible to the medium when the membranes were everted. I t was for t h i s reason that membrane p a r t i c l e s , rather than EDTA-lysozyme spheroplasts, were selected as the s t a r t i n g material for the present investigation. Conditions of assay pH optimum of the enzyme The energy-independent transhydrogenase reaction measured on membrane p a r t i c l e s from E. c o l i exhibited maximum a c t i v i t y at pH 7.0 (Fig. 5). There was no a c t i v i t y at pH 6.0 and only 36% of the maximal a c t i v i t y was shown at pH 9.0. E f f e c t of b a c t e r i a l s t r a i n type on the s p e c i f i c a c t i v i t y of the membrane-bound enzyme The a c t i v i t y of the enzyme i n membrane p a r t i c l e s from several d i f f e r e n t strains of E. c o l i was measured using the F i g . 5. E f f e c t o f pH on the a c t i v i t y of the energy-independent transhydrogenase. Washed membrane p a r t i c l e s from E. c o l i ML308-225 were suspended i n 50 mM TM b u f f e r , pH 7.8, and the energy-independent transhydrogenase r e a c t i o n measured a t 22°C as d e s c r i b e d i n MATERIALS AND METHODS except t h a t KCN was omitted from the assay mixture. The c o n c e n t r a t i o n of mem-brane p a r t i c l e p r o t e i n was 0.115 mg/ml assay mixture. The c o n c e n t r a t i o n s o f NADPH and APNAD + were those f o r the "standard assay". The potassium phosphate b u f f e r i n the assay mixture was r e p l a c e d by MES, pH 6.0 (•); PIPES, pH 6.5 and 7.0 (O); potassium phosphate, pH 7.0 (•); HEPES, pH 7.5 (•); TES, pH 7.6 (©); t r i c i n e , pH 8.0 and 8.5 «» ; T r i s - H 2 S 0 n , pH 8.0 and 8.5.(B) and g l y c y l glycine-NaOH, pH 8.5 and 9.0 (•) , r e s p e c t i v e l y . A l l b u f f e r s were a t f i n a l c o n c e n t r a t i o n s o f 50 mM. A c t i v i t y i s expressed as units/mg p r o t e i n . 7 6 . "standard" assay procedure a t 22°C and pH 7.0 as o u t l i n e d i n the MATERIALS AND METHODS s e c t i o n . Table 3 shows average s p e c i f i c a c t i v i t i e s f o r the energy-independent transhydrog-enase of membrane p a r t i c l e s from E. c o l i s t r a i n s NRC-482, ML308-225 and W6 measured i n v a r i o u s b u f f e r s . Although the enzyme a c t i v i t y i n s t r a i n s NRC-482 and ML308-225 (230-260 nmol per min per mg p r o t e i n ) d i d not appear to be s i g n i f i -c a n t l y d i f f e r e n t under these c o n d i t i o n s , t h a t i n s t r a i n W6 showed on the average a hig h e r s p e c i f i c a c t i v i t y (7 80 nmol per min per mg p r o t e i n ) . E f f e c t of b u f f e r s Since the p r e v i o u s r e s u l t s i n d i c a t e d t h a t the energy-independent transhydrogenase a c t i v i t y of membrane p a r t i c l e s of E. c o l i W6 had a higher s p e c i f i c a c t i v i t y than the other s t r a i n s i n v e s t i g a t e d , i t was used to t e s t the e f f e c t of d i f f e r e n t b u f f e r s on the maximal v e l o c i t y (V) and f o r both s u b s t r a t e s (NADPH and APNAD+) of the r e a c t i o n . The k i n e t i c parameters were determined g r a p h i c a l l y from Lineweaver-Burk r e c i p r o c a l p l o t s (1/v versus 1/[S]) and t a b u l a t e d as mean valu e s i n Table 4. Although the range of V was f a i r l y broad (464-1450 nmol per min per mg p r o t e i n ) , the b u f f e r medium used to suspend the membrane p a r t i c l e s had no apparent i n f l u e n c e upon V and K m f o r the r e a c t i o n . 78. Table 3. The E f f e c t of B a c t e r i a l S t r a i n Type on The S p e c i f i c A c t i v i t y of Energy-Independent Transhydrogenase i n Membrane P a r t i c l e s S t r a i n S p e c i f i c A c t i v i t y (units/mg p r o t e i n ) Mean ± S.D.* Range E. c o l i NRC-482 (12)** E. c o l i ML308-225 (23) E. c o l i W6 (55) 0.23 ± 0.057 0.26 ± 0.059 0.78 ± 0.232 0.13 - 0.31 0.15 - 0.37 0.42 - 1.45 * Standard d e v i a t i o n ** F i g u r e s i n parentheses i n d i c a t e the number of pr e p a r a -t i o n s assayed. Membrane p a r t i c l e s from the i n d i c a t e d s t r a i n s of E. c o l i were suspended i n one of the f o l l o w i n g b u f f e r s : 50 mM TM, 100 mM TEGD, 2 mM TGD-1, 20 mM TGD-2, 20 mM TED-2, 50 mM TED-1, 50 mM sodium borate or 5 0 mM TGDS/KCl/cholate. With the e x c e p t i o n of TGDS/KCl/cholate (pH 7.5), a l l other b u f f e r s were a t pH 7.8. Energy-independent transhydrogenase a c t i v i t y was assayed as d e s c r i b e d i n MATERIALS AND METHODS. Table 4. The E f f e c t of B u f f e r s on Energy-Independent Trans-hydrogenase A c t i v i t y B u f f e r K m V (yM) (units/mg p r o t e i n ) APNAD + NADPH 50 mM TED-1 59 .4 ± 20. 95 38 .2 ± 9.85 1. 02 + 0 . 307 50 mM Sodium borate 49 .0 ± 22. 03 56 .6 ± 8.56 0. 85 + 0 . 220 50 mM TM 73. 3* 46 .0* 0. 73 + 0 .171 Washed membrane p a r t i c l e s from E. c o l i W6 were suspended i n the" i n d i c a t e d b u f f e r and the energy-independent t r a n s -hydrogenase a c t i v i t y measured as d e s c r i b e d i n MATERIALS AND METHODS. The pH of each b u f f e r was 7.8. The r e s u l t s are expressed as a r i t h m e t i c means ± standard d e v i a t i o n s . * Since the K m v a l u e f o r each s u b s t r a t e i n 50 mM TM b u f f e r was a mean of onl y two o b s e r v a t i o n s , standard d e v i a t i o n s c o u l d not be c a l c u l a t e d i n these cases. Effect- of substrates • Kinetic studies of the energy-independent transhydrogen-ase, i t s mechanism of reaction, and the active s i t e of the enzyme were carried out on membrane p a r t i c l e s rather than on a s o l u b i l i z e d form of the enzyme. The membrane-bound enzyme i s more l i k e l y to resemble the "native" state of the enzyme in the in t a c t c e l l than would a s o l u b i l i z e d enzyme, stripped of i t s surrounding l i p i d and protein environment. The e f f e c t of variations i n the concentration of the two substrates APNAD+ and NADPH on the a c t i v i t y of energy-independent transhydrogenase of washed membrane p a r t i c l e s of E. c o l i W6 i s shown i n F i g . 6. The maximum v e l o c i t y was i n the range of 0.83 to 1.0 units per mg membrane p a r t i c l e protein. K values for APNAD+ and NADPH were 59 uM and m 4 3.5 uM, respectively. The ri g h t panel of F i g . 6 shows that high l e v e l s of NADPH caused substrate i n h i b i t i o n of the enzyme. This e f f e c t was observed with a l l of the strains of E. c o l i investigated i n t h i s study. Houghton et a l . (68) observed the same e f f e c t using E. c o l i membrane part-i c l e s from c e l l s grown on glucose-yeast extract, but were unable to demonstrate t h i s e f f e c t with membrane p a r t i c l e s from c e l l s grown on glucose-salts, s i m i l a r to those used i n the present investigation. They„ attributed t h i s e f f e c t to a discrete control mechanism related to the requirements for NADPH under these two d i f f e r e n t growth conditions. A more F i g . 6. E f f e c t of c o n c e n t r a t i o n s of APNAD' and NADPH on the a c t i v i t y o f energy-independent transhydrog-enase . Washed membrane p a r t i c l e s from E. c o l i W6 c e l l s were prepared i n 50 mM TM b u f f e r , pH 7.8, and suspended i n 50 mM TED-1 b u f f e r , pH 7.8. Energy-independent transhydrogen-ase a c t i v i t y was measured u s i n g 46 ug p r o t e i n per assay as d e s c r i b e d i n MATERIALS AND METHODS, as a f u n c t i o n of APNAD + c o n c e n t r a t i o n ( l e f t panel) or NADPH c o n c e n t r a t i o n ( r i g h t p a n e l ) . The c o n c e n t r a t i o n of the other s u b s t r a t e was h e l d c o n s t a n t a t the "standard c o n c e n t r a t i o n " . A c t i v i t y " 1 i s expressed as (units/mg p r o t e i n ) - 1 . S u b s t r a t e c o n c e n t r a t i o n [ S ] - 1 i s expressed as mM-1. 83. d e t a i l e d i n v e s t i g a t i o n of the phenomenon was undertaken and w i l l be d i s c u s s e d subsequently i n r e l a t i o n t o the mechanism of a c t i o n of the energy-independent transhydrogenase. 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 . o f p y r i d i n e n u c l e o t i d e t r a n s - hydrogenase In t h i s study an attempt was made to e x t r a c t and p u r i f y the p y r i d i n e n u c l e o t i d e transhydrogenase of E. c o l i . The e x t r a c t i o n procedure i n v o l v e d treatment of the membrane p a r t i c l e s w i t h detergent and sedimentation of the non-s o l u b i l i z e d f r a c t i o n by u l t r a c e n t r i f u g a t i o n . Membrane m a t e r i a l was c o n s i d e r e d as being " s o l u b i l i z e d " i f i t was not s e d i -mentable by two hours of c e n t r i f u g a t i o n a t 120 000 x g. The c o n d i t i o n s o f s o l u b i l i z a t i o n were i n v e s t i g a t e d . T h i s i n v o l v e d s e l e c t i o n of the detergent, a n a l y s i s of the s i z e of the s o l u b i l i z e d fragment, and p r e v e n t i o n of r e a g g r e g a t i o n of the s o l u b i l i z e d enzyme. S o l u b i l i z a t i o n o f the energy-dependent transhydrogenase S o l u b i l i z a t i o n o f membrane p a r t i c l e s of E. c o l i w i t h detergent r e s u l t e d i n the complete l o s s of ATP-driven energy-dependent transhydrogenase a c t i v i t y . T h i s f a c t i m p l i e s the need of an i n t a c t membrane f o r measurement of the energy-dependent transhydrogenase a c t i v i t y . Membrane p a r t i c l e s from E. c o l i NRC-482 were suspended i n b u f f e r , c o n t a i n i n g 0 t o 0.5% (w/v) T r i t o n X-100. As 8 4 . shown i n Table 5 supernatants of the 0.5% (w/v) T r i t o n X--100-t r e a t e d membrane p a r t i c l e s e x h i b i t e d 3 0% of the energy-independent a c t i v i t y and 97% of the p r o t e i n c o n c e n t r a t i o n present i n the s t a r t i n g m a t e r i a l . No ATP-driven energy-dependent transhydrogenase a c t i v i t y c o u l d be d e t e c t e d e i t h e r i n the supernatants or i n the p e l l e t s r e s u l t i n g from cen-t r i f u g a t i o n o f the d e t e r g e n t - t r e a t e d m a t e r i a l . Energy-independent transhydrogenase a c t i v i t y was absent from the supernatant of the membrane p a r t i c l e suspension t h a t had not been t r e a t e d w i t h T r i t o n X-100, i n d i c a t i n g t h a t the enzyme i s t i g h t l y bound i n the membrane. Since the energy-independent transhydrogenase a c t i v i t y c o u l d be measured i n supernatant f r a c t i o n s o f T r i t o n X-100-t r e a t e d membranes, i t was c l e a r t h a t detergents c o u l d be used f o r the s o l u b i l i z a t i o n o f t h i s enzyme from E. c o l i membrane p a r t i c l e s . The remaining r e s u l t s presented w i l l thus be l i m i t e d t o the study of the energy-independent transhydrogenase, i t s p r o p e r t i e s and mode of a c t i o n . The t o t a l p r o t e i n r e covered i n the supernatants and p e l l e t s was not compatible w i t h t h a t measured on the u n t r e a t e d membrane p a r t i c l e suspension. T h i s was due to i n t e r f e r e n c e by the detergent i n the Lowry assay f o r p r o t e i n s . The problem was f r e q u e n t l y encountered whenever detergents were used even though the standard curves f o r p r o t e i n assays were c o n s t r u c t e d i n the presence of e q u i v a l e n t amounts of detergent as those i n the samples. Table 5. D i s t r i b u t i o n of Transhydrogenase A c t i v i t y F o l l o w i n g Treatment of Mem-brane P a r t i c l e s w i t h T r i t o n X-100 P e l l e t Supernatant T r i t o n X-100 % (w/v) Transhydrogenase Transhydrogenase P r o t e i n Energy- Energy- , . Energy- Energy-ependent independent g dependent independe (units) (units) (units) (units) 0.1 5.5 0 1.9 9.2 0 0.43 0.2 4.5 0 1.3 12 0 0.71 0.5 4 0 1.0 15 0 0.81 Membrane p a r t i c l e s were suspended i n 0.1 M TEDG b u f f e r , pH 7.8 at a p r o t e i n c o n c e n t r a t i o n o f 15.5 mg/ml. The energy-independent and ATP-driven energy-dependent transhydrogenase a c t i v i t i e s of t h i s suspension were 2.7 and 0.5 u n i t s / ml, r e s p e c t i v e l y . One ml of the suspension was made up to 10 ml wit h 0.1 M TEDG, b u f f e r pH 7.8, c o n t a i n i n g 0.1-0.5% (w/v) T r i t o n X-100. The mixtures were s t i r r e d a t 0°C f o r 30 minutes and c e n t r i f u g e d a t 120 000 x g f o r 2 hours. The p e l l e t s were resuspended i n 1 ml 0.1M TED-1 buffer, pH 7.8. Transhydrogenase a c t i v i t y was measured u s i n g 0.4 ml supernatants and 0.05 ml p e l l e t suspension as d e s c r i b e d i n MATERIALS AND METHODS. 86. S o l u b i l i z a t i o n of the energy-independent transhydrogenase  Choice of d e t e r g e n t Many d i f f e r e n t detergents have been used f o r the e x t r a c -t i o n and p u r i f i c a t i o n of membrane-bound enzymes. T r i t o n X-100 (145-150), sodium deoxycholate (137,151,152), Ammonyx LO (dimethyl l a u r y l amine oxide) (153,154), sodium c h o l a t e ( i n the presence of ammonium sulphate) (88,136,155-158), are among the detergents used f o r the s o l u b i l i z a t i o n of transmembrane p r o t e i n s . L y s o l e c i t h i n has been s u c c e s s f u l l y used to s o l u b i l i z e the energy-independent transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s (7,85). These detergents were used i n the p r e s e n t study t o s o l u b i l i z e the energy-independent transhydrogenase from E. c o l i membrane p a r t i c l e s . Membrane p a r t i c l e s from E. c o l i NRC-4 82 were suspended i n 0.1 M TEGD b u f f e r , pH 7.8, and t r e a t e d w i t h T r i t o n X-100 or l y s o l e c i t h i n , r e s p e c t i v e l y , as d e s c r i b e d i n MATERIALS AND METHODS. S o l u b i l i z a t i o n w i t h Ammonyx LO (dimethyl l a u r y l amine oxide) f o l l o w e d the same procedure but w i t h the omis-s i o n of d i t h i o t h r e i t o l from the b u f f e r . Membrane p a r t i c l e s were a l s o s o l u b i l i z e d by sodium c h o l a t e i n the presence of 3 0% s a t u r a t e d ammonium sulphate (.13 6) , or by sodium deoxy-c h o l a t e (137). T a b l e 6 shows the degree of s o l u b i l i z a t i o n of energy-independent transhydrogenase and p r o t e i n by the v a r i o u s Table 6. S o l u b i l i z a t i o n of Energy-Independent Transhy-drogenase of E. c o l i by V a r i o u s Detergents Detergent Concentra-t i o n (w/v) % * D:P o. P r o t e i n S o l u b i l i z e d .Transhydrogenase Ammonyx LO 0.33 2.2 - 0 T r i t o n X-100 0.2 1.4 54 65 Sodium c h o l a t e 1.5 1.9 54 83 L y s o l e c i t h i n 0. 08 0.52 43 67 Sodium deoxy- 0.2 0.1 _ 123 c h o l a t e Detergent t o p r o t e i n r a t i o . Membrane p a r t i c l e s from E. c o l i NRC-48 2 were suspended a t p r o t e i n c o n c e n t r a t i o n s of 1.5 mg/ml i n 0.1 M TEG b u f f e r , pH 7.8, and a t 1.3 mg/ml and 1.5 mg/ml i n 0.1 M TEGD b u f f e r , pH 7.8, f o r s o l u b i l i z a t i o n w i t h Ammonyx LO, T r i t o n X-100 and l y s o l e c i t h i n , r e s p e c t i v e l y . Membrane p a r t i c l e s from E. c o l i ML308-225 a t 13.5 mg p r o t e i n / m l i n 50 mM TGDS/KCl/ c h o l a t e b u f f e r , pH 7.5, or a t 20 mg p r o t e i n / m l i n 50 mM TMDG b u f f e r , pH 7.5, c o n t a i n i n g 1 M KCl , were s o l u b i l i z e d w i t h sodium c h o l a t e or sodium deoxycholate, r e s p e c t i v e l y . The s o l u b i l i z a t i o n c o n d i t i o n s are giv e n i n the MATERIALS AND METHODS s e c t i o n . 88. detergents tested. No enzyme a c t i v i t y was observed.in the supernatant when Ammonyx LO was used. I t i s possible that the enzyme was s o l u b i l i z e d but destroyed by t h i s detergent since energy-independent transhydrogenase a c t i v i t y i n the material sedimented by centrifugation at 120 000 x g decreased with increasing detergent concentration. The extent of s o l u b i l i z a t i o n of the enzyme by Tr i t o n X-100 and l y s o l e c i t h i n appeared to be sim i l a r , although l y s o l e c i t h i n was somewhat more sel e c t i v e as indicated by the lower (43%) amount of protein s o l u b i l i z e d by lyso-l e c i t h i n as opposed to that s o l u b i l i z e d by Tri t o n X-100 (54%). Conditions of s o l u b i l i z a t i o n S o l u b i l i z a t i o n of the energy-independent transhydrogenase by Triton X-100 or l y s o l e c i t h i n was investigated i n more d e t a i l to determine the concentration of detergent necessary to produce the optimum y i e l d of s o l u b i l i z e d enzyme. Membrane p a r t i c l e s from E. c o l i NRC-482 or ML308-225 were treated with Tr i t o n X-100 or l y s o l e c i t h i n (Table 7). In some cases EDTA was omitted from the buffer i n which the membrane p a r t i c l e s were suspended. At moderate Tri t o n X-100 concentrations (0.5 to 1.0% (w/v)), more than 50% of the enzyme was.solubilized i f the detergent to protein r a t i o was kept between 4.5 and 8.9. For large-scale preparations, where higher concentrations of membrane p a r t i c l e protein were used, maintenance of t h i s r a t i o required a much greater Table 7. E f f e c t of V a r y i n g Detergent and P r o t e i n Concen-t r a t i o n s on the S o l u b i l i z a t i o n of Energy-Independent Transhydrogenase Detergent D:P* Supernatant P e l l e t % (w/v) Transhy- P r o t e i n Transhy- P r o t e i n drogenase % drogenase % . . .%.*.* a T r i t o n X-100: 0.1 0.89 36 67 45 31 0.5 4.5 67 89 7.4 22 1.0 8.9 52 68 2.8 22 3.0 1.3 17 38 17 38 6.0 2.0 11 66 25 13 L y s o l e c i t h i n : 0.02 0.13 24 36 59 33 0.05 0.33 58 40 27 21 0.08 0.52 67 43 7.8 19 0.10 0.66 53 43 0 18 0.6 0.5 63 55 22 8.4 3.0 2.30 3.2*** 83 0 51 * Detergent t o p r o t e i n r a t i o . ** Values are expressed as a percentage of the enzyme a c t i v i t y and p r o t e i n c o n c e n t r a t i o n i n the membrane p a r t i c l e suspensions. *** Assayed i n the presence .of'-0.1 % (w/v) sodium c h o l a t e . 0.1-1.0% (w/v) T r i t o n X-100 was used t o s o l u b i l i z e membrane p a r t i c l e s from E. c o l i NRC-482 (1.1 mg protein/ml) i n 0.1 M TEGD b u f f e r , pH 7.8. Membrane p a r t i c l e s from E. c o l i ML308-225 (23 mg p r o t e i n / m l and 30 mg p r o t e i n / m l , r e s p e c t i v e l y ) i n 2 mM TGD-1 b u f f e r , pH 7.8, were s o l u b i l i z e d w i t h 3% (w/v) and 6% (w/v) T r i t o n X-100, r e s p e c t i v e l y . 0.02-0.1% (w/v) l y s o l e c i t h i n was used t o s o l u b i l i z e membrane p a r t i c l e s from E. c o l i NRC-482 (1.5 mg protein/ml) Table 7 (cont'd) i n 0.1 M TEGD b u f f e r , pH. 7.8. Membrane p a r t i c l e s from E. c o l i ML308-225 a t 12 mg p r o t e i n / m l i n 0.1 M TEGD b u f f e r , pH 7.8, and a t 13 mg p r o t e i n / m l i n 2 0 mM TED-2 b u f f e r , pH 8.0, were s o l u b i l i z e d w i t h 0.6% (w/v) and 3% (w/v) l y s o -l e c i t h i n , r e s p e c t i v e l y . The c o n d i t i o n s f o r s o l u b i l i z a t i o n are d e s c r i b e d i n MATERIALS AND METHODS. 91. concentration of Tri t o n X-100. High concentrations of Tri t o n X-100 (3 to 6% (w/v)) resulted i n a decreased y i e l d of enzyme (less than 20%), either due to i n h i b i t i o n of . enzyme a c t i v i t y by excess detergent, denaturation or s o l -u b i l i z a t i o n of other membrane components (e.g. phospho-l i p i d s ) necessary for the functional conformation of the enzyme. S o l u b i l i z a t i o n of the energy-independent transhydrogenase with l y s o l e c i t h i n was optimal (more than 60% yield) at a detergent to protein r a t i o of 0.52 and 0.08% (w/v) of lyso-l e c i t h i n . Similar y i e l d s of enzyme were obtained when the detergent to protein r a t i o was maintained at 0.5 and the l y s o l e c i t h i n concentration increased to 0.6% (w/v). How-ever, increasing the l y s o l e c i t h i n concentration to 3% (w/v) at a detergent to protein r a t i o of 2.3, completely i n h i b i t e d the enzyme a c t i v i t y of the supernatant. The presence of 10 3 % (w/v) sodium cholate i n the assay mixture activated the enzyme a c t i v i t y s l i g h t l y to 3.2% of the o r i g i n a l s t a r t -ing material, although t h i s figure may be an underestimate due to masked enzymic a c t i v i t y . Thus, although there seems to be a rel a t i o n s h i p between the detergent to protein r a t i o s used and the y i e l d of enzyme obtained, at moderately low detergent concentrations, t h i s r e l a t i o n s h i p may not hold at high concentrations (greater than 1% (w/v) Triton X-100 or 0.6% (w/v) l y s o l e c i t h i n ) . In such, cases, detergent to p r o t e i n r a t i o s are r e l a t i v e l y un-important and the enzyme a c t i v i t y y i e l d e d i s more dependent upon the c o n c e n t r a t i o n of d e t e r g e n t per se. Since supernatants of membrane p a r t i c l e s t r e a t e d w i t h h i g h c o n c e n t r a t i o n s of detergent appeared to have lower energy-independent transhydrogenase a c t i v i t y , i t was of i n t e r e s t t o determine how s t a b l e the enzyme was i n the presence of detergent, over the p e r i o d of time spanned by some of the s o l u b i l i z a t i o n procedures used i n t h i s study. Membrane p a r t i c l e s from E. c o l i ML308-225 i n 0.1 M TEGD b u f f e r , pH 7.8, were t r e a t e d w i t h detergent and samples withdrawn a t timed i n t e r v a l s . The a d d i t i o n of 1% (w/v) T r i t o n X-100 to the membrane p a r t i c l e suspension caused an immediate l o s s of 60% of the energy-independent transhydrog-enase a c t i v i t y . T h e r e a f t e r the enzyme a c t i v i t y remained s t a b l e over a p e r i o d of 26 hours ( F i g . 7, panel X). Lyso-l e c i t h i n (0.4% (w/v)), used i n a s i m i l a r experiment ( F i g . 7, panel Y ) , caused an immediate (3 0%) l o s s of enzyme a c t i v i t y w i t h no f u r t h e r decrease up to 150 minutes. In ot h e r experiments (not shown), assay of energy-independent t r a n s -hydrogenase a c t i v i t y i n supernatants from membrane p a r t i c l e s t r e a t e d w i t h up t o 6% (w/v) l y s o l e c i t h i n remained constant over a 6-day p e r i o d . Thus, although these d e t e r g e n t s were d e t r i m e n t a l t o the enzyme, the a c t i v i t y f o l l o w i n g the i n i t i a l l o s s was f a i r l y s t a b l e i n the presence of dete r g e n t a t 0°C du r i n g the p e r i o d of time t e s t e d . F i g . 7. S t a b i l i t y of energy-independent transhydrogenase i n the presence of detergent. Membrane p a r t i c l e s from E. c o l i ML308-225 were sus-pended i n 0.1 M TEGD buffer, pH 7.8, at 2.7 mg protein/ml. The suspension was kept at 0°C and a sample was withdrawn for energy-independent transhydrogenase assay. T r i t o n X-100 or l y s o l e c i t h i n , to give f i n a l concentrations of 1% (w/v) or 0.4% (w/v), respectively, were then added to the membrane p a r t i c l e suspension and a sample was immedi-ately withdrawn for the enzyme assay. Samples were there-after withdrawn at timed i n t e r v a l s and the energy-independent transhydrogenase a c t i v i t y was measured. A c t i v i t y i s expressed as units/mg protein. 95. . Molecular size of. the s o l u b i l i z e d enzyme The material r e s u l t i n g from the treatment of E. c o l i membrane parti c l e s . w i t h T r i t o n X-100 or l y s o l e c i t h i n was subjected to analysis by sucrose density gradient centrifuga-t i o n i n order to determine the molecular size of the energy-independent transhydrogenase species s o l u b i l i z e d from the membranes by these detergents. Some of the s o l u b i l i z e d enzyme applied to 10 to 20% sucrose density gradients sedimented to the bottom of the gradients during centrifugation. Thus, sucrose gradients of a greater density range (10 to 50%) were selected to examine the molecular size of the detergent-solubilized enzyme. Thyroglobulin (molecular weight, 669 000), with a sedimentation c o e f f i c i e n t of 19.4S "and bovine catalase (molecular weight, 247 500) with a sedimentation c o e f f i c i e n t of 11.3S (133), were used as markers for sedimentation c o e f f i c i e n t s i n the sucrose density gradients. In some of the buffers used, both i n the sucrose gradients and sub-sequent preparations, low concentrations of detergents, e.g. B r i j 58, Tri t o n X-100, or Tween 80 were included i n the hope of preventing reaggregation of the s o l u b i l i z e d material. The membrane p a r t i c l e s of -E. c o l i ML308-225 i n 20 mM TED-2 buffer, pH 8, were s o l u b i l i z e d with 3% (w/v) lyso-l e c i t h i n at a detergent to protein r a t i o of 2.3 and then separated on g r a d i e n t s of 10 t o 50% sucrose i n 20 mM TED-2 b u f f e r , pH 7.4 ( F i g . 8). B r i j 58, 0.2% (w/v), was i n c l u d e d i n one of the g r a d i e n t s ( F i g . 8, r i g h t p a n e l ) . A l l o f the energy-independent transhydrogenase a c t i v i t y r ecovered was p r e s e n t i n the f r a c t i o n s c o l l e c t e d from the 10 to 50% sucrose d e n s i t y g r a d i e n t s . There was no enzyme a c t i v i t y i n the r e s i d u e sedimenting to the bottom of the g r a d i e n t as had been found when 10 to 20% sucrose g r a d i e n t s were used. In the absence of d e t e r g e n t i n the g r a d i e n t , two peaks of energy-independent transhydrogenase a c t i v i t y were de-t e c t e d . These had sedimentation c o e f f i c i e n t s of 19S and 11.5S, r e s p e c t i v e l y . The presence o f 0.2% (w/v) B r i j 58 i n the sucrose d e n s i t y g r a d i e n t caused a s h i f t i n the sedimen-t a t i o n p o s i t i o n s of the energy-independent transhydrogenase to 16.5S and 7.3S, r e s p e c t i v e l y . In t h i s case the 7.3S peak, together w i t h a shoulder a t 11.8S, rep r e s e n t e d 62% of the enzyme a c t i v i t y recovered from the g r a d i e n t , compared wi t h 51% f o r the 11.5S peak of a c t i v i t y o b t a i n e d i n the absence of B r i j 58. The i n c r e a s e i n the 7.3S peak of the B r i j 5 8 -containing sucrose d e n s i t y g r a d i e n t may r e f l e c t the d i s a g g r e g a t i o n by the detergent of a peak of g r e a t e r sedimentation c o e f f i c i e n t , o r the p r e v e n t i o n of r e a g g r e g a t i o n of m a t e r i a l of lower sedimentation c o e f f i c i e n t . Almost twice as much energy-F i g . 8. S e p a r a t i o n of membrane p a r t i c l e s of E. c o l i s o l u b i l i z e d w i t h 3% (w/v) l y s o l e c i t h i n on 10-50% sucrose d e n s i t y g r a d i e n t s . Membrane p a r t i c l e s from E. c o l i ML308-225 were sus-pended i n 20 mM TED-2 b u f f e r , pH 8, a t 13 mg p r o t e i n / m l . They were s o l u b i l i z e d w i t h 3% (w/v) l y s o l e c i t h i n as des-c r i b e d i n MATERIALS AND METHODS. The supernatant volume was reduced by mixing f o r 3 0 minutes w i t h 1.5 g of Sephadex G-25 a t 0°C f o l l o w e d by c e n t r i f u g a t i o n a t 2000 x g f o r 10 min. A sample of the c o n c e n t r a t e d supernatant (8 mg pro-t e i n ) was a p p l i e d t o a 10-50% sucrose g r a d i e n t i n 50 mM T r i s - C H 3 C O O H b u f f e r , pH 7.4, c o n t a i n i n g 1.5 mM EDTA, 2 mM DTT and 0.2% (w/v) B r i j 58 ( r i g h t p a n e l ) . Another sample (11 mg p r o t e i n ) of the same supernatant was a p p l i e d t o a s i m i l a r sucrose g r a d i e n t but without B r i j 58 ( l e f t p a n e l ) . The g r a d i e n t s were c e n t r i f u g e d a t 0°C f o r 12 h a t 138 000 x g. F r a c t i o n s (20 drops) were c o l l e c t e d and assayed f o r energy-independent transhydrogenase (TH), s u c c i n a t e de-hydrogenase (SDH) and A 2 8 o • Transhydrogenase and s u c c i n a t e dehydrogenase a c t i v i t i e s are expressed as u n i t s / f r a c t i o n and n mol/min/fraction, r e s p e c t i v e l y . The p o s i t i o n s of t h y r o g l o b u l i n (T) and c a t a l a s e (C) markers are i n d i c a t e d i n the F i g u r e s . 98. V o l u m e — ml independent transhydrogenase a c t i v i t y was recovered from the sucrose density gradient containing B r i j 58 than that without detergent (Table 8). This suggests that B r i j 58 may be an activator of the s o l u b i l i z e d energy-independent transhydrog-enase. This p o s s i b i l i t y i s discussed i n a l a t e r section. Treatment of membrane p a r t i c l e s of E. c o l i with deter-gents often y i e l d s large fragments of the b a c t e r i a l res-piratory chain. B a i l l i e et a l . (136), by treatment of membrane p a r t i c l e s of E. c o l i with sodium cholate and ammonium sulphate and p u r i f i c a t i o n on Sepharose 6B and Sepharose 4B columns of the f r a c t i o n p r e c i p i t a t i n g at 0.3 3 to 0.6 saturation of ammonium sulphate, obtained a stable moiety which they named the "soluble respiratory complex". This complex of molecular weight 2 x 10 6 was found to con-ta i n a l l the c a r r i e r s detected i n the membrane-bound res-piratory chain, including some cytochromes, f l a v i n s , NADH oxidase, succinate oxidase and succinate dehydrogenase. In order to determine whether the energy-independent transhydrogenase a c t i v i t y i n the present study had been s e l e c t i v e l y s o l u b i l i z e d by detergents, or whether i t was a component of a larger moiety comprising the group of enzymes constituting the respiratory chain, succinate de-hydrogenase a c t i v i t y was measured as a marker enzyme for t h i s larger complex. Two peaks of succinate dehydrogenase a c t i v i t y were obtained i n the 10 to 50% sucrose density gradient fractions Table 8. Recovery of Energy-Independent Transhydrogenase A c t i v i t y Following S o l u b i l i z a -t i o n with Various Detergents and Separation on Sucrose Gradients Detergent (w/v) Membrane A c t i v i t y units P a r t i c l e s S p e c i f i c A c t i v i t y units/mg protein Supernatant A c t i v i t y units %*'* P e l l e t A c t i v i t y units % Sucrose Density Gradient Detergent A c t i v i t y (w/v) units % 3% Lyso- 49 0.38 1.6 3.2* 0 0 3.8 7.7 l e c i t h i n 0 .2% B r i j 58 7.4 15 3% Tr i t o n 58 0.31 26 45 12 21 _ 12 21 X-100 0 .5% B r i j 58 13 22 2% Sodium 63 0.24 15 24 1.6 2.6 _ 4.6 7.2 cholate 0 .1% Aso- 7.2 11 l e c t i n Assayed i n the presence of 0.1% (w/v) sodium cholate. ** % of a c t i v i t y i n membrane p a r t i c l e suspension. Membrane p a r t i c l e s from E. c o l i ML308-225 were suspended i n 20 mM TED-2 buffer, pH 7.8, at a protein concentration of 13 mg/ml, 37 mg/ml or 44 mg/ml for s o l u b i l i z a t i o n with 3% (w/v) l y s o l e c i t h i n , 3% (w/v) Triton or 2% (w/v) sodium cholate (in the presence of 10% saturated ammonium sulphate),. respectively. During s o l u b i l i z a t i o n with lyso-l e c i t h i n or T r i t o n X-100, the supernatant volumes were reduced by gentle mixing with 1.5 g Sephadex G-25 f o r !-2 h at 0°C followed by c e n t r i f ugation at 2000 x g f o r 10 min. The concentrated supernatants were then applied to 10-50% sucrose density gradients in 2 0 mM TED-2 buffer, pH 7.4. Gradients containing detergents or phospholipids are indicated i n the table. 11 mg and 8 mg protein of l y s o l e c i t h i n - s o l u b i l i z e d material were applied to sucrose density gradients without and with 0.2% (w/v) B r i j 58, Table 8 (cont'd) r e s p e c t i v e l y . 5 mg p r o t e i n was a p p l i e d t o each g r a d i e n t i n the experiment u s i n g T r i t o n X-100. In the experiment u s i n g 2% (w/v) sodium c h o l a t e (in the presence of 10% s a t u r a -ted ammonium sulphate) 7.3 mg and 5.8 mg p r o t e i n were a p p l i e d t o g r a d i e n t s without and with 0.1% (w/v) a s o l e c t i n , r e s p e c t i v e l y . The c o n d i t i o n s f o r s o l u b i l i z a t i o n are des-c r i b e d i n MATERIALS AND METHODS. 102. of l y s o l e c i t h i n - s o l u b i l i z e d membrane p a r t i c l e s . o f E. c o l i ( F i g . 8, l e f t p a n e l ) . The peaks were c l o s e t o but not c o i n c i d e n t a l w i t h those of the energy-^ independent t r a n s -hydrogenase, suggesting t h a t the fragment r e s u l t i n g from the s o l u b i l i z a t i o n by l y s o l e c i t h i n i s not a homogeneous complex, but r a t h e r a s e r i e s of s p e c i e s i n which the s o l u -b i l i z e d enzyme was aggregated to v a r i o u s e x t e n t s . The m a t e r i a l s o l u b i l i z e d from membrane p a r t i c l e s of E. c o l i ML308-225 by 3% (w/v) T r i t o n X-100 at a detergent to p r o t e i n r a t i o of 1.6 was a l s o analyzed on 10 t o 50% sucrose d e n s i t y g r a d i e n t s . In o t h e r experiments (not shown) c o n c e n t r a t i o n s of up to 10% (w/v) T r i t o n X-100 were used and y i e l d e d s i m i l a r r e s u l t s . The procedure was the same as t h a t employed u s i n g l y s o l e c i t h i n as d e t e r g e n t . Sucrose d e n s i t y g r a d i e n t s were a l s o run w i t h and without 0.5% (w/v) B r i j 58. In c o n t r a s t t o the r e s u l t s w i t h l y s o -l e c i t h i n , o n l y one main peak of energy-independent t r a n s -hydrogenase a c t i v i t y was d e t e c t e d . A shoulder at 11S was a l s o p r e s e n t ( F i g . 9, panels l^and 3). T h i s had a sedimenta-t i o n c o e f f i c i e n t of 24.5S, somewhat g r e a t e r than the l a r g e s p e c i e s o b tained w i t h l y s o l e c i t h i n (19S). The presence of B r i j 58 made l i t t l e d i f f e r e n c e e i t h e r i n the p o s i t i o n s of the energy-independent transhydrogenase a c t i v i t y peaks (24.2S and 8.2S) or i n t h e i r r e l a t i v e abundance. The 24.2-24.5S peak c o n s t i t u t e d 82% and 80% of the energy-independent 103. F i g . 9. A n a l y s i s by sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of membrane p a r t i c l e s from E. c o l i s o l u b i l i z e d w i t h 3% (w/v) T r i t o n X-100 or 2% (w/v) sodium c h o l a t e ( i n the presence of ammonium s u l p h a t e ) . Membrane p a r t i c l e s were suspended i n 20 mM TED-2 b u f f e r , pH 8, a t 18 mg p r o t e i n / m l , and s o l u b i l i z e d u s i n g 3% (w/v) T r i t o n X-100 as d e s c r i b e d i n MATERIALS AND METHODS. Samples of the concentrated supernatant (0.5 mg p r o t e i n ) were ap-p l i e d t o 10-50% sucrose d e n s i t y g r a d i e n t s c o n t a i n i n g 0.5% (w/v) B r i j 58 (panel 1) or not c o n t a i n i n g detergent (panel 3). With the e x c e p t i o n of the c o n c e n t r a t i o n of B r i j 58 i n the sucrose d e n s i t y g r a d i e n t , the procedure f o l l o w e d was s i m i l a r to t h a t d e s c r i b e d i n the legend t o F i g . 8. For s o l u b i l i z a t i o n w i t h 0.2% (w/v) sodium c h o l a t e ( i n the presence of 10% s a t u r a t e d ammonium sulphate) (panels 2 and 4), the membrane p a r t i c l e s were suspended a t 44 mg pr o t e i n / m l i n 20 mM TED-2 b u f f e r , pH 8, and processed as d e s c r i b e d i n MATERIALS AND METHODS. The (33-43P) f r a c t i o n was suspended i n 2 0 mM TED-2 b u f f e r , pH 8, a t 15 mg p r o t e i n / ml. Samples c o n t a i n i n g 6 mg and 7.5 mg p r o t e i n r e s p e c t i v e l y were a p p l i e d to 10-50% sucrose g r a d i e n t s i n panels 2 and 4. The g r a d i e n t s c o n t a i n e d 10-50% sucrose i n 50 mM T r i s - C H 3 C O O H b u f f e r , pH 7.4, c o n t a i n i n g 1.5% (w/v) sodium c h o l a t e , 1.5 mM EDTA and 2 mM DTT. The g r a d i e n t i n panel 2 cont a i n e d , i n a d d i t i o n , 0.1% (w/v) a s o l e c t i n . The sucrose d e n s i t y g r a d i e n t s were c e n t r i f u g e d a t 0°C f o r 12 h a t 138 000 x g. F r a c t i o n s (20 drops) were c o l l e c t e d and assayed f o r energy-independent transhydrogenase (TH) ( p <m ) r s u c c i n a t e de-hydrogenase (SDH) ( o—O ) and A 2so (. * - r ). Transhydrogenase and s u c c i n a t e dehydrogenase a c t i v i -t i e s are expressed as u n i t s / f r a c t i o n and nmol / m i n / f r a c t i o n , r e s p e c t i v e l y . The p o s i t i o n s o f t h y r o g l o b u l i n (T) and c a t a l a s e (C) markers are i n d i c a t e d i n the F i g u r e s . 104 . 105. transhydrogenase a c t i v i t y r ecovered i n the g r a d i e n t s , w i t h and without B r i j 58, r e s p e c t i v e l y . The t o t a l u n i t s recov-ered from each of these g r a d i e n t s represented 102% and 95% r e s p e c t i v e l y , o f the energy-independent transhydrogenase a c t i v i t y a p p l i e d t o the g r a d i e n t s (Table 8 ) . Only one peak of s u c c i n a t e dehydrogenase a c t i v i t y was recovered i n e i t h e r g r a d i e n t . I t s p o s i t i o n c o i n c i d e d w i t h t h a t of the 8.2S peak of energy-independent transhydrogenase i n the sucrose d e n s i t y g r a d i e n t c o n t a i n i n g B r i j 58. In the absence of B r i j 58, the s u c c i n a t e dehydrogenase a c t i v i t y peak appeared to be s h i f t e d towards the top of the g r a d i e n t . I t was of i n t e r e s t t o analyze the s i z e of the s o l u b i -l i z e d f r a c t i o n of membrane p a r t i c l e s i n p r e p a r a t i o n s known to y i e l d the " s o l u b l e r e s p i r a t o r y complex" (136). Since the c o n d i t i o n s y i e l d i n g the complex are s i m i l a r t o those employed by Rydstrbm e t a l . (88) t o s o l u b i l i z e the energy-independent transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s , i t was decided t o use a m o d i f i c a t i o n of the l a t t e r c o n d i t i o n s o f s o l u b i l i z a t i o n i n t h i s p a r t i c u l a r experiment. Membrane p a r t i c l e s from E. c o l i ML308-225 were s o l u b i -l i z e d w i t h 2% (w/v) sodium c h o l a t e ( i n the presence of 10% (w/v) ammonium sulphate) a t a dete r g e n t t o p r o t e i n r a t i o of 0.46. The f r a c t i o n o f s o l u b i l i z e d m a t e r i a l p r e c i p i t a t i n g between 33% and 43% s a t u r a t i o n (33-43P) and between 43% 106. and 60% s a t u r a t i o n of ammonium sulphate (43-60P) were sus-pended s e p a r a t e l y i n 20 mM TED-2 b u f f e r , pH 8, f o r a n a l y s i s on the sucrose d e n s i t y g r a d i e n t s . Since the r e s u l t s ob-served w i t h both f r a c t i o n s were s i m i l a r , o n l y one s e t of r e s u l t s w i l l be d i s c u s s e d . The 10-50% sucrose d e n s i t y g r a d i e n t s c o n t a i n e d 1.5% (w/v) sodium c h o l a t e . In a d d i t i o n , one g r a d i e n t c o n t a i n e d 0.1% (w/v) soybean p h o s p h o l i p i d ( a s o l e c t i n ) . The s e p a r a t i o n of the (33-43P) f r a c t i o n on sucrose d e n s i t y g r a d i e n t s i n the presence or absence of a s o l e c t i n , i s shown i n F i g . 9 (panels 2 and 4, r e s p e c t i v e l y ) . As w i t h T r i t o n X-100, two peaks of energy-independent transhydrogenase a c t i v i t y were d e t e c t e d i n the g r a d i e n t s , w i t h most of the enzyme (88% and 85%) m i g r a t i n g as the s p e c i e s of h i g h sedimentation c o e f f i c i e n t s (22.8S and 25.4S) i n sucrose g r a d i e n t s w i t h and without a s o l e c t i n , r e s p e c t i v e l y . The s p e c i f i c a c t i v i t y of the f a s t - s e d i m e n t i n g peaks was 0.52 u n i t s per mg p r o t e i n and 2.0 u n i t s per mg p r o t e i n , r e p r e s e n t i n g 2 - f o l d and 8 - f o l d p u r i f i c a t i o n , r e s p e c t i v e l y , over the membrane p a r t i c l e suspension. In the sucrose d e n s i t y g r a d i e n t c o n t a i n i n g a s o l e c t i n , 41% of the energy-independent transhydrogenase a c t i v i t y t h a t was a p p l i e d to the g r a d i e n t was recovered. The corresponding y i e l d i n the g r a d i e n t without a s o l e c t i n was 31% (Table 8). In t h i s experiment, i n c o n t r a s t to the r e s u l t s w i t h T r i t o n X-100, most of the s u c c i n a t e dehydrogenase a c t i v i t y sedimented w i t h the 22.8S peak of the energy-independent transhydrogenase. 107. Each, of the detergents used i n these experiments y i e l d e d two peaks of energy-independent transhydrogenase a c t i v i t y by sucrose d e n s i t y g r a d i e n t a n a l y s i s . However, the d i s t r i b u t i o n of enzyme between these peaks v a r i e d with the detergent used f o r s o l u b i l i z a t i o n . Thus, the f a s t -sedimenting peak co n t a i n e d 49%, 80% and 85% of the a c t i v i t y r e c o vered from the g r a d i e n t w i t h l y s o l e c i t h i n , T r i t o n X-100 and sodium c h o l a t e ( i n the presence of 0.33 s a t u r a t i o n of ammonium s u l p h a t e ) , r e s p e c t i v e l y . S u c c i n a t e dehydrogenase was separated from the peak of high sedimentation c o e f f i c i e n t when l y s o l e c i t h i n and T r i t o n X-100 were used, but t h i s enzyme cosedimented w i t h the energy-independent transhydrogenase on d e n s i t y g r a d i e n t s s e p a r a t i n g the sodium c h o l a t e - s o l u b i l i z e d m a t e r i a l . The sedimentation c o e f f i c i e n t s of the major peaks obtained by s o l u b i l i z a t i o n of membrane p a r t i c l e s w i t h T r i t o n X-100 were g r e a t e r than those obtained u s i n g l y s o l e c i t h i n . I t i s not c l e a r i f t h i s i s due to a d i f f e r e n c e i n b i n d i n g of the v a r i o u s detergents i n these two cases or whether th e r e i s a d i f f e r e n c e i n the molecular s i z e of the fragments formed. The presence of detergents (e.g. B r i j 58) i n the sucrose g r a d i e n t s i n some cases caused a s h i f t i n the peaks of the energy-independent transhydrogenase .. a c t i v i t y t o lower s e d i -mentation c o e f f i c i e n t s ( F i g . 8), p o s s i b l y by p r e v e n t i n g the r e a g g r e g a t i o n of the s o l u b i l i z e d enzyme to l a r g e r s p e c i e s . 108 . It was of inte r e s t to determine whether the energy-independent transhydrogenase could be prevented from re-aggregation by various detergents and whether these could cause further disaggregation :of the s o l u b i l i z e d enzyme com-plex. The detergents selected for t h i s purpose were B r i j 35, Tween 8 0 and T r i t o n X-100. In order to compare the effects of these three detergents, the same procedure for the s o l u b i l i z a t i o n of the energy-independent transhydrogen-ase was followed i n each case. Membrane p a r t i c l e s from E. c o l i W6 were s o l u b i l i z e d with sodium cholate (in the presence of 0.33 saturation of ammonium sulphate) at a detergent to protein r a t i o of 3.7 (136). The material p r e c i p i a t i n g between 0.33 and 0.6 saturation of ammonium sulphate (hereafter referred to as (0.33-0.6P) f r a c t i o n ) , was dissolved i n 50 mM TGD-2 buffer, pH 7.8, containing 0.2% (w/v) B r i j 35. The solution was applied to a column of Sepharose 6B. Chromatography was effected by elu t i o n with 50 mM TGD-2 buffer, pH 7.8, contain-ing 0.2% (w/v) B r i j 35. F i g . 10 shows the elu t i o n pattern of the (0.33-0.6P) f r a c t i o n on Sepharose 6B. Energy-independent transhydrog-enase was eluted as a single peak included by the column but.close to the void volume (V"o = 85 ml) and migrating ahead of catalase. Cytochrome b^ and the main peak of succinate dehydrogenase comigrated with the energy-independent transhydrogenase. 109. F i g . 10. Chromatography of the (0.33-0.6P) f r a c t i o n s on Sepharose 6B i n the presence of 0.2% (w/v) B r i j 35. The (0.33-0.6P) f r a c t i o n was prepared from membrane p a r t i c l e s from 9.5 g of E. c o l i W6 c e l l s as d e s c r i b e d i n MATERIALS AND METHODS. I t was suspended i n 50 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.2% (w/v) B r i j 35. Bovine c a t a l a s e (5 mg) was d i s s o l v e d i n 8.2 ml of t h i s f r a c t i o n , c o n t a i n i n g 24 u n i t s of energy-independent transhydrogenase a c t i v i t y , and the suspension was a p p l i e d t o a column of Sepharose 6B (2.5 x 38.5 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h 50 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.2% (w/v) B r i j 35. The f r a c t i o n s were e l u t e d w i t h the same b u f f e r . Ten ml f r a c t i o n s were c o l l e c t e d and assayed f o r energy-independent transhydrogenase (TH) (•—••.), s u c c i n a t e dehydrogenase (SDH) ( cr—O )/ c a t a l a s e ( Q — • )r cytochrome b-L (• •) and A 2 8 o ( ) • Energy-independent transhydrogenase and s u c c i n a t e dehydrogenase a c t i v i t i e s are expressed as u n i t s / f r a c t i o n and n mol/min/fraction, r e s p e c t i v e l y , and c a t a l a s e as the r a t e of change i n absorbance a t 240 n m / f r a c t i o n and cytochrome b_i as uM c o n c e n t r a t i o n . 110. V o l u m e - m l The e l u t i o n p r o f i l e from the Sepharose 6B chromatography of the (0.33-0.6P) f r a c t i o n o f a s i m i l a r experiment i s shown i n F i g . 11. In t h i s case, however, 1% (w/v) Tween 8 0 i n s t e a d of B r i j 35 was used i n 50 mM TGD-2 b u f f e r , pH 7.8, f o r suspending the (0.33-0.6P) f r a c t i o n and e l u t i n g i t from the Sepharose 6B column. The dete r g e n t t o p r o t e i n r a t i o d u r i n g the s o l u b i l i z a t i o n o f the membrane p a r t i c l e s w i t h sodium c h o l a t e and ammonium sulphate was 2.8. The f r a c t i o n s e l u t e d from the column r e v e a l e d one peak of energy-independent transhydrogenase a c t i v i t y . In t h i s case, as i n the p r e c e d i n g experiment i n which B r i j 3 5 was used, the energy-independent transhydrogenase a c t i v i t y peak was e l u t e d a f t e r the v o i d volume (V = 8 5 ml) but pr e c e d i n g the peak of c a t a l a s e a c t i v i t y . As b e f o r e , cytochrome b^ migrated w i t h the energy-independent transhydrogenase. The main peak of s u c c i n a t e dehydrogenase, however, c o i n c i d e d w i t h t h a t of c a t a l a s e . The e f f e c t of T r i t o n X-100 on the e l u t i o n p a t t e r n of the (0.33-0.6P) f r a c t i o n by Sepharose 6B chromatography was a l s o i n v e s t i g a t e d ( F i g . 12). In t h i s case membrane p a r t i c l e s from E. c o l i ML308-225 were used and the detergent t o p r o t e i n r a t i o was 1.65. The procedure was the same as t h a t f o l l o w e d f o r B r i j 35 and Tween 80, but the suspension and e l u t i o n b u f f e r c o n t a i n e d 0.5% (w/v) T r i t o n X-100 i n s t e a d of B r i j 35 or Tween 80. T h i s enzyme was e l u t e d from Sepharose 6B as a F i g . 11. Chromatography of the (0.33-0.6P) f r a c t i o n on Sepharose 6B i n the presence of 1% (w/v) Tween 80. The (0.33-0.6P) f r a c t i o n was prepared from membrane p a r t i c l e s of 8.4 g of E. c o l i W6 c e l l s as d e s c r i b e d i n MATERIALS AND METHODS. I t was suspended i n 50 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 1% (w/v) Tween 80. Bovine c a t a l a s e (5 mg) was d i s s o l v e d i n 5 ml of t h i s f r a c t i o n , con-t a i n i n g 18 u n i t s of energy-independent transhydrogenase a c t i v i t y , and the suspension was a p p l i e d t o a column of Sepharose 6B (2.5 x 38.5 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h 50 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 1% (w/v) Tween 80, and e l u t e d w i t h the same b u f f e r . F r a c t i o n s (9 ml) were c o l -l e c t e d and assayed f o r energy-independent transhydrogenase (TH) ( #—# ), s u c c i n a t e dehydrogenase (SDH) (o—O )/ c a t a -l a s e (•—a )r cytochrome b j (• •) and A 2 8 0 ( ) . Energy-independent transhydrogenase and s u c c i n a t e dehydrog-enase a c t i v i t i e s are expressed as u n i t s / f r a c t i o n and nmol/ m i n / f r a c t i o n , r e s p e c t i v e l y , c a t a l a s e as the r a t e of change i n absorbance a t 24 0 n m / f r a c t i o n , and cytochrome b^ as uM c o n c e n t r a t i o n . Energy-independent transhydrogenase a c t i v i t y was assayed i n the presence of 0.0014% (w/v) l i p i d e x t r a c t e d from E. c o l i K-12. 113. V o l u m e — ml 114 F i g . 12. Chromatography of the (0.33-0.6P) f r a c t i o n on Sepharose 6B i n the presence of 0.5% (w/v) T r i t o n X-100. The (0.33-0.6P) f r a c t i o n was prepared from membrane p a r t i c l e s of 8.1 g of E. c o l i ML308-225 as described i n MATERIALS AND METHODS. It was suspended i n 50 mM TGD-2 buffer, pH 7.8, containing 0.5% (w/v) Triton X-100. Bovine catalase (5 mg) was dissolved i n 5.5 ml of t h i s f r a c t i o n , containing 21 units of energy-independent transhydrogenase a c t i v i t y , and the suspension was applied to a column of Sepharose 6B (2.5 x 38.5 cm) previously equilibrated with 50 mM TGD-2 buffer, pH 8, containing 0.5% (w/v) Tr i t o n X-100. The fractions were eluted with the same buffer. Thirteen ml fractions were co l l e c t e d and assayed for energy-independent transhydrogenase (TH) (#—• ), succinate dehydrogenase (SDH) (O-O ), catalase (•-• ), cytochrome bj_ (•-•-•) and t o t a l protein — + ) . Energy-independent transhydrogenase and succinate dehydrogenase a c t i v i t i e s are expressed as u n i t s / f r a c t i o n and nmol/min/fraction, respectively, catalase as the rate of change i n absorbance at 24 0 nm/fraction, cyto-chrome bj_ as yM concentration and t o t a l protein as mg/ f r a c t i o n . Energy-independent transhydrogenase a c t i v i t y was assayed i n the presence of 0.025% (w/v) B r i j 35. 115. 116. single peak following the void volume .(V =70 ml) and preceding the catalase. -In contrast to the experiments i n the presence of B r i j 35 or Tween 80, the energy-independent transhydrog-enase i n t h i s case was separated from the cytochromes and succinate dehydrogenase a c t i v i t y . However, the s p e c i f i c a c t i v i t y of the peak f r a c t i o n of energy-independent trans-hydrogenase was 0.61 units per mg protein and represented only on 2.3-fold p u r i f i c a t i o n over the membrane p a r t i c l e suspension (Table 9). The t o t a l units of energy-independent transhydrogenase a c t i v i t y recovered from the Sepharose 6B column represented 46% of t h i s enzyme a c t i v i t y applied to the column, and 15% of the a c t i v i t y present i n the membrane p a r t i c l e suspension. The corresponding figures for the experiment using B r i j 35 were 151% and 37%, while those using Tween 80 were 113% and 17%, respectively. In a l l three experiments, the peak of energy-independent transhydrogenase a c t i v i t y was eluted at approximately the same pos i t i o n from the Sepharose 6B columns. This occurred at about 100 ml for columns with void volumes of 70 to 85 ml. Since catalase (molecular weight, 247 500) was eluted af t e r the energy-independent transhydrogenase i n a l l cases, t h i s implies that the l a t t e r enzyme i s part of a larger fragment whose molecular weight must be i n the range of 247 500 to 4 x 10 6. Thus the energy-independent transhydrogenase s o l u b i l i z e d by sodium cholate (in the presence of 0.33 Table 9. P u r i f i c a t i o n of Energy-Independent Transhydrogenase i n the (0.33-0.6P) Fraction, by Chromatography on Sepharose 6B i n the Presence of Trit o n X-100 Energy-independent ^ . c . F r a c t i o n T o t a l P r o t e i n Transhydrogenase* ( x - f S l d " mg/ml % A c t i v i t y % S p e c i f i c u n i t s A c t i v i t y units/mg Membrane p a r t i c l e s 16 100 86 100 0.27 1 Ammonium sulphate f r a c t i o n s 0-0.33 s a t u r a t i o n p e l l e t 12 43 32 37 0.23 0.85 supernatant 3.9 37 52 60 0.42 1.6 (0.33-0.6P) f r a c t i o n 17 32 26 33 0.26 0.96 Sepharose 6B Pool 0.87 32 11 15 0.12 0.44 Sepharose 6B f r a c t i o n 7 0.52 2.4 4.2 5.6 0.61 2.3 * Energy-independent transhydrogenase a c t i v i t y assayed in the presence of 0.025% (w/v) B r i j 35. Membrane p a r t i c l e s from 8 g of E. c o l i ML308-225 were suspended in 50 mM TGDS/KCl/cholate buffer, pH 7.8. S o l u b i l i z a t i o n with sodium cholate was as des-cribed i n MATERIALS AND METHODS. The (0.33-0.6P) fr a c t i o n was suspended i n 50 mM TGD-2 buffer, pH 7.8 containing 0.5% (w/v) Triton X-100, and a portion of i t (91 mg protein) was applied to a column of Sepharose 6B equilibrated with 5 0 mM TGD-2 buffer, pH 7.8, containing 0.5% (w/v) Triton X-100. Elution was effected with the same buffer. The Sepharose 6B pool represented fractions eluted between 44-149 ml of ef f l u e n t . Fraction #7 was eluted at 96 ml of effl u e n t . s a t u r a t i o n of ammonium sulphate) i s p a r t of a l a r g e fragment, or c o n c e i v a b l y an aggregate, which cannot be d i s a g g r e g a t e d by B r i j 35, Tween 80 or T r i t o n X-100 to a monomeric form. D i s a g g r e g a t i o n by l y s o l e c i t h i n A n a l y s i s of s o l u b i l i z e d .energy-independent t r a n s h y -drogenase by sucrose d e n s i t y g r a d i e n t s presented i n the p r e c e d i n g s e c t i o n r e v e a l e d t h a t s o l u b i l i z a t i o n w i t h T r i t o n X-100 or sodium c h o l a t e ( i n the presence of 0.33 s a t u r a t i o n of ammonium sulphate) produced a r a p i d l y - s e d i m e n t i n g s p e c i e s of energy-independent transhydrogenase (24.5S and 25.4S, r e s p e c t i v e l y ) , which was not a s s o c i a t e d w i t h the m a j o r i t y of p r o t e i n s i n the sucrose d e n s i t y g r a d i e n t s . S o l u b i l i z a -t i o n by e i t h e r of these d e t e r g e n t s , t h e r e f o r e , might y i e l d a f a v o u r a b l e s t a r t i n g m a t e r i a l f o r f u r t h e r p u r i f i c a t i o n of the enzyme. S o l u b i l i z a t i o n by l y s o l e c i t h i n on the other hand, pro-duced two s p e c i e s of the enzyme (19S and 11.5S). The 19S s p e c i e s was l i k e l y e q u i v a l e n t t o the fragment s o l u b i l i z e d by T r i t o n X-100 and probably r e p r e s e n t e d an aggregate of the 11.5S m a t e r i a l . T h i s hypothesis was t e s t e d by s o l u b i l i z i n g membrane p a r t i c l e s of E. c o l i w i t h T r i t o n X-100, t r e a t i n g the s o l u -b i l i z e d m a t e r i a l w i t h l y s o l e c i t h i n , and determining, by the use of sucrose d e n s i t y g r a d i e n t s , whether l y s o l e c i t h i n c o u l d reduce the T r i t o n X - 1 0 0 - s o l u b i l i z e d m a t e r i a l t o s m a l l e r fragments. 119. In order to have s u f f i c i e n t l y large quantities of the T r i t o n X-100-solubilized energy-independent transhydrogenase species, i s o l a t i o n of t h i s fragment was achieved by chrom-atography on DEAE-cellulose columns instead of sucrose den-s i t y gradient centrifugation. Membrane p a r t i c l e s from E. c o l i ML308-225 were solu-b i l i z e d with 3% (w/v) Triton X-100 at a detergent to protein r a t i o of 1.3. The supernatant was applied to a column of DEAE-cellulose equilibrated with 2 mM TGD-1 buffer, pH 7.8. The applied material was eluted with sequential additions of 2 to 1000 mM TGD-1 buffer, pH 7.8. F i g . 13 i s the elution pattern of the fractions c o l -lected from the DEAE-cellulose column. Energy-independent transhydrogenase a c t i v i t y was found i n several of the fractions eluted from the column. The t o t a l units of t h i s enzyme recovered from the fractions were 8.1 units. This was equivalent to 20% of the a c t i v i t y i n the membrane p a r t i c l e suspension and 114% of the supernatant f r a c t i o n s o l u b i l i z e d by Triton X-100. Fraction 3, eluting at 15 0 mM TGD-1, was the r i c h e s t i n a c t i v i t y and contained no succinate dehydrogenase a c t i v i t y . The s p e c i f i c a c t i v i t y of the energy-independent transhydrogenase i n t h i s f r a c t i o n was 0.35 units per mg protein (Table 10), representing a two-fold p u r i f i c a t i o n over the membrane p a r t i c l e suspension. 120. F i g . 13. Chromatography of T r i t o n X - 1 0 0 - s o l u b i l i z e d mem-brane p a r t i c l e s of E. c o l i on D E A E - c e l l u l o s e . Membrane p a r t i c l e s from E. c o l i ML308-225 were sus-pended i n 2 mM TGD-1 b u f f e r , pH 7.8, a t 23 mg p r o t e i n / m l . 8.5 ml of the supernatant (81 mg p r o t e i n ) was a p p l i e d t o a column of D E A E - c e l l u l o s e (1.9 cm x 5.7 cm) p r e v i o u s l y e q u i l i b r a t e d w i t h 2 mM TGD-buffer, pH 7.8. The sample was washed i n t o the column w i t h 15 ml of the same b u f f e r and e l u t e d by the s e q u e n t i a l a d d i t i o n of 15 ml each of 100 mM, 150 mM, 200 mM, 250 mM, 500 mM and 1 M TGD-1 b u f f e r , pH 7.8. F r a c t i o n s (15 ml) were c o l l e c t e d and assayed f o r energy-independent transhydrogenase (0), s u c c i n a t e dehy-drogenase (•) and A 2 8 0 (_0. The procedure was c a r r i e d out at 0°C. Enzyme a c t i v i t i e s are expressed as u n i t s / f r a c t i o n . 121. [Tris] Table 10. E f f e c t o f L y s o l e c i t h i n on T r i t o n X-100-Solubi-l i z e d Membrane P a r t i c l e s a f t e r Chromatography on DEAE-Cellulose F r a c t i o n Energy-independent Transhydrogenase T o t a l P r o t e i n U n i t s Q. (mg) .....%. Membrane p a r t i c l e s 41 - 225 T r i t o n supernatant 7.1 100- 86 100 D E A E - c e l l u l o s e F r a c t i o n F F r a c t i o n L 2.9 0. 61 41 8.6 8.3 9.7 4.9 4.7 Sucrose d e n s i t y g r a d i e n t s 22.3S peak (gradient F) 19.4S peak (gradient L) 8.4S peak (gradient L) 2.4 0.39 0.29 34 5.5 4.0 -Membrane p a r t i c l e s from E. c o l i ML308-225 were suspended at 45 mg protein/ml i n 2 mM TGD-1 buffer, pH 7.8, and solu-b i l i z e d with 3% (w/v) Triton X-100. A f r a c t i o n of the super-natant (81 mg protein) was applied to a column of DEAE-ce l l u l o s e and eluted sequentially with 0.2 mM-1.0 M TGD-1 buffer, pH 7.8. The f r a c t i o n eluted by 150 mM TGD-1 buffer, pH 7.8, was concentrated (F) and a portion of i t was treated with l y s o l e c i t h i n . The preparation was then centrifuged and the supernatant (L) (0.23 mg protein) was applied to a 10-50% sucrose density gradient. A portion (0.42 mg protein) of f r a c t i o n F was also applied to a 10-50% sucrose density gradient. The conditions for s o l u b i l i z a t i o n and sucrose density centrifugation are described i n the text. 123. F r a c t i o n 3 was c o n c e n t r a t e d by u l t r a f i l t r a t i o n and then d i v i d e d i n t o two p o r t i o n s . One p o r t i o n was l e f t as such (termed F) and the other was t r e a t e d w i t h 0.09% (w/v) l y s o l e c i t h i n a t a detergent to p r o t e i n r a t i o o f 0.7, f o r 30 minutes a t 0°C, and then c e n t r i f u g e d a t 138 000 x g f o r 2 hours. The supernatant (termed L ) , and f r a c t i o n F were each a p p l i e d to 10-50% sucrose d e n s i t y g r a d i e n t s c o n t a i n i n g 0.5% (w/v) B r i j 58 and c e n t r i f u g e d as b e f o r e . F i g . 14 i s the p r o f i l e of energy-independent t r a n s -hydrogenase a c t i v i t y o b t a i n e d . Treatment w i t h l y s o l e c i t h i n r e s u l t e d i n the formation of two d i s t i n c t peaks of enzyme a c t i v i t y (19.4S and 8.4S) as opposed to the one peak of a c t i v i t y observed w i t h the o r i g i n a l m a t e r i a l (22.3S). About 42% of the a c t i v i t y r ecovered from the g r a d i e n t was found i n the 8.4S peak. Approximately 113% and 88% of the energy-independent transhydrogenase a c t i v i t y t h a t was a p p l i e d was recovered from sucrose g r a d i e n t s L and F, r e s p e c t i v e l y . Attempts t o analyze the peak f r a c t i o n s by 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 f o l l o w i n g e x t e n s i v e d i a l y s i s proved u n s u c c e s s f u l , due to the d i f f i c u l t y of removing the T r i t o n X-100 and l y s o l e c i t h i n completely. These detergents caused smearing of the p r o t e i n bands d u r i n g e l e c t r o p h o r e s i s . The a b i l i t y of l y s o l e c i t h i n t o produce s m a l l e r f r a g -ments from T r i t o n X - 1 0 0 - s o l u b i l i z e d m a t e r i a l was a l s o examined i n another s i m i l a r experiment. In t h i s case the F i g . 14. S e p a r a t i o n of f r a c t i o n s F and L on 10-50% sucrose d e n s i t y g r a d i e n t s . F r a c t i o n F (obtained by chromatography of s o l u b i l i z e d membrane p a r t i c l e s on DEAE-cellulose) and i t s l y s o l e c i t h i n -t r e a t e d c o u n t e r p a r t , f r a c t i o n L, were prepared as d e s c r i b e d i n the t e x t . 0.3 ml of f r a c t i o n F (0.42 mg p r o t e i n ) and 0.3 ml of f r a c t i o n L (0.23 mg p r o t e i n ) were a p p l i e d t o s u c r o s e ' d e n s i t y g r a d i e n t s c o n t a i n i n g 10-50% sucrose i n 50 mM T r i s - H C l , pH 7.4, 1.5 mM EDTA, 2 mM DTT and 0.5% (w/v) B r i j 58. They were c e n t r i f u g e d a t 138 000 x g and 0°C f o r 12 h. Twenty drop f r a c t i o n s were c o l l e c t e d and assayed f o r energy-independent transhydrogenase a c t i v i t y . A c t i v i t y i s expressed as nmol / m i n / f r a c t i o n . Sedimentation p o s i t i o n s of t h y r o g l o b u l i n (T) and c a t a l a s e (C) markers are shown on the f i g u r e s . 125. V o l u m e - ml membrane p a r t i c l e s were s o l u b i l i z e d w i t h 6% (w/v) T r i t o n X-100 and the f r a c t i o n t r e a t e d w i t h l y s o l e c i t h i n a f t e r D E A E - c e l l u l o s e chromatography was a p p l i e d (without p r i o r c e n t r i f u g a t i o n ) t o 10-50% sucrose g r a d i e n t s c o n t a i n i n g 0.5% B r i j 58. A s i m i l a r r e s u l t to t h a t of the p r e v i o u s experiment was observed. The degree of p u r i f i c a t i o n a chieved by the peak f r a c t i o n s of sucrose g r a d i e n t L were not estimated due t o i n s u f f i c i e n t m a t e r i a l being a v a i l a b l e f o r a c c u r a t e d e t e r m i n a t i o n of p r o t e i n c o n c e n t r a t i o n . How-ever, the a b i l i t y of l y s o l e c i t h i n t o f u r t h e r c l e a v e T r i t o n X - 1 0 0 - s o l u b i l i z e d membrane p a r t i c l e s t o s m a l l e r fragments w i t h energy-independent transhydrogenase a c t i v i t y , c o u l d be the p o t e n t i a l b a s i s f o r the p u r i f i c a t i o n of t h i s enzyme. The disadvantage of t h i s procedure f o r p u r i f i c a t i o n would be the sma l l q u a n t i t y of enzyme ob t a i n e d from the 10-50% sucrose d e n s i t y g r a d i e n t f r a c t i o n . An a l t e r n a t e procedure f o r the p u r i f i c a t i o n of the energy-independent transhydrogenase, y i e l d i n g more enzyme and g r e a t e r p u r i f i c a t i o n was undertaken and i s d i s c u s s e d i n the f o l l o w i n g s e c t i o n . P u r i f i c a t i o n o f energy-independent transhydrogenase Hojeberg and Rydstrom (6) have p u r i f i e d the energy-independent transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s to homogeneity. The procedure i n v o l v e d s o l u b i l i z a -t i o n by 2% sodium c h o l a t e i n the presence of 10% s a t u r a t e d 127. ammonium sulphate. The f r a c t i o n p r e c i p i t a t i n g between 0.38 and 0.43 saturation of ammonium sulphate was chromatographed on DEAE-Sepharose CL-6B i n the presence of 0.05% Tr i t o n X-100. The active fractions from the DEAE-Sepharose CL-6B column were pooled. The p u r i f i c a t i o n achieved i n the pooled f r a c -tions was 8.2-fold that i n the submitochondrial p a r t i c l e suspension. P u r i f i c a t i o n of 40-fold was achieved by sub-sequent procedures which yielded a single homogeneous poly-peptide. It seemed desirable to follow the i n i t i a l procedure undertaken by Hojeberg and Rydstrom (6) for the p u r i f i c a t i o n of the energy-independent transhydrogenase from E. c o l i . Membrane p a r t i c l e s from E. c o l i ML308-225 were solu-b i l i z e d with sodium cholate i n the presence of 33% saturated ammonium sulphate at a detergent to protein r a t i o of 1.7 (136). The f r a c t i o n p r e c i p i t a t i n g between 0.33 and 0.6 saturation of ammonium sulphate (0.33-0.6P) was suspended in 50 mM TGD-2 buffer, pH 8, containing 0.2% (w/v) Trit o n X-100. The suspension was then dialyzed at 0°C for 4 hours against three changes of 50 mM TGD-2 buffer, pH 8. A sample of the dialyzed material was applied to a column of DEAE-Sepharose CL-6B equilibrated with 50 mM TGD-2 buffer contain-ing 0.2% (w/v) Trit o n X-100. Elution'of the fractions was accomplished by means of a l i n e a r s a l t gradient of 50 to 400 mM TGD-2 buffer, pH 8, containing 0.2% (w/v) Trit o n X-100. 128. Energy-independent transhydrogenase was eluted i n two main peaks of a c t i v i t y (Fig. 15). Only one peak was due to material absorbed by the column and represented 18% of t h i s enzyme a c t i v i t y present i n the chromatography f r a c t i o n s . The f r a c t i o n eluting at 549 ml of eff l u e n t and 360 mM s a l t (Fraction 32) was the ric h e s t i n energy-independent transhydrogenase a c t i v i t y . The s p e c i f i c a c t i v i t y i n thi s f r a c t i o n was 5 units per mg protein and represented a 16-f o l d p u r i f i c a t i o n over the membrane p a r t i c l e suspension (Table 11). The highest p u r i f i c a t i o n attained, however, was in Fraction 35 (eluting at 595 ml of eff l u e n t and 380 mM s a l t ) . This had a s p e c i f i c a c t i v i t y of 7.9 units per mg protein and corresponded to a 25-fold p u r i f i c a t i o n . The t o t a l units of energy-independent transhydrogenase a c t i v i t y recovered from the column comprised 62% of the a c t i v i t y of the dialysate and 20% that of the membrane p a r t i c l e suspension. Measurement of ATPase and succinate dehydrogenase ac-t i v i t y revealed peaks of a c t i v i t y eluting at 549 ml and 595 ml, respectively. Since these enzymes were present i n the fr a c t i o n containing the energy-independent transhydrogenase, several attempts were made to improve the separation. These involved the use of 0.5% (w/v) Trit o n X-100 i n the buffer, instead of 0.2% (w/v), and changing the gradient i n the eluting buffer from 150 to 500 mM T r i s to the broader range, 50 to 400 mM T r i s . These changes did not improve the separa-ti o n . The energy-independent transhydrogenase, which was F i g . 15. Chromatography of the (0.33-0.6P) f r a c t i o n on DEAE-Sepharose CL-6B i n the presence of 0.2% (w/v) T r i t o n X-100. The (0.33-0.6P) f r a c t i o n was prepared from membrane p a r t i c l e s from 8.9 g of E. c o l i ML308-225 as d e s c r i b e d i n MATERIALS AND METHODS. I t was d i a l y z e d as d e s c r i b e d i n the t e x t . A p o r t i o n of the d i a l y z e d m a t e r i a l (6.2 ml con-t a i n i n g 91 mg p r o t e i n ) was a p p l i e d t o a column of DEAE-Sepharose CL-6B (1.9 x 19.5 cm) e q u i l i b r a t e d with 50 mM TGD-2 b u f f e r , pH 8, c o n t a i n i n g 0.2% (w/v) T r i t o n X-100. The f r a c t i o n s were e l u t e d by a l i n e a r g r a d i e n t formed from 2 50 ml each of 50 mM and 4 00 mM TGD b u f f e r , pH 8, c o n t a i n -i n g 0.2% (w/v) T r i t o n X-100, f o l l o w e d by 100 ml of the l a t t e r b u f f e r . F r a c t i o n s (16 ml) were c o l l e c t e d and assayed f o r energy-independent transhydrogenase (•—• ), s u c c i n a t e dehydrogenase (O—O ), ATPase ( A—A ), t o t a l p r o t e i n (•-• ) and T r i s c o n c e n t r a t i o n ( — ). The c o n c e n t r a t i o n of T r i s i n each f r a c t i o n was obtained by measuring the c o n d u c t i v i t y and determining the c o n c e n t r a t i o n from a standard curve c o n s t r u c t e d f o r t h a t b u f f e r . Enzyme a c t i v i t i e s are ex-pressed as u n i t s / f r a c t i o n , t o t a l p r o t e i n i n m g / f r a c t i o n and T r i s c o n c e n t r a t i o n i s i n mM. Energy-independent transhydrogenase a c t i v i t y was assayed i n the presence of 0.025% (w/v) B r i j 35. SDH [Tris] Total protein •OCT Table 11. P u r i f i c a t i o n of Energy-Independent Transhydrogenase from Fraction (0.33-0.6P) by Chromatography on DEAE-Sepharose CL-6B i n the Pres-ence of Triton X-100 Energy-independent . f. Fraction T o t a l P r o t e i n Transhydrogenase P u ^ n a t i o n mg/ml % A c t i v i t y % Specific units A c t i v i t y units/mg Membrane p a r t i c l e s 15 100 113 100 0.32 1 Ammonium sulphate fractions 0-0.33 saturation p e l l e t 20 45 19 17 0.12 0.37 supernatant 4.3 43 64 57 0.41 1.3 (0.33-0.6P) f r a c t i o n 15 31 33 33 0.34 1.1 DEAE-Sepharose CL-6B fractions 32 . 036 0.19 2.8 2.9 5.0 16 35 .014 0.072 1.7 1.8 7.9 25 Pooled .030 2.0 16 17 2.7 8.4 Membrane p a r t i c l e s from 8.9 g of E. c o l i ML308-225 were suspended in 50 mM TGDS/KCl/cholate buffer, pH 7.5. S o l u b i l i z a t i o n with sodium cholate was as des-cribed i n MATERIALS AND METHODS. The (0.33-0.6P) fr a c t i o n was suspended i n 50 mM TGD-2 buffer, pH 8.0, containing 0.2% (w/v) Triton X-100 and dialyzed. A sample of the dialysate (91 mg protein) was applied to a column of DEAE-Sepharose CL-6B equ i l i b r a t e d with 50 mM TGD-2 buffer, pH 8.0, containing 0.2% (w/v) T r i t o n X-100. El u t i o n was effected by a l i n e a r gradient of 50-400 mM TGD-2 buffer, pH 8.0, containing 0.2% (w/v) T r i t o n X-100. The pooled fractions are those which were eluted between 436-630 ml of effluent (282-400 mM T r i s ) . Fraction 32 and 35 were eluted at 549 ml (360 mM Tris) and 595 ml (380 mM T r i s ) , respectively. A l l fractions were assayed i n the presence of 0.025% (w/v) B r i j 35. 132. eluted at 310 to 33 5 mM T r i s was s t i l l associated with the other enzymes and the a c t i v i t y recovered was lower than before, possibly due to the increased concentration of Tr i t o n X-100. In one such experiment, fractions r i c h i n energy-independent transhydrogenase a c t i v i t y eluted from the DEAE-Sepharose CL-6B column, were applied to a column of hydroxylapatite, e q u i l i -brated with 100 mM PDGX buffer, pH 7.5. Elu t i o n was effected with a li n e a r gradient of 100 to 500 mM PDGX buffer, pH 7.5 (Fig. 16). The energy-independent transhydrogenase a c t i v i t y i n the eluted fractions was s t i l l associated with succinate dehydrogenase and other proteins. The s p e c i f i c a c t i v i t y (2.9 units per mg protein) of the peak f r a c t i o n of energy-independent transhydrogenase was not improved, possibly because the protein concentrations were overestimated due to interference by phosphate i n the Lowry assays for protein. Analysis of the active fractions from the hydroxylapatite column by electrophoresis i n the presence of 0.1% (w/v) SDS (Fig. 17) revealed many bands staining for protein, indicating the presence of impurities. The peak f r a c t i o n of energy-independent transhydrogenase a c t i v i t y from the hydroxyl-apatite column showed at least 24 polypeptide bands on the gel. Polypeptide bands that appeared enriched i n t h i s f r a c t i o n were those with molecular weights of about 90 000 (.2 bands) 57 000, 50 000 and 40 000, together with six minor 133. F i g . 16. Chromatography of p a r t i a l l y p u r i f i e d energy-independent transhydrogenase on h y d r o x y l a p a t i t e . Membrane p a r t i c l e s from 11 g of E. c o l i W6 c e l l s were used t o prepare the (0.33-0.6P) f r a c t i o n . T h i s was d i a -l y z e d and chromatographed as d e s c r i b e d i n the legend t o F i g . 15 but u s i n g 150 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.5% (w/v) T r i t o n X-100 to suspend the f r a c t i o n and to e q u i l i b r a t e the DEAE-Sepharose CL-6B column. E l u t i o n was with 500 ml of a l i n e a r g r a d i e n t of 150-500 mM TGD-2 b u f f e r s , pH 7.8, c o n t a i n i n g 0.5% (w/v) T r i t o n X-100. The a c t i v e f r a c t i o n s (6.5 u n i t s of energy-independent t r a n s -hydrogenase a c t i v i t y and 4.1 mg p r o t e i n ) were pooled and a p p l i e d t o a (1.9 x 15 cm) column of h y d r o x y l a p a t i t e e q u i l i b r a t e d w i t h 100 mM PDGX b u f f e r , pH 7.5. The f r a c -t i o n s were e l u t e d by a l i n e a r g r a d i e n t of 100-500 mM (PDGX) b u f f e r , pH 7.5 u s i n g a t o t a l volume of 500 ml. F r a c t i o n s (8.5 ml) were c o l l e c t e d and assayed f o r energy-independent transhydrogenase (TH) ( +-# ), and s u c c i n a t e dehydrogenase (SDH) (O—O ) a c t i v i t i e s and t o t a l p r o t e i n ( # - + ) Enzyme a c t i v i t i e s are expressed as u n i t s / f r a c t i o n , t o t a l p r o t e i n i s i n m g / f r a c t i o n . Energy-independent transhydrogenase and s u c c i n a t e de-hydrogenase assays were performed i n the presence of 0.0028% (w/v) and 0.0014% (w/v). E. c o l i l i p i d s , r e s p e c -t i v e l y . The bar above the peak i n d i c a t e s the f r a c t i o n s (13-18) t h a t were s u b j e c t e d t o SDS-polyacrylamide g e l e l e c t r o -p h o r e s i s ( F i g . 17). 134. Vol u m e _ ml 135. Fig . 17. SDS-polyacrylamide gel electrophoresis of part-i a l l y p u r i f i e d energy-independent transhydrogenase. Fractions 13-18 from the hydroxylapatite column (Fig. 16) were prepared and resolved by polyacrylamide gel elec-trophoresis as described i n MATERIALS AND METHODS. The separating gel contained 9% (w/v) acrylamide. Electrophor-esis was c a r r i e d out for 1.25 h. Gels a-f representing fractions 13-18 contained 0.16, 1.4, 1.5, 1.6, 2.3 and 2.0 ug protein, respectively. Bovine serum albumin (molecular weight, 66 000 (216)), phosphoryl-ase a (molecular weight, 92 500 (217)) and p u r i f i e d ATPase (molecular weights, 13 200, 20 700, 32 000, 51 800 and 56 800 (218)) were applied to gels g - i at protein concentra-tions of 1, 2.5 and 5 ug, respectively to act as molecular weight markers. 136. 137. bands with molecular weights between 13 000 and 40 000. The molecular weight of the energy-independent transhydrogenase from b e e f - h e a r t mitochondria i s 97 000 to 120 000 (6,7). The v a r i a t i o n i n the i n t e n s i t y of s t a i n i n g of the 90 000 molecu-l a r weight p o l y p e p t i d e bands and some of those of low molecu-l a r weight i n the samples s u b j e c t e d to e l e c t r o p h o r e s i s , p a r a l l e l s the energy-independent transhydrogenase a c t i v i t y p r o f i l e i n these same samples. In r e c e n t s t u d i e s , i t has been r e p o r t e d t h a t proteases contaminating the p r e p a r a t i o n s of some i n t e g r a l membrane p r o t e i n s have been r e s p o n s i b l e f o r the d e g r a d a t i o n of these p r o t e i n s to s m a l l e r p o l y p e p t i d e s (94-96). For example, an almost homogeneous p r e p a r a t i o n of ATPase from E. c o l i , e x t e n s i v e l y p u r i f i e d by ion-exchange chromatography and sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , was s t i l l contamin-ated with proteases which caused i n c r e a s i n g d e g r a d a t i o n of the enzyme wit h time (159). Thus i t i s not u n l i k e l y t h a t some of the low molecular weight bands i n F i g . 17 are a c t u -a l l y cleavage products of p o l y p e p t i d e s w i t h h i g h e r molecular weights. I f t h i s were the case, p u r i f i c a t i o n of the energy-independent transhydrogenase to homogeneity, as assessed by e l e c t r o p h o r e s i s , would be i m p o s s i b l e without the presence of the a p p r o p r i a t e protease i n h i b i t o r s . P u r i f i c a t i o n of the energy-independent transhydrogenase was a l s o attempted u s i n g sodium deoxycholate i n s t e a d of 138. T r i t o n X-100 i n the b u f f e r suspending the (0.33-0.6P) f r a c -t i o n . T h i s m o d i f i c a t i o n , together w i t h the use of 0.1% (w/v) B r i j 3 5 as d i saggregant i n the b u f f e r s d u r i n g chrom-atography on DEAE-Sepharose CL-6B, y i e l d e d a g r e a t e r degree of p u r i f i c a t i o n . A t y p i c a l example of such an experiment i s d e s c r i b e d below. Membrane p a r t i c l e s from E. c o l i ML308-225 were t r e a t e d w i t h sodium c h o l a t e i n the presence of 33% s a t u r a t e d ammonium sulphate a t a d e t e r g e n t to p r o t e i n r a t i o of 1.9 and the f r a c t i o n p r e c i p i t a t i n g between 0.33 and 0.6 s a t u r a t i o n of ammonium sulphate (0.33-0.6P) was suspended i n 20 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.!4% (w/v) sodium deoxycholate. The suspension was d i a l y z e d a t 0°C a g a i n s t 20 mM TGD-2 b u f f e r , pH 7.8, f o r 16 hours. The d i a l y z e d m a t e r i a l was c e n t r i f u g e d a t 120 000 x g f o r 2 hours and the supernatant was a p p l i e d to a column of DEAE-Sepharose CL-6B, e q u i l i b r a t e d w i t h 20 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.1% (w/v) B r i j 35. E l u t i o n was e f f e c t e d by a l i n e a r g r a d i e n t of 20 t o 500 mM TGD-2 b u f f e r , pH 7.8 c o n t a i n i n g 0.1% (w/v) B r i j 35 ( F i g . 18). Two peaks of energy-independent transhydrogenase a c t i v i t y were de t e c t e d . One peak, r e p r e s e n t i n g 31% of the a c t i v i t y recovered from the chromatography, was e l u t e d i n the v o i d volume. The enzyme r e t a i n e d i n the column was e l u t e d a t 240 mM s a l t and migrated w i t h a major peak of s u c c i n a t e dehydrogenase a c t i v i t y but was separate from the two peaks 139. Fig. 18. Chromatography of the (0.33-0.6P) f r a c t i o n , treated with 0.4% (w/v) sodium deoxycholate, on DEAE-Sepharose CL-6B i n the presence of 0.1% (w/v) B r i j 35. The (0.33-0.6P) f r a c t i o n was prepared from membrane p a r t i c l e s from 11 g of E. c o l i ML308-225, as described i n MATERIALS AND METHODS. It was dialyzed and centrifuged as described i n the text. The supernatant containing 181 mg protein was subjected to chromatography on DEAE-Sepharose CL-6B. The column (1.9 x 19.5 cm) was previously e q u i l i -brated with 2 0 mM TGD-2 buffer, pH 7.8, containing 0.1% (w/v) B r i j 35. The fractions (13 ml) were eluted by a l i n -ear gradient formed from 250 ml each of 20 mM and 500 mM TGD-2 buffer, pH 7.8, containing 0.1% (w/v) B r i j 35. They were assayed for energy-independent transhydrogenase (TH) ( ), succinate dehydrogenase (SDH) (O—O ), ATPase ( A — A ) and A 2 8 Q ( - - - ) . The concentration of T r i s i n each f r a c t i o n was obtained by measuring the conductivity and determining the concentration from a standard curve constructed for that buffer. Enzyme a c t i v i t i e s are in u n i t s / f r a c t i o n , and T r i s con-centration i s mM. 140. Vol ume...mI 141. of ATPase a c t i v i t y . The s p e c i f i c a c t i v i t y of the energy-independent transhydrogenase i n the peak f r a c t i o n (number 28) eluting at 390 ml and 240 mM s a l t , was 4.3 units per mg protein. This represented an 18-fold p u r i f i c a t i o n over the membrane p a r t i c l e suspension. The highest s p e c i f i c a c t i v i t y (8.8 units per mg protein), however, was obtained i n f r a c t i o n 25 (eluted at 356 ml and 220 mM s a l t ) , and represented a 37-f o l d p u r i f i c a t i o n over the membrane p a r t i c l e suspension (Table 12). In a si m i l a r experiment, a p u r i f i c a t i o n of 21-fold over the membrane p a r t i c l e suspension was attained i n the peak f r a c t i o n from the DEAE-Sepharose CL-6B column (eluted at 270 mM salt) with a maximum p u r i f i c a t i o n of 68-fold and a s p e c i f i c a c t i v i t y of 15.7 units per mg protein i n one of the fr a c t i o n s . Electrophoresis on 13% polyacrylamide gels i n the presence of 0.1% (w/v) SDS, gave a p r o f i l e of major bands somewhat si m i l a r to that shown previously. Thus, while a considerable improvement i n p u r i f i c a t i o n was achieved by t h i s procedure over that involving Sepharose 6B chromato-graphy, p u r i f i c a t i o n to homogeneity was not observed. The e f f e c t of l i p i d s and detergents on s o l u b i l i z e d energy-independent transhydrogenase Energy-independent transhydrogenase from submitochondrial p a r t i c l e s i s highly sensitive to reagents (e.g., organic Table 12. P u r i f i c a t i o n of Energy-Independent Transhydrogenase from Fraction (0.33-0.6P) by Treatment with 0.4% (w/v) Sodium Deoxycholate and Chromatography on DEAE-Sepharose CL-6B i n the Presence of B r i j 35 Fraction Total Protein mg/ml % Energy-independent Transhydrogenase A c t i v i t y % Specific units A c t i v i t y units/mg P u r i f i c a t i o n (X-fold) Membrane p a r t i c l e s 14 100 95 100 0.24 1 • Ammonium sulphate fractions 0-0.33 saturation p e l l e t 18 44 0 0 0 0 supernatant 4.6 54 79 83 0.37 1.5 (0.33-0.6P) f r a c t i o n , dialyzed p e l l e t 7.1 10 6.4 7.6 0.18 0.75 supernatant 15 54 73 87 0.39 1.6 DEAE-Sepharose CL-6B fractions 25 0.02 0.39 2.0 2.5 8.8 37 28 0.12 0.068 5.7 7.2 4.3 18 Pooled .097 1.9 23 29 3.6 15 Membrane p a r t i c l e s from 11 g of E. c o l i ML308-225 were suspended in 50 mM TGDS/KCl/cholate buffer, pH 7.5. The (0.33-0.6P) fractio n was prepared and treated as described in the text. The f r a c t i o n applied to the column of DEAE-Sepharose CL-6B contained 181 mg protein. Fractions were eluted from the column by a l i n e a r gradient of 20-500 mM TGD-2 buffer, pH 7.8, containing 0.1% (w/v) B r i j 35. The DEAE-Sepharose CL-6B pool represents fractions eluted between 345-412 ml of ef f l u e n t at 220-270 mM TGD-2. Fractions 25 and 28 were eluted at 356 ml (200 mM TGD-2) and 390 ml (240 mM TGD-2), respectively. 143. s o l v e n t s , d e t e r g e n t s and phospholipases which e i t h e r remove or a l t e r p h o s p h o l i p i d s . R e a c t i v a t i o n of p a r t i a l l y p u r i f i e d or l i p i d - d e p l e t e d p r e p a r a t i o n s of t h i s enzyme has been achieved by the a d d i t i o n of p h o s p h o l i p i d s , e i t h e r i n d i v i d u a l l y (as p u r i f i e d p r e p a r a t i o n s ) , or as crude e x t r a c t s from mitochon-d r i a (7,101). Soybean p h o s p h o l i p i d ( a s o l e c t i n ) and E. c o l i p h o s p h o l i p i d s have been s u c c e s s f u l l y used to r e s t o r e energy-independent transhydrogenase a c t i v i t y i n p a r t i a l l y p u r i f i e d p r e p a r a t i o n s from E. c o l i membrane p a r t i c l e suspensions (8 9, 90). Non-ionic d e t e r g e n t s of the Tween or T r i t o n s e r i e s have a l s o been r e p o r t e d t o r e s t o r e the a c t i v i t y of other p u r i f i e d membrane-bound enzymes from t h i s organism (147,148,156,160,161). In t h i s i n v e s t i g a t i o n , having p a r t i a l l y p u r i f i e d the energy-independent transhydrogenase, i t was of i n t e r e s t t o determine the e f f e c t of s e v e r a l p h o s p h o l i p i d s and detergents on the a c t i v i t y of t h i s enzyme i n some of the p r e p a r a t i o n s obtained. E f f e c t of l i p i d s and d e t e r g e n t s on the enzyme a c t i v i t y  i n s o l u b l e r e s p i r a t o r y complex The type of p r e p a r a t i o n s e l e c t e d f o r t h i s purpose was the (0.33-0.6P) f r a c t i o n (used i n the e a r l i e r experiments) a f t e r i t had been su b j e c t e d t o chromatography on Sepharose 6B and Sepharose 4B. The a c t i v e f r a c t i o n s thus obtained (termed the p u r i f i e d " s o l u b l e r e s p i r a t o r y complex" (136)) were then t r e a t e d w i t h p h o s p h o l i p i d s or d e t e r g e n t s . A t y p i c a l p r e p a r a t i o n i s d e s c r i b e d below. Membrane p a r t i c l e s from E. c o l i ML308-225 were s o l u b i -l i z e d w i t h sodium c h o l a t e i n the presence of 33% s a t u r a t e d ammonium sulphate a t a detergent to p r o t e i n r a t i o of 2.2. The f r a c t i o n p r e c i p i t a t i n g between 0.33 and 0.6, s a t u r a t i o n of ammonium s u l p h a t e (0.33-0.6P) was suspended i n 50 mM TGDS/KCl/cholate b u f f e r , pH 7.5. The suspension was a p p l i e d to a column of Sepharose 6B coupled to a column of Sepharose 4B, e q u i l i b r a t e d w i t h 50 mM TGDS/KCl/cholate b u f f e r , pH 7.5, and e l u t e d w i t h the same b u f f e r . Energy-independent t r a n s -hydrogenase emerged from the columns as a s i n g l e peak of a c t i v i t y ( F i g . 19) separated.from the m a j o r i t y of the pro-t e i n s . A sample of the most a c t i v e f r a c t i o n was then assayed f o r energy-independent transhydrogenase a c t i v i t y i n the presence of detergents or p h o s p h o l i p i d s (Table 13). At the c o n c e n t r a t i o n t e s t e d (18.8 uM), p h o s p h o l i p i d e x t r a c t s from E. c o l i had no e f f e c t on energy-independent transhydrogenase a c t i v i t y whereas l y s o l e c i t h i n (3840 uM) had an a c t i v a t i n g e f f e c t . T r i t o n X-100 (3180 uM) , sodium c h o l a t e (4890 uM) and a s o l e c t i n (255 uM) were i n h i b i t o r y . Low c o n c e n t r a t i o n s of p a l m i t i c a c i d (156 uM) and detergents of the Tween or B r i j s e r i e s (15 t o 40.8 uM) s t i m u l a t e d the enzyme a c t i v i t y approximately 1.5 to 3 - f o l d over t h a t of the c o n t r o l v a l u e . I t was of i n t e r e s t , t h e r e f o r e , t o i n v e s t i -gate the a c t i v a t i n g e f f e c t of these .detergents more t h o r -oughly over a broad range of c o n c e n t r a t i o n s . F i g . 19. Preparation of soluble respiratory complex. Membrane p a r t i c l e s from 16 g of E. c o l i ML308-225 were used to prepare the (0.33-0.6P) f r a c t i o n , which was sus-pended (at 23 mg protein/ml) i n 50 mM TGDS/KCl/cholate buffer, pH 7.5. A sample of the suspension (12 units of energy-independent transhydrogenase a c t i v i t y and 225 mg protein) was applied to a column of Sepharose 6B coupled to one of Sepharose 4B and eluted with the same buffer as described i n MATERIALS AND METHODS. Fractions (12 ml) were co l l e c t e d and assayed for energy-independent transhydrog-enase a c t i v i t y (TH) (•—• ), succinate dehydrogenase a c t i v i t y (SDH) (O—O) and A 2 8 o ( A—_, ). Enzyme a c t i v i t i e s are expressed as u n i t s / f r a c t i o n . 147. Table 13. E f f e c t of P h o s p h o l i p i d s and Detergents on Trans-hydrogenase A c t i v i t y i n S o l u b l e R e s p i r a t o r y Complex A d d i t i v e . F i n a l Concentra-t i o n . uM A c t i v i t y (% of c o n t r o l value) E. c o l i p h o s p h o l i p i d s 18.8 95 A s o l e c t i n 255 50 L y s o l e c i t h i n 3840 165 P a l m i t i c a c i d 156 146 Sodium c h o l a t e 4890 43 T r i t o n X-100 3180 81 Tween 20 16 187 Tween 40 15.4 193 Tween 6 0 15.4 222 Tween 8 0 15 177 B r i j 35 21 277 B r i j 3 6T 40.8 300 B r i j 58 22.3 204 S o l u b l e r e s p i r a t o r y complex was prepared as d e s c r i b e d i n the legend to F i g . 19. In the experiments u s i n g E. c o l i p h o s p h o l i p i d s , p a l m i t i c a c i d and dete r g e n t s of the B r i j s e r i e s , the energy-independent transhydrogenase a c t i v i t y of the c o n t r o l sample was 0.4 8 units/mg p r o t e i n and the samples assayed c o n t a i n e d 16 ug p r o t e i n . In the experiments w i t h other a d d i t i v e s , the enzyme a c t i v i t y of the c o n t r o l sample was 0.55 units/mg p r o t e i n and the samples assayed c o n t a i n e d 75 yg p r o t e i n . E. c o l i p h o s p h o l i p i d s , a s o l e c t i n and p a l -m i t i c a c i d were prepared as d e s c r i b e d i n MATERIALS AND METHODS, and added to the r e a c t i o n mixture j u s t p r i o r to assay of the enzyme a c t i v i t y . 148. Detergents of the B r i j s e r i e s are compounds t h a t are polyoxyethylene e t h e r s of l a u r y l or c e t y l a l c o h o l s . B r i j 3 5 (polyoxyethylene (23) l a u r y l e t h e r ) , B r i j 3 6T (polyoxyethylene (10) l a u r y l ether) and B r i j 58 (polyoxyethylene (20) c e t y l ether) are the three detergents o f t h i s s e r i e s t h a t were t e s t e d i n t h i s experiment. T h e i r e f f e c t s on p a r t i a l l y p u r i -f i e d energy-independent transhydrogenase a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex p r e p a r a t i o n s i s shown i n F i g . 20. A l l thr e e d e t e r g e n t s appeared t o have an a c t i v a t i n g e f f e c t on the enzyme over a broad range of dete r g e n t c o n c e n t r a t i o n . The maximum a c t i v i t y was obtained i n the presence of 4 0 t o 62 uM dete r g e n t i n the assay mixture. The maximum s t i m u l a -t i o n i n enzyme a c t i v i t y achieved was 336% of the c o n t r o l v a l u e (measured i n the absence of detergent) and was obtained i n the presence of B r i j 35. The corresponding f i g u r e s o b t ained i n the presence of B r i j 36T and B r i j 58 were 292% and 217%, r e s p e c t i v e l y . The amount of B r i j 35, B r i j 36T and B r i j 58 r e q u i r e d f o r half-maximal a c t i v a t i o n of the enzyme was 0.95, 0.51 and 0.17 umol detergent per mg p r o t e i n , r e s p e c t i v e l y . Thus, although B r i j 58 d i d not s t i m u l a t e the energy-independent transhydrogenase t o the same extent as B r i j 35, lower c o n c e n t r a t i o n s of i t were r e q u i r e d f o r h a l f -maximal a c t i v a t i o n of the enzyme. Detergents of the Tween s e r i e s . a r e f a t t y a c i d e s t e r s of polyoxyethylene s o r b i t o l . Those co n s i d e r e d i n t h i s 149. Fi g . 20. E f f e c t of B r i j on energy-independent transhydrog-enase a c t i v i t y i n soluble respiratory complex fr a c t i o n s . The soluble respiratory complex was prepared as des-cribed i n the legend to F i g . 19. The energy-independent transhydrogenase a c t i v i t y of the f r a c t i o n investigated i n the absence of detergent was 0.4 8 units/mg protein. Samples (16 yg protein) were assayed i n the presence of variable concentrations of B r i j 35 (top panel), B r i j 36T (centre panel), and B r i j 58 (bottom panel). Energy-independent transhydrogenase a c t i v i t y i s i n units/mg protein. Detergent concentration i s expressed as mM (based on molecular weights of 1184, 612 and 1122 for B r i j 35, B r i j 36T and B r i j 58, res p e c t i v e l y ) . 2 Brij 35 [Detergent] 151. experiment were Tween 20 (polyoxyethylene (20) s o r b i t a n mono-l a u r a t e ) , Tween 40 (polyoxyethylene (20) s o r b i t a n monopalmitate), Tween 60 (polyoxyethylene (20) s o r b i t a n monostearate) and Tween 80 (polyoxyethylene (2 0) s o r b i t a n monooleate). An experiment was undertaken to study the e f f e c t of these f o u r d e t e r g e n t s on p a r t i a l l y p u r i f i e d energy-independent transhydrogenase of s o l u b l e r e s p i r a t o r y complex p r e p a r a t i o n s ( F i g . 21). Tween 20, Tween 40, Tween 60 and Tween 80 each had an a c t i v a t i n g e f f e c t on the energy-independent transhydrogenase a c t i v i t y . The a c t i v i t y appeared to reach a p l a t e a u at 15 to 3 0 yM d e t e r -gent i n the assay mixture. The dete r g e n t g i v i n g the g r e a t e s t s t i m u l a t i o n of enzyme a c t i v i t y (238%. of c o n t r o l a c t i v i t y ) i n t h a t range of c o n c e n t r a t i o n s was Tween 60. The correspond-i n g f i g u r e s f o r Tween 20, Tween 40 and Tween 80 were 187%, 217% and 193%, r e s p e c t i v e l y . Half-maximal s t i m u l a t i o n of enzyme a c t i v i t y u s i n g Tween 20, Tween 40, Tween 60 or Tween 8 0 was a t t a i n e d at 0.12, 0.11, 0.12 and 0.14 ymol detergent per mg p r o t e i n , r e s p e c t i v e l y . Compounds of the B r i j or Tween s e r i e s c o n t a i n f a t t y a c i d s or f a t t y a l c o h o l s i n t h e i r s t r u c t u r e s . I t i s p o s s i b l e t h a t these d e t e r g e n t s , besides t h e i r a b i l i t y to form m i c e l l e s , may be s t i m u l a t i n g the enzyme a c t i v i t y by the s p e c i f i c i n t e r -a c t i o n of t h e i r f a t t y a c i d s w i t h s i t e s on the enzyme molecule. With t h i s i n mind, the e f f e c t of p a l m i t i c a c i d , a s o l e c t i n and l y s o l e c i t h i n on the energy-independent transhydrogenase was examined. F i g . 2 1 . E f f e c t of Tween on energy-independent transhy-drogenase a c t i v i t y i n soluble respiratory complex fr a c t i o n s . The soluble respiratory complex was prepared as des-cribed i n the legend to F i g . 1 9 . Samples of t h i s f r a c t i o n ( 7 5 ug protein) were assayed for energy-independent trans-hydrogenase a c t i v i t y i n the presence of variable concentra-tions of Tween 2 0 (panel 1 ) , Tween 40 (panel 2 ) , Tween 60 (panel 3) and Tween 80 (panel 4 ) . Enzyme a c t i v i t y i n the absence of detergent was 0 . 5 9 , 0 . 5 8 , .0.58 and .0.47 units/mg protein, respectively. Enzyme a c t i v i t y i s i n units/mg protein. Detergent concentration i s expressed as mM (based on molecular weights of 1 2 4 4 , 1 3 0 0 , 1 3 2 8 and 1 3 2 6 for Tween 2 0 , Tween 4 0 , Tween 60 and Tween 8 0 , r e s p e c t i v e l y ) . 153. [Detergent ] 154. The a d d i t i o n of up to 300 uM p a l m i t i c a c i d to assay mixtures, c o n t a i n i n g s o l u b l e r e s p i r a t o r y complex prepara-t i o n s , i n c r e a s e d energy-independent transhydrogenase a c t i v i t y ( F i g . 22). The maximum a c t i v i t y was ob t a i n e d a t 156 uM p a l m i t i c a c i d and represented 146% of the c o n t r o l a c t i v i t y . The c o n c e n t r a t i o n r e q u i r e d f o r half-maximal s t i m u l a t i o n of the enzyme a c t i v i t y was 1.6 Umol p a l m i t i c a c i d per mg p r o t e i n . The presence of a s o l e c t i n i n assay mixtures c o n t a i n i n g s o l u b l e r e s p i r a t o r y complex p r e p a r a t i o n s i n h i b i t e d energy-independent transhydrogenase a c t i v i t y a t c o n c e n t r a t i o n s of p h o s p h o l i p i d g r e a t e r than 13 uM ( F i g . 22). In c o n t r a s t to a s o l e c t i n , l y s o l e c i t h i n s t i m u l a t e d transhydrogenase a c t i v i t y ( F i g . 22). The a c t i v a t i o n reached a maximum (171% of c o n t r o l a c t i v i t y ) a t 3.8 mM l y s o l e c i t h i n i n the assay mixture. H a l f -maximal s t i m u l a t i o n o f enzyme a c t i v i t y was produced by 27 umol l y s o l e c i t h i n per mg p r o t e i n . T h i s f i g u r e i s high com-pared to the p h o s p h o l i p i d c o n c e n t r a t i o n (0.2 to 0.5 Umol per mg p r o t e i n ) r e q u i r e d f o r half-maximal s t i m u l a t i o n of the energy-independent transhydrogenase i n p r e p a r a t i o n s from mi t o c h o n d r i a (101). T h i s may be due t o the f a c t t h a t the l a t t e r p r e p a r a t i o n was l i p i d - d e p l e t e d whereas the s o l u b l e r e s p i r a t o r y complex p r e p a r a t i o n used i n t h i s and the preceding experiments s t i l l c o n t a i n e d . l i p i d (136). The e f f e c t o f p h o s p h o l i p i d s on l i p i d - d e p l e t e d p r e p a r a t i o n s from E.: c o l i membrane p a r t i c l e s was thus undertaken and i s d i s c u s s e d below. 155. F i g . 22. E f f e c t of palmitic acid and phospholipids on energy-independent transhydrogenase a c t i v i t y i n soluble respiratory complex f r a c t i o n s . The soluble respiratory complex was prepared as des-cribed i n the legend to F i g . 19. Samples of t h i s f r a c t i o n (16, 65 and 65 yg protein, respectively) were assayed for energy-independent transhydrogenase a c t i v i t y i n the presence of varying concentrations of palmitic acid (9 9 ), a s o l e c t i n (O-O ) and l y s o l e c i t h i n ( o—O ), respectively. Enzyme a c t i v i t y i n the absence of palmitic acid, a s o l e c t i n and l y s o l e c i t h i n was 0.48, 0.88 and 0.51 units/mg protein, re-spectively. Palmitic acid and a s o l e c t i n solutions were prepared as described i n MATERIALS AND METHODS. The con-centrations of as o l e c t i n and l y s o l e c i t h i n were based on approximate molecular weights of 785 and 521, respectively. A c t i v i t y i s expressed as a percentage of the enzyme a c t i v i t y i n the absence of f a t t y acid or phospholipid. Palmitic acid or phospholipid conentration i s i n mM. * 9 S T 157. E f f e c t of p h o s p h o l i p i d s on l i p i d - d e p l e t e d p r e p a r a t i o n s  of the enzyme The s t i m u l a t i o n of enzyme a c t i v i t y i n p a r t i a l l y p u r i f i e d p r e p a r a t i o n s of energy-independent transhydrogenase by some p h o s p h o l i p i d s , f a t t y a c i d s and d e t e r g e n t s , w i t h f a t t y a c i d s i d e - c h a i n s , t h a t was r e p o r t e d i n the p r e c e d i n g s e c t i o n , suggests t h a t the enzyme depends on l i p i d f o r i t s a c t i v i t y . However, the p o s s i b i l i t y t h a t the a c t i v a t i o n was p a r t l y due to d i s a g g r e g a t i o n of the enzyme complex by detergent should not be overlooked. In order t o e s t a b l i s h whether a l i p i d -dependence by the enzyme does e x i s t , an i n v e s t i g a t i o n was c a r r i e d out i n which p h o s p h o l i p i d was added t o a p a r t i a l l y p u r i f i e d p r e p a r a t i o n of the enzyme which had been d e p l e t e d of l i p i d . Membrane p a r t i c l e s from E. c o l i ML308-225 were s o l u b i -l i z e d w i t h sodium c h o l a t e i n the presence of 33% s a t u r a t e d ammonium sulphate a t a detergent to p r o t e i n r a t i o of 1.56. The f r a c t i o n p r e c i p i t a t i n g between 0.33 and 0.6 s a t u r a t i o n of ammonium sulphate (0.33-0.6P) was suspended i n 20 mM TGD-2 b u f f e r pH, 7.8, c o n t a i n i n g 0.4% (w/v) sodium deoxy-c h o l a t e . The suspension was d i a l y z e d f o r 16 hours a g a i n s t 20 mM TGD-2 b u f f e r , pH 7.8, and a sample of i t was a p p l i e d to a column of DEAE-Sepharose CL-6B, e q u i l i b r a t e d w i t h 20 mM TGD-2 b u f f e r , pH 7.8, c o n t a i n i n g 0.1% (w/v) B r i j 35, The f r a c t i o n s were e l u t e d by a l i n e a r g r a d i e n t of 20 to 500 mM 158. TGD-2 buffer, pH 7.8 containing 0.1% (w/v) B r i j 35. The fractions active i n energy-independent transhydrogenase a c t i v i t y were pooled and delipidated by treatment with 1% (w/v) sodium cholate i n the presence of 50% saturation of ammonium sulphate (89) as described In the MATERIALS AND METHODS section. The f r a c t i o n p r e c i p i t a t i n g between 0 and 0.5 saturation of ammonium sulphate was suspended and di a -lyzed against 50 mM TED-1 buffer, pH 7.8. The dialyzed f r a c t i o n was then assayed for energy-independent trans-hydrogenase a c t i v i t y i n the presence of varying concentrations of a s o l e c t i n or E. c o l i phospholipids. The l a t t e r preparation was an acetone extract of t o t a l l i p i d s from E. c o l i K-12 c e l l s . I t i s referred to as "phospholipid" since about 90% of the l i p i d content of E. c o l i i s phospholipid, 7 0% of which i s phosphatidyl ethanolamine (162). Both as o l e c t i n and E. c o l i phospholipids stimulated the energy-independent transhydrogenase a c t i v i t y of the d e l i p i -dated preparation (Fig. 23). The presence of 563 uM E. c o l i phospholipids i n the assay mixture resulted i n a maximum stimulation of energy-independent transhydrogenase a c t i v i t y , equivalent to an eleven-fold increase over the a c t i v i t y i n the absence of phospholipid. The concentration of. E. c o l i phospholipid required for half-maximal stimulation of the enzyme a c t i v i t y was 15 umol phospholipid per mg protein. In the stimulation of energy-independent transhydrogenase by asolectin, the a c t i v i t y of the enzyme did not reach a 159 F i g . 23. E f f e c t of p h o s p h o l i p i d s on energy-independent transhydrogenase a c t i v i t y i n p a r t i a l l y p u r i f i e d and d e l i p i d a t e d (0.33-0.6P) f r a c t i o n . Membrane p a r t i c l e s from 9.7 g of E. c o l i ML308-225 were used t o prepare the (0.33-0.6P) f r a c t i o n . A sample was a p p l i e d t o a column of DEAE-Sepharose CL-6B and e l u t e d as d e s c r i b e d i n the t e x t . F r a c t i o n s e l u t e d between 200 and 305 mM s a l t were pooled and d e l i p i d a t e d as d e s c r i b e d i n MATERIALS AND METHODS. Samples of the d e l i p i d a t e d f r a c t i o n s ( c o n t a i n i n g 6.6 yg p r o t e i n ) were assayed f o r energy-inde-pendent transhydrogenase a c t i v i t y i n the presence o f v a r y -i n g c o n c e n t r a t i o n s of a s o l e c t i n (•—• ) and E. c o l i phos-p h o l i p i d s (•-• ). The a c t i v i t y i n the absence of phos-p h o l i p i d was 0.3 units/mg p r o t e i n . A s o l e c t i n and E. c o l i p h o s p h o l i p i d s were prepared as d e s c r i b e d i n MATERIALS AND METHODS. The c o n c e n t r a t i o n s were based on approximate molecular weights of 785 and 744 f o r a s o l e c t i n and E. c o l i p h o s p h o l i p i d s , r e s p e c t i v e l y . Enzyme a c t i v i t y i s i n u n i t s / mg p r o t e i n . P h o s p h o l i p i d c o n c e n t r a t i o n i s i n mM. 160. 161. p l a t e a u w i t h i n c r e a s i n g c o n c e n t r a t i o n s of the p h o s p h o l i p i d . The maximum a c t i v i t y a t t a i n e d was i n the presence of 5 mM a s o l e c t i n and represented an e i g h t - f o l d i n c r e a s e over t h a t measured i n the absence of p h o s p h o l i p i d . The f a c t t h a t the energy-independent transhydrogenase a c t i v i t y i n t h i s prep-a r a t i o n was s t i m u l a t e d i n the presence of 5 mM a s o l e c t i n , while t h a t i n the s o l u b l e r e s p i r a t o r y complex f r a c t i o n was i n h i b i t e d by a s o l e c t i n a t c o n c e n t r a t i o n s as low as 0.025 mM ( F i g . 22) c o u l d be r e l a t e d t o the r o l e of a s o l e c t i n i n p a r t -i a l l y f u l f i l l i n g the p h o s p h o l i p i d requirements i n the former p r e p a r a t i o n . The l a t t e r p r e p a r a t i o n a l r e a d y contained 3 0% (w/v) p h o s p h o l i p i d (136). Of the two p h o s p h o l i p i d s t e s t e d , the E. c o l i phospho-l i p i d e x t r a c t was more e f f i c i e n t than a s o l e c t i n i n s t i m u l a t i n g the energy-independent transhydrogenase a c t i v i t y , a t a much lower c o n c e n t r a t i o n of p h o s p h o l i p i d . Thus, 0.5 mM phospho-l i p i d from E . c o l i r e s u l t e d i n a g r e a t e r a c t i v a t i o n than 5 mM a s o l e c t i n . T h i s i s probably due to the former phospho-l i p i d p r o v i d i n g a more n a t i v e environment t o the enzyme than t h a t p r o v i d e d by a s o l e c t i n . K i n e t i c constants o f p a r t i a l l y p u r i f i e d energy-independent  t r an s hydr o'ge nase I t was of i n t e r e s t to determine whether s o l u b i l i z a t i o n and p a r t i a l p u r i f i c a t i o n of the energy-independent t r a n s -hydrogenase had a f f e c t e d i t s k i n e t i c parameters and whether the s u b s t r a t e i n h i b i t i o n by NADPH t h a t had been observed on the enzyme i n membrane p a r t i c l e suspensions c o u l d a l s o be seen i n p a r t i a l l y p u r i f i e d p r e p a r a t i o n s of energy-independent transhydrogenase. The p r e p a r a t i o n s e l e c t e d f o r t h i s purpose was the s o l u b l e r e s p i r a t o r y complex. The e f f e c t of v a r i a t i o n s i n the c o n c e n t r a t i o n of the s u b s t r a t e s APNAD + and NADPH i n the a c t i v i t y of the enzyme i s shown i n F i g . 24. Lineweaver-Burk p l o t s f o r each sub-s t r a t e gave a maximum v e l o c i t y i n the range of 0.29 to 0.4 0 u n i t s per mg p r o t e i n . The K m v a l u e f o r APNAD + was 109 uM. The p l o t o b tained over the range of APNAD + c o n c e n t r a t i o n s t e s t e d (0.066 t o 2.2 mM) was l i n e a r . The p l o t o b tained f o r v a r y i n g NADPH c o n c e n t r a t i o n s was l i n e a r a t c o n c e n t r a t i o n s of 0.047 to 0.88 mM but a t hi g h e r c o n c e n t r a t i o n s (1.2 mM) th e r e was a marked decrease i n the enzyme a c t i v i t y . The value f o r NADPH d e r i v e d from the l i n e a r p o r t i o n of the p l o t was 9 2 uM. T h i s value i s high e r than the corresponding K , 43.5 uM, i n membrane p a r t i c l e s . The K f o r APNAD i n mem-c m brane p a r t i c l e suspensions (59 uM) was a l s o lower than t h a t i n the p a r t i a l l y p u r i f i e d enzyme p r e p a r a t i o n . Thus, although the v a l u e f o r NADPH was high e r i n the p a r t i a l l y p u r i f i e d p r e p a r a t i o n than i n the i n t a c t membrane p a r t i c l e suspension, i n h i b i t i o n by h i g h c o n c e n t r a t i o n s of t h i s s u b s t r a t e was observed i n both cases. That t h i s phenomenon c o u l d be observed i n two such d i f f e r e n t types of p r e p a r a t i o n s , made 163. F i g . 24. E f f e c t o f APNAD and NADPH c o n c e n t r a t i o n s on the a c t i v i t y of energy-independent transhydrogenase i n s o l u b l e r e s p i r a t o r y complex. The s o l u b l e r e s p i r a t o r y complex was prepared as des-c r i b e d i n the legend t o F i g . 19. F r a c t i o n s c o n t a i n i n g 7 5 ug p r o t e i n were assayed f o r energy-independent t r a n s h y d r o g -enase a c t i v i t y i n the presence of v a r i a b l e c o n c e n t r a t i o n s of APNAD + ( l e f t panel) or NADPH ( r i g h t p a n e l ) . The oth e r s u b s t r a t e c o n c e n t r a t i o n was hel d c o n s t a n t a t the c o n c e n t r a -t i o n i n the "standard assay" (see MATERIALS AND METHODS). A c t i v i t y - 1 i s expressed as (units/mg p r o t e i n ) - 1 . S u b s t r a t e c o n c e n t r a t i o n [ S ] - 1 i s expressed as mM-1. CTl 165. i t d e s i r a b l e to i n v e s t i g a t e the k i n e t i c r e a c t i o n of the energy-independent transhydrogenase i n g r e a t e r d e t a i l . T h i s w i l l be d e s c r i b e d i n the remaining s e c t i o n o f t h i s thesis." S t e a d y - s t a t e k i n e t i c s of the membrane-bound energy-independent  transhydrogenase E f f e c t of s i t e - s p e c i f i c i n h i b i t o r s on the a c t i v i t y of the enzyme The energy-independent transhydrogenase r e a c t i o n was c a r r i e d out u s i n g phosphoadenosine d e r i v a t i v e s as competitors w i t h r e s p e c t t o the s u b s t r a t e s APNAD4" or NADPH. 2'-AMP and 51-AMP were chosen as they are s t r u c t u r a l l y r e l a t e d to NADPH and APNAD + r e s p e c t i v e l y . Rydstrom (83), demonstrated, u s i n g the energy-independent transhydrogenase from beef-h e a r t m i t o c h o n d r i a , t h a t the r e a c t i o n was i n h i b i t e d com-p e t i t i v e l y with r e s p e c t to NADPH by 2'-AMP and i n h i b i t e d c o m p e t i t i v e l y w i t h r e s p e c t to NAD by 5'—AMP. T h i s r e s u l t was used t o support h i s argument t h a t the two s u b s t r a t e -b i n d i n g s i t e s of the enzyme are d i f f e r e n t and can d i s -c r i m i n a t e between adenosine m o i e t i e s w i t h and without a 2'-phosphate s u b s t i t u e n t . These s i t e s w i l l subsequently be r e f e r r e d t o as the NADPH and the APNAD + s i t e , r e s p e c t i v e l y . In the presen t study the k i n e t i c p a t t e r n s obtained u s i n g low c o n c e n t r a t i o n s of i n h i b i t o r s d i f f e r e d from those u s i n g 166. h i g h c o n c e n t r a t i o n s . These r e s u l t s are thus, presented . s e p a r a t e l y . Low c o n c e n t r a t i o n s of adenosine phosphates The e f f e c t of adding up to 11.7 8 mM 2'-AMP i n the energy-independent transhydrogenase assay mixture u s i n g washed membrane p a r t i c l e s from E. c o l i was examined ( F i g . 25). These Dixon p l o t s show t h a t w i t h i n c r e a s i n g concen-t r a t i o n s of 2'-AMP a t two l e v e l s of APNAD + (Panel X), the l i n e s i n t e r s e c t a t the a b c i s s a , i n d i c a t i n g t h a t 21-AMP i s a non-competitive i n h i b i t o r (K^, 2 5 mM) with r e s p e c t to the APNAD + s i t e . In an analogous experiment w i t h NADPH (Panel Y) the l i n e s i n t e r s e c t on the l e f t of the o r d i n a t e , above the a b c i s s a , showing a c o m p e t i t i v e r e l a t i o n s h i p be-tween 2'-AMP and NADPH {K^r 3.9 mM). The e f f e c t of adding up t o 9.6 mM 51-AMP i n the energy-independent transhydrogenase assay mixture u s i n g washed membrane p a r t i c l e s from E. c o l i i s shown i n F i g . 26. Dixon p l o t s obtained a t v a r i o u s l e v e l s of APNAD + (Panel X) or NADPH (Panel Y) are l i n e a r and show a c o m p e t i t i v e r e l a t i o n -s h i p between 5'-AMP and APNAD (K^, 0.8 mM), and a non-c o m p e t i t i v e r e l a t i o n s h i p w i t h NADPH (K., 2 0 mM). 167. F i g . 25. E f f e c t of low concentrations of 21-AMP on energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i W6 were suspended i n 50 mM sodium borate, buffer pH 7.8, at a concentration of 2.9 mg protein/ml. Variable concentrations of 21-AMP were added to the assay mixture, at constant concentrations of APNAD+ ( O , 270 uM; f , 1000 uM) (panel X), or NADPH ( • , 98 uM; • , 489 yM) (panel Y), respectively. The concentration of the second substrate was that for the "standard assay" i n each case. 73 yg protein was used i n each assay. A c t i v i t y - 1 i s expressed as (units/mg p r o t e i n ) - 1 . 2'-AMP concentration i s expressed as mM. 168. F i g . 26. E f f e c t of low c o n c e n t r a t i o n s of 51-AMP on energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM TED-1 b u f f e r , pH 7.8. 51-AMP was added t o the assay mixture a t constant c o n c e n t r a t i o n s of APNAD + ( • , 237 yM; A , 305 yM; A , 930 yM) (panel X) or NADPH ( • , 29 yM; O , 41 yM; • , 54 yM; O , 446 yM) (panel Y), r e s p e c t i v e l y . "Standard assay" c o n c e n t r a -t i o n s were used f o r the second s u b s t r a t e i n each case. 63 yg p r o t e i n (panel X) and 58 yg p r o t e i n (panel Y) r e s p e c t i v e l y , was used i n each assay. A c t i v i t y - 1 and 5'-AMP c o n c e n t r a t i o n s are expressed as (units/mg p r o t e i n ) - 1 and mM, r e s p e c t i v e l y . ' O L l 171. High concentration of adenosine phosphates The e f f e c t of adding up to 56.4 mM 2'-AMP to the energy-independent transhydrogenase assay mixture was examined. Washed membrane p a r t i c l e s from E. c o l i W6 were used. Dixon plots i l l u s t r a t i n g the k i n e t i c behaviour of 2'-AMP with the APNAD+ s i t e (Panel X) and NADPH s i t e (Panel Y) are shown i n F i g . 27. In panel X the lin e s deviate from l i n e a r i t y at higher concentrations of 2'-AMP, indicating i n -creasing i n h i b i t i o n at these concentrations of nucleotide. This could be due to the binding of 21-AMP at more than one s i t e on the enzyme. At lower concentrations of 21-AMP the plots appear to be l i n e a r , with the lin e s intersecting above the abcissa showing an apparent competitive r e l a t i o n -ship between 21-AMP and APNAD for the APNAD s i t e of the enzyme (K^, 17.85 mM). This point i s discussed i n greater d e t a i l l a t e r . Panel Y of F i g . 27 examines the ef f e c t of 2'-AMP on the NADP(H) s i t e of the enzyme. Non-linearity was again observed at higher concentrations of 2'-AMP, but i n contrast to the re s u l t s with the APNAD+ s i t e , there was no additional i n h i b i t i o n . Indeed, there appeared to be a r e l a t i v e en-hancement, i n the rate of reaction as the concentrations of NADPH were decreased i n the assay mixture. Linear and competitive relationships were observed at lower concentra-tions of 2'-AMP (K., 4.3 mM). F i g . 27. E f f e c t of high concentrations of 2'-AMP on the energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM TED-1 buffer, pH 7.8, at a concentration of 2.7 mg protein/ml. The procedure was s i m i l a r to that described i n the legend to F i g . 25. Concentrations of APNAD+ were ( O , 970 yM; • , 320 yM; o ,13 5 yM) (panel X). Concentrations of NADPH were ( • , 51 yM; • , 65 yM; + , 490 yM) (panel Y), respectively. 68 yg protein was used i n each assay. A c t i v i t y " 1 i s ex-pressed as (units/mg p r o t e i n ) - 1 . 2'-AMP concentration i s expressed as mM. 17 4. The e f f e c t o f high c o n c e n t r a t i o n s of 51-AMP on the APNAD + and NADP(H) s i t e s of the enzyme was a l s o examined. F i g . 28 Panel X shows the e f f e c t of 51-AMP on the APNAD+ s i t e w h i l e panel Y shows t h a t on the NADP(H) s i t e . Both Dixon p l o t s i n d i c a t e d i n c r e a s e d i n h i b i t i o n of enzyme a c t i v i t y at h i g h e r c o n c e n t r a t i o n s of 5'-AMP. T h i s e f f e c t was not apparent w i t h the lower s u b s t r a t e c o n c e n t r a t i o n s . The p l o t s tended t o be l i n e a r a t low c o n c e n t r a t i o n s of i n -h i b i t o r and i n d i c a t e d a c o m p e t i t i v e r e l a t i o n s h p between 5'-AMP and APNAD + (K^, 2 mM) and a non-competitive r e l a t i o n s h i p w i t h NADPH (K^,28 mM). Increased i n h i b i t i o n by hi g h c o n c e n t r a t i o n s of phospho-adenosine d e r i v a t i v e s , e s p e c i a l l y a t low s u b s t r a t e concen-t r a t i o n s , was a l s o observed u s i n g ADP. Co n c e n t r a t i o n s of up t o 48.85 mM ADP i n the energy-independnet transhydrogenase assay mixture were t e s t e d u s i n g washed membrane p a r t i c l e s of E. c o l i ML308-225. F i g . 29 i s a Dixon p l o t showing the e f f e c t of ADP upon the APNAD + (panel X) and NADPH (panel Y) s i t e s of the enzyme, r e s p e c t i v e l y . In both cases, the p l o t s were n o n - l i n e a r at low s u b s t r a t e and hig h i n h i b i t o r concen-t r a t i o n s . N o n - l i n e a r i t y became more apparent as the con-c e n t r a t i o n s of ADP were i n c r e a s e d i n the assay mixture. From the l i n e a r p o r t i o n of the p l o t s , ADP appeared t o be com-p e t i t i v e w i t h r e s p e c t to APNAD + (K^, 4 mM) and non-competitive w i t h r e s p e c t to NADP(H) (K±r 24 mM). F i g . 28. E f f e c t of high concentrations of 5'-AMP on energy-independent transhydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i ML308-225 i n 50 mM TM buffer, pH 7.8, were suspended at a protein concen-t r a t i o n of 9 mg/ml. The procedure was s i m i l a r to that des-cribed i n the legend to F i g . 26. Concentrations of APNAD"1" were ( O, 0.3 mM; • , 1 mM) (panel X), those of NADPH were ( • , 0.1 mM; • , 0.5 mM) (panel Y), respectively. 230 u g protein was used i n each assay. A c t i v i t y - 1 i s ex-pressed as (units/mg p r o t e i n ) - 1 . 5'-AMP concentration i s expressed as mM. 176. 177. F i g . 29. E f f e c t of ADP on energy-independent transhydrog-enase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i ML308-225 were suspended i n 50 mM TM buffer, pH 7.8 at a protein concentration of 11 mg/ml. ADP was added to the assay mixture at varying concentrations. The concentration of APNAD+ were ( • , 0.35 mM; _ , 1.1 mM) (panel X), those of NADPH were ( • ,0.11 mM; O ,0.56 mM) (panel Y), respectively. 275 ug protein was used i n each assay. The second substrate was used at the "standard concentration" (see MATERIALS AND METHODS). A c t i v i t y " i s expressed as (units/mg p r o t e i n ) - 1 . ADP concentration i s expressed as mM. 179. E f f e c t of s a l t on the a c t i v i t y of the enzyme The s o l u t i o n s of adenosine phosphates used i n the experiments d e s c r i b e d i n the preceding s e c t i o n had been n e u t r a l i z e d w i t h potassium hydroxide. The p o s s i b i l i t y t h a t the i n c r e a s e d i n h i b i t i o n of the enzyme a t high c o n c e n t r a t i o n s of i n h i b i t o r was, i n f a c t , due to the comcomitant i n t r o -d u c t i o n of hig h c o n c e n t r a t i o n s of K + i n the assay mixture was examined. The h i g h e s t c o n c e n t r a t i o n of 2'-AMP t e s t e d as an i n -h i b i t o r of energy-independent transhydrogenase, i n the pre v i o u s s e c t i o n , was 56.4 mM. T h i s s o l u t i o n was 127.5 mM with r e s p e c t t o K +. S i m i l a r l y , 3 8.4 mM 5'-AMP s o l u t i o n c o n c o m i t a n t l y i n t r o d u c e d 108.34 mM K + i n t o the assay mix-t u r e . A s i m i l a r experiment t o t h a t i n the pr e v i o u s s e c t i o n was c a r r i e d out, i n which the energy-independent transhy-drogenase a c t i v i t y was measured both i n the presence and absence of 2'- and 51-AMP. In the absence of adenosine monophosphate, a c o n c e n t r a t i o n of K + (as KC1) e q u i v a l e n t to t h a t i n t r o d u c e d by these n u c l e o t i d e s , was present. F i g s . 30 and 31 are Dixon p l o t s of the data r e s u l t i n g from such experiments. I t i s c l e a r t h a t K + i n t r o d u c e d i n the system comcom-i t a n t l y with 2'-AMP or 5'-AMP was not r e s p o n s i b l e f o r the in c r e a s e d i n h i b i t i o n seen a t e l e v a t e d c o n c e n t r a t i o n s of phosphonucleotides. Thus the curved p l o t s observed w i t h these n u c l e o t i d e s are probably due to the i n h i b i t o r s 180. F i g . 30. E f f e c t of 2'-AMP and KCl on energy-independent transhydrogenase a c t i v i t y . Washed membrane p a r t i c l e s from E. c o l i W6 c e l l s were taken up i n 50 mM TED-1 b u f f e r , pH 7.8 a t a p r o t e i n con-c e n t r a t i o n of 2.7 mg/ml. The i n h i b i t o r i s 21-AMP i n panel X (curves a and b) and i n panel Y (curves a and b ) , re s p e c -t i v e l y . The i n h i b i t o r i s KCl i n panel X (curves c and d) and i n panel Y (curves c and d ) , r e s p e c t i v e l y . The APNAD + c o n c e n t r a t i o n f o r curves a and c (panel X) was 135 uM. The NADPH c o n c e n t r a t i o n f o r curves a and c (panel Y) was 65 uM. Standard assay c o n c e n t r a t i o n s were employed i n the o t h e r cases. Membrane p a r t i c l e s were used a t 63 ug p r o t e i n per assay. A c t i v i t y - 1 i s gi v e n as (units/mg p r o t e i n ) The c o n c e n t r a t i o n s o f KCl and 21-AMP are expressed as mM. 181. J m C O ! a i j a n o v F i g . 31. E f f e c t of 51-AMP and KCl on energy-independent transhydrogenase a c t i v i t y . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM TED-1 b u f f e r , pH 7.8 a t a p r o t e i n concen-t r a t i o n of 4.2 mg/ml. The i n h i b i t o r i s 5'-AMP i n panel X (curves a and b) and i n panel Y (curves a and b ) , res p e c -t i v e l y . The i n h i b i t o r i s KCl i n panels X (curves c and d) and i n panel Y (curves c and d ) , r e s p e c t i v e l y . The APNAD + c o n c e n t r a t i o n f o r curves a and c (panel X) was 241 uM. The NADPH c o n c e n t r a t i o n f o r curves a and c (panel Y) was 57 uM. Standard assay c o n c e n t r a t i o n s were employed i n the oth e r c a s e s . Membrane p a r t i c l e s were used a t 105 ug p r o t e i n per assay. A c t i v i t y - 1 i s g i v e n as (units/mg p r o t e i n ) - 1 . The c o n c e n t r a t i o n s of KCl and 5'-AMP are ex-pressed as mM. 184. themselves b i n d i n g a t more than one s i t e on the enzyme. I t i s l i k e l y t h a t h i g h e r c o n c e n t r a t i o n s of 2'-AMP bind a t the APNAD + s i t e as w e l l as the NADP(H) s i t e . In the same manner, 51-AMP c o u l d p o s s i b l y b i n d a t the NADP(H) s i t e as w e l l as the APNAD + s i t e when in t r o d u c e d t o the system i n s u f f i c i e n t l y h i g h c o n c e n t r a t i o n s . Although KCl c o n c e n t r a t i o n s of up to 127.5 mM d i d not appear t o a f f e c t the energy-independent transhydrogenase r e a c t i o n , i t was of i n t e r e s t t o examine the e f f e c t s , i f any, of higher c o n c e n t r a t i o n s of K C l , s i n c e the p h y s i o l o g i c a l c o n c e n t r a t i o n s of K + i n the growing c e l l s of E. c o l i i s 200 mM (163) and i n c r e a s e d K + c o n c e n t r a t i o n s have been shown to a f f e c t the k i n e t i c parameters of other enzymes, such as the membrane-bound ATPase of E. c o l i (164). The e f f e c t of 2 00 mM KCl upon the K and maximal m v e l o c i t y (V) of the energy-independent transhydrogenase r e a c t i o n was s t u d i e d , u s i n g washed membrane p a r t i c l e s from E. c o l i W6 ( F i g . 32). M i c h a e l i s constants f o r APNAD + and NADPH were measured from Lineweaver-Burk p l o t s i n the presence and absence of the s a l t . A d d i t i o n of 200 mM KCl was i n h i b i t o r y a t a l l the c o n c e n t r a t i o n s of APNAD + t e s t e d . The apparent K f o r APNAD+ was a l t e r e d from 62.5 uM to 179 c c m uM, however, the maximal v e l o c i t y (V, 0.8 5 u n i t s per mg p r o t e i n ) was u n a f f e c t e d by s a l t . 185. F i g . 32. E f f e c t of 200 mM KC1 on the APNAD+- and NADPH-binding s i t e s of the energy-independent trans-hydrogenase . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM TM buffer, pH 7.8 at a protein concentra-tion of 4.6 mg/ml. The APNAD+ (panel X) or NADPH (panel Y) concentration was varied at a fixed (standard assay) con-centration of the other substrate. Enzyme a c t i v i t i e s were assayed using 40 yg protein per assay, i n the presence or absence of 200 mM KC1. A c t i v i t y - 1 i s expressed as (units/ mg p r o t e i n ) - 1 . Substrate concentration i s expressed as mM. 186. 187. In the absence of KC l , the d o u b l e - r e c i p r o c a l p l o t f o r the NADPH s i t e was l i n e a r , showing s u b s t r a t e i n h i b i t i o n at e l e v a t e d c o n c e n t r a t i o n s of NADPH (Fig..3 2, panel Y ). Add-i t i o n of 200 mM KCl had r e l a t i v e l y l i t t l e e f f e c t on the k i n e t i c s of the r e a c t i o n a t h i g h (378 uM) c o n c e n t r a t i o n s of NADPH, but caused a marked i n h i b i t i o n a t low (7.6 uM) con-c e n t r a t i o n s , g i v i n g a t r i p h a s i c appearance t o the p l o t , r a t h e r than the b i p h a s i c one observed i n the absence of KC l . T h i s may i n d i c a t e the presence of more than one b i n d -i n g s i t e on the enzyme f o r NADPH ( i n c l u d i n g p o s s i b l y an a l l o s t e r i c s i t e ) , the s i t e s being i n f l u e n c e d t o d i f f e r e n t e xtents by K C l . Mechanism of a c t i o n of the enzyme  I n i t i a l v e l o c i t y p a t t e r n s The o b s e r v a t i o n s made w i t h KCl i n the preceding s e c t i o n , as w e l l as the s u b s t r a t e i n h i b i t i o n a t h i g h NADPH co n c e n t r a -t i o n s ( F i g . 6), i n d i c a t i n g the presence of more than one NADPH-binding s i t e on the enzyme, made i t d e s i r a b l e t o study these s i t e s and t o i n v e s t i g a t e the mechanism of the energy-independent transhydrogenase r e a c t i o n i n membrane p a r t i c l e s of E. c o l i . F i g s . 33 and 34 are d o u b l e - r e c i p r o c a l p l o t s of the v e l o c i t i e s of the energy-independent transhydrogenase r e a c -t i o n at v a r y i n g c o n c e n t r a t i o n s of s u b s t r a t e . When the APNAD + c o n c e n t r a t i o n was v a r i e d ( F i g . 33), at d i f f e r e n t F i g . 33. E f f e c t of the c o n c e n t r a t i o n of APNAD + on the ac-t i v i t y of the energy-independent transhydrogenas at c o nstant c o n c e n t r a t i o n s of NADPH. Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM TED-1 b u f f e r , pH 7.8 a t a p r o t e i n concen-t r a t i o n of 3.9 mg/ml. Constant c o n c e n t r a t i o n s of NADPH ( • — • , 10 yM; 4 - + , 24 yM; •-• , 48 yM; O-O , 97 yM; , 241 yM; A—A , 482 yM; , 965 yM) were used. 98 yg p r o t e i n was used i n each assay. A c t i v i t y - 1 i s ex-pressed as (units/mg p r o t e i n ) - 1 . The c o n c e n t r a t i o n of APNAD4" i s expressed as mM. •681 190. F i g . 34. E f f e c t o f the c o n c e n t r a t i o n o f NADPH on the ac-t i v i t y o f the energy-independent transhydrogenase a t c o nstant c o n c e n t r a t i o n s o f APNAD+. Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM TED-1 b u f f e r , pH 7.8 a t a p r o t e i n concen-t r a t i o n o f 3.6 mg/ml. Constant c o n c e n t r a t i o n s of APNAD + ( , 20 yM; Q-O , 49 yM; » — • , 98 yM; , 491 yM; A.—A i 982 yM; • - - • , 3872 yM) were used. 90 yg p r o t e i n was used i n each assay. A c t i v i t y - 1 i s expressed as ( u n i t s / mg p r o t e i n ) - 1 . The c o n c e n t r a t i o n of NADPH i s expressed as mM. *T6T 192. f i x e d c o n c e n t r a t i o n s of NADPH, the p l o t s obtained at ve r y low c o n c e n t r a t i o n s of NADPH (10, 24 uM) appeared t o be almost p a r a l l e l , but became ; i n c r e a s i n g l y convergent at hig h e r c o n c e n t r a t i o n s of NADPH (97, 241 uM), i n t e r s e c t i n g on the l e f t of the o r d i n a t e . On a s i m i l a r type of enzyme p r e p a r a t i o n , the f a m i l y of l i n e s obtained u s i n g v a r i a b l e NADPH c o n c e n t r a t i o n s at f i x e d APNAD + c o n c e n t r a t i o n s ( F i g . 34), l i k e w i s e i n d i c a t e d convergent p l o t s a t c o n c e n t r a t i o n s of 4 9 to 4 91 uM APNAD . The convergent nature of both s e t s of data at these l e v e l s are d i a g n o s t i c of a r e a c t i o n i n -v o l v i n g a t e r n a r y complex (119). The slop e s of the l i n e s decreased t o a minimum va l u e b e f o r e i n c r e a s i n g once again as the c o n c e n t r a t i o n of f i x e d s u b s t r a t e was i n c r e a s e d y e t f u r t h e r . T h i s o c c u r r e d a t 482 uM NADPH ( F i g . 33) and 982 uM APNAD + ( F i g . 34), r e s p e c t i v e l y . At these c o n c e n t r a t i o n s and above, i n t e r s e c t i o n of the l i n e s o c c u r r e d on the o r d i n a t e , i n d i c a t i n g a c o m p e t i t i v e r e l a t i o n s h i p between the f i x e d s u b s t r a t e and the v a r i a b l e one. T h i s type of k i n e t i c p l o t i s c h a r a c t e r i s t i c of a r a p i d e q u i l i b r i u m random b i r e a c t a n t system w i t h two dead-end complexes (120). Analyzed i n the l i g h t of t h i s i n t e r p r e t a t i o n , h i g h c o n c e n t r a t i o n s of NADPH would cause i t t o bind a t the APNAD + s i t e as w e l l as i t s own s i t e , thus competing w i t h APNAD+ f o r the second s i t e . In the same manner, but to a l e s s e r extent, h i g h c o n c e n t r a -t i o n s of APNAD + would tend t o compete f o r the NADP(H) s i t e on the enzyme. The n o n - l i n e a r i t y of the l i n e s i n Fi g s . 34 and 6 (panel Y) a t h i g h NADPH c o n c e n t r a t i o n i n d i c a t e sub-s t r a t e i n h i b i t i o n , p o s s i b l y due to competition f o r the APNAD + s i t e . T h i s i n h i b i t i o n i s overcome by the presence of h i g h c o n c e n t r a t i o n s of APNAD+, as i n d i c a t e d by the shallower c u r v a t u r e of the p l o t a t 98 uM APNAD+ as opposed to t h a t a t 20 uM, a t hig h c o n c e n t r a t i o n s of NADPH. Sub-s t r a t e i n h i b i t i o n by 129 uM NADPH was thus overcome by con-c e n t r a t i o n s of 491 uM APNAD+ or g r e a t e r . I f A and B are two s u b s t r a t e s , b i n d i n g i n a r a p i d e q u i l -i b r i u m random system, and i f the b i n d i n g of one s u b s t r a t e changes the d i s s o c i a t i o n c o n s t a n t f o r the other s u b s t r a t e by a f a c t o r a, the system may be d e s c r i b e d by the f o l l o w i n g e q u i l i b r i a (120) E + B T EB + + A A A K A aK ak A B _ " k EA + B ^  , EAB E + P where K_ , K_, are the d i s s o c i a t i o n constants f o r A and B, r e s p e c t i v e l y , k^ i s the v e l o c i t y constant f o r the c o n v e r s i o n of EAB to product. A value of a g r e a t e r than u n i t y i n d i c a t e s t h a t the b i n d i n g of one l i g a n d decreases the a f f i n i t y of the enzyme f o r the other l i g a n d . For such i n t e r a c t i o n s , the f a m i l y of r e c i p r o c a l p l o t s i n t e r s e c t a t a p o i n t below the 194. a b c i s s a . T h i s was the tendency observed i n the data pre-sented i n F i g s . 33 and 34 a t i n t e r m e d i a t e l e v e l s of f i x e d s u b s t r a t e , i n which no competition w i t h the second sub-s t r a t e o c c u r r e d . The v e l o c i t y equations f o r r a p i d e q u i l i b r i u m random systems may be g i v e n as: v = k [EAB] and _v_ = k p [ E A B ] [ E ] t [E]+ [EA]+ [EB]+ [EAB] s u b s t i t u t i n g V = k ^ t E ] ^ and r e a r r a n g i n g the e q u a t i o n g i v e s For v a r i a b l e A: 1 ^ A [ K B ) 1 . i f , . a K B ) v " V \ TBI/ [A] + V V 1 + [B]/ For v a r i a b l e B: 1 ^ B / K A ^ 1 i f , a K A ) v _ V { 1 + [A] J [B] + V V [A]/ These equations can be more simply expressed as: f o r v a r i a b l e . A f o r v a r i a b l e B 1 _ 1 A.app V V app [A] and 1 _ 1 , Bapp V V P 1 [B] where V = 7 — — - T F ~ \ ' V = , V v . A P P A + A a p p ^ + a A 1 .+. K A [B] 1 x T TA] K = aK * L ^ J / K - rvTC v [A] V P . : M app y x + " K A 195. From the 1 versus 1 p l o t v [A] _____ _ _ _ _ _ . ! V a P P " V [ B ] V and from the 1 versus 1 plot v [B] _____ - a K A 1 , 1 V V [A] V app The k i n e t i c constants V, K_^ , K B and may be obtained from replots of the 1 versus 1 and 1. versus 1 data, respectively, v [A] v [B] From the 1 versus 1 data, a replot of the intercepts v [B] 1 as a function of the second substrate 1 y i e l d s a V a P P [ A ] aK l i n e with a slope of A and an intercept on the ordinate V of 1. Whereas a replot of the slopes as a function of 1 V [A] cxK K y i e l d s a l i n e with a slope of B A and an intercept on V the ordinate of a KB. V Kinetic constants were calculated from the data of Figs. 33 and 34 from intercept and slope replots. The replots i n F i g . 35 are constructed from the family of r e c i p r o c a l plots shown i n F i g . 33. The ordinate intercept of F i g . 35, panel X, represents 1, from which V was determined to V be 1.34 units per mg protein. Dividing the slope i n F i g . 35, panel Y a K A K B by that of Fi g . 35, panel X y i e l d s V V K A (the d i s s o c i a t i o n constant for APNAD+). This was deter-mined to be 3.6 uM. The slope i n F i g . 35 / panel X, divided 196. F i g . 35. Determination of kin e t i c constants from replots of 1 versus 1 at d i f f e r e n t fixed concen-v [APNAD+] trati o n s of NADPH. The experimental conditions are the same as those des-cribed i n the legend to F i g . 33. Top panel, replot of ordinate intercepts i n F i g . 33 as a function of NADPH con-centration. Bottom panel, replot of the slopes of the l i n e s as a function of NADPH concentration. The intercepts , 1 x are expressed as (units/mg protein) 1. The concentration' of NADPH i s expressed as uM. S l o p e Intercept O o 198. by the ordinate intercept 1 yi e l d s the value aK D. This V B value was found to be 106.7 yM and corresponds to the Michaelis constant (K ) for NADPH. m Fig. 36 i s the intercept and slope replot of Fig . 34. V was determined to be 0.95 units per mg protein. Kinetic constants calculated as above gave a d i s s o c i a t i o n constant for NADPH of 16.2 yM, K m for APNAD+ of 45.6 yM, and a value for a of 2.2. The calculated values of d i s s o c i a t i o n constants for the substrates were found to be i n agreement with those deter-mined graphically from Hanes-Woolf pl o t s , where the x-coordinate of the point of in t e r s e c t i o n of the li n e s y i e l d s the d i s s o c i a t i o n constant. The values obtained from Figs. 37 and 38 are 14.5 yM for NADPH and 3 yM for APNAD+, respectively. Product i n h i b i t i o n The mechanism of the reaction was also examined using + the products NADP and APNADH. Fig. 39 i s a Dixon p l o t showing the e f f e c t of NADP+ on the APNAD+ s i t e (panel X) and NADPH s i t e (panel Y) of the energy-independent trans-hydrogenase studied using washed membrane p a r t i c l e s from E. c o l i W6. The plots were non-linear at high con-centrations of NADP+ (> 0.55 mM). Below th i s concentra-t i o n of NADP+, straight l i n e s could be drawn which indicated a non-competitive relationship between NADP+ and APNAD+ F i g . 36. Determination of k i n e t i c c o n s t a n t s from r e p l o t s of 1 versus 1 a t d i f f e r e n t f i x e d concen-v [NADPH] t r a t i o n s o f APNAD+. The experimental c o n d i t i o n s are the same as those de c r i b e d i n the legend t o F i g . 34. Top p a n e l , r e p l o t of o r d i n a t e i n t e r c e p t s i n F i g . 34 as a f u n c t i o n o f APNAD+ c o n c e n t r a t i o n . Bottom panel, r e p l o t of the s l o p e s of the l i n e s as a f u n c t i o n o f APNAD + c o n c e n t r a t i o n . The i n t e r -cepts ^ 1 j are expressed as (units/mg p r o t e i n ) - 1 . The v a p p c o n c e n t r a t i o n o f APNAD + i s expressed as y M . S l o p e Intercept CO o en 201. F i g . 3 7 . Determination of the d i s s o c i a t i o n c o n s t a n t f o r NADPH by a Hanes-Woolf p l o t . The experimental c o n d i t i o n s are the same as those d e s c r i b e d i n the legend to F i g . 3 4 . NADPH c o n c e n t r a t i o n [ S ] i s expressed as y M . [ N A D P H ] 203. F i g . 38. Determination of the d i s s o c i a t i o n c onstant f o r APNAD + by a Hanes-Woolf p l o t . The experimental c o n d i t i o n s are the same as those d e s c r i b e d i n the legend t o F i g . 33. APNAD + c o n c e n t r a t i o n [ S ] i s expressed as y M . 204 . [ A P N A D * ] 205. 4-F i g . 39. E f f e c t of NADP on the energy-independent trans-hydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM sodium borate buffer, pH 7.8 at a protein concentration of 3.1 mg/ml (panel X) and 5.2 mg/ml (panel Y) , respectively. Variable concentrations of NADP4" were added to the assay mixture at constant concentrations of APNAD+ ( •-• , 134 yM; O-O , 268 yM; A - A , 93 9 yM) (panel X), or NADPH ( , 103 yM; « - # , 501 yM) (panel Y). 77 yg protein (panel X) and 13 0 yg protein (panel Y) were used i n each assay. The second substrate was used at the "standard concentration" (see MATERIALS AND METHODS). A c t i v i t y - 1 i s expressed as (units/mg p r o t e i n ) - 1 . NADP4" concentration i s expressed as mM. A c t i v i t y • 902 207. ( K i f 440 yM) and a c o m p e t i t i v e one w i t h NADPH. (K^, 110 yM) . The n o n - l i n e a r i t y o f the p l o t s a t high e r c o n c e n t r a t i o n s of NADP + c o u l d be i n d i c a t i v e of i t s b i n d i n g a t more than one s i t e on the enzyme. A s i m i l a r type o f membrane p r e p a r a t i o n was used t o determine the product i n h i b i t i o n p a t t e r n s w i t h APNADH. F i g . 4 0 (panel X) i n d i c a t e d a c o m p e t i t i v e r e l a t i o n s h i p between APNADH and APNAD + (K.^, 3 6 yM). In order t o e l i m i n a t e p o s s i b l e i n t e r f e r e n c e from e l e v a t e d c o n c e n t r a t i o n s of NADPH, the r e a c t i o n was c a r r i e d out u s i n g o n e - t h i r d of the NADPH c o n c e n t r a t i o n normally used i n a "standard assay" ( i . e . 0.16 yM). The r e s u l t s , however, were the same as those o b t a i n e d u s i n g 0.5 mM NADPH g i v i n g a f o r APNADH of 38 yM. (not shown). I n h i b i t i o n of the NADPH s i t e by APNADH was non-competitive (K^, 280 yM) (panel Y ). The p a t t e r n ob-t a i n e d a t e l e v a t e d c o n c e n t r a t i o n s of NADPH was su g g e s t i v e of a c o m p e t i t i v e r e l a t i o n s h i p between APNADH and NADPH, however. T h i s was not u n l i k e the tr e n d observed i n the i n i t i a l v e l o c i t y data ( F i g . 33) i n which APNAD + was the v a r i a b l e s u b s t r a t e a t f i x e d , h i g h c o n c e n t r a t i o n s of NADPH. The c o m p e t i t i v e r e l a t i o n s h i p s between NADP + and NADPH and between APNADH and APNAD + as w e l l as the non-competitive r e l a t i o n s h i p s between NADP + and APNAD + and between APNADH and NADPH, seems t o e l i m i n a t e ordered b i - b i mechanisms (165) and i s d i a g n o s t i c of e i t h e r a r a p i d e q u i l i b r i u m random 208. F i g . 40. E f f e c t of APNADH on the energy-independent trans-hydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n 50 mM sodium borate buffer, pH 7.8 at a protein concentration of 2.56 mg/ml. APNADH was added to the assay mixture at varying concentrations. The concentrations of APNAD+ were ( o-O / HO UM; •—• > 220 UM; O-O , 880 yM). (panel X) , those of NADPH were (. Q_Q , 7 0 yM; A—<4 , 90 yM; _W_ , 180 yM; , 540 yM; •-# 1080 yM) (panel Y) , respectively. 64 yg protein was used i n each, assay. The NADPH concentration (panel X) was 162 yM. The APNAD+ con-centration (panel Y) was 970 yM. A c t i v i t y - 1 i s expressed as (units/mg p r o t e i n ) - 1 . APNADH concentrations i s ex-pressed as mM. 210. bireactant reaction or a The'orell-Chance bireactant mechanism (119,120) . The e f f e c t of NADH on the energy-independent trans-hydrogenase was also studied. Although NADH i s not a pro-duct of the reaction when APNAD+ i s used as a substrate, i t is. a product of the transhydrogenation occurring with NAD+. F i g . 41 i s a Dixon plot showing the e f f e c t of NADH on the ki n e t i c s of the enzyme using washed membrane part-i c l e s from E. c o l i W6. 'Standard' substrate concentrations were used i n plot 1. In plots 2 and 3 low concentrations (0.3 58 mM and 0.116 mM) of APNAD+ and NADPH respectively, were used. Although concentrations of 0.2 mM NADH or more were i n h i b i t o r y , low concentrations (0.104 mM or less) appeared to stimulate the reaction. The stimulatory e f f e c t of low concentrations of NADH on the reaction was i n v e s t i -gated to determine i f t h i s phenomenon occurred over a broad range of substrate concentrations. Fi g . 42 shows the e f f e c t of adding 0.104 mM NADH to the reaction mixture when the concentrations of APNAD4" ( l e f t panel) or NADPH4" (right panel) were varied. The NADPH concentration was held constant in the former case, while the APNAD+ concentration was held constant i n the l a t t e r . Over the range of APNAD+ and NADPH concentrations tested (up to 1.5 mM and 0.85 mM, respe c t i v e l y ) , NADH stimulated the energy-independent transhydrogenase reaction. The substrate i n h i b i t i o n observed at 0.85 mM NADPH (right panel) 211. Fi g . 41. E f f e c t of NADH on the energy-independent trans-hydrogenase a c t i v i t y i n membrane p a r t i c l e s . Washed membrane p a r t i c l e s from E. c o l i W6 were sus-pended i n sodium borate buffer, pH 7.8 at a protein concen-t r a t i o n of 2 mg/ml. NADH was added at varying concentra-tions . i n the reaction mixture. Curve 1: 1.08 mM APNAD+, 0.58 mM NADPH. Curve 2: 1.08 mM APNAD+, 0.11.6 mM NADPH. Curve 3: 0.358 mM APNAD+, 0.58 .-mM NADPH. 50 yg protein was used i n each assay. The concentration of NADH i s ex-pressed as. mM. A c t i v i t y - 1 i s expressed as (units/mg pro-tein) - 1 . 212. 213. F i g . 42. E f f e c t of NADH on the energy-independent t r a n s -hydrogenase a c t i v i t y a t v a r i o u s c o n c e n t r a t i o n s of s u b s t r a t e . Washed membrane p a r t i c l e s from E. c o l i W6 were suspen-ded i n 50 mM sodium borate b u f f e r , pH 7.8 a t a p r o t e i n con-c e n t r a t i o n of 2 mg/ml. The c o n c e n t r a t i o n s of NADPH ( l e f t panel) and APNAD + ( r i g h t panel) were 0.5 and 1.0 mM, r e -s p e c t i v e l y . NADH, where p r e s e n t ( s o l i d p o i n t s ) , was a t a c o n c e n t r a t i o n o f 104 yM. 50 yg p r o t e i n were used i n each assay. A c t i v i t y i s expressed as (nmol/min per mg p r o t e i n ) ~ x 1 0 - 2 . Su b s t r a t e c o n c e n t r a t i o n i s expressed as mM. 215. was not overcome by the presence of NADH. Both the K m values as well as the maximum v e l o c i t y of the reaction were affected by NADH. Apparent values i n the presence of 0.104 mM NADH were 192 yM and 31 yM for APNAD+ and NADPH, respectively. These re s u l t s suggest that there i s an a l l o -s t e r i c s i t e on the energy-independent transhydrogenase which i s capable of binding NADH. Possibly, low concentrations of NADH bind at the a l l o s t e r i c s i t e causing a conformational change i n the enzyme re s u l t i n g i n an increased a c t i v i t y , whereas high concentrations (above 0.2 mM) compete with substrate at the APNAD+ s i t e . Evidence for the coenzyme-induced conformational change of the energy-independent transhydrogenase w i l l be presented i n a subsequent section of t h i s thesis. Alternate substrates Further investigation of the k i n e t i c mechanism of the energy-independent transhydrogenase was undertaken i n order to d i s t i n g u i s h between the Theorell-Chance and the rapid equilibrium random bireactant mechanisms. Rudolph and Fromm (166) have reported than random mechanisms and ordered sequential ones, such as the Theorell-Chance mechanism may be distinguished by the use of alternate substrates of the enzyme. In the l a t t e r case the order of binding of the substrates may also be determined. If A and B are the two substrates for the reaction (with A b i n d i n g f i r s t i n the case of ordered r e a c t i o n s ) and i f A' and B 1 are a l t e r n a t e s u b s t r a t e s t o A and B r e s p e c t i v e l y , then f o r a r a p i d e q u i l i b r i u m random r e a c t i o n , A' w i l l a c t as a c o m p e t i t i v e i n h i b i t o r f o r A and a non-competitive i n h i b i t o r f o r B. In the same manner, B' w i l l be c o m p e t i t i v e r e l a t i v e t o B and non-competitive r e l a t i v e t o A. In Theorell-Chance mechanisms, however, double r e c i p r o c a l p l o t s would i n d i c a t e a c o m p e t i t i v e r e l a t i o n s h i p between A 1 and A, but a p a r a b o l i c one between A' and B. Competitive i n h i b i t i o n would occur between B' and B, but would appear to be non-competitive r e l a t i v e t o A. In t h i s i n v e s t i g a t i o n an attempt was made to determine which of these two mechanisms governs the r e a c t i o n o f the energy-independent transhydrogenase of E. c o l i u s i n g NAD + (as an a l t e r n a t e s u b s t r a t e to APNAD+) and deamino-NADPH (as an a l t e r n a t e s u b s t r a t e t o NADPH). F i g . 43 i s a f a m i l y of double r e c i p r o c a l p l o t s obtained v a r y i n g the c o n c e n t r a t i o n of NADPH (panels 1 and 2) or APNAD (panels 3 and 4) a t f i x e d c o n c e n t r a t i o n s of NAD + (panels 1 and 4) or deamino-NADPH (panels 2 and 3), r e s p e c t i v e l y . A c o m p e t i t i v e r e l a t i o n s h i p between the p a i r s NAD + and APNAD + ( K i f 0.14 9 mM) and deamino-NADPH and NADPH (K i, 0.47 mM) was observed. Non-competitive i n h i b i t i o n e x i s t e d between the p a i r s NAD + and NADPH (K ±, 3.2 mM) and deamino-NADPH and APNAD + (K i, 2.1 mM), r e s p e c t i v e l y . 217. F i g . 4 3 . I n h i b i t i o n of energy-independent transhydrogenase a c t i v i t y by a l t e r n a t e s u b s t r a t e s NAD + and deamino-NADPH. In panels 1 and 2 the v a r i a b l e s u b s t r a t e [S] i s NADPH with 0 . 9 mM APNAD+. In panels 3 and 4 the v a r i a b l e s u b s t r a t e [S] i s APNAD"1" w i t h 0 . 2 5 mM NADPH. The c o n c e n t r a t i o n . of the v a r i a b l e s u b s t r a t e i s expressed as mM. A c t i v i t y ' " 1 i s ex-pressed as (units/mg p r o t e i n ) - 1 . . P a n e l - l . NAD* concentra-t i o n : O - O , 0 yM; • — » , 4 7 5 yM; • — O , 8 5 5 yM; +—Q , 3 4 4 0 yM. Panel 2 . Deamino-NADPH c o n c e n t r a t i o n : , 0 yM; • — • , 5 3 0 yM; • — • , 7 9 6 yM. Panel 3 . Deamino-NADPH c o n c e n t r a t i o n : • , 0 yM; o—o / 2 93 u M ' * • ' 7 7 0 yM; • — Q , 1 2 9 0 yM. Panel 4 . NAD"1" c o n c e n t r a t i o n : O — O , 0 yM; A-_> , 3 4 8 yM; A — _ ^ 5 2 2 yM. The membranes, prepared i n 5 0 mM TED-1 , pH 7 . 8 , were from E. c o l i W6 c e l l s . 5 2 - 6 3 yg p r o t e i n was used i n each assay. 218. 219. The r e s u l t s are c o n s i s t e n t w i t h a r a p i d e q u i l i b r i u m random b i r e a c t a n t mechanism. Had the r e a c t i o n proceeded by way of a Theorell-Chance mechanism wit h APNAD + as the f i r s t s u b s t r a t e to bin d to the enzyme as p o s t u l a t e d by Rydstrom ( 8 3 ) , then the i n h i b i t i o n between NAD + ( s u b s t r a t e A') and NADPH (s u b s t r a t e B) would have been p a r a b o l i c i n s t e a d of non-competitive. T h i s was not the case as i n -d i c a t e d i n F i g . 4 3 . In panel 3 of F i g . 4 3 , the l i n e ob-t a i n e d u s i n g 1290 uM deamino-NADPH d i d not i n t e r s e c t the a b c i s s a a t the same p o i n t as t h a t f o r the lower concentra-t i o n s , but tended more towards the o r d i n a t e . T h i s suggests a t r e n d f o r a c o m p e t i t i v e r e l a t i o n s h i p with APNAD + a t high c o n c e n t r a t i o n s of deamino-NADPH. T h i s i s not u n l i k e the p a t t e r n observed i n F i g . 3 3 when hig h f i x e d c o n c e n t r a t i o n s of NADPH were used wi t h APNAD + as the v a r i a b l e s u b s t r a t e . Competitive i n h i b i t i o n a t e l e v a t e d c o n c e n t r a t i o n s of e i t h e r s u b s t r a t e gave i n i t i a l v e l o c i t y p l o t s c h a r a c t e r i s t i c of r a p i d e q u i l i b r i u m random b i r e a c t a n t mechanisms wit h two dead-end products (120). Thus, the data e l i m i n a t e s the Theorell-Chance mechanism i n favour of the r a p i d e q u i l i b r i u m random b i r e a c t a n t mechanism to d e s c r i b e the r e a c t i o n under-gone by the energy-independent transhydrogenase of E. c o l i . 220. S t u d i e s on the a c t i v e s i t e of the enzyme by chemical Mod- i f i c a t i o n I n a c t i v a t i o n by 2, 3-butanedione and phenyl g l y o x a l A r g i n y l r e s i d u e s have been found t o p l a y an important r o l e i n the b i n d i n g o f a n i o n i c s u b s t r a t e s and c o f a c t o r s t o a c t i v e s i t e s of many enzymes (98,124,125,167-177). The p a r t i c i p a t i o n of a r g i n y l r e s i d u e s was demonstrated by the use of a - d i c a r b o n y l reagents which c h e m i c a l l y modify the guanidino group of a r g i n y l r e s i d u e s and r e s u l t i n l o s s of enzyme a c t i v i t y . Two such reagents are 2,3-butanedione i n borate b u f f e r , and phenyl g l y o x a l . The r e a c t i o n of 2,3-butanedione w i t h a r g i n y l r e s i d u e s proceeds v i a the formation of a 4,5-dimethyl-4,5-dihydroxy-2-imidazoline d e r i v a t i v e (I) . T h i s i n t e r m e d i a t e then forms a more s t a b l e complex (II) w i t h borate as shown below (167). CH 3 H 2 N ^ + CH 3 C__ 0 + > C_NHR ^ _ HO-9-NH-^. i=_0 H 2N HO-C-NH-^ CH 3 CH 3 (I) V CH 3 HO. - .0-C-NH. ^ B ^ I J^C—NR HO^ 0-C-MT CH 3 ( I D 221. In the present i n v e s t i g a t i o n , the presence of an a r g i n y l r e s i d u e a t or near the c a t a l y t i c s i t e of the energy-independent transhydrogenase was demonstrated as f o l l o w s . Washed membrane p a r t i c l e s from E. c o l i W6 were incubated w i t h e i t h e r 2,3-butanedione or phenyl g l y o x a l i n borate b u f f e r . Samples were withdrawn a t timed i n t e r v a l s and the energy-independent transhydrogenase a c t i v i t y was measured. F i g . 4 4 shows the time-dependent i n a c t i v a t i o n of the enzyme by 2,3-butanedione (top panel) and phenyl g l y o x a l (bottom p a n e l ) . The p s e u d o - f i r s t order r a t e c o n s t a n t s (K 1) were c a l c u l a t e d from the slope of each l i n e . P l o t t i n g the l o g a r i t h m of the p s e u d o - f i r s t - o r d e r r a t e c onstants ob-t a i n e d , versus the l o g a r i t h m of the corresponding i n h i b i t o r c o n c e n t r a t i o n used, y i e l d e d the i n s e t s shown i n F i g . 44. The slope of the p l o t i n the i n s e t i s e q u i v a l e n t t o the number of molecules of i n a c t i v a t o r r e a c t i n g a t the c a t a -l y t i c s i t e of the enzyme t o produce i n a c t i v a t i o n . T h i s v a l u e was c a l c u l a t e d t o be 0.8 and 1.1 f o r i n a c t i v a t i o n w i t h 2,3-butanedione and phenyl g l y o x a l , r e s p e c t i v e l y . T h i s suggests t h a t one a r g i n y l r e s i d u e i s m o d i f i e d per a c t i v e s i t e of the enzyme. P r o t e c t i o n a g a i n s t i n a c t i v a t i o n by 2,3-butanedione I f the a r g i n y l r e s i d u e m o d i f i e d by theeC-diketones i s r e a l l y a t the c a t a l y t i c s i t e of the energy-independent transhydrogenase, then m o d i f i c a t i o n of t h i s r e s i d u e by F i g . 44. K i n e t i c s of i n a c t i v a t i o n of the energy-independent transhydrogenase by 2,3-butanedione and phenyl g l y o x a l . Washed membrane p a r t i c l e s from E. c o l i W6 were suspen-ded i n 50 mM sodium borate, b u f f e r , pH 7.8 and incubated a t 22°C a t a c o n c e n t r a t i o n of 1.1 and 1.96 mg p r o t e i n / m l w i t h the i n d i c a t e d c o n c e n t r a t i o n s of 2,3-butanedione (BD) (upper panel) and phenyl g l y o x a l (PG) (lower p a n e l ) , r e s p e c t i v e l y . Samples were withdrawn and assayed a t timed i n t e r v a l s . A c t i v i t y i s expressed as a percentage of the c o n t r o l ac-t i v i t y taken a t the onset of i n c u b a t i o n . The i n s e t s show the r e l a t i o n s h i p between the p s e u d o - f i r s t - o r d e r r a t e con-s t a n t of i n a c t i v a t i o n (k 1) and the i n h i b i t o r c o n c e n t r a t i o n expressed as mM. k 1 i s expressed as m i n - 1 . 2 2 3 log PG 20 40 Minutes 224. 2,3-butanedione might be decreased by the simultaneous presence of the substrate (s). The substrate should pro-tect the enzyme against i n a c t i v a t i o n by reducing the access of the 2,3-butanedione to the arg i n y l residue at the active s i t e . This has been observed with several enzymes (124,178). As shown i n Figs. 45 and 46, the substrates APNAD+ or NAD , the product NADP , or the competitive i n h i b i t o r s 2*-AMP and 5'-AMP, a l l afforded some protection against i n a c t i v a t i o n of the energy-independent transhydrogenase by 2,3-butanedione. In a l l cases, however, the enzyme was not completely protected against i n a c t i v a t i o n at the con-centrations of substrates or analogues used. Djavadi-Ohaniance and Hatefi (113) have shown that 50 mM NAD+ or 50 mM NADP+ afforded some protection against i n a c t i v a t i o n by 2,3-butanedione of the energy-independent transhydrog-enase from beef-heart submitochondrial p a r t i c l e s . However, the addition of both NAD+ and NADP+, to the incubation mixture, offered better protection than either nucleotide alone, even at the same t o t a l concentration. This r e s u l t was interpreted as indicating that the modified a r g i n y l residue was at the active s i t e of the enzyme and that the binding of ligands at both of the nucleotide-binding s i t e s caused a cooperative in t e r a c t i o n between the s i t e s , r e s u l t -ing i n greater protection against i n a c t i v a t i o n . F i g . 45. E f f e c t of APNAD on the i n a c t i v a t i o n of the energy-independent transhydrogenase by 2,3-butanedione. Washed membranes from E. c o l i W6 were suspended i n 50 mM sodium b o r a t e , pH 7.8 a t a p r o t e i n c o n c e n t r a t i o n of 6.3 mg/ml. They were incubated a t 22°C at a c o n c e n t r a t i o n of 2.5 mg p r o t e i n / m l with. 11.5 mM 2.3-butanedione i n the ab-sence o r presence of 21 mM APNAD4". . Samples were withdrawn a t timed i n t e r v a l s f o r assay. A c t i v i t y i s expressed as a percentage of the c o n t r o l a c t i v i t y taken a t the onset of i n c u b a t i o n . F i g . 46. E f f e c t of s u b s t r a t e s and s u b s t r a t e analogues on the i n a c t i v a t i o n of the energy-independent transhydrogenase by 2,3-butanedione. Membrane p a r t i c l e s were prepared from E. c o l i W6 and suspended i n 50 mM sodium borate b u f f e r , pH 7.8. They were incubated a t 22°C a t a c o n c e n t r a t i o n of 1.0 (top panels) and 1.4 (bottom panels) mg p r o t e i n / m l i n 50 mM sodium b o r a t e , pH 7.8 w i t h 53.7 mM 2,3-butanedione, i n the absence or presence of the i n d i c a t e d m i l l i m o l a r con-c e n t r a t i o n s of s u b s t r a t e s and s u b s t r a t e analogues. Samples were withdrawn a t timed i n t e r v a l s f o r assay. A c t i v i t y i s expressed as a percentage of the c o n t r o l a c t i v i t y taken a t the onset of i n c u b a t i o n . 228 . 229. The combined protective e f f e c t of NAD and NADP was tested on the energy-independent transhydrogenase of E. c o l i W6 using washed membrane p a r t i c l e s . A mixture containing 15.6 mM NAD+ plus 14.2 mM NADP+ afforded greater protection against i n a c t i v a t i o n by 2,3-butanedione than did either 31.1 mM NAD+ ( l e f t panel) or 28.4 mM NADP+ (right panel) alone (Fig. 47). Thus, the pseudo-first-order rate constant was lower for the mixture of NAD plus NADP (3.7 x 10 ) than for NAD+ (6.9 x 10~ 3) or NADP+ (8.8 x 10~ 3), respec-t i v e l y . Experiments testing the capacity of NADH and NADPH as potent i a l protectors against i n a c t i v a t i o n by 2,3-butanedione of the enzyme were also c a r r i e d out. The 2,3-butanedione was found to cause photooxidation of the reduced pyridine nucleotides (179) and thus a l l the reactions involving NADH or NADPH as protecting agents were carr i e d out i n the absence of l i g h t . The light-dependent oxidation of these cofactors by 2,3-butanedione i s discussed i n a subsequent section of t h i s thesis. The e f f e c t of NADH or NADPH upon the in a c t i v a t i o n of the energy-independent transhydrogenase of E. c o l i W6 by 2,3-butanedione i s shown i n F i g . 48. Washed membrane p a r t i c l e s i n borate buffer were incubated at 22°C with 2,3-butanedione i n the presence or absence of NADPH ( l e f t panel) or NADH (right panel). Samples were withdrawn at 230. F i g . 47. E f f e c t o f NAD and NADP4" on the i n h i b i t i o n of energy-independent transhydrogenase by 2,3-butanedione. Washed membrane p a r t i c l e s from E.. c o l i W'6 i n 50 mM sodium borate b u f f e r , pH 7.8 (1.84 mg protein/ml)., were incubated a t 22°C w i t h 53.7 mM 2,3-butanedione in the p r e s -ence or absence ( l i n e s a) of the i n d i c a t e d c o n c e n t r a t i o n s of s u b s t r a t e s . L i n e s c, 15.5 mM NAD4" + 14 mM NADP4"; ( l e f t panel) l i n e b, 31 mM NAD4"; ( r i g h t panel) l i n e b, 28 mM NADP4". The experiment was performed as d e s c r i b e d i n MATERIALS AND METHODS. Log a c t i v i t y 232. F i g . 48. E f f e c t of NADPH and NADH on the I n h i b i t i o n of the energy-independent transhydrogenase by 2,3-butanedione. Washed membranes from E. c o l i W6 at a c o n c e n t r a t i o n of 2.3 ( l e f t panel) and 1.74 ( r i g h t panel) mg p r o t e i n / m l of 50 mM sodium borate b u f f e r , pH 7.8, were incubated at 22°C wit h 53.7 mM 2,3-butanedione i n the presence and absence of the i n d i c a t e d m i l l i m o l a r c o n c e n t r a t i o n s of NADPH or NADH. Samples were withdrawn and assayed a t timed i n t e r v a l s . A c t i v i t y i s expressed as a percentage of the c o n t r o l ac-t i v i t y a t the onset of i n c u b a t i o n . Minutes timed i n t e r v a l s and the enzyme a c t i v i t y measured. Where NADPH was used i n the incubation medium, the amount of substrate for the enzyme assay was correspondingly decreased to maintain a f i n a l NADPH concentration of 0.5 mM i n the assay mixture. In contrast to the protecting e f f e c t against the i n -act i v a t i o n with 2,3-butanedione, seen with the oxidized coenzymes, low concentrations of NADPH and NADH appeared to enhance rather than to decrease the i n h i b i t o r y e f f e c t of 2,3-butanedione. In the presence of 10 mM NADPH, only 2 0% of the i n i t i a l enzyme a c t i v i t y remained afte r 45 minutes of incubation with 2,3-butanedione whereas i n the absence of NADPH, 3 9% of the i n i t i a l a c t i v i t y remained. S i m i l a r l y , in the presence of 2.4 mM NADH, 37% of the i n i t i a l a c t i v i t y remained after 45 minutes as opposed to 62% i n the absence of NADH. In other experiments, high concentrations (17 mM) of NADH reversed t h i s trend and completely protected the enzyme against i n a c t i v a t i o n by 2,3-butanedione. NADPH at high concentrations could not be tested as a protecting agent, due to the substrate i n h i b i t i o n i t caused during the enzymatic assay. The enhancing e f f e c t of NADH or NADPH upon enzyme in a c t i v a t i o n by 2,3-butanedione could be due to conforma-t i o n a l changes i n the transhydrogenase. Presumably, binding of low concentratons of NADPH or NADH to the enzyme render 235. the a r g i n y l r e s i d u e more a c c e s s i b l e t o the 2,3-butanedione. T h i s c o u l d be taken as f u r t h e r evidence f o r an a l l o s t e r i c s i t e ( s ) on the enzyme, capable of b i n d i n g NADPH or NADH. P r o t e c t i o n a g a i n s t i n a c t i v a t i o n by TPCK-trypsin Conformational changes i n enzyme s t r u c t u r e can o f t e n be d e t e c t e d by d i f f e r e n t i a l p r o t e o l y t i c i n a c t i v a t i o n i n the presence and absence of l i g a n d s . Blazyk and F i s h e r (115) demonstrated t h a t the energy-independent transhydrogenase from r a t - l i v e r s u b m i t o c h o n d r i a l p a r t i c l e s underwent pro-t e o l y t i c i n a c t i v a t i o n by t r y p s i n . P r e i n c u b a t i o n of the sub m i t o c h o n d r i a l p a r t i c l e s w i t h NADPH s i g n i f i c a n t l y pro-moted p r o t e o l y t i c i n a c t i v a t i o n of the enzyme, w h i l e pre-i n c u b a t i o n w i t h NADP + had l i t t l e or no e f f e c t . Since t r y p s i n c l e a v e s p o l y p e p t i d e s at l y s y l and a r g i n y l r e s i d u e s they i n t e r p r e t e d the enhancement of i n a c t i v a t i o n by NADPH t o i n d i c a t e a c o n f o r m a t i o n a l rearrangement due t o NADPH, thus r e n d e r i n g a t l e a s t one a r g i n y l or l y s y l r e s i d u e on the enzyme more a c c e s s i b l e t o t r y p s i n . In t h i s study, the e f f e c t of NADP+, NADPH, NAD + and NADH upon t r y p t i c i n a c t i v a t i o n of the energy-independent transhydrogenase was i n v e s t i g a t e d . Washed membranes from E. c o l i W6 were incubated a t 22°C w i t h TPCK-trypsin, i n the presence and absence of each p y r i d i n e n u c l e o t i d e . Samples were withdrawn a t timed i n t e r v a l s and the d i g e s t i o n stopped by the a d d i t i o n of t r y p s i n i n h i b i t o r . The samples were 236. maintained a t 0°C and the enzyme a c t i v i t y measured a t the end of each experiment. As seen i n F i g . 49, low conc e n t r a -t i o n s o f the reduced coenzymes (7.8 mM NADPH and 2 mM NADH, r e s p e c t i v e l y ) promoted t r y p t i c i n a c t i v a t i o n of the enzyme. Low c o n c e n t r a t i o n s of NAD + (2 mM) d i d not produce t h i s e f f e c t , w h i l e 1.3 mM NADP + had ve r y s l i g h t , i f any, en-hancing e f f e c t on the p r o t e o l y t i c i n a c t i v a t i o n of the enzyme. P r o t e c t i o n a g a i n s t t r y p t i c d i g e s t i o n of the enzyme was demonstrated at h i g h c o n c e n t r a t i o n s of l i g a n d , namely 15.7 mM NADH, 15.6 mil NAD + and 5.2 mM NADP+, r e s p e c t i v e l y . The t e s t i n g of hig h c o n c e n t r a t i o n s of NADPH was not t e c h n i c a l l y f e a s i b l e . Thus, the e f f e c t of these coenzymes upon t r y p t i c d i g e s t i o n of the energy-independent transhydrogenase seems to be s i m i l a r t o t h a t observed w i t h the 2,3-butanedione-m o d i f i e d enzyme. Whereas the o x i d i z e d p y r i d i n e n u c l e o t i d e s a f f o r d e d p r o t e c t i o n of the enzyme a g a i n s t i n a c t i v a t i o n , the reduced coenzymes pr o v i d e d p r o t e c t i o n o n l y a t high e r con-c e n t r a t i o n s . Low c o n c e n t r a t i o n s of reduced coenzymes caused s t r u c t u r a l changes i n the enzyme. T h i s i n c r e a s e d the a c c e s s i b i l i t y of the a r g i n y l r e s i d u e t o modifying reagents and enhanced the i n a c t i v a t i n g e f f e c t of the t r y p s i n or 2,3-butanedione. F i g . 49. E f f e c t of s u b s t r a t e s on the i n a c t i v a t i o n of the energy-independent transhydrogenase by TPCK-t r y p s i n . Washed membrane p a r t i c l e s from E. c o l i W6 i n 50 mM TED-1 b u f f e r , pH 7.8, were incubated a t 22°C a t a TPCK-t r y p s i n : p r o t e i n r a t i o (ug/mg) of 0.9, 0.42, 0.84 and 0.42 f o r the experiments u s i n g NADP+, NADPH, NAD+ and NADH, r e s p e c t i v e l y . Samples were withdrawn f o r assay,and d i g e s -t i o n stopped by a d d i t i o n of t r y p s i n i n h i b i t o r . The sample were kept a t 0°C and assayed a t the end of each experiment A c t i v i t y i s expressed as a percentage of the c o n t r o l ac-t i v i t y a t the onset of i n c u b a t i o n . The c o n c e n t r a t i o n of s u b s t r a t e i s expressed as mM. 2 3 8 . Minutes The r e s u l t s of the t r y p t i c i n a c t i v a t i o n of the enzyme support those of the 2,3-butanedione m o d i f i c a t i o n experiments as w e l l as the other k i n e t i c s t u d i e s of the e f f e c t of NADH. The da t a p r o v i d e evidence f o r an a l l o s t e r i c s i t e ( s ) on the energy-independent transhydrogenase capable of b i n d -in g NADPH or NADH s e p a r a t e l y from the c a t a l y t i c s i t e of the enzyme. DISCUSSION S o l u b i l i z a t i o n of the energy-independent transhydrogenase  of E. c o l i Many attempts have been made to s o l u b i l i z e the membrane-bound energy-independent transhydrogenase from d i f f e r e n t organisms s i n c e i t s d i s c o v e r y i n 1952 by Kaplan and co-workers (1,25,70,85,88,89,92,100,180). Although p u r i f i c a -t i o n t o homogeneity was achieved w i t h the enzyme from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s (6,7), s i m i l a r pur-i f i c a t i o n of the energy-independent transhydrogenase from E. c o l i has not been r e p o r t e d t o the present date. In the p r e s e n t study, a comparison was made of the a b i l i t y of s e v e r a l d e t e r g e n t s to s o l u b i l i z e the energy-independent transhydrogenase from E. c o l i membrane p a r t -i c l e s . Of these, T r i t o n X-100, l y s o l e c i t h i n and sodium c h o l a t e ( i n the presence of ammonium sulphate) were the most e f f e c t i v e . The y i e l d of energy-independent t r a n s h y -drogenase was g r e a t e s t when 0.5% (w/v) (8 mM) T r i t o n X-100 or 0.08% (w/v) (1.54 mM) l y s o l e c i t h i n r e s p e c t i v e l y , were used (Table 7). However, at these c o n c e n t r a t i o n s of d e t e r -gent o n l y 7 5% of the enzyme a c t i v i t y c o u l d be accounted f o r . The a c t i v i t y of the d e t e r g e n t - s o l u b i l i z e d enzyme was found t o depend on s p e c i f i c l i p i d s . Removal of some of the l i p i d by these d e t e r g e n t s i s l i k e l y t o be the cause of t h e i r i n a c t i v a t i o n (150,181). These t h r e e d e t e r g e n t s , T r i t o n X-100 (85,92), l y s o l e c i t h i n (7,53,85,86,101), and sodium 241. c h o l a t e ( i n the presence of ammonium sulphate) (.88) , have been used i n o t h e r l a b o r a t o r i e s , t o s o l u b i l i z e energy-independent transhydrogenase. The molecular s i z e of the e n z y m i c a l l y a c t i v e f r a c t i o n o b t ained by s o l u b i l i z a t i o n , however, was not r e p o r t e d . In t h i s study, the f r a c t i o n r e s u l t i n g from the s o l u b i l i z a t i o n of membrane p a r t i c l e s of E. c o l i w i t h 3% (w/v)Triton X-100, 3% (w/v) l y s o l e c i t h i n or 2% (w/v) sodium c h o l a t e ( i n the presence of 10% s a t u r a t e d ammonium sulphate) was analyzed on 10-50% sucrose d e n s i t y g r a d i e n t s . In a l l cases t h e r e appeared to be two s p e c i e s of energy-independent transhydrogenase, a l a r g e , f a s t -sedimenting (19.5 t o 25.4S) peak, and a slow-sedimenting peak or shoulder with a sedimentation c o e f f i c i e n t of 7.5 t o 11.5S. The f a s t - s e d i m e n t i n g peak obtained w i t h l y s o -l e c i t h i n appeared t o be s m a l l e r than t h a t w i t h T r i t o n X-100 or sodium c h o l a t e (90). T h i s may i n d i c a t e t h a t s o l u b i l i z a -t i o n w i t h l y s o l e c i t h i n produces a s m a l l e r fragment from the membrane. I t i s known t h a t p r o t e i n s are r e l e a s e d from membranes as l i p o p r o t e i n - d e t e r g e n t complexes and t h a t sucrose d e n s i t y g r a d i e n t s can remove T r i t o n X-100 from such complexes, c a u s i n g the enzyme t o aggregate. Thus, the d i f f e r e n c e i n molecular s i z e of the f a s t - s e d i m e n t i n g peaks c o u l d a l s o be due to d i f f e r e n t degrees of aggregation or t o b i n d i n g of some l y s o l e c i t h i n t o the p r o t e i n , r e s u l t i n g i n d i f f e r e n c e s i n -the sedimentation c o e f f i c i e n t of the p r o t e i n s (181). The presence of B r i j 58 i n the sucrose 242. d e n s i t y g r a d i e n t s caused the l y s o l e c i t h i n - s o l u b i l i z e d f r a g -ments t o s h i f t towards lower sedimentation c o e f f i c i e n t s . T h i s was not observed when T r i t o n X-100 or sodium c h o l a t e were used t o s o l u b i l i z e the enzyme. T h i s may mean t h a t the fragment s o l u b i l i z e d by the l a t t e r two d e t e r g e n t s was l a r g e to s t a r t w i t h , r a t h e r than an aggregate of s m a l l e r s p e c i e s . Hanson (92) r e p o r t e d t h a t B r i j 35 (which i s s i m i l a r i n i t s p r o p e r t i e s t o B r i j 58) i s a more e f f e c t i v e agent i n p r e -v e n t i n g the r e a g g r e g a t i o n of the transhydrogenase than T r i t o n X-100. Thus, i f the 24.5S peak ( F i g . 9; panel 3) were e n t i r e l y due to a g g r e g a t i o n of a s m a l l e r s p e c i e s due to removal of T r i t o n X-100 by the sucrose d e n s i t y g r a d i e n t , then i t would be expected t h a t the presence of B r i j 58 ( F i g . 9; panel 1) would prevent t h i s and r e s u l t i n a peak wit h a lower sedimentation c o e f f i c i e n t . Since the presence of B r i j 58 (or a s o l e c t i n ) caused o n l y minor changes i n the slow-sedimenting peaks of the T r i t o n X-100 or sodium c h o l a t e -s o l u b i l i z e d membrane p a r t i c l e s , t h i s may be taken as evidence t h a t most of the energy-independent transhydrogenase a c t i v i t y s o l u b i l i z e d by these two d e t e r g e n t s i s p a r t of a l a r g e complex of p r o t e i n s . T h i s was supported by the r e s u l t s of experiments i n which the (0.33-0.6P) f r a c t i o n of membrane p a r t i c l e s s o l u b i l i z e d by sodium c h o l a t e i n the presence >of ammonium sulphate was a p p l i e d t o a column of Sepharose 6B i n the presence of 0.2% (w/v) B r i j 35, 1% (w/v) Tween 80 or 0.5% (w/v) Triton X-100 (Figs. 10, 11 and 12). In a l l cases, the energy-independent transhydrog-enase a c t i v i t y was associated with a peak of molecular weight between 247 500 (the molecular weight of the marker protein catalase) • and 4 x 10 5 the exclusion l i m i t of Sepharose 6B. Other enzymes of the respiratory chain were also present (136). Hanson (92) showed that energy-independent transhy-drogenase a c t i v i t y r e s u l t i n g from the s o l u b i l i z a t i o n of E. c o l i membrane p a r t i c l e s with 15% Tr i t o n X-100 was re-tained on a Sepharose 4B column i n the presence of 0.1% B r i j 35, but appeared at the void volume of the column when the B r i j 3 5 was replaced by 0.1% Triton X-100. This suggests the enzyme i n his preparation also exists as a large moiety. The implication that s o l u b i l i z a t i o n by l y s o l e c i t h i n may produce a smaller fragment than that produced by Triton X-100, was tested by treating p u r i f i e d Triton X-100-s o l u b i l i z e d membrane p a r t i c l e s with a 0.09% (w/v) lyso-l e c i t h i n (Fig. 14). Analysis on sucrose density gradients revealed the appearance of a new and major peak of a c t i v i t y at 8.4S, confirming the hypothesis that l y s o l e c i t h i n can further cleave T r i t o n - s o l u b i l i z e d fragments into smaller fragments. A comparative study between the molecular size of the species obtained by both of these detergents has not been reported elsewhere. 244. P u r i f i c a t i o n of energy-independent transhydrogenase. In t h i s study, energy-independent transhydrogenase was p u r i f i e d by a v a r i e t y of methods. The method y i e l d i n g the highest s p e c i f i c a c t i v i t y (8.8 to 15.7 units per mg protein) and 37- to 68-fold p u r i f i c a t i o n involved s o l u b i l i z a t i o n of the enzyme with sodium cholate i n the presence of 33% sat-urated ammonium sulphate, treatment of the p r e c i p i t a t i n g f r a c t i o n with sodium deoxycholate, and ion-exchange chromato-graphy on columns of DEAE-Sepharose CL-6B i n the presence of 0.1% (w/v) B r i j 35. Hanson (92) has p a r t i a l l y p u r i f i e d the enzyme from E. c o l i membrane p a r t i c l e s . He reported a s p e c i f i c a c t i v i t y of 10 units per mg protein and a 71-f o l d p u r i f i c a t i o n over the membrane p a r t i c l e suspension. The membrane p a r t i c l e suspension tested contained 15% Tri t o n X-100. This detergent possibly masked some of the enzyme a c t i v i t y i n the s t a r t i n g material since the s p e c i f i c a c t i v i t y of the membrane p a r t i c l e suspension was only 0.14 units per mg protein (92) . Houghton et al_. (8 9) and Liang and Houghton (7 0) also p a r t i a l l y p u r i f i e d the enzyme from E. c o l i membrane p a r t i c l e s . The maximum p u r i f i c a t i o n ob-tained was 10- and 16.8-fold that of the sta r t i n g material, respectively. The s p e c i f i c a c t i v i t i e s were 13.6 and 20.2 units per.mg protein, respectively, and were obtained by assaying the enzyme i n the presence of phospholipids. In none of these cases has the enzyme from E. c o l i been p u r i -f i e d to homogeneity. On the other hand, homogeneous p r e p a r a t i o n s of energy-independent transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s have been obtained w i t h a s p e c i f i c a c t i v i t y of 4.4 to 15 u n i t s per mg p r o t e i n (6,7) and a p u r i f i c a t i o n f a c t o r of 23.3 (7) to 40 (6). The enzyme appeared as a s i n g l e p o l y p e p t i d e band on p o l y a c r y l a -mide g e l s , i n the presence of SDS, of molecular weight 97 000 (6) to 120 000 (7). The p u r i f i c a t i o n f a c t o r o b t ained f o r the enzyme from E. c o l i membrane p a r t i c l e s i s w i t h i n the same range as t h a t r e p o r t e d f o r the m i t o c h o n d r i a l enzyme. Since the former enzyme i s not completely pure a t t h i s l e v e l of p u r i f i c a t i o n , i t must .constitute a s m a l l e r f r a c t i o n of the t o t a l membrane p r o t e i n , than does the m i t o c h o n d r i a l enzyme. F u r t h e r p u r i f i c a t i o n of the energy-independent t r a n s -hydrogenase from E. c o l i by chromatography on h y d r o x y l -a p a t i t e , and a n a l y s i s by e l e c t r o p h o r e s i s i n the presence of 0.1% (w/v) SDS ( F i g . 17) r e v e a l e d bands s t a i n i n g f o r p r o t e i n w i t h molecular weights of about 90 000, 57 000, 50 000, 40 000 and a few minor bands i n the range of 13 000 t o 4 0 000. The v a r i a t i o n i n the i n t e n s i t y of s t a i n i n g of these bands i n v a r i o u s f r a c t i o n s seemed to p a r a l l e l the a c t i v i t y of the energy-:independent transhydrogenase i n the samples, suggesting t h a t some of the bands of low mo l e c u l a r weight are cleavage products of the 90 000 molecular weight band. Recently, Liang and Houghton (70) induced the energy-independent transhydrogenase i n E. c o l i membranes by t r a n s -f e r r i n g the c e l l s from a medium c o n t a i n i n g amino a c i d s , i n which the enzyme i s r e p r e s s e d , to one l a c k i n g amino a c i d s . By d u a l l a b e l l i n g experiments, they were able to r e c o g n i z e the newly s y n t h e s i z e d p o l y p e p t i d e s of the transhydrogenase i n SDS-polyacrylamide g e l s . A band w i t h a molecular weight of 94 000 was i d e n t i f i e d as the p y r i d i n e n u c l e o t i d e t r a n s -hydrogenase. However, two other newly s y n t h e s i z e d bands of about 50 000 were seen. Moreover, f u r t h e r f r a c t i o n a t i o n of the l i p i d - d e p l e t e d enzyme t h a t they had p r e v i o u s l y i s o -l a t e d on a column of agarose A5 0M i n the presence of potassium deoxycholate, r e s u l t e d i n peaks of a c t i v i t y which, by e l e c -t r o p h o r e s i s on SDS-polyacrylamide g e l s , again r e v e a l e d the 94 000 molecular weight band as w e l l as the p o l y p e p t i d e s of m o l e c u l a r weight 50 00 0. T h i s i s i n agreement wi t h the r e s u l t s obtained i n the present study. I t i s l i k e l y t h a t the bands of molecular weights of 90,000, 57 000 and 50 000 observed i n F i g . 17 correspond t o the bands at 94 000 and a t about 50 000 d e s c r i b e d by Liang and Houghton (70). A p o s s i b l e e x p l a n a t i o n f o r these o b s e r v a t i o n s may be t h a t the low molecular weight components are p r o t e o l y t i c products of the high molecular weight component. That t h i s occurs i n p u r i f i e d p r e p a r a t i o n s of the enzyme from E. c o l i but not i n those from m i t o c h o n d r i a , may be due to the higher s e n s i t i v i t y t o t r y p s i n of t h e former enzyme than the l a t t e r (2) or to the presence of h i g h l e v e l s of proteases i n the b a c t e r i a l membrane (94-96, 159). E f f e c t of d e t e r g e n t s and p h o s p h o l i p i d s 1 on the energy- independent transhydrogenase The a c t i v i t y of the p a r t i a l l y p u r i f i e d energy-independent transhydrogenase was a f f e c t e d by l i p i d s and d e t e r g e n t s . P h o s p h o l i p i d s i s o l a t e d from E. c o l i , l y s o -l e c i t h i n , p a l m i t i c a c i d and d e t e r g e n t s of the Tween and B r i j s e r i e s had an a c t i v a t i n g e f f e c t on the enzyme, w h i l e a s o l e c t i n (soy bean p h o s p h o l i p i d s ) , sodium c h o l a t e and T r i t o n X-100 were i n h i b i t o r y . The molecular b a s i s f o r the i n t e r p l a y between membrane-bound p r o t e i n s , t h e i r l i p i d environment :and d e t e r g e n t s i s s t i l l v i r t u a l l y unknown (182). In s e l e c t i n g a s u i t a b l e d e t e r g e n t f o r s o l u b i l i z a t i o n or r e a c t i v a t i o n of the enzyme, c e r t a i n p h y s i c a l and chemical p r o p e r t i e s of the d e t e r g e n t are c o n s i d e r e d to be c r u c i a l , i n c l u d i n g i t s charge, c r i t i c a l m i c e l l a r c o n c e n t r a t i o n and an e m p i r i c a l q u a n t i t y c a l l e d the h y d r o p h i l e - l i p o p h i l e balance (HLB). T h i s i s a measure f o r the s t r e n g t h of opposing h y d r o p h i l i c and hydrophobic groups on amphiphiles. The most hydro-phobic m a t e r i a l s have an HLB v a l u e of 1 to 10 while i n -c r e a s i n g HLB v a l u e s denote more h y d r o p h i l i c c h a r a c t e r . The u s e f u l n e s s of a n o n - i o n i c d e t e r g e n t may be p r e d i c t e d from 248. i t s HLB v a l u e . The s o l u b i l i z i n g potency i s g r e a t e r f o r detergents w i t h an HLB range of 12.5 t o 14.5 (183-187). The e f f e c t of dete r g e n t s as a c t i v a t o r s of enzyme a c t i v i t y , however, i s more dependent on the c r i t i c a l m i c e l l a r concen-t r a t o r Non-ionic detergent a c t i v a t o r s are o f t e n more e f f e c t i v e at or near the c r i t i c a l m i c e l l a r c o n c e n t r a t i o n but may be i n h i b i t o r y when added a t c o n c e n t r a t i o n s above the c r i t i c a l m i c e l l a r c o n c e n t r a t i o n (160). The r e l a t i o n s h i p between the p h y s i c a l p r o p e r t i e s of some of the detergents used i n t h i s study and t h e i r e f f e c t s on energy-independent transhydrogenase a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex p r e p a r a t i o n s i s summarized i n Table 14. Non-ionic d e t e r g e n t s of the Tween s e r i e s s t i m u l a t e d the enzyme a c t i v i t y at con-c e n t r a t i o n s of about the same order of magnitude as t h e i r c r i t i c a l m i c e l l a r c o n c e n t r a t i o n s . T h i s was e s p e c i a l l y t r u e of Tween 4 0 and Tween 60. The extent of a c t i v a t i o n by these d e t e r g e n t s d i d not seem to be r e l a t e d to the l e n g t h or composition of t h e i r f a t t y a c i d s i d e c h a i n s . T h i s c o n t r a s t s w i t h the l i p i d requirements of cytochrome c oxidase where amphiphiles of t h i s type regenerated higher a c t i v i t i e s i n l i p i d - d e p l e t e d p r e p a r a t i o n s , when the d e t e r -gent c o n t a i n e d u n s a t u r a t e d f a t t y a c i d s , f o r example Tween 80 (188). The d e t e r g e n t s T r i t o n X-100 and sodium c h o l a t e i n -h i b i t e d energy-independent transhydrogenase a c t i v i t y i n s o l u b l e r e s p i r a t o r y complex p r e p a r a t i o n s . Although T r i t o n Table 14. The R e l a t i o n s h i p Between P h y s i c a l P r o p e r t i e s of Some Detergents and t h e i r E f f e c t on Energy-independent Transhydrogenase A c t i v i t y i n S o l u b l e Respiratory' Complex P h y s i c a l P r o p e r t i e s E f f e c t on Transhydrogenase HLB* CMC* * Concen- A c t i v i t y mM t r a t i o n % of C o n t r o l mM Value T r i t o n X-100 13. 5 0.24 3. 2 81 L y s o l e c i t h i n - 0.019-0.19 3. 8 165 Sodium c h o l a t e 18 45 4. 9 43 B r i j 35 — _ 0. 062 336 36T - - 0. 04 292 58 15.7 0.077 0. 04 217 Tween 2 0 16.7 0.0482 0. 016 187 40 15.6 0.0223 0. 023 217 60 14. 9 0.0203 0. 03 238 80 15 0.0098 0. 025 193 * CMC: C r i t i c a l m i c e l l a r c o n c e n t r a t i o n ** HLB: H y d r o p h i l e - l i p o p h i l e balance HLB and CMC va l u e s f o r sodium c h o l a t e were taken from r e f . 187. Those f o r the other d e t e r g e n t s were from r e f . 181. The experiments are d e s c r i b e d i n the RESULTS s e c t i o n . 250. X-100 does not appear t o induce c o n f o r m a t i o n a l changes i n p r o t e i n s (189-192) the c o n c e n t r a t i o n used i n the prese n t study (3.2 mM) was w e l l above the c r i t i c a l m i c e l l a r con-c e n t r a t i o n s and wit h an HLB of 13.5 which i s f a v o u r a b l e f o r l i p i d e x t r a c t i o n , i t may have i n h i b i t e d the enzyme a c t i v i t y by removing e s s e n t i a l l i p i d (s) remaining i n the p r e p a r a t i o n . The i n a c t i v a t i o n of the enzyme by sodium c h o l a t e at a c o n c e n t r a t i o n f a r below i t s c r i t i c a l m i c e l l a r c o n c e n t r a t i o n i s of i n t e r e s t . There i s evidence t h a t b i l e s a l t s may cause l y s i s of membranes a t c o n c e n t r a t i o n s f a r below t h e i r c r i t i c a l m i c e l l a r c o n c e n t r a t i o n s (193,194), suggesting t h a t some b i n d i n g must occur a t these c o n c e n t r a -t i o n s . The co n n e c t i o n between c r i t i c a l m i c e l l a r c o n c e n t r a -t i o n s of b i l e s a l t s and t h e i r r e l a t i o n t o b i n d i n g w i t h membranes i s s t i l l an >open q u e s t i o n . Rydstrom e t a_l. (101) r e p o r t e d the i r r e v e r s i b l e i n a c t i v a t i o n of energy-independent transhydrogenase p r e p a r a t i o n s ( c o n t a i n i n g 0.04 umol phospho-l i p i d per mg p r o t e i n ) from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s by h i g h c o n c e n t r a t i o n s of T r i t o n X-100 and sodium c h o l a t e . On the other hand, l y s o l e c i t h i n and other phospho-l i p i d s a c t i v a t e d the enzyme a c t i v i t y , which, they suggested, may r e f l e c t a.hydrophobic i n t e r a c t i o n between the p r o t e i n and the p h o s p h o l i p i d r a t h e r than a d i s p e r s i o n phenomenon. In the prese n t study, l y s o l e c i t h i n (even a t concen-t r a t i o n s f a r above i t s c r i t i c a l m i c e l l a r c o n c e n t r a t i o n ) 251. s t i m u l a t e d the energy-independent transhydrogenase a c t i v i t y i n the s o l u b l e r e s p i r a t o r y complex. T h i s e f f e c t has a l s o been r e p o r t e d by o t h e r s u s i n g d e l i p i d a t e d p r e p a r a t i o n s of the enzyme (86,101,195). T h i s suggests t h a t l y s o l e c i t h i n , i n c o n t r a s t to T r i t o n X-100 and sodium c h o l a t e , i s not o n l y an e f f i c i e n t d e t ergent, but a l s o a p h o s p h o l i p i d , capable of s a t i s f y i n g a l i p i d requirement f o r the enzyme. Sin c e the common f e a t u r e between l y s o l e c i t h i n and the n o n i o n i c detergents of the Tween and B r i j s e r i e s i s the presence of f a t t y a c i d s i d e c h a i n s , the s t i m u l a t o r y e f f e c t of p a l m i t i c a c i d on energy-independent transhydrogenase a c t i v -i t y , i n :. s o l u b l e r e s p i r a t o r y complex i s c o n s i s t e n t w i t h the need f o r s p e c i f i c i n t e r a c t i o n s of f a t t y a c y l chains with the enzyme. S t i m u l a t i o n by p a l m i t i c a c i d has not been r e p o r t e d elsewhere f o r energy-independent transhydrogenase, but has been found to occur i n other enzymes w i t h phospho-l i p i d dependence (160). Soybean p h o s p h o l i p i d ( a s o l e c t i n ) s t i m u l a t e d the energy-independent transhydrogenase a c t i v i t y i n a p a r t i a l l y p u r i f i e d l i p i d - d e p l e t e d p r e p a r a t i o n of the enzyme ( F i g . 23) but not i n s o l u b l e r e s p i r a t o r y complex ( F i g . 22). In the l a t t e r p r e p a r a t i o n , the enzyme a c t i v i t y was i n h i b i t e d at a s o l e c t i n c o n c e n t r a t i o n s above 13 yM. The reason f o r t h i s i s not c l e a r . One p o s s i b l e e x p l a n a t i o n c o u l d be the d i f f e r e n c e i n the amount and composition of r e s i d u a l 252. p h o s p h o l i p i d p r e s e n t i n the enzyme p r e p a r a t i o n . The aso-l e c t i n was not incubated f o r any l e n g t h of time w i t h the enzyme p r e p a r a t i o n . In some i n s t a n c e s , i n c u b a t i o n of aso-l e c t i n w i t h enzyme p r e p a r a t i o n s f o r long p e r i o d s of time i s c r u c i a l i n order f o r the f u l l e x p r e s s i o n of enzymatic a c t i v i t y (155). Anderson and F i s h e r (7) r e p o r t e d t h a t s o n i c a t i o n of p u r i f i e d transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s w i t h soybean p h o s p h a t i d y l c h o l i n e i n i t i a l l y r e s u l t e d i n a s l i g h t i n a c t i v a t i o n , f o l l o w e d by a slow r e c o n s t i t u t i o n of enzyme a c t i v i t y . Another p o s s i b i l i t y f o r the d i f f e r e n c e observed f o r the e f f e c t of a s o l e c t i n on the two types of p r e p a r a t i o n s i s the presence of sodium c h o l a t e i n the b u f f e r c o n t a i n i n g the s o l u b l e r e s p i r a t o r y complex. A f t e r d i l u t i o n i n t o the assay mixture t h i s would o n l y amount t o 50 u g per ml. However, i n r e c o n s t i t u t i o n experiments u s i n g p h o s p h a t i d y l c h o l i n e , Ragan and Racker r e p o r t e d t h a t d i a l y s i s t o remove a l l t r a c e s of c h o l a t e was an a b s o l u t e requirement f o r r e c o n s t i t u t i o n of enzyme a c t i v i t y t o occur (196). The d e l i p i d a t e d p r e p a r a t i o n of energy-independent transhydrogenase had been d i a l y z e d p r i o r t o a c t i v a t i o n with a s o l e c t i n . Energy-independent transhydrogenase a c t i v i t y i n a d e l i p i d a t e d p r e p a r a t i o n of the enzyme was most e f f e c t i v e l y s t i m u l a t e d by E. c o l i p h o s p h o l i p i d s ( F i g . 23). T h i s e f f e c t was also observed by other laboratories for the enzyme from E. c o l i (89). Mixed mitochondrial phospholipids are the most e f f e c t i v e l i p i d preparations for stimulating the beef-heart transhydrogenase (7,101). In t h i s investigation, r e a c t i v a t i o n of energy-independent transhydrogenase a c t i v i t y by s p e c i f i c p u r i f i e d phospho-l i p i d s was not attempted. Data from other studies i n d i -cate that c a r d i o l i p i n extracts from the same source as the enzyme stimulate the a c t i v i t y at lower concentrations than any other phospholipid (89,101). At higher concentrations, beef-heart c a r d i o l i p i n was i n h i b i t o r y probably because of the increased negative charge i n the environment of the enzyme. Mixtures of phospholipids, however, rather than i n d i v i d u a l ones,appeared to be more e f f i c i e n t with respect to the amount required for half-maximal a c t i v a t i o n , i n d i -cating that the acti v a t i n g e f f e c t s of the in d i v i d u a l phospholipids was additive. Rydstrom et a_l. (101) suggest that the ro l e of the phospholipids in t h i s respect may be as agents to s t a b i l i z e an active conformation of the trans-hydrogenase, by occupying hydrophobic surfaces on the molecule, which are normally exposed to the hydrophobic i n t e r i o r of the intact membrane. 254. Ste a d y - s t a t e k i n e t i c s of the energy-independent t r a n s - hydrogenase r e a c t i o n The transhydrogenase of E. c o l i (68), s i m i l a r t o the transhydrogenase from b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s (8,9,7 9)possesses separate b i n d i n g s i t e s on the enzyme f o r the s u b s t r a t e s NAD(H) and NADP(H). Both the energy-independent and ATP-driven energy-dependent r e a c t i o n s are s t e r e o s p e c i f i c w i t h regards t o the 4B hydrogen atom of NADPH and the 4A hydrogen atom of NADH. In t h i s study, the p r o p e r t i e s of these t w o - s i t e s and the mechanism of a c t i o n of the enzyme were i n v e s t i g a t e d . E a r l i e r s t u d i e s on the k i n e t i c mechanism of the energy-independent transhydrogenases of be e f - h e a r t m i t o c h o n d r i a (117,118) and E. c o l i (68) had concluded t h a t the r e a c t i o n proceeded by way of a Theorell-Chance mechanism. In these s t u d i e s the l i n e s i n Lineweaver-Burk p l o t s i n t e r s e c t e d below the a b c i s s a f o r the r e a c t i o n i n the forward d i r e c t i o n and on the a b c i s s a f o r t h a t i n the r e v e r s e d i r e c t i o n (118). These r e s u l t s are c o n s i s t e n t w i t h a random s e q u e n t i a l mechanism and c l e a r l y r u l e out a Theorell-Chance mechanism. Had the r e a c t i o n indeed proceeded by a Theorell-Chance mechanism, the l i n e s i n t e r s e c t i n g below the a b c i s s a f o r the r e a c t i o n i n one d i r e c t i o n should have i n t e r s e c t e d at an equal d i s t a n c e above the a b c i s s a f o r t h a t i n the r e v e r s e d i r e c t i o n (197). In the presen t i n v e s t i g a t i o n product i n h i b i t i o n s t u d i e s ( F i g s . 3 9 and 4 0) r e v e a l e d t h a t APNADH was c o m p e t i t i v e w i t h r e s p e c t t o APNAD*1" but non-^competititve with r e s p e c t t o NADPH. S i m i l a r l y , NADP + was c o m p e t i t i v e w i t h r e s p e c t t o NADPH but non-competitive w i t h r e s p e c t t o APNAD +. T h i s p a t t e r n of i n h i b i t i o n i s d i a g n o s t i c of both a r a p i d e q u i l -i b r i u m random b i r e a c t a n t mechanism (12 0) and a T h e o r e l l -Chance mechanism (119,165). S i m i l a r p a t t e r n s of i n h i b i t i o n were obtained by others f o r the E. c o l i enzyme (68,92) and the m i t o c h o n d r i a l enzyme (117,118) although these workers e l i m i n a t e d the p o s s i b i l i t y of a r a p i d e q u i l i b r i u m random b i r e a c t a n t mechanism i n favour of a Theorell-Chance mechan-ism. The b a s i s f o r such an i n t e r p r e t a t i o n of t h e i r r e s u l t s was an e a r l i e r r e p o r t by C l e l a n d (165) which s t a t e d t h a t the former mechanism would show no product i n h i b i t i o n be-tween a s u b s t r a t e - p r o d u c t p a i r , but t h i s assumption has r e c e n t l y been shown t o be i n c o r r e c t (198), The r e s u l t s of product i n h i b i t i o n s t u d i e s are c o n s i s t e n t w i t h e i t h e r one of the two p o s s i b l e mechanisms. In t h i s t h e s i s , t h e r e -f o r e , an attempt was made to d i s t i n g u i s h between both of these mechanisms, and determine which one governed the mechanism of tr a n s h y d r o g e n a t i o n . D o u b l e - r e c i p r o c a l p l o t s of the v e l o c i t i e s of the r e a c t i o n s w i t h v a r y i n g c o n c e n t r a t i o n s of e i t h e r s u b s t r a t e 256. at d i f f e r e n t f i x e d c o n c e n t r a t i o n s of the other s u b s t r a t e ( F i g s . 33 and 34) were c o n s t r u c t e d . The convergent nature of the l i n e s o b tained a t i n t e r m e d i a t e f i x e d l e v e l s of NADPH (97 t o 241 yM) and APNAD + (48 t o 491 yM) i n d i c a t e d t h a t the energy-independent transhydrogenase r e a c t i o n proceeds by way of a s e q u e n t i a l mechanism (119,120). Competition between each s u b s t r a t e at hig h c o n c e n t r a t i o n s of the other f i x e d s u b s t r a t e was suggested by the i n t e r -s e c t i o n on the o r d i n a t e by the l i n e s obtained a t 482 yM NADPH and 982 yM APNAD + or g r e a t e r . The p a t t e r n of l i n e s i n both of these p l o t s are t y p i c a l of a r a p i d e q u i l i b r i u m random b i r e a c t a n t system i n which each s u b s t r a t e can a c t at the b i n d i n g s i t e of the other s u b s t r a t e t o form a dead-end complex (120). T h i s i s not improbable i n view of the s t r u c t u r a l s i m i l a r i t i e s between APNAD + and NADPH. F u r t h e r support f o r the hypothesis t h a t NADPH can occupy the b i n d -i n g s i t e of APNAD + i s pro v i d e d by the i n h i b i t i o n obtained at e l e v a t e d c o n c e n t r a t i o n of NADPH i n F i g . 34. T h i s e f f e c t was a b o l i s h e d as the c o n c e n t r a t i o n of APNAD + was i n c r e a s e d . S u b s t r a t e i n h i b i t i o n by NADPH was observed i n both the membrane-bound enzyme ( F i g . 6, panel Y) and i n the p a r t i a l l y p u r i f i e d enzyme from the s o l u b l e r e s p i r a t o r y complex ( F i g . 24, panel Y ) . I t was r e p o r t e d i n other s t u d i e s t o occur i n the membrane-bound enzyme of E. c o l i grown on 2 5 7 . glucose-yeast extract (68), but was interpreted to be related to a unique control mechanism, related to the NADPH requirements, under these conditions of growth. The trans-hydrogenase from A. v i n e l a n d i i has also shown substrate i n h i b i t i o n at high concentrations of NADPH and has been reported to proceed by way of a rapid equilibrium random bireactant mechanism (24). In order to di s t i n g u i s h further between the rapid equilibrium random bireactant mechanism and the Theorell-Chance mechanism, the energy-independent transhydrogenase reaction was ca r r i e d out i n the presence of the alternate substrates, NAD+ and deamino-NADPH (Fig. 43). A competitive re l a t i o n s h i p was demonstrated between NAD+ and APNAD+ and between deamino-NADPH and NADPH, while a non-competitive re l a t i o n s h i p was shown to ex i s t between NAD+ and NADPH and between deamino-NADPH and APNAD4". These re s u l t s are consistent with a rapid equilibrium random bireactant mechanism. Had the reaction been dictated by a Theorell-Chance mechanism, there would have existed a competitive re l a t i o n s h i p between the alternate substrate and the (normal) f i r s t substrate, but parabolic double-reciprocal plots would have been observed for i t s r e l a t i o n s h i p to the second substrate (166). 258. The e f f e c t s of a c o m p e t i t i v e i n h i b i t o r f o r each sub-s t r a t e a l l o w s e q u e n t i a l mechanisms t o be c l a s s i f i e d as being ordered or random. ADP and 51-AMP were c o m p e t i t i v e i n h i b i t o r s w i t h r e s p e c t t o APNAD + ( F i g s . 26, 28 and 29) and non-competitive i n h i b i t o r s w i t h r e s p e c t t o NADPH. On the other hand, 21-AMP was a c o m p e t i t i v e i n h i b i t o r w i t h r e s p e c t t o NADPH and a non-competitive i n h i b i t o r w i t h r e s p e c t t o APNAD + ( F i g . 25). These p a t t e r n s e s t a b l i s h t h a t the energy-independent transhydrogenase r e a c t i o n i s d i c t a t e d by a random mechanism. I f the mechanism were ordered (as i n the case of a Theorell-Chance mechanism, a c o m p e t i t i v e i n h i b i t o r of the second s u b s t r a t e adding to the enzyme would be uncompetitive w i t h r e s p e c t t o the f i r s t s u b s t r a t e (120). The p l o t s f o r the i n h i b i t i o n of the m i t o c h o n d r i a l enzyme by 2'-AMP versus NAD + c o n c e n t r a t i o n s , p u b l i s h e d by Rydstrom (83) are c l e a r l y converging and y e t the i n h i b i t i o n was i n t e r p r e t e d as being uncompetitive by t h i s author, who regarded the l i n e s as being p a r a l l e l . T h i s data was used t o support h i s p o s t u l a t e d mechanism as being a Theorell-Chance one. The r e s u l t s of Hanson (92) and Houghton e t a l . (68), u s i n g the same i n h i b i t o r s , were s i m i l a r t o those o b t a i n e d i n the pr e s e n t s t u d i e s . The former authors concluded, as I have, t h a t the mechanism of energy-independent t r a n s h y d r o g e n a t i o n i n E. c o l i i s a r a p i d e q u i l i b r i u m random b i r e a c t a n t one. The hypothesis that each substrate, at high concentra-tions, i s capable of binding at the s i t e of the other sub-strate, may be used to explain some of the anomalous res u l t s obtained using high concentrations of 51-AMP and ADP. It i s possible that these nucleotides, although s p e c i f i c for the APNAD4" s i t e , can also bind at the NADPH s i t e at very high concentrations of i n h i b i t o r and r e l a t i v e l y low concen-trations of substrate. This may be the reason for the curved Dixon plots observed in Figs. 28, 29 and 31 at higher concentrations of ADP and 5'-AMP. This i n h i b i t i o n was not due to the introduction of K + concomitant with high concentrations of i n h i b i t o r s . Interaction of the i n h i b i t o r s at the s i t e of the other substrate may also be the reason for the apparently competitive r e l a t i o n s h i p between 21-AMP and APNAD4" (Fig. 27, panel X). The change from a non-competitive rel a t i o n s h i p (Fig. 25) to a com-p e t i t i v e one at higher concentrations of 21-AMP i s rem-iniscent of the change i n rel a t i o n s h i p between APNAD4" and NADPH (Figs. 3 3 and 34) as well as between APNADH and NADPH (Fig. 4 0) at elevated concentrations of the substrates. The e f f e c t of NADH upon the energy-independent transhydrogenase i s of in t e r e s t . The reaction was i n -hibi t e d at a concentration greater than 0.2 mM NADH, possibly due to competition, with APNAD4*. At lower c o n c e n t r a t i o n s (0.1 mM or l e s s ) the enzyme a c t i v i t y was s t i m u l a t e d ( F i g s . 41 and 42). The s t i m u l a t i o n observed i s not due to the r e d u c t i o n of APNAD + by NADH, s i n c e the r a t e f o r such a r e a c t i o n i n b e e f - h e a r t s u b m i t o c h o n d r i a l p a r t i c l e s i s l e s s than 1% t h a t of the r e d u c t i o n of APNAD + by NADPH (199). In E. c o l i membrane p a r t i c l e p r e p a r a t i o n s no other enzyme can c a t a l y z e the same r e a c t i o n as t h a t of the transhydrogenase (7 6 ) . The presence of low c o n c e n t r a t i o n s of NADH i n the r e a c t i o n mixture a f f e c t e d both the apparent v a l u e s and the maximum v e l o c i t y of the r e a c t i o n ( F i g . 42). T h i s suggests t h a t t h e r e may be an a l l o s t e r i c s i t e on the transhydrogenase capable of b i n d i n g NADH, which a t low c o n c e n t r a t i o n s causes a c o n f o r m a t i o n a l change i n the enzyme r e s u l t i n g i n i n c r e a s e d a c t i v i t y . Evidence f o r such a c o n f o r m a t i o n a l change w i l l be d i s c u s s e d i n the next s e c t i o n . The a c t i v e s i t e of the energy-independent transhydrogenase The energy-independent transhydrogenase was i n a c t i v a t e d by phenyl g l y o x a l or 2,3-butanedione i n borate b u f f e r ( F i g . 44) suggesting the presence of a r g i n y l groups a t or near the a c t i v e s i t e of the enzyme. From the p l o t s of the p s e u d o - f i r s t - o r d e r r a t e constant versus the l o g a r i t h m of the c o n c e n t r a t i o n of the modifying reagent, i t was estimated t h a t the i n a c t i v a t i o n was due t o the m o d i f i c a t i o n of one a r g i n y l r e s i d u e per a c t i v e s i t e ' of the enzyme. The i n h i b i t o r s 21-AMP and 5'-AMP a f f o r d e d some p r o t e c t i o n a g a i n s t i n a c t i v a t i o n of the enzyme by 2,3-butanedione ( F i g . 46). In the case where the bound i n h i b i t o r renders the e s s e n t i a l a r g i n i n e completely i n a c c e s s i b l e f o r m o d i f i c a -t i o n by 2,3-butanedione k_ = K j k Q " K . + [I] where k and k° are the p s e u d o - f i r s t - o r d e r i n a c t i v a t i o n r a t e c o n s t a n t s i n the presence and absence r e s p e c t i v e l y , of i n h i b i t o r . i s the en z y m e - i n h i b i t o r d i s s o c i a t i o n con-s t a n t , and [I] i s the i n h i b i t o r c o n c e n t r a t i o n (200). Using t h i s e q u a t i o n , the i L v a l u e s f o r 2'-AMP and 51-AMP were c a l c u l a t e d t o be 38 and 23 mM, r e s p e c t i v e l y . The v a l u e s f o r the b i n d i n g of these n u c l e o t i d e s a t the NADPH s i t e and APNAD + s i t e i n the absence of 2,3-butanedione were 3.9 and 2.0 mM, r e s p e c t i v e l y . I t i s p o s s i b l e t h a t the m o d i f i c a -t i o n of a n o n - e s s e n t i a l r e s i d u e near the a c t i v e s i t e l e d to i n a c t i v a t i o n due t o s t e r i c hindrance of the a c t i v e c e n t r e , by the i n t r o d u c t i o n of the 2,3-butanedione. Im-po r t a n t a l t e r a t i o n s of charge or c o n f o r m a t i o n a l changes may have o c c u r r e d as a r e s u l t of the m o d i f i c a t i o n s (201,202). T h i s , i n t u r n , c o u l d p o s s i b l y a f f e c t the enzy m e - i n h i b i t o r (or enzyme-substrate) d i s s o c i a t i o n c o n s t a n t s . 262. P a r t i a l protection against i n a c t i v a t i o n of the enzyme by 2,3-butanedione, was also afforded by the substrates APNAD4" (Fig. 4 5) and NAD+ as well as the product NADP+ (Fig. 4 6). A mixture of NAD and NADP afforded greater protection against i n a c t i v a t i o n than did each pyridine nucleotide alone at the same t o t a l concentration of the pyridine nucleotides i n the mixture (Fig. 47). The d i f f e r -ence i n pseudo-first-order rate constants between the ind i v i d u a l substrates and the mixture may be due to s t e r i c or cooperative i n t e r a c t i o n between both s i t e s . These res u l t s are similar to those observed for the i n h i b i t i o n by 2,3-butanedione of the enzymes from beef-heart submito-chondrial p a r t i c l e s (113) and R. rubrum chromatophores (98). - In contrast to the e f f e c t of NAD+ and NADP4", low con-centrations of NADH and NADPH enhanced the in a c t i v a t i n g a f f e c t of the 2,3-butanedione on the transhydrogenase a c t i v i t y (Fig. 48). High concentrations (17 mM) of NADH, however, completely protected the enzyme against i n a c t i v a -t i o n by the 2,3-butanedione. This i s evidence that binding of low concentrations of NADH (2.4 mM) or NADPH (6 mM) cause a conformational change i n the enzyme, rendering the argi n y l group more accessible to modification. The presence of an a l l o s t e r i c s ite(s) on the enzyme, for which the reduced pyridine nucleotides have a greater a f f i n i t y than 263. for the substrate binding s i t e would be i n agreement with the stimulating e f f e c t of low concentrations of NADH on the energy-independent transhydrogenase reaction, mentioned i n the previous section. The concept of an a l l o s t e r i c s i t e on the transhydrog-enase, with a greater a f f i n i t y than the active s i t e for NADPH, i s somewhat similar to that proposed by Fisher and Gu i l l o r y for the enzyme of R. rubrum (97,98). In t h i s case binding of NADP or NADPH, but not of NAD , a l t e r s the conformation of the membrane component of the enzyme (114). Further evidence that binding of low concentrations of NADPH or NADH cause a conformational change i n the energy-independent transhydrogenase i s seen from the inac-t i v a t i o n of the enzyme by TPCK-trypsin i n the presence of oxidized or reduced pyridine nucleotides (Fig. 49). The resul t s were sim i l a r to those observed with 2,3-butanedione in that low concentrations of NADH or NADPH enhanced the ina c t i v a t i n g e f f e c t of try p s i n upon the enzyme, whereas protection against i n a c t i v a t i o n was afforded by high con-centrations of NADH. The oxidized pyridine nucleotides did not s i g n i f i c a n t l y enhance the t r y p t i c digestion of the enzyme, but protected i t from i n a c t i v a t i o n . The re s u l t s of the t r y p t i c digestion experiments are similar to those reported for the energy-independent transhydrogenase from beef-heart submitochondrial p a r t i c l e s , where i n a c t i v a t i o n was enhanced by the presence of NADPH but not by NADP (7,115,121). However, i n t h i s case, NADH d i d not s t i m u l a t e t r y p t i c - d i g e s t i o n , but a f f o r d e d p r o t e c t i o n a g a i n s t i n a c -t i v a t i o n , i n d i c a t i n g t h a t i n the m i t o c h o n d r i a l t r a n s -hydrogenase, the a c c e s s i b l e l y s y l . o r a r g i n y l r e s i d u e s may be a t the NAD-binding s i t e (7). F u r t h e r evidence t h a t the NADP +-enzyme complex has a d i f f e r e n t conformation from the NADPH-enzyme complex i s e v i d e n t from r e p o r t s t h a t NADP + l a b i l i z e s the enzyme to thermal i n a c t i v a t i o n , w h i l e NADPH s t a b i l i z e s i t (115,121). A l s o , s u l p h y d r y l group m o d i f i c a t i o n by N - e t h y l maleimide w i t h e i t h e r the mito-c h o n d r i a l enzyme or the enzyme from E. c o l i was enhanced by NADPH, whereas NADP + a f f o r d e d p r o t e c t i o n a g a i n s t m o d i f i c a t i o n (8 9,122). i n the case of the enzyme f o r E. c o l i , i n c o n t r a s t to t h a t from m i t o c h o n d r i a , NAD + a l s o a f f o r d e d p r o t e c t i o n a g a i n s t i n h i b i t i o n by N-ethylmaleimide. Thus, the r e s u l t s presented i n t h i s study, although d i f f e r e n t i n d e t a i l from those of the m i t o c h o n d r i a l enzyme, i n d i c a t e t h a t the energy-independent transhydrogenase of E. c o l i undergoes d i f f e r e n t c o n f o r m a t i o n a l changes. To-gether with the r e s u l t s of Houghton e t a l . (68), i t appears t h a t b i n d i n g of low c o n c e n t r a t i o n of reduced p y r i d i n e n u c l e o t i d e s a t an a l l o s t e r i c s i t e ( s ) on the enzyme causes a c o n f o r m a t i o n a l change such t h a t the a r g i n y l and s u l p h y d r y l r e s i d u e s become more exposed and thus a c c e s s i b l e t o modifying agents. C o n c l u s i o n In c o n c l u s i o n , t h i s work has demonstrated t h a t the s t e a d y - s t a t e k i n e t i c s of tr a n s h y d r o g e n a t i o n i s governed by a r a p i d e q u i l i b r i u m random b i r e a c t a n t mechanism, r a t h e r than a Theorell-Chance one. Chemical m o d i f i c a t i o n of the enzyme i n d i c a t e s the presence of an a r g i n y l r e s i d u e a t or near the a c t i v e s i t e . Conformational changes occur upon b i n d i n g of reduced s u b s t r a t e s t o the enzyme d u r i n g the course of the r e a c t i o n . F u r t h e r s t u d i e s r e q u i r e the i s o l a t i o n of the enzyme i n the pure form. Although p a r t i a l p u r i f i c a t i o n of the p y r i d i n e n u c l e o t i d e transhydrogenase was obta i n e d , the high i n s t a b i l i t y of the enzyme prevented the s u c c e s s f u l com-p l e t i o n of t h i s p r o j e c t . Work from other l a b o r a t o r i e s has r e c e n t l y proved the presence of p r o t e o l y t i c enzymes i n s i m i l a r p r e p a r a t i o n s as those i n v e s t i g a t e d i n the prese n t study. Since p r o t e o l y s i s seems to have been r e s p o n s i b l e f o r the d e g r a d a t i o n of the enzyme, f u t u r e p u r i f i c a t i o n attempts should take t h i s f a c t o r i n t o c o n s i d e r a t i o n . 2 6 6 . PART I I 267. P h o t o o x i d a t i o n of reduced p y r i d i n e n u c l e o t i d e s by 2,3- butanedione and phenyl g l y o x a l During the course of i n v e s t i g a t i n g the involvement of a r g i n y l r e s i d u e s i n the a c t i v e s i t e of the energy-independent transhydrogenase w i t h 2,3-butanedione, an o b s e r v a t i o n i n -d i c a t i n g the p o s s i b l e r e a c t i o n between 2,3-butanedione and NADPH was made. When membrane p a r t i c l e s f o r E. c o l i W6 were incubated (under ambient l a b o r a t o r y l i g h t i n g ) w i t h 2,3-butanedione i n sodium b o r a t e b u f f e r i n t h e presence o f NADPH, o x i d a t i o n of the l a t t e r o c c u r r e d as evidenced by a decrease i n the absorbance at 34 0 nm. To t e s t whether t h i s e f f e c t was enzyme-mediated or due t o other f a c t o r s , an experiment was s e t up i n which NADPH was incubated a t 22°C w i t h 2,3-butanedione i n 50 mM sodium borate b u f f e r , pH 7.8, or i n 50 mM potassium phosphate b u f f e r , pH 7.0 i n the presence or absence of the membrane p a r t i c l e suspension. The a b s o r p t i o n spectrum was scanned between 28 0 and 400 nm a t the onset of the experiment and a f t e r 3 0 minutes, the r e s u l t s are shown i n Table 15. The absorbance a t 340 nm, due t o NADPH, decreased by 63 t o 7 0% i n the i n c u b a t i o n mixtures c o n t a i n i n g 2,3-butanedione. The presence of membrane p a r t i c l e s d i d not make a s i g n i f i c a n t c o n t r i b u t i o n t o t h i s decrease. The o x i d a t i o n was not dependent on the type of b u f f e r (sodium borate or potassium phosphate) used. Table 1 5 . E f f e c t of Membrane P a r t i c l e s and 2,3-Butanedione on the Oxidation of NADPH Buffer Membrane P a r t i c l e s yg Protein Phosphate mM Borate mM 2,3-butanedione mM NADPH ox i -dized* 58 0 5 0 1 3 7 0 0 0 5 0 1 3 63 58 0 5 0 0 3 . 1 0 0 5 0 0 2 . 8 0 5 0 0 0 3 . 4 0 5 0 0 1 3 6 9 % of t o t a l NADPH present i n i t i a l l y i n assay mixture. The reaction mixture contained 0 . 1 0 6 mM NADPH i n 5 0 mM sodium borate buffer, pH 7 . 8 , or 5 0 mM potassium phos-phate buffer, pH 7 . 8 , i n a f i n a l volume of 2 ml. In some cases the reaction mixture contained 1 3 mM 2 , 3 -butanedione or 58 yg of membrane p a r t i c l e protein as i n -dicated. The solutions were scanned between 28 0 nm and 4 0 0 nm and incubated at 2 2 ° C under ambient laboratory l i g h t i n g for 3 0 min, as described i n MATERIALS AND METHODS. The amount of NADPH oxidized a f t e r 3 0 min of i r r a d i a t i o n i s reported as a percentage of the amount of NADPH i n i t i a l l y presented i n the assay mixture. The reduced p y r i d i n e n u c l e o t i d e , NADH, was a l s o o x i -d i z a b l e by 2 , 3-butanedione. The o x i d a t i o n of NADH by 2 , 3-butanedione i n 50 mM sodium borate b u f f e r , pH 7 . 8 , i s shown i n F i g . 5 0 . Incubation of NADH w i t h 2 , 3-butanedione f o r 3 0 minutes under ambient l a b o r a t o r y l i g h t i n g , r e s u l t e d i n the o x i d a t i o n of about one-half of the NADH prese n t . The NADH was completely o x i d i z e d i n other experiments, i n which the r e a c t i o n was allowed t o proceed f o r longer p e r i o d s . The r e a c t i o n showed p s e u d o - f i r s t - o r d e r k i n e t i c s w i t h r e s p e c t t o the c o n c e n t r a t i o n of NADH. I f , however, the i n c u b a t i o n was c a r r i e d out i n the absence of l i g h t (scan 3 0 ' , F i g . 5 0 ) , no o x i d a t i o n o f NADH oc c u r r e d . T h i s may be taken as evidence t h a t the r e a c t i o n between 2 , 3 -butanedione and NADH (or NADPH) i n v o l v e s p h o t o o x i d a t i o n . P h o t o o x i d a t i o n of NADH was a l s o observed i n the presence of phenyl g l y o x a l ( F i g . 51) although the r e a c t i o n was much slower than t h a t w i t h 2 , 3-butanedione. The r a t e of NADH o x i d a t i o n i n the presence o f 13 mM phenyl g l y o x a l was 14 nmol per hour compared w i t h 521 nmol per hour i n the presence of 13 mM 2 , 3-butanedione. I t i s f o r t h i s reason t h a t f u r t h e r i n v e s t i g a t i o n o f the p h o t o o x i d a t i o n of NADH was c o n f i n e d t o t h a t mediated by 2 , 3-butanedione i n s t e a d of phenyl g l y o x a l . Riordan (167) observed t h a t when 1.5 ml of 2 , 3-butanedione was mixed w i t h 8 . 5 ml of 50 mM sodium borate b u f f e r , the pH changed from 8 . 6 t o 3 . 8 . F i g . 50. P h o t o o x i d a t i o n of NADH by 2,3-butanedione. The r e a c t i o n mixture c o n t a i n e d 0.12 mM NADH, 6.7 mM 2,3-butanedione and 50 mM sodium borate b u f f e r , pH 7.8 i n a f i n a l volume of 2 ml. The experiment was performed as d e s c r i b e d i n MATERIALS AND METHODS. The time of ex-posure t o ambient l i g h t i s g i v e n i n minutes. 30', the r e a c t i o n mixture was kept f o r 30 min i n the dark. 0 I I 1 1 400 360 320 280 Wavelength -nm F i g . 51. P h o t o o x i d a t i o n o f NADH by phenyl g l y o x a l . The r e a c t i o n mixture c o n t a i n e d 0.12 mM NADH, and 50 mM sodium borate b u f f e r , pH 7.8 i n a f i n a l volume of 2 ml. The r e a c t i o n was c a r r i e d out i n the presence ( l e f t panel) or absence ( r i g h t panel) o f 13.3 mM phenyl g l y o x a l , r e -s p e c t i v e l y . The experiment was performed as d e s c r i b e d f o r 2,3-butanedione i n MATERIALS AND METHODS. The time of exposure t o ambient l i g h t i s g i v e n i n h. 2 7 3 . Wavelength ._ nm 274. Furthermore, the a b s o r p t i o n maximum at 408 nm (e = 1.15 M ^cm" 1) due t o 2,3-butanedione was a b o l i s h e d by the a d d i t i o n of 0.5 M sodium borate b u f f e r , pH 7.5, w h i l e the i n t e n s i t y of the peak at 284 nm (e = 9.75 M ^cm 1) was reduced by about 75%. These r e s u l t s i n d i c a t e d t h a t borate i n t e r a c t e d w i t h the dione. I t was of i n t e r e s t , t h e r e f o r e , i n the present study, to determine whether sodium borate b u f f e r , pH 7.8, a t the c o n c e n t r a t i o n used i n the photo-o x i d a t i o n s t u d i e s , a f f e c t e d the a b s o r p t i o n spectrum of 2,3-butanedione. The a b s o r p t i o n spectrum of a s o l u t i o n of 2,3-butanedione i n water showed two maxima, one a t 408 nm (e = 5.47 M '-cm 1) and another a t 283 nm (e = 25.74 M~ 1•cm" 1). In the presence of 50 mM sodium borate b u f f e r , pH 7.8, t h e r e was no a l t e r -a t i o n i n the p o s i t i o n of the a b s o r p t i o n maxima of the 2,3-butanedione, and the molar e x t i n c t i o n c o e f f i c i e n t s were only s l i g h t l y decreased from 5.47 t o 4.86 M 1•cm 1 f o r the band at 408 nm and from 25.74 t o 24.65 M - 1-cm - 1 f o r t h a t a t 283 nm. These v a l u e s are 89% and 96%, r e s p e c t i v e l y , of the v a l u e s obtained u s i n g water i n s t e a d of borate b u f f e r . Thus i t seems u n l i k e l y t h a t t h e r e i s s i g n i f i c a n t complex formation between 2,3-butanedione and borate under the c o n d i t i o n s of the p h o t o o x i d a t i o n experiments. Since the p h o t o o x i d a t i v e r e a c t i o n between 2,3-' butanedione and NADH were assessed on the b a s i s of the absorbance changes a t 3 40 nm due t o NADH, i t was e s s e n t i a l to ensure t h a t the absorbance a t 4 08 nm due t o 2,3-butanedione, d i d not i n t e r f e r e w i t h the measurements at 340 nm. A s o l u t i o n of 53 yM NADH was mixed w i t h 27 mM 2,3-butanedione i n the presence of 50 mM sodium borate b u f f e r , pH 7.8. The mixture, scanned between 3 00 and 500 nm ( F i g . 52), r e v e a l e d an a b s o r p t i o n maximum a t 340 nm due to NADH and a shoulder a t about 400 nm, due t o the 2,3-butanedione. I r r a d i a t i o n o f the mixture f o r lh hours under ambient l a b o r a t o r y l i g h t i n g r e s u l t e d i n the a b o l i -t i o n o f more than 90% of the absorbance a t 3 40 nm and i n the appearance of a w e l l - d e f i n e d peak a t 400 nm. T h i s i n d i c a t e s t h a t although the NADH had masked the absorbance, due t o 2,3-butanedione, the c o n t r i b u t i o n , i f any, of the l a t t e r t o the absorbance a t 340 nm was l e s s than 10%. The e f f e c t o f the c o n c e n t r a t i o n of 2,3-butanedione on the r a t e of the p h o t o o x i d a t i o n of NADH i s shown i n F i g . 53 ( l e f t p a n e l ) . The r a t e of p h o t o o x i d a t i o n was found t o be l i n e a r l y r e l a t e d t o the c o n c e n t r a t i o n of the 2,3-butanedione over the range of c o n c e n t r a t i o n s t e s t e d (2.7 t o 12 mM). In order t o t e s t the e f f e c t of pH on the r a t e of the r e a c t i o n , the sodium borate b u f f e r was r e -pl a c e d by potassium phosphate b u f f e r , as the l a t t e r has a s u p e r i o r b u f f e r i n g c a p a c i t y over a broader range of pH. 276. F i g . 52. A b s o r p t i o n spectrum of a mixture of 2,3-butanedione and NADH bef o r e and a f t e r i r r a d i a t i o n . The r e a c t i o n mixture contained 53 uM NADH, 27 mM 2,3-butanedione and 50 mM sodium borate b u f f e r , pH 7.8 i n a f i n a l volume of 2 ml. The experiment was performed as d e s c r i b e d i n the t e x t . Curve X: A b s o r p t i o n spectrum of the r e a c t i o n mixture b e f o r e i r r a d i a t i o n . Curve Y: Absorp-t i o n spectrum o f the r e a c t i o n mixture a f t e r lh h of i r r a d -i a t i o n . Curve Z.:. B a s e l i n e r e a d i n g f o r spectrophotometer. 2 7 7 . W a v e i e n g th.._nm 278. F i g . 53. E f f e c t of pH and c o n c e n t r a t i o n of 2,3-butanedione on the r a t e of p h o t o o x i d a t i o n of NADH. NADH (0.12 mM) was incubated w i t h 2.7-12 mM 2,3-butane-dione i n 50 mM sodium borate b u f f e r , pH 7.8 i n a f i n a l volume of 2 ml ( l e f t panel) or w i t h 6.7 mM 2,3-butanedione i n 50 mM potassium phosphate b u f f e r (pH 6-8.5} i n a f i n a l volume of 2 ml ( r i g h t p a n e l ) . The experiments were performed as d e s c r i b e d i n MATERIALS AND METHODS. Rate i s expressed as nmol NADH o x i d i z e d per minute. mM butanedlone pH - J 280. As shown i n F i g . 53 (right panel) the reaction rate was found to be pH-dependent over the range of pH 6 to 8.5 with the maximum rate of photooxidation occurring at pH 7. An experiment to determine the e f f e c t of the wavelength of i r r a d i a t i o n on the rate of photooxidation was carried out as follows. Solutions, of NADH were mixed with 2,3-butanedione i n the presence of 50 mM potassium phosphate buffer, pH 7.0 and ir r a d i a t e d for equal lengths of time i n the c e l l compartment of a spectrofluorometer. The i r r a d i -ating beam of l i g h t was adjusted to d i f f e r e n t wavelengths between 340 and 48 0 nm. The rate of photooxidation of NADH i n each case was plotted against the wavelength of i r r a d i a t i n g l i g h t (Fig. 54, curve X). The reaction rate was optimal at an excit a t i o n wavelength of 410 nm. This wavelength corresponds to the absorption maximum of 2,3-butanedione i n the v i s i b l e region of the spectrum (Fig. 54, curve Y). Since there appeared to be no photooxidation when l i g h t at 340 nm, the wavelength of maximum l i g h t absorption by NADH, was used, i t i s l i k e l y that photooxi-dation i s i n i t i a t e d by absorption of l i g h t by 2,3-butanedione, not NADH. The product of the 2,3-butanedione-dependent photooxidation  of NADH The reaction product of the light-dependent oxidation of NADH by 2,3-butanedione has not been characterized (179). 281. F i g . 54. E f f e c t of the wavelength of the i r r a d i a t i n g l i g h t on the r a t e of p h o t o o x i d a t i o n of NADH by 2,3-butanedione. The r e a c t i o n mixture c o n t a i n e d 0.109 mM NADH, 7.3 mM 2,3-butanedione and 50 mM potassium phosphate b u f f e r , pH 7.0 i n a f i n a l volume of 1.1 ml (curve X). The samples were contained i n a stoppered quartz c u v e t t e w i t h a 1 cm l i g h t path. They were i r r a d i a t e d f o r 3 0 min a t the i n d i -c a t e d wavelengths i n the c e l l compartment of a Turner model 420 s p e c t r o f l u o r o m e t e r . The a b s o r p t i o n spectrum a t 400-280 nm was measured f o r each sample b e f o r e and a f t e r i r r a d i a t i o n . The r a t e i s expressed as nmol. of NADH o x i d i z e d per min. The a b s o r p t i o n spectrum of a 134 mM s o l u t i o n of 2,3-butane-dione i n 50 mM potassium phosphate b u f f e r , pH 7.0, i s a l s o shown (curve Y ) . 28 I n i t i a l experiments were undertaken t o determine the s t o -c h i o m e t r i c r e l a t i o n s h i p between the r e a c t a n t s i n the ph o t o o x i d a t i o n of NADH by 2,3-butanedione ( F i g . 55). NADH (0.24 umol) was mixed w i t h 2,3-butanedione (0.11 umol) i n 50 mM sodium b o r a t e b u f f e r , pH 7.8, and the r e a c t i o n allowed to proceed t o e q u i l i b r i u m under continuous i l l u m i n a t i o n . The r e a c t i o n was c o n s i d e r e d as being complete ( a f t e r 141 hours) when the r a t e of change i n absorbance a t 3 40 nm, due t o o x i d a t i o n of the NADH was n e g l i g i b l e . At t h i s p o i n t , the amount of NADH o x i d i z e d by the 2,3-butanedione was 0.094 umol i n d i c a t i n g t h a t one molecule of NADH i s o x i d i z e d per molecule of 2 , 3-butanedione aaeacting. I t has been r e p o r t e d t h a t i r r a d i a t i o n of an aqueous s o l u t i o n of 2,3-butanedione u s i n g a 220 V mercury arc r e s u l t e d i n the p h o t o l y s i s of t h i s reagent, with the forma-t i o n of acetaldehyde and a c e t i c a c i d (203). My i n i t i a l experiment suggested t h a t acetaldehyde was an i n t e r m e d i a t e i n the p h o t o o x i d a t i o n , s i n c e the NADH remaining a f t e r 3 0 minutes of i l l u m i n a t i o n w i t h 2,3-butanedione was immediately and completely o x i d i z e d upon the a d d i t i o n of ye a s t a l c o h o l dehydrogenase (Table 16). In or d e r t o determine whether acetaldehyde was indeed the p h o t o l y t i c product r e s p o n s i b l e f o r the o x i d a t i o n of NADH, a 407-fold molar excess of acetaldehyde was allowed t o r e a c t w i t h 0.11 mM NADH i n 50 mM potassium phosphate b u f f e r , pH 7.0 (Table 16). A f t e r Table 16. E f f e c t of Yeast A l c o h o l Dehydrogenase on the O x i d a t i o n of NADH by Acetaldehyde or 2,3-Butanedione i n the Presence or Absence of L i g h t A d d i t i v e Concen-t r a t i o n ymol L i g h t Reaction Time I n t e r v a l (min) NADH Oxidized ymol Subsequent A d d i t i o n R e a c t i o n Time I n t e r v a l (min) NADH Ox i d i z e d ymol 2,3-butanedione 27 - 0 to 30 0 ADH* 30 t o 31 0.12 2,3-butanedione 27 + 0 to 3 0 0.018 ADH 30 t o 31 0.035 Acetaldehyde 90 - 0 to 1 1 to 31 31 to 61 0. 019 0.054 0.001 acetaldehyde 61 t o 71 0. 048 Acetaldehyde 90 + 0 t o 1 1 to 31 0.015 0.053 ADH 31 t o 32 0.15 Yeast a l c o h o l dehydrogenase. The r e a c t i o n mixture contained 0.12 mM NADH i n 50 mM potassium phosphate b u f f e r , pH 7.0 and e i t h e r 2,3-butanedione (13.4 mM) or acetaldehyde (45 mM) i n a f i n a l volume of 2 ml. In some cases, 5 y l of acetaldehyde or an excess of yeast a l c o h o l dehydrogenase were added subsequently t o the r e a c t i o n mixture, as i n d i c a t e d . The experiment was performed as d e s c r i b e d i n MATERIALS AND METHODS. F i g . 55. Stochiometry of p h o t o o x i d a t i o n o f NADH by 2,3-butanedione. NADH (0.12 mM) i n 50 mM sodium borate b u f f e r , pH 7.8 was incubated i n the presence (curve Y) or absence (curve X) of 0.054 mM 2,3-butanedione, r e s p e c t i v e l y , i n a f i n a l volume of 2 ml. The experiment was performed as d e s c r i b e d i n MATERIALS AND METHODS. 286 . 287. 3 0 minutes, 32 t o 33% of the NADH was o x i d i z e d , but, i n c o n t r a s t t o the r e a c t i o n observed w i t h 2,3-butanedione, the o x i d a t i o n took p l a c e i n the dark as w e l l as i n the l i g h t . The NADH was never completely o x i d i z e d when the r e a c t i o n was allowed to proceed f o r a g r e a t e r l e n g t h of time. The o x i d a t i o n was e s s e n t i a l l y complete a f t e r 31 minutes. A d d i t i o n of acetaldehyde t o the r e a c t i o n mixture a f t e r the r e a c t i o n was completed promoted f u r t h e r o x i d a t i o n of NADH. Since the p a t t e r n f o r the NADH o x i d a t i o n r e a c t i o n u s i n g 2,3-butanedione was d i f f e r e n t from that u s i n g a c e t a l d e -hyde, i t seems u n l i k e l y t h a t acetaldehyde i s the photo-l y t i c i n t e r m e d i a t e r e s p o n s i b l e f o r the r e a c t i o n s of 2,3-butanedione. However, the p r e l i m i n a r y o b s e r v a t i o n t h a t a d d i t i o n of ye a s t a l c o h o l dehydrogenase t o the photo-l y s i s mixture r e s u l t e d i n o x i d a t i o n of NADH needs t o be ex p l a i n e d . A d d i t i o n of yeast a l c o h o l dehydrogenase t o a mixture of 2,3-butanedione and NADH i n the dark, caused r a p i d o x i d a t i o n o f the reduced p y r i d i n e n u c l e o t i d e (Table 16). T h i s i n d i c a t e s t h a t 2,3-butanedione i s a s u b s t r a t e f o r y e a s t a l c o h o l dehydrogenase. Thus the o x i d a t i o n of NADH i n the presence of 2,3-butanedione a f t e r i l l u m i n a t i o n i s most l i k e l y due t o the r e d u c t i o n of 2,3-butanedione ( c a t a l y z e d by ye a s t a l c o h o l dehydrogenase) r a t h e r than t o r e d u c t i o n of acetaldehyde. 288. An experiment t o determine the stochiometry of the o x i d a t i o n of NADH by 2,3-butandione, c a t a l y z e d by y e a s t a l c o h o l dehydrogenase, i n the dark i s shown i n F i g . 56. The r e a c t i o n appeared t o be completed a f t e r 11 hours a t which time 0.094 ymol of NADH had been o x i d i z e d by 0.11 ymol of 2,3-butanedione, i n d i c a t i n g t h a t the r e a c t i o n between NADH and 2,3-butanedione, c a t a l y z e d by yeast a l c o h o l dehydrogenase, f o l l o w s a 1:1 stochiometry s i m i l a r to the p h o t o ] y t i c r e a c t i o n . D i s c u s s i o n P h o t o o x i d a t i o n of NADH (or NADPH) by 2,3-butanedione o c c u r r e d i n potassium phosphate or sodium borate b u f f e r . I t was pH-dependent and e x h i b i t e d p s e u d o - f i r s t - o r d e r k i n e t i c s with r e s p e c t t o the c o n c e n t r a t i o n of NADH. The r e a c t i o n f o l l o w e d a 1:1 stochiometry. The r a t e of photo-o x i d a t i o n was maximal at an i r r a d i a t i n g wavelength of 410 nm. In aqueous s o l u t i o n , 2,3-butanedione i s predominantly i n the hydrated form and e x h i b i t s an a b s o r p t i o n maximum i n the v i s i b l e r e g i o n of 405 t o 408 nm (167,204). In the pre s e n t study, the wavelength of maximum a b s o r p t i o n i n the v i s i b l e r e g i o n f o r the dik e t o n e i n aqueous s o l u t i o n was found t o be 410 nm, which corresponds t o the wave-l e n g t h f o r the r a t e o f maximal p h o t o o x i d a t i o n . Thus, a b s o r p t i o n of l i g h t by 2,3-butanedione i s necessary f o r 289. F i g . 56. Stochiometry of o x i d a t i o n of NADH by 2,3-butanedione c a t a l y z e d by ye a s t a l c o h o l dehydrogenase. The r e a c t i o n mixture contained 0.13 mM NADH, 0.054 mM 2,3-butanedione and 50 mM potassium phosphate b u f f e r , pH 7.0 and an excess o f ye a s t a l c o h o l dehydrogenase to a f i n a l volume of 2 ml. The mixture was incubated a t 22°C i n the dark. Absorbance measurements a t 34 0 nm were taken between 0 and 10 h. of i n c u b a t i o n . A r e a c t i o n mixture i d e n t i c a l to t h a t above, but l a c k i n g 2,3-butanedione was t r e a t e d s i m i l a r l y . The decrease i n A 3 i t 0 due to decomposition of NADH i n the c o n t r o l r e a c t i o n mixture a f t e r 10 h was 0.178. Hours 10 V.O O the p h o t o o x i d a t i o n of NADH. Ph o t o o x i d a t i o n of NADH has a l s o been r e p o r t e d t o occur w i t h methylene b l u e a t the wavelength of maximum a b s o r p t i o n f o r t h i s dye ( 2 0 5 ) . P h o t o s e n s i t i z i n g r e a c t i o n s of 2,3-butanedione have been r e p o r t e d t o take p l a c e w i t h a-amino a c i d s and a-chymotryps ( 2 0 6 ) but the products of the r e a c t i o n s or the optimum wavelength f o r i r r a d i a t i o n have not been r e p o r t e d . The f a c t t h a t 2,3-butanedione w i l l undergo photo-chemical changes has been observed p r e v i o u s l y ( 2 0 3 , 2 0 4 , 2 0 7 , 2 0 8 ) . P o r t e r e t a l . ( 2 0 3 ) r e p o r t e d the formation of acetaldehyde and a c e t i c a c i d when 2,3-butanedione was i l l u m i n a t e d w i t h a 2 2 0 V mercury a r c . Lemaire ( 2 0 4 ) found t h a t the h i g h - i n t e n s i t y i r r a d i a t i o n of aqueous s o l u t i o n s of 2,3-butanedione u s i n g a 500-W h i g h p r e s s u r e mercury lamp r e s u l t e d i n p h o t o e n o l i z a t i o n of the compound, con-comitant w i t h the appearance of a new a b s o r p t i o n maximum at 27 5 nm i n the spectrum of the d i k e t o n e . The presence of oxygen d i d not prevent the formation of the e n o l a t h i g h - i n t e n s i t y i r r a d i a t i o n ( 1 0 1 6 photons per second i n the e n t i r e c e l l ) and the c o n t r i b u t i o n of the e n o l to the absorbance at 2 6 0 nm was 7 1 % t h a t a t 27 5 nm. At low-i n t e n s i t y i r r a d i a t i o n ( 1 0 1 3 photons per second per c e l l ) , oxygen i n h i b i t e d the e n o l formation more e f f e c t i v e l y . The e n o l of 2,3-butanedione, which was not i d e n t i f i e d , was named "compound I" and was r e p o r t e d to resemble the 292. e n o l of a c e t y l a c e t o n e i n i t s absorbance p r o p e r t i e s . The e x t i n c t i o n c o e f f i c i e n t a t 275 nm was g r e a t e r than 5000 M 1-cm 1 and was a s s i g n e d t o the a b s o r p t i o n of an unsat-u r a t e d c a r b o n y l group, conjugated t o another double bond. A c e t y l a c e t o n e i s known t o e x i s t as an e q u i l i b r i u m mixture of d i k e t o and e n o l forms, y C H 2 ^ C H V CH 3-C C-CH 3 v CH 3-C C-CH 3 the e n o l form predominates (7 0 t o 9 0%) i n or g a n i c s o l v e n t s , but comprises o n l y 12% of the mixture i n aqueous s o l u t i o n (209). I t seems u n l i k e l y t h a t acetaldehyde i s the product of p h o t o o x i d a t i o n by 2,3-butanedione s i n c e i t has l i m i t e d r e a c t i v i t y w i t h NADH, even at a 407 - f o l d molar excess. Furthermore, i n c o n t r a s t t o the r e a c t i o n w i t h 2,3-butanedione, o x i d a t i o n of NADH by acetaldehyde stopped a f t e r 3 0 minutes of r e a c t i o n time i n the l i g h t or i n the dark and was o n l y resumed upon f u r t h e r a d d i t i o n of acetaldehyde. On the other hand, i f the product of p h o t o l y s i s of 2,3-butanedione i s a co r r e s p o n d i n g e n o l , i t would support the f i n d i n g o f the presen t i n v e s t i g a t i o n , t h a t o n l y one molecule of NADH i s o x i d i z e d by the 2,3-butanedione i n the presence o f l i g h t , and not two as would have been expected had both the c a r b o n y l groups been reduced by the 293. p y r i d i n e n u c l e o t i d e . The p h o t o l y t i c product of 2,3-butanedione would thus l i k e l y be reduced by NADH as f o l l o w s . C H 2«C-C-CH 3 "•+ NADH >- CH2-=C — C H - C H 3 + NAD + I I I I OH 0 OH OH The c o n d i t i o n s of l i g h t i n g used f o r the photochemical o x i d a t i o n experiments i n the present study were q u i t e d i f -f e r e n t from those employed by Lemaire (204). Ambient l a b o r a t o r y l i g h t i n g , comprising i n d i r e c t s u n l i g h t and f l u o r e s c e n t l i g h t i n g , was used t o simulate experimental c o n d i t i o n s under which 2,3-butanedione had been used t o modify a c t i v e - s i t e a r g i n y l r e s i d u e s i n enzymes, i n the presence of reduced p y r i d i n e n u c l e o t i d e s (113,168,210) and t o assess the extent of s i d e - r e a c t i o n s due to photo-l y s i s . I t i s c l e a r t h a t a r g i n y l m o d i f i c a t i o n r e a c t i o n s i n which reduced p y r i d i n e n u c l e o t i d e s are used t o p r o t e c t the s u b s t r a t e - b i n d i n g s i t e s , must take i n t o c o n s i d e r a t i o n t h a t the m o d i f ying reagent can be removed i n the presence of l i g h t . Thus, such experiments should be c a r r i e d out i n the dark. I t i s of i n t e r e s t t h a t 2,3-butanedione c o u l d o x i d i z e NADH i n the dark i n the presence of ye a s t a l c o h o l dehy-drogenase. The stochiometry under these circumstances appeared t o be one molecule of NADH o x i d i z e d per molecule of 2,3-butanedione. 294. The acceptance of 2,3-butanedione, as a s u b s t r a t e , by yeast a l c o h o l dehydrogenase i s a novel o b s e r v a t i o n . A l -though y e a s t a l c o h o l dehydrogenase has been shown to be a c t i v e towards a v a r i e t y of a l c o h o l s , ketones and a l d e -hydes, i t d i f f e r s i n many ways and e x h i b i t s g r e a t e r sub-s t r a t e s p e c i f i c i t y than-horse l i v e r a l c o h o l dehydrogenase (211.212) . Yeast a l c o h o l dehydrogenase shows a g r e a t e r a f f i n i t y f o r primary a l c o h o l s than secondary a l c o h o l s (211.213) . A l c o h o l s become b e t t e r s u b s t r a t e s with de-c r e a s i n g c h a i n l e n g t h (212). Secondary a l c o h o l s , i n which both a l k y l groups are l a r g e r than methyl are not u t i l i z e d by y e a s t a l c o h o l dehydrogenase. The enzyme i s a c t i v e w i t h o n l y one isomer of butan-2-ol and o c t a n - 2 - o l . T h i s i s i n c o n t r a s t t o horse l i v e r a l c o h o l dehydrogenase which i s a c t i v e w i t h a l l of these s u b s t r a t e s (214). On the b a s i s of t h i s i n f o r m a t i o n i t i s u n l i k e l y t h a t 2,3-butanediol (or 2,3-butanedione) would be a s u b s t r a t e f o r yeast a l c o h o l dehydrogenase. Furthermore, a ketone of the same c h a i n l e n g t h (methyl e t h y l ketone) was r e p o r t e d not t o be u t i -l i z e d by a l c o h o l dehydrogenase from horse l i v e r (211,215). I t i s not c l e a r then how 2,3-butanedione can be a s u b s t r a t e f o r y e a s t a l c o h o l dehydrogenase. I t has been shown i n amino a c y l m o d i f i c a t i o n experiments t h a t 2,3-butanedione binds a t the NADH-site of y e a s t and horse l i v e r a l c o h o l dehydrogenases (168), p o s s i b l y a t the arg-47 r e s i d u e (212). 295. M o d i f i c a t i o n of ye a s t a l c o h o l dehydrogenase w i t h 2,3-butanedione r e s u l t e d i n the l o s s of 2.1 of the 8 a r g i n y l r e s i d u e s per subunit of enzyme. P r o t e c t i o n a g a i n s t mod-i f i c a t i o n was o n l y p a r t i a l l y a f f o r d e d by NADH (168). Thus, i t i s c o n c e i v a b l e t h a t m o d i f i c a t i o n by 2,3-butanedione of a r g i n y l r e s i d u e s , a t s i t e s other than the NADH-binding s i t e s on the enzyme, c o u l d a l t e r the s u b s t r a t e s p e c i f i c i t y o f the a l c o h o l dehydrogenase and permit b i n d i n g of 2,3-butanedione a t the s u b s t r a t e - b i n d i n g s i t e . 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