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Pyridine nucleotide transhydrogenase of Escherichia coli: nucleotide sequence of the pnt gene and characterization.. 1986

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PYRIDINE NUCLEOTIDE TRANSHYDROGENASE OF Escher i c h i a NUCLEOTIDE SEQUENCE OF THE pnt_ GENE AND CHARACTERIZATION OF THE ENZYME COMPLEX by DAVID MORGAN CLARKE M.Sc, McMaster University, 1980 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORATE OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF BIOCHEMISTRY We accept t h i s thesis as conforming to the required standard. THE UNIVERSITY OF BRITISH COLUMBIA February 1986 copyright by David Clarke, 1986 8 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 of the requirements f o r an advanced degree a t the 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 the 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 study. I f u r t h e r agree 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 purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying 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 not be allowed without my w r i t t e n p e r m i s s i o n . Department of jSsfypLe.Mt&'tr f- The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 79) ABSTRACT Based on the r a t i o n a l e that Escherichia c o l i c e l l s harboring plasmids containing the pnt gene would contain elevated l e v e l s of enzyme, three clones were i s o l a t e d bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to r e s t r i c t i o n endonuclease a n a l y s i s . A 10.4-kilobase r e s t r i c t i o n fragment which overlapped a l l three plasmids was cloned into pUC13. Examination of several d e l e t i o n d e r i v a t i v e s of the r e s u l t i n g plasmids and subsequent treatment with exonuclease BAL31 revealed that enhanced transhydrogenase expression was l o c a l i z e d within a 3.05-kilobase segment. This segment was located at 35.4 min i n the E_. c o l i genome. Plasmid pDC21 conferred on i t s host 70-fold overproduction of transhydrogenase. The pr o t e i n products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide g e l electrophoresis of membranes from c e l l s containing the plasmids and by i n v i t r o t r a n s c r i p t i o n / t r a n s l a t i o n of pDC21. Two polypeptides of molecular weights 52,000 and 48,000 were coded by the 3.05-kilobase fragment of pDC21. Both polypeptides were required for expression of transhydrogenase a c t i v i t y . The transhydrogenase was p u r i f i e d from cytoplasmic membranes of E_. c o l i by pre-extraction of the membranes with sodium cholate and T r i t o n X-100, s o l u b i l i z a t i o n of the enzyme with sodium deoxycholate i n the presence of 1 M potassium c h l o r i d e , and c e n t r i f u g a t i o n through a 1.1 M sucrose s o l u t i o n . The p u r i f i e d enzyme consists of two subunits, cx and 6, of molecular weights 52,000 and 48,000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the p u r i f i e d enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient i s established across the membrane as indicated by the quenching of fluorescence of 9-aminoacridine. It was concluded that E_. c o l i transhydrogenase acts as a proton pump which i s regulated primarily by a pH gradient rather than a membrane p o t e n t i a l . Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide r e s u l t s i n an i n h i b i t i o n of proton pump a c t i v i t y and transhydrogenation, suggesting that proton t r a n s l o c a t i o n and c a t a l y t i c a c t i v i t i e s are o b l i g a t o r i l y linked. [ 1 "*C]Dicyclohexylcarbodiimide p r e f e r e n t i a l l y l a b e l l e d the a subunit. The transhydrogenase-catalyzed reduction of 3-acetylpyridine adenine dinucleotide by NADPH was stimulated over three-fold by NADH. It was concluded that NADH binds to an a l l o s t e r i c binding s i t e on the enzyme. The nucleotide sequences of the pntA and pntB genes, coding for the transhydrogenase a and 6 subunits r e s p e c t i v e l y , were est a b l i s h e d . The molecular masses of 53,906 (a) and 48,667 (B) and the N-terminal sequences of the predicted polypeptides agree well with the data obtained by analysis of the p u r i f i e d subunits. Several hydrophobic regions large enough to span the cytoplasmic membrane were observed for each subunit. - i v - TABLE OF CONTENTS TITLE PAGE i ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES i x ABBREVIATIONS x i i ACKNOWLEDGEMENTS x i i i INTRODUCTION Linkage with the Oxidative Phosphorylation System 3 P u r i f i c a t i o n of Transhydrogenase • 5 Reconstitution of Transhydrogenase 9 Transhydrogenase as a Proton Pump 11 E f f e c t of Electrochemical P o t e n t i a l on Reconstituted Transhydrogenase 12 Chemical Mod i f i c a t i o n with DCCD 13 Reaction Mechanism of Transhydrogenase 14 Phy s i o l o g i c a l Role of Transhydrogenase 19 Objective of th i s Study 20 MATERIALS AMD METHODS Chemicals and Isotopes 21 Strains and B a c t e r i a l Growth 21 Preparation of Membranes 23 S o l u b i l i z a t i o n of Membrane Vesicles with Detergents 23 P u r i f i c a t i o n of Transhydrogenase from S t r a i n W6 23 -v- Screening of Clarke and Carbon C o l l e c t i o n for Plasmids Carrying the pnt Gene 25 Preparation of C o l i c i n E l 25 Preparation of Plasmid DNA 27 Preparation of Nucleic Acids 29 Digestion with R e s t r i c t i o n Enzymes 30 Ligations with T4-DNA Ligase 30 Transformations 30 Electrophoresis of DNA 31 Dephosphorylation of DNA 32 Nuclease BAL31 Digestion 33 P u r i f i c a t i o n of Transhydrogenase from JM83 pDC21 33 Peptide Mapping 34 Polyacrylamide Gel Electrophoresis 35 Reconstitution of Transhydrogenase 35 In v i t r o Protein Synthesis 36 DNA Sequence Determination 37 Is o l a t i o n of M13 Phage 38 Sequence Reaction 38 Iso l a t i o n of Transhydrogenase c* and IB Subunits 40 Is o l a t i o n of Transhydrogenase Subunits using the Prep-Gel Apparatus 40 Protein Assay • 41 Assay of Energy-Independent Transhydrogenase A c t i v i t y 41 Assay of Energy-Dependent Transhydrogenase A c t i v i t y 42 Fluorescence Assays 43 Preparation of RNase that i s Free of DNase A c t i v i t y 44 - v i - Glutamate Dehydrogenase Assay 43 PI Transduction 44 Lab e l l i n g of Membrane Vesicles with [ll,C]DCCD 45 Crossed Immunoelectrophoresis 45 Reaction of E. c o l i Transhydrogenase with Mitochondrial Anti-Transhydrogenase 46 RESULTS I. P h y s i o l o g i c a l Role of Transhydrogenase 48 II. P u r i f i c a t i o n of Transhydrogenase from Str a i n W6 51 Growth of C e l l s 51 Selection of Detergent 55 P u r i f i c a t i o n of Transhydrogenase 58 II I . Cloning of the pnt Gene 64 I d e n t i f i c a t i o n of the pnt Plasmids 64 R e s t r i c t i o n Endonuclease Analysis of the pnt Plasmids 66 Subcloning of the pnt Gene into pUCl3 66 Lo c a l i z a t i o n of the pnt Gene i n pDC3 68 I d e n t i f i c a t i o n of the pnt Gene Products 71 Complementation of Transhydrogenase A c t i v i t y 76 Morphological E f f e c t s of pnt Overproduction 77 IV. P u r i f i c a t i o n of Transhydrogenase from Strain JM83 pDC21 81 V. Properties of Transhydrogenase 90 Kin e t i c Parameters 90 Inact i v a t i o n by Trypsin..... 90 - v i i - VI. Transhydrogenase as a Proton Pump 90 Proteoliposome Energization 90 Interaction of Transhydrogenase with a pH Gradient or Membrane Potential 95 In h i b i t i o n by DCCD 101 Iso l a t i o n of Transhydrogenase Subunits by Ex c i s i o n from Polyacrylamide Gels 114 VII. I s o l a t i o n of the Transhydrogenase Subunits f o r Amino Acid Sequence Analysis 110 P u r i f i c a t i o n of Subunits Using Polyacrylamide Slab Gels.... 110 I s o l a t i o n of the Transhydrogenase Subunits Using a Commercial Preparative Gel Electrophoresis System 110 VIII. Nucleotide Sequencing of the pnt Gene 116 DISCUSSION P h y s i o l o g i c a l Role 124 Cloning and Expression of Transhydrogenase 125 P u r i f i c a t i o n and Characterization of Transhydrogenase 129 Nucleotide Sequence of the pnt Gene 135 REFERENCES 141 - v i i i - LIST OF TABLES Table 1. B a c t e r i a l Strains 22 2. E f f e c t of transhydrogenase a c t i v i t y on aerobic growth r a t e s . . . . 49 3. Role of transhydrogenase i n the a s s i m i l a t i o n of ammonia 53 4. E f f e c t of transhydrogenase mutation on the growth rate of glutamate synthase mutants 54 5. P a r t i a l p u r i f i c a t i o n of transhydrogenase from E_. c o l i s t r a i n W6 59 6. Transhydrogenase a c t i v i t y i n membranes of selected s t r a i n s from the Clarke-Carbon colony bank 65 7. Complementation of chromosomal pnt::Tn5 by various pnt a l l e l e s on plasmids 78 8. Growth c h a r a c t e r i s t i c s of JM83 carrying various plasmids 82 9. P u r i f i c a t i o n of transhydrogenase from E. c o l i s t r a i n JM83pDC21 86 10. Treatment of membrane v e s i c l e s prepared from E. c o l i JM83pDC21 with various levels of TPCK-trypsin 9 2 11. Treatment of E. c o l i W6 pDC21 membranes with EDC 106 12. E f f e c t of substrates on the i n h i b i t i o n of transhydrogenase a c t i v i t y by DCCD 108 13. Amino acid compositions of the transhydrogenase subunits 123 14. Codon usage i n the E. c o l i pnt genes 137 - i x - LIST OF FIGURES Figure 1. Proposed proton pump mechanisms for mitochondrial transhydrogenase 18 2. Pathways of nitrogen a s s i m i l a t i o n i n E. c o l i 50 3. E f f e c t of exogenous NH^Cl on glutamate dehydrogenase and transhydrogenase a c t i v i t i e s i n E. c o l i W6 52 4. S o l u b i l i z a t i o n of membrane-bound transhydrogenase with various detergents 57 5. SDS-polyacrylamide gel electrophoresis of f r a c t i o n s at various stages of the transhydrogenase p u r i f i c a t i o n from E. c o l i s t r a i n W6 60 6. Separation of transhydrogenase by ion-exchange chromatography 61 7. P u r i f i c a t i o n of transhydrogenase by a f f i n i t y chromatography.. 63 8. Comparison of r e s t r i c t i o n endonuclease maps of Co l E l plasmid i n s e r t s with a region of the E. c o l i genome 67 9. Subcloning of DNA carrying the pnt gene 69 10. R e s t r i c t i o n endonuclease maps of plasmids containing the pnt gene and transhydrogenase a c t i v i t i e s of membranes prepared from c e l l s harboring each of the plasmids 70 11. SDS-polyacrylamide gel electrophoresis of membranes of JM83 containing e i t h e r pUC13 or pDCll 73 12. Autoradiograph of SDS-polyacrylamide electrophoresis gel of [ 3 sSjmethionine-labeled in vivo t r a n s c r i p t i o n / t r a n s l a t i o n products 74 13. SDS-polyacrylamide gel electrophoresis of membrane f r a c t i o n s of JM83 carrying hybrid plasmids 75 14. Agarose gel electrophoresis of plasmids prepared from E. c o l i AB1450 containing transhydrogenase subunits on separate replicons 79 15. SDS-polyacrylamide gel electrophoresis of membranes prepared from E. c o l i AB1450 pnt::Tn5 and AB1450 pnt: :Tn5 pDC9, pDC50 80 16. Microphotographs of E. c o l i JM83 c e l l s containing plasmids... 83 -x- 17. Thin section electron micrographs of JM83 pDC21 c e l l s 84 18. SDS-polyacrylamide gel electrophoresis of various f r a c t i o n s obtained during the p u r i f i c a t i o n of transhydrogenase from E. c o l i s t r a i n JM83 pDC21 88 19. P a r t i a l p r o t e o l y s i s of the 100,000-molecular-weight protein and the c* and 6 subunits of the transhydrogenase 89 20. K i n e t i c parameters of transhydrogenase 91 21. SDS-polyacrylamide gel electrophoresis of membrane v e s i c l e s prepared from E. c o l i JM83 pDC21 treated with t r y p s i n 93 22. Inactivation of membrane-bound transhydrogenase by TPCK-trypsin i n the presence of various l e v e l s of nucleotides 94 23. E f f e c t of FCCP on reverse and forward transhydrogenation 96 24. Quenching of the fluorescence of 9-aminoacridine during the reduction of AcNAD by NADPH catalyzed by e i t h e r membrane-bound or reconstituted transhydrogenase 97 25. Influence of a transmembrane pH gradient on transhydrogenation 99 26. Influence of a membrane po t e n t i a l on transhydrogenation 100 27. E f f e c t of ionophores on the reduction of AcNAD by NADPH by membrane-bound transhydrogenase i n the presence of a membrane p o t e n t i a l 102 28. K i n e t i c s of i n h i b i t i o n of membrane-bound and p u r i f i e d transhydrogenase by DCCD 103 29. E f f e c t of DCCD on proton tr a n s l o c a t i o n and c a t a l y t i c a c t i v i t i e s of membrane-bound transhydrogenase 105 30. [ l l ,C]DCCD l a b e l l i n g of membrane-bound transhydrogenase....... 107 31. E f f e c t of NADH and AcNADH on the reduction of AcNAD by NADPH catalyzed by p u r i f i e d transhydrogenase 109 32. SDS-polyacrylamide gel electrophoresis of samples from f r a c t i o n s obtained during the separation of transhydrogenase subunits using the preparative gel electrophoresis system 112 - x i - 33. SDS-polyacrylamide gel electrophoresis of samples from f r a c t i o n s obtained during the separation of transhydrogenase subunits using the preparative gel electrophoresis system 113 34. SDS-polyacrylamide gel electrophoresis of transhydrogenase subunits p u r i f i e d by excision of the protein bands from a gel 115 35. Amino acid sequences of transhydrogenase a and 8 subunits.... 117 36. Nucleotide sequence of the pnt gene region 118 37. Structure of the M13 mpl8 and mpl9 cloning regions 120 38. Summary of the clones used to e s t a b l i s h the nucleotide sequence 122 39. Proposed mechanism of transhydrogenase i n in t a c t E. c o l i c e l l s 134 40. Hydropathy pl o t of the transhydrogenase a subunit 139 41. Hydropathy plot of the transhydrogenase 8 subunit 140 - x i i - ABBREVIATIONS Ac NAD 3-Acetylpyridine adenine dinucleotide AG-NAD NAD coupled to agarose resin. Brij 35 Polyoxyethylene(23) lauryl ether DCCD N,N'-dicyclohexylcarbodiimide DNase Deoxyribonuclease DTT Dithiothreitol EDTA (Ethylenedinitrilo)-tetraacetic acid EDC l-Ethyl-3(3-dimethyl-amino-propyl)carbodiimide EGTA [Ethylenebis(oxyethylenenitrilo]-tetraacetic acid HEPES N-2-hydroxyethylpiperazine N1-2-ethanesulphonic ac MES 2-(N-morpholino)ethanesulphonic acid MOPS 3-(N-morpholino)propanesulphonic acid PEG Polyethyleneglycol RNase Ribonuclease SDS Sodium dodecyl sulphate TEMED N,N,N',N'-tetramethylethylenediamine TPCK-trypsin Trypsin treated with L-(tosylamido 2-phenyl)ethyl chloromethyl ketone Tris Tris (hydroxymethyl)-aminoethane Triton X-100 Polyoxyethyleneglycol(9-10)p-t-octylphenol Triton X-114 Polyoxyethyleneglyco1(7-8)p-t-octylpheno1 U Unit aw Membrane potential ApH Difference in pH across the membrane ACKNOWLEDGEMENTS To my supervisor and teacher, Dr. P.D. Bragg, I am deeply obliged. His scientific and organizing ability and his never failing interest in this work have made a deep impression on me. His continuous support during these years has been invaluable. I am especially grateful to my friend Tip Loo. His assistance with nearly a l l aspects of this work greatly contributed to its success. His positive attitude to lif e and science has meant much to me during these studies. To Helga Stan-Lotter, I want to express my warmest thanks for the excellent assistance. Her knowledge and technical s k i l l has made it a pleasure to work together with her. I am particulary grateful to Dr. Shirley Gillam for the use of her facilities to do the nucleotide sequencing studies. My sincere thanks to my collaborators, Dr. Ross MacGillivray for his patience to teach me the basics in molecular biology and gifts of plasmid and enzymes, Dr. Pat Dennis for his help in techniques of molecular biology and performing the Sl-mapping studies, Dr. Bob Molday for doing the electron microscopy studies and Keith Withers for his help with computer analysis. I thank Dr. J. Weiner of the University of Alberta fo the gift of the Clarke and Carbon colony bank. I wish to thank Dr. Pat Dennis and Dr. Peter Candido for their constructive criticism of this thesis. My sincere thanks are also due to Masako Williams for her untiring help of typing the manuscripts. Finally I wish to thank Dr. Ted Sedgwick and Cynthia Hou for their assistance. - x i v - F i n a n c i a l support for t h i s work was provided by a Medical Research Council Studentship, for which I express my gratitude. 1. INTRODUCTION Pyridine nucleotide transhydrogenases (EC 1.6.1.1) catalyze the di r e c t and r e v e r s i b l e transfer of a hydride ion equivalent between NAD and NADP according to the equation: NADH + NADP ±~ NAD + NADPH An enzyme possessing transhydrogenase a c t i v i t y was f i r s t discovered by Colowick et a l . (1) i n extracts from Pseudomonas fluorescens. Soon a f t e r the discovery of the Pseudomonas enzyme, Kaplan and co-workers (2) reported that transhydrogenase a c t i v i t y was found i n bovine heart preparations. I t became apparent that there were s i g n i f i c a n t differences between the Pseudomonas and bovine heart transhydrogenases. Today these enzymes are known to be representatives of two d i s t i n c t classes of transhydrogenases. Both classes of transhydrogenases have been recently reviewed (3,4,5). The f i r s t c l a s s , termed BB-specific transhydrogenases, and represented by the Pseudomonas enzyme, catalyze the transfe r of a hydride ion equivalent between the 4B locus of both NADH and NADPH. The BB-specific transhydrogenases are soluble, FAD-containing enzymes which are under a l l o s t e r i c r e g u l a t i o n by nucleotides such as 2'-AMP. They are found i n some heterotrophic bacteria such as Pseudomonas fluorescens (1), Pseudomonas aeruginosa (6), Azotobacter v i n e l a n d i i (7), Azotobacter chroococcum, and Azotobacter a g i l e (8). The transhydrogenase enzymes of Pseudomonas aeruginosa (9) and Azotobacter v i n e l a n d i i (10,11) have been p u r i f i e d to homogeneity. The p u r i f i e d enzymes were i s o l a t e d as large filamentous aggregates with molecular weights of several m i l l i o n . In the presence of 2'-AMP or NADP the Pseudomonas enzyme d i s s o c i a t e d into smaller 2. fragments of molecular weight 900,000, composed of 20 polypeptides of molecular weight 40,000 to 45,000 (12). The Azotobacter transhydrogenase also disaggregated in the presence of NADP into fragments of molecular weight 58,000 (10). FAD was found as a prosthetic group in both enzymes (9,10). The second class, termed AB-specific transhydrogenases, and represented by the bovine heart mitochondrial enzyme, catalyze the transfer of a hydride ion equivalent between the 4B locus of NADPH and the 4A locus of NADH. These enzymes are found in the cytoplasmic membrane of certain bacteria and in the inner membrane of mitochondria. They are not allosterically regulated by 2'-AMP nor do they require FAD insofar as is known. The AB-specific transhydrogenases are an interesting class of enzymes because upon membrane energization by respiration or ATP hydrolysis the rate of reduction of NADP by NADH is increased up to ten-fold (3). This energy-linked transhydrogenase is widespread; it is found in the mitochondria of heart, kidney, liver, arterial and muscle tissue, in heterotrophic bacteria such as Escherichia coli (13), Micrococcus denitrificans (14), Bacillus megaterium (15), Salmonella typhimurium (16) and Benekea natriegens (17), and in photosynthesizing bacteria including Rhodospirilium rubrum (18), Rhodopseudomonas spheroides (19), Rhodopseudomonas palustris and Rhodospirilium molischianum (18). This thesis focused on the study of the energy-linked transhydrogenase of E. coli. The E. coli enzyme, along with the transhydrogenases of bovine heart mitochondria and R. rubrum are the most studied AB-specific transhydrogenases. The following will focus on advancements that have contributed to an understanding of the structure, function and properties of the aforementioned transhydrogenases. 3. Linkage with the Oxidative Phosphorylation System AB-specific transhydrogenases are integral membrane proteins found in the inner mitochondrial membrane (20-22), the cytoplasmic membrane of E. coli (23), and R. rubrum chromatophore membranes (13,24). Energy-linked transhydrogenases are functionally linked to the energy-transfer system of the membrane in which they are located (4). The energy required may be generated through any of the coupling sites of the respiratory chain in mitochondria or respiring bacteria. In E. coli, the transhydrogenase may be driven by ATP hydrolysis or respiration (25-27). Energy to drive the mitochondrial enzyme may be furnished by hydrolysis of ATP or by oxidation of NADH, succinate or reduced cytochrome c (21,22). With photosynthetic bacteria such as R. rubrum, energy can be generated either by light-induced electron transport or by the hydrolysis of pyrophosphate, ATP or GTP (18,24). The presence of an energy source results in an energy dependent increase in both the rate (5) and the extent (28,29) of reduction of NADP by NADH. In mitochondria, the apparent equilibrium constant for the reaction is increased from unity to about 500 and the rate of reduction of NADP by NADH is stimulated about 10-fold in the presence of an energy source such as ATP. The effect of ATP is mediated by the energy-transducing ATPase (3). In mutants of E. coli lacking ATPase, ATP does not drive the transhydrogenase reaction (30), although respiration is s t i l l effective (31). An antibody to purified ATPase inhibits the stimulation by ATP of transhydrogenase activity as well as inhibiting ATPase activity (32). In addition, ATPase can be extracted from the membrane with loss of the ATP stimulation and reconstituted to restore the effect (27,33). Energy-linked transhydrogenation driven by any of the energy sources is inhibited by oxidative phosphorylation uncouplers (28,34). The ATP-dependent reaction i s s p e c i f i c a l l y i n h i b i t e d by the phosphorylation i n h i b i t o r s , oligomycin and dicyclocarbodiimide (3,18,35). The av a i l a b l e information suggests that the energy-linked transhydrogenase reaction and electron transport-linked phosphorylation u t i l i z e a common energy pool. A consequence of t h i s assumption would be that a reversal of the transhydrogenase reaction ( i . e . , NADPH+NAD transhydrogenation) should r e s u l t i n the conservation of free energy. Dontsov and co-workers (36) showed that energy-linked transhydrogenation i s r e v e r s i b l e i n studies on the d i s t r i b u t i o n of the l i p o p h i l i c anion phenyl dicarbaundecarborane (PCB ), across submitochondrial p a r t i c l e and R. rubrum chromatophore membranes. NADPH+NAD transhydrogenation was linked to PCB uptake which i s i n d i c a t i v e of the formation of a membrane po t e n t i a l , p o s i t i v e on the inside of the v e s i c l e s (3). On the other hand, NABH+NADP transhydrogenation caused an e f f l u x of PCB . Similar r e s u l t s were obtained using E_. c o l i v e s i c l e s (37). By using tightly-coupled submitochondrial p a r t i c l e s , Van de Stadt et a l . (38) were able to couple ATP synthesis to NADPH^NAD transhydrogenation. Based on these observations i t was proposed that the transhydrogenase functions as a rev e r s i b l e proton pump (39-42), consistent with the chemiosmotic hypothesis (43,44). One of the major goals of research on AB-specific transhydrogenases i s to determine the mechanism of the transhydrogenase reaction and how the reac t i o n i s coupled to the energy conservation system. These studies require a p u r i f i e d enzyme which can be reconstituted into a r t i f i c i a l membranes. 5. P u r i f i c a t i o n of Transhydrogenase Transhydrogenase i s an i n t e g r a l membrane protein which must be released from the membranes for p u r i f i c a t i o n purposes. However, release of transhydrogenase from the membrane i s complicated by the fac t that d e l i p i d a t i o n inactivates the enzyme. Lipid-removing agents such as acetone and b i l e s a l t s i n a c t i v a t e mitochondrial transhydrogenase (2). Rydstrom has demonstrated that bovine heart mitochondrial transhydrogenase i s i nactivated by ammonium s u l f a t e p r e c i p i t a t i o n i n the presence of sodium cholate (45). Addition of phospholipids such as phosphatidylcholine, phosphatidylethanolamine or lysophosphatidylcholine to t h i s preparation restored a c t i v i t y . Treatment of E. c o l i transhydrogenase with cholate and ammonium sulphate also caused i n a c t i v a t i o n of the enzyme (46). The preparation was reactivated by various phospholipids, p a r t i c u l a r l y b a c t e r i a l c a r d i o l i p i n and phosphatidylglycerol. Therefore, s o l u b i l i z a t i o n of transhydrogenase requires a detergent that e i t h e r s o l u b i l i z e s without s t r i p p i n g away e s s e n t i a l l i p i d s or can e f f e c t i v e l y substitute for native l i p i d s . Transhydrogenase was f i r s t p u r i f i e d from beef heart mitochondria i n the laboratories of Rydstrom (47) and Fisher (48). Rydstrom 1s group extracted transhydrogenase from beef heart submitochondrial p a r t i c l e s using sodium cholate i n the presence of ammonium sulphate. P u r i f i c a t i o n of such an extract by chromatography on DEAE-Sepharose and hydroxyapatite yielded a homogeneous preparation of transhydrogenase having a molecular weight of 97,000. No prosthetic group was detected. Fisher's group p u r i f i e d transhydrogenase from beef heart submitochondrial p a r t i c l e s using a six-step procedure. Submitochondrial p a r t i c l e s were f i r s t extracted with sodium perchlorate to remove peripheral proteins and then transhydrogenase was s o l u b i l i z e d using 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 of the s o l u b i l i z e d enzyme on alumina g e l , calcium phosphate g e l , and by chromatography on NAD a f f i n i t y columns. The p u r i f i e d transhydrogenase was reported to have a molecular weight of 110,000. The enzyme was free of f l a v i n , cytochromes, NADPH-dichlorophenolindophenol reductase, NADPH-ferricyanide reductase and NADH+NAD transhydrogenase a c t i v i t i e s . The stereochemistry of hydrogen transfer by p u r i f i e d transhydrogenase was shown to be i d e n t i c a l to the submitochondrial enzyme with no exchange of protons with medium water (3). Both of the above procedures were laborious and yiel d e d small amounts of transhydrogenase. A much simpler p u r i f i c a t i o n procedure for mitochondrial transhydrogenase was developed by Wu et a l . (49). Bovine heart submitochondrial p a r t i c l e s were washed with 2 M NaCl to remove peripheral proteins. This was followed by extraction of the membranes with 1.5% T r i t o n X-100. The extract was then applied to an a f f i n i t y column of NAD immobilized on agarose and the enzyme eluted with NADH. The enzyme preparation was judged to be homogeneous by analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis. This procedure resulted i n a high y i e l d of enzyme (47.4%). However, t h i s p u r i f i c a t i o n procedure i s not reproducible. Persson et a l . (50), despite repeated attempts, could not obtain y i e l d s of transhydrogenase greater than 5% when using methods employing immobilized NAD. In addition, the s p e c i f i c a c t i v i t y of t h e i r preparations was only about 15-20 umol/min/mg of protein, which was much less than the s p e c i f i c a c t i v i t y of 62.3 umol/min/mg of protein reported by Wu et a l . Persson et a l . (50) p u r i f i e d the mitochondrial transhydrogenase by cholate-ammonium sulphate fr a c t i o n a t i o n followed by DEAE-Sepharose chromatography and fast protein 7. l i q u i d chromatography. The advantages of this preparation, as compared to other preparations, i s i t s superior pu r i t y , the r e p r o d u c i b i l i t y of the method, and the a b i l i t y to obtain large amounts of p u r i f i e d transhydrogenase. Preparations of the p u r i f i e d mitochondrial transhydrogenase have shown that the minimal molecular weight of the enzyme i s 97,000 to 115,000. The amino acid composition of the mitochondrial transhydrogenase has been determined (49,50). The p o l a r i t y index (percentage of Asx, Glx, Ser, Thr, His, Lys and Arg residues) i s about 40% (49). This i s somewhat more nonpolar than the t y p i c a l water-soluble protein (51). The subunit structure of p u r i f i e d bovine heart transhydrogenase was investigated using c r o s s - l i n k i n g reagents (52). Reaction of p u r i f i e d bovine heart transhydrogenase with the b i f u n c t i o n a l c r o s s - l i n k i n g reagents dimethyl adipimidate, dimethyl pimelimidate, dimethylsuberimidate and d i t h i o b i s (succinimidy1 propionate) r e s u l t s i n the appearance of a dimer band on sodium dodecyl sulphate polyacrylamide gels with no higher oligomers being formed. Treatment of the enzyme with 6 M urea led to i n a c t i v a t i o n of transhydrogenase and prevented c r o s s - l i n k i n g . The subunit structure of membrane-bound mitochondrial transhydrogenase was also investigated using c r o s s - l i n k i n g reagents (53). It was concluded that transhydrogenase ex i s t s i n the native membrane primarily as a dimeric species. Attempts have been made by several laboratories to i s o l a t e or i d e n t i f y the polypeptide composition of the E. c o l i enzyme (46,54,55,56). Liang and Houghton (55) p a r t i a l l y p u r i f i e d the E. c o l i transhydrogenase by deoxycholate e x t r a c t i o n of membranes followed by ion-exchange and gel f i l t r a t i o n chromatography. Sodium dodecyl s u l f a t e polyacrylamide gel electrophoresis of the preparation showed two major pr o t e i n bands of 8. molecular weights 94,000 and 50,000, and several minor bands. When E. c o l i c e l l s are grown on complex media containing high l e v e l s of amino acids, the synthesis of transhydrogenase i s repressed (57). Using t h i s observation, Liang and Houghton (55) attempted to determine which polypeptides were components of transhydrogenase by i n c o r p o r a t i n g 3 H - l a b e l l e d Casamino acids i n the i n i t i a l repressive growth phase and nonrepressive l e v e l s of [ 1 ^ C ] l e u c i n e in the induction phase. Sodium dodecyl sulphate polyacrylamide gels of the preparation i n d i c a t e d that the protein bands with the highest l l fC/ 3H r a t i o corresponded to polypeptides of molecular weights 94,000 and 50,000. The r e l a t i o n s h i p of the two polypeptides i s not known. The largest polypeptide may represent an unusually stable dimer of the 50,000-molecular weight polypeptide. A l t e r n a t i v e l y , the smaller component may represent a p r o t e o l y t i c fragment of the larger component. This i s consistent with the s e n s i t i v i t y of the enzyme to p r o t e o l y t i c degradation and with the f a c t that the mitochondrial transhydrogenase i s a s i n g l e polypeptide of molecular weight 97,000 to 120,000. A t h i r d p o s s i b i l i t y i s that the enzyme i s composed of two components. Chromatophores prepared from R. rubrum contain a transhydrogenase complex which i s r e a d i l y separable by d i l u t i o n and c e n t r i f u g a t i o n into a soluble protein f a c t o r having a molecular weight of about 70,000 and a membrane-bound component (58,59). Neither the soluble f a c t o r nor the membrane-bound component alone e x h i b i t s transhydrogenase a c t i v i t y (60). A 2000-fold p u r i f i c a t i o n of the soluble factor was obtained using ammonium sulphate p r e c i p i t a t i o n followed by chromatography on DEAE-Sephadex (61). The membrane component was s u c c e s s f u l l y extracted from both R. rubrum chromatophores and soluble factor-depleted membranes using 9. lysophosphatidylcholine (62). Transhydrogenase a c t i v i t y can be reconstituted 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 (62,63) i n the presence of low concentrations of NADP or NADPH. Reconstitution of Transhydrogenase In order to test the hypothesis that transhydrogenase couples the transfer of protons across membranes to the transfer of a hydride ion equivalent between the substrates, the p u r i f i e d enzyme must be reconstituted into liposomes. P u r i f i e d bovine heart transhydrogenase was f i r s t reconstituted into phosphatidylcholine v e s i c l e s by Hojeberg and Rydstrom (47). Reduction of NAD by NADPH catalyzed by reconstituted transhydrogenase generated an uncoupler-sensitive uptake of l i p o p h i l i c anion i n d i c a t i v e of the formation of a membrane p o t e n t i a l , p o s i t i v e inside the v e s i c l e . The rate of reduction of NAD by NADPH was enhanced over 10-fold by uncouplers. Other work showed that p u r i f i e d mitochondrial transhydrogenase could be reconstituted by d i a l y s i s of mixtures of transhydrogenase, sodium cholate and phosphatidylcholine to form small unilamellar proteoliposomes (64-66). Experiments with reconstituted transhydrogenase demonstrated that NADPH+NAD transhydrogenase i s coupled to 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 n a l space and that transhydrogenation i s stimulated s e v e r a l - f o l d i n both d i r e c t i o n s upon addit i o n of uncoupler (64-66). The i n h i b i t i o n of transhydrogenation i n both di r e c t i o n s upon r e c o n s t i t u t i o n r e s u l t s from the rapid establishment of a pH gradient across the membrane (3). Uncouplers allow the c y c l i n g of protons across the membrane and r e l i e v e the i n h i b i t i o n of transhydrogenation. 10. The r e s p i r a t o r y c o n t r o l r a t i o i s the r a t i o of transhydrogenase a c t i v i t y i n the presence to that i n the absence of uncoupler (65). The respiratory control r a t i o serves as an i n d i c a t o r of f u n c t i o n a l r e c o n s t i t u t i o n of transhydrogenase. Respiratory c o n t r o l was abolished by detergents such as T r i t o n X-100 or lysophosphatidylcholine (65,66). No latent transhydrogenase a c t i v i t y was observed when reconstituted transhydrogenase was treated with detergents. Since pyridine nucleotides cannot cross the membranes, the enzyme molecules in the reconstituted v e s i c l e s must be oriented asymmetrically with t h e i r a c t i v e s i t e s exposed to the external medium. Pennington and Fisher (67) demonstrated that reconstituted mitochondrial transhydrogenase i s a transmembrane p r o t e i n . P u r i f i e d bovine heart mitochondrial transhydrogenase was asymmetrically inserted into phosphatidylcholine liposomes by c h o l a t e - d i a l y s i s procedure. N-(4-Azido-2-nitrophenyl)-2-aminoethy1sulfonate, a membrane-impermeant photoprobe, when encapsulated i n the v e s i c l e s , covalently modified the enzyme and i n h i b i t e d transhydrogenation. External AcNAD (3-acetylpyridine analog of NAD) increased the rate of i n a c t i v a t i o n s e v e r a l - f o l d , whereas NADPH, NADP and NADH were without e f f e c t . L a b e l l i n g of the enzyme by the i s o t o p i c a l l y l a b e l l e d photoprobe was enhanced by AcNAD and NADP, decreased by NADH, and not s i g n i f i c a n t l y affected by NADPH. These r e s u l t s indicate that reconstituted mitochondrial transhydrogenase spans the membrane and that substrate binding a l t e r s the conformation of the enzyme. Neither the £. c o l i nor R. rubrum enzyme have been p u r i f i e d and reconstituted into membranes. 11. Transhydrogenase as a Proton Pump Several l i n e s of evidence indicate that reconstituted transhydrogenase functions as a proton pump. Direct evidence for proton translocation coupled to transhydrogenation has been provided by the use of pH probes such as 9-aminoacridine (50,64,65) and 9-amino-6-chloro-2-methoxyacridine (66). A decrease i n i n t r a v e s i c u l a r pH causes the uptake of the probes with a resultant quenching of fluorescence (68). During NADPH*NAD transhydrogenation by the reconstituted enzyme, the fluorescence of these probes was s u b s t a n t i a l l y quenched, i n d i c a t i n g uptake of the probe i n response to a decrease i n i n t r a v e s i c u l a r pH. Similar r e s u l t s were obtained using the nonpermeant pH i n d i c a t o r , f l u o r e s c e i n isothiocyanate-dextran trapped within the v e s i c l e (65). A d i r e c t demonstration of proton translocation coupled to transhydrogenation in reconstituted v e s i c l e s was c a r r i e d out by Earle and Fisher (3,69). Transhydrogenase was reconstituted into potassium loaded phosphatidylcholine v e s i c l e s and the v e s i c l e s were suspended i n potassium-free buffer. The rate of NADPth-NAD transhydrogenation was measured using the NAD analogue AcNAD. Proton t r a n s l o c a t i o n was measured using a pH electrode. When NADPH*AcNAD transhydrogenation was c a r r i e d out i n the presence of valinomycin a concomitant uptake of protons from the medium was demonstrated by electrode measurements. About one proton was translocated for each hydride ion equivalent transferred between the substrates. Addition of valinomycin i n the absence of substrates did not r e s u l t i n the uptake of protons i n d i c a t i n g that transhydrogenase does not act as a proton pore. 12. E f f e c t of an Electrochemical Potential on Reconstituted Transhydrogenase Rydstrom (66) found that both pH gradients and membrane potentials influence the transhydrogenase reaction. When the reduction of AcNAD by NADPH was catalyzed by transhydrogenase v e s i c l e s with an i n t e r n a l pH of 8 i n a medium of pH 6, a transient phase of high i n i t i a l a c t i v i t y was observed which r a p i d l y declined to a lower a c t i v i t y . This implied that the a r t i f i c i a l l y imposed pH gradient promoted NADPH*AcNAD transhydrogenation. Interactions between transhydrogenase and imposed membrane potentials were investigated with potassium gradients i n the presence of valinomycin. A high external concentration of potassium chloride (150 mM), generating a minimal membrane p o t e n t i a l of 100 mV, po s i t i v e inside the v e s i c l e s , caused an i n h i b i t i o n of the reduction of AcNAD by NADPH. The reduction of AcNAD by NADPH was stimulated when a membrane p o t e n t i a l of the same size but negative inside the v e s i c l e s was generated by the presence of valinomycin and the same concentration of potassium chloride i n s i d e the v e s i c l e s . Thus, these r e s u l t s i n d i c a t e that both a pH gradient and a membrane potential regulate the transhydrogenase reaction i n v e s i c l e s . Earle and Fisher (65) studied the influence of pH gradients and membrane potentials on reconstituted transhydrogenase using valinomycin and n i g e r i c i n . They found that creation of pH gradients, either a c i d i c inside the v e s i c l e s , by addition of n i g e r i c i n to proteoliposomes prepared with appropriate potassium gradients, had l i t t l e e f f e c t on the transhydrogenase rate i n eit h e r d i r e c t i o n . However, valinomycin-dependent movement of potassium ions, i n a d i r e c t i o n opposite to proposed transhydrogenase-coupled proton movements, stimulated the rate of transhydrogenation markedly. These r e s u l t s i n d i c a t e that reconstituted transhydrogenase i s influenced primarily by membrane pot e n t i a l s with only a l i m i t e d contribution by the pH gradient. 13. Chemical Modification with Dicyclohexylcarbodiimide NjN'-Dicyclohexylcarbodiimide (DCCD) has been known as a potent covalently i n t e r a c t i n g i n h i b i t o r of a number of enzymes involved i n proton translocation across b i o l o g i c a l membranes. DCCD i n h i b i t s proton-linked ATP synthase (70), ubiquinol-cytochrome c reductase from mammalian and yeast mitochondria (71,72), and cytochrome oxidase (73). In these systems DCCD i n h i b i t s proton t r a n s l o c a t i o n primarily rather than the h y d r o l y t i c or redox reactions catalyzed by these enzymes. Since transhydrogenase acts as a proton pump, i t i s an t i c i p a t e d that DCCD may modify the proton-binding domain i n an analogous way. Treatment of bovine heart submitochondrial p a r t i c l e s with DCCD does cause i n a c t i v a t i o n of transhydrogenase (74-76). The k i n e t i c s of i n a c t i v a t i o n suggest that the reaction of 1 mol of DCCD per ac t i v e enzyme complex r e s u l t s i n complete i n a c t i v a t i o n . Pennington and Fisher (74) found that NADPH and NADP stimulated i n a c t i v a t i o n of transhydrogenase by DCCD, whereas AcNAD and NADH afforded no protection. They concluded that DCCD-modified the transhydrogenase outside the active s i t e , p o s s i b l y i n a proton-binding domain that functions to translocate protons across the membrane. However, Phelps and Hate f i (75,76) reported that AcNAD and NADH protected the enzyme from i n a c t i v a t i o n and came to the conclusion that DCCD binds at, or near, the NAD(H)-binding s i t e on transhydrogenase. Both p u r i f i e d and submitochondrial transhydrogenases were l a b e l e l e d with [i',C]DCCD i n a manner which p a r a l l e l e d the extent of i n h i b i t i o n (74). By contrast Persson et a l . (50) found that treatment of reconstituted transhydrogenase with DCCD resulted i n an i n h i b i t i o n of proton pump a c t i v i t y without an e f f e c t on uncoupled c a t a l y t i c a c t i v i t y , suggesting that proton t r a n s l o c a t i o n and c a t a l y t i c a c t i v i t i e s are not o b l i g a t o r i l y 14. linked or that this agent separates proton pumping from the c a t a l y t i c a c t i v i t y . S i milar r e s u l t s had been observed by Pennington and Fisher (74). In experiments with transhydrogenase reconstituted potassium-loaded phosphatidylcholine v e s i c l e s , DCCD i n h i b i t e d the rate of proton uptake into the liposomes to a s i g n i f i c a n t l y greater extent than transhydrogenation. These r e s u l t s support the hypothesis that DCCD may modify the proton-binding domain of transhydrogenase. Reaction Mechanism of Transhydrogenase The mechanism by which transhydrogenation i s coupled to the energized state of the membrane i s unknown. M i t c h e l l has proposed a loop mechanism for the mitochondrial transhydrogenase (44). In t h i s mechanism, the enzyme i s reduced by a hydride ion equivalent donated by NADPH and a proton from the matrix side of the membrane to form a reduced-enzyme intermediate. This i s followed by the transfer of a hydride ion to NAD and the release of the proton to the c y t o s o l i c side of the membrane. Skulachev (85) suggested an al t e r n a t i v e to M i t c h e l l ' s scheme based on ligand-induced conformational changes. In the Skulachev model, the transhydrogenase was proposed to have c a t a l y t i c and proton t r a n s l o c a t i n g subunits. A p o s i t i v e charge on the proton t r a n s l o c a t i n g subunit was envisioned to be near the NADPH binding s i t e of the c a t a l y t i c subunit. The proton binding s i t e would reorient from one side of the membrane to the other when NADP, formed by the oxidation of bound NADPH by NAD, occupied the NADPH binding s i t e . Ligand-induced conformational changes i n the transhydrogenase have been detected i n p r o t e o l y t i c , thermostability and chemical modification studies. Both the bovine heart (86) and rat l i v e r (87) mitochondrial transhydrogenases were protected from thermal i n a c t i v a t i o n by NADPH, and became more thermally l a b i l e i n the presence of NADP. Neither NADH nor NAD affected thermostability. The rate of i n a c t i v a t i o n of mitochondrial transhydrogenase by t r y p s i n i s affected by the presence of ligands. Bovine heart transhydrogenase was protected by low concentrations of NAD or NADH (86), whereas these substrates did not a f f e c t t r y p s i n i n a c t i v a t i o n of the l i v e r enzyme. The tr y p s i n i n a c t i v a t i o n of both enzymes was stimulated i n the presence of NADPH. NADP had l i t t l e e f f e c t on the rate of i n a c t i v a t i o n . Hence, at least three d i f f e r e n t conformations of the mitochondrial transhydrogenase have been detected: unliganded enzyme, the NADPH-enzyme complex and the NADP-enzyme complex. Phenylglyoxal and 2,3-butanedione i n borate buffer i n h i b i t E. c o l i transhydrogenase a c t i v i t y (88). NADP, NAD and high concentrations of NADPH and NADH protected the enzyme against i n h i b i t i o n by 2,3-butanedione. Low concentratins of NADPH and NADH increased the rate of i n h i b i t i o n by 2,3-butanedione. Similar e f f e c t s were observed for the i n a c t i v a t i o n of E_. c o l i transhydrogenase by t r y p t i c digestion i n the presence of these coenzymes. It was concluded that there were at least two conformations of the a c t i v e s i t e of E. c o l i transhydrogenase. Separate binding s i t e s for the NAD(H) and NADP(H) substrates at the active s i t e are indicated by k i n e t i c studies (54,88,89,90), the existence oi i n h i b i t o r s s p e c i f i c a l l y competitive for binding with e i t h e r NAD or NADP (91,92), and by d i r e c t hydride ion transfer between the 4A locus of NADH and the 4B locus of NADPH. Kozlov et a l . (96) presented evidence that there i s a short distance between the NADP(H) and NAD(H) binding s i t e s of mitochondrial transhydrogenase. They found that the 7-nitrobenzofurazan-4-y1 d e r i v a t i v e of dephospho-CoA i s a competitive 16. i n h i b i t o r with regard to both binding s i t e s . The k i n e t i c s of the i n h i b i t i o n indicated that one molecule of the i n h i b i t o r binds simultaneously to both the NADP(H) and NAD(H) binding s i t e s of the enzyme. The presence of separate binding s i t e s indicated that p a r t i a l transhydrogenase reactions could take place at the NAD(H) and NADP(H) binding s i t e s . Bovine heart mitochondrial transhydrogenase was shown to catalyze an exchange r e a c t i o n between NADH and NAD, but only i n the presence ot NADPH (93). The stereochemistry of the NADH*NAD re a c t i o n was the transfer of hydride ion equivalent d i r e c t l y from the 4A locus of NADH to the 4A locus of the NADH product. The stereochemistry of hydride ion transfer between NADH and NAD provided evidence against NADH binding to the NADP s i t e . Because transhydrogenation involves only the 4B locus of NADPH, binding of NADH to the NADP s i t e would r e s u l t i n the s p e c i f i c removal of the 4B hydrogen of NADH. It was proposed that NADH+NAD transhydrogenation represents a p a r t i a l reaction of NADPH+NAD transhydrogenation which involves the p a r t i c i p a t i o n of a reduced enzyme intermediate. Bovine heart transhydrogenase was also shown to catalyze NADPH+NADP transhydrogenation (94,95). Wu and Fisher (95) demonstrated that during NADPH+NADP transhydrogenation the NADP was reduced e x c l u s i v e l y at the 4B locus and that oxidation of NADPH was predominantly at the 4B locus. Wu and Fisher (95) proposed that NADPH+NADP transhydrogenation represents a p a r t i a l reaction of NADH+NADP transhydrogenation which a l s o involves the p a r t i c i p a t i o n of a reduced enzyme intermediate. More recent r e s u l t s obtained by Enander and Rydstrom (97) do not support these conclusions. They reported that the reduction of NADP by NADPH by bovine heart mitochondrial transhydrogenase requires c a t a l y t i c amounts of NADH. This argues against the involvement of a reduced enzyme intermediate. Other evidence against the p a r t i c i p a t i o n of a reduced enzyme intermediate i s the lack of exchange between substrate hydrogen and water hydrogen (93,97,98) and the lack of reducible groups (47,48). The k i n e t i c mechanism of the transhydrogenase from E_. c o l i (54,88) and bovine heart mitochondria (97) have been determined. In both cases double r e c i p r o c a l plots of i n i t i a l v e l o c i t i e s for the reduction of NAD by NADPH versus substrate concentrations were convergent and i n t e r s e c t i n g i n d i c a t i n g a ternary complex mechanism. The 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 indicated that the order of addition of the substrates to the enzyme was random. Enander and Rydstrom (97) used the knowledge that mitochondrial transhydrogenase was a dimer and a proton pump to propose a model for transhydrogenation shown i n F i g . l a . The model i s based on the proposal that oligomeric proteins i n general may exert the so - c a l l e d h a l f of the s i t e s r e a c t i v i t y , i . e . , only h a l f of the subunits are c a t a l y t i c a l l y a ctive at a given time (99,100). In A ( F i g . l a ) , subunit I i s involved i n exchange of the products NADH and NADP for the substrates NAD and NADPH, and binding of a proton from the side of the enzyme facing the e x t e r i o r of the v e s i c l e s . Simultaneously, subunit II i s active i n pumping one proton from the proton-binding s i t e to the i n t e r i o r of the v e s i c l e s , driven by the reduction of NAD by NADPH. A s i m i l a r mechanism had been proposed by Pennington and Fisher (74). As shown i n F i g . l b , they proposed that the transhydrogenase dimer forms a proton channel which spans the inner mitochondrial membrane. In the unliganded native transhydrogenase conformation (CQ), the proton binding domain on each subunit i s inaccessible to protons on e i t h e r side of the 18. F i g . 1. Proposed proton pump mechanisms for mitochondrial transhydrogenase as outlined by Enander and Rydstrom (97)(a) and Pennington and Fisher (74)(b). A B N A D P * N A D P H \ N A D H ^ N A D V H * NAD*-NADH NAOPH-NADP* 3>C II - + H * NADPH-> NADP* NAD**NADH H * N A D * V i NADH ' NADPH NADP* 5>C ! I • * H + 17 1 9 . membrane. The binding of NADPH and NAD to either a c t i v e s i t e induces the formation of conformation , forming and exposing the proton hydrophobic binding domain to the matrix side of the membrane. Subsequent to protonation, hydride ion transfer generates a second ternary complex having conformation , i n which the proton binding domain i s exposed to the c y t o s o l i c side of the membrane. Products and protons are released and the enzyme returns to conformation CQ. This mechanism r e f l e c t s the stoichiometry of protons translocated for each turnover of the enzyme (H /H = 1 ) as determined with homogenous transhydrogenase reconstituted into phospholipid v e s i c l e s ( 6 9 ) . P h y s i o l o g i c a l Role of Transhydrogenase The p h y s i o l o g i c a l function of transhydrogenase i s unclear. Several phy s i o l o g i c a l roles for the enzyme i n mitochondria have been suggested such as supplying NADPH for biosynthesis and hydroxylation reactions ( 5 ) or p a r t i c i p a t i n g i n a pathway which inactivates hydroperoxides ( 7 7 , 7 8 ) . In E. c o l i , energy-linked transhydrogenase has been associated with the supply of NADPH for the biosynthesis of amino acids since the presence of the l a t t e r i n the growth medium repressed the l e v e l of the enzyme i n the c e l l s ( 5 7 ) . If the c e l l s are i n i t i a l l y cultured i n a medium containing high l e v e l s of amino acids and then washed and placed i n a medium containing low l e v e l s of amino acids, the l e v e l of transhydrogenase a c t i v i t y w i l l increase. Incorporation of chloramphenicol i n the induction medium i n h i b i t s the increase i n transhydrogenase a c t i v i t y i n d i c a t i n g that de novo protein synthesis i s required for the induction of transhydrogenase ( 7 9 ) . Gerolimatos and Hanson ( 8 0 ) presented evidence that the transhydrogenase of IS. c o l i may play a ro l e i n branched-chain 20. amino acid transport as they found that leucyl-tRNA functions as a regulator of the enzyme. However, the i r hypothesis could not explain the repressive e f f e c t s of the other amino acids on i t s formation. The E. c o l i transnydrogenase may function as a component of the ammonia as s i m i l a t i o n pathway. When E. c o l i was grown on glucose and various concentrations of NH^Cl, a s i m i l a r i t y i n the regu l a t i o n of transhydrogenase and glutamate dehydrogenase was observed (81). In the range of 0.5 to 20 mM NH^Cl both transhydrogenase and glutamate denydrogenase a c t i v i t i e s increased two- to threefold. NH^Cl concentrations of 20 to 60 mM resulted in r e l a t i v e l y constant s p e c i f i c a c t i v i t i e s f o r both enzymes. Higher exogenous NH^Cl, however, led to a decline i n both a c t i v i t i e s . The coregulation of transhydrogenase and glutamate dehydrogenase a c t i v i t i e s may indicate that transhydrogenase may act as a d i r e c t source of NADPH in the ammonia a s s i m i l a t i o n system. Mutants of E_. c o l i lacking transhydrogenase a c t i v i t y have been is o l a t e d (82-84). Such mutants grow normally under growth conditions so tar tested leading to the conclusion that under normal growth conditions an active transhydrogenase i s not es s e n t i a l to c e l l v i a b i l i t y . Objective of th i s Study Comparatively l i t t l e work has been done on the transhydrogenase from £. c o l i despite advantages that genetic manipulation of th i s system can o f f e r . One of the major goals of the work described i n t h i s thesis was to purify this enzyme, determine i t s subunit composition, and examine i t s possible r o l e as a proton pump. MATERIALS AND METHODS Chemicals and Isotopes The chemicals and column materials used in this work were the highest grade obtainable from commercial suppliers. Radioactive materials were purchased from Amersham International Corp. Restriction endonucleases were from Amersham International Corp., Boehringer Mannheim Biochemicals or Pharmacia P-L Biochemicals. Exonuclease BAL31, T4-DNA ligase, calf intestinal phosphatase and DNA polymerase I (Klenow fragment) were obtained from Boehringer Mannheim Biochemicals. Strains and Bacterial Growth Table 1 lists the strains of bacteria used in this study. These strains were stored at -50° to -70°C in 25% glycerol. Strains were prepared for storage by adding an equal amount of 50% glycerol to exponentially growing bacterial cultures. Cells used in this study were grown on one of three types of medium. LB medium: 1% Bacto-tryptone, 1% NaCl and 0.5% yeast extract; YT medium: 0.8% Bacto-tryptone, 0.5% NaCl and 0.5% yeast extract; M9 medium: 0.7% Na2HP04, 0.3% KH2P04, 0.1% NH4C1, 0.05% NaCl, 0.4% glucose, 100 ug/ml thiamine and supplemented with 40 ug/ml of the appropriate amino acids. Cells were grown with shaking (at 250 rpm) at 37°C to an absorbance of 1.2 at 600 nm. Larger batches of 4.5 1 were grown at 37°C with vigorous aeration (at 25 1/min) in a Lab-Line/S.M.S. Hi-Density Fermentor. The cells were harvested, washed with TED buffer (50 mM Tris-HCl, pH 7.8, 1 mM dithiothreitol, 1 mM EDTA) or 0.9% NaCl and either used immediately or stored at -70°C. 22. Table 1. B a c t e r i a l Strains STRAIN CHARACTERISTICS RH-5 F* pnt::Tn5 argE3 l a c Y l galK2 mtl-1 rpsL700 A." supE44 AB1450 JT thi-1 ilvD-16 argHl metBl hi s G l l a c Y l or lacZ4 malAl mtl-2 xyl-7 ara-13 gal-6 strA8 A9 or Al_7 tonA2 tsx-7 _X" supE44 gltB13 W6 pro MV-12 F + trpA thr, leu recA JM83 ara alac pro strA t h i I80d lacZ AM15 JM103 F 1 Alac pro supE t h i strA endA sbcB15 hsdR4 traD36 proAB lacIJl ZAM15 X1197 F" thr leu arg lacY gal minA minB t h i T6 re c A l s t r A 1 9 Ml. r"a-19 his-95 r e l A l metBl spoTl W1485 F + supE l i p GMS343 F" argE3 l a c Y l galK2 mtl-1 rpsL700 A_* supE44 Strains were supplied by B. Bachmann (E. c o l i Genetic Stock Centre). 23. Preparation of Membranes A l l steps were performed at 0-4°C. The c e l l s were suspended i n TED buffer (50 mM Tris-HCl, pH 7.8, 1 mM d i t h i o t h r e i t o l , 1 mM EDTA) and MgSO^ and DNasel were added to 5 mM and 1 uM, r e s p e c t i v e l y . The c e l l s were lysed by passage through an i c e - c o l d French pressure c e l l at 1400 kg/cm*. Unbroken c e l l s were removed by c e n t r i f u g a t i o n at 12,000 x g for 10 min. The supernatant was centrifuged at 180,000 x g for 2 h. The supernatant obtained i s referred to as the cytoplasmic f r a c t i o n . The membranes were suspended i n TED buffer. S o l u b i l i z a t i o n of Membrane Vesicles with Detergents The s o l u b i l i z a t i o n c h a r a c t e r i s t i c s of various detergents were determined as follows. Membranes of E. c o l i ML308-225 were prepared as described previously and suspended i n TED buffer at a protein concentration of 5 mg/ml. The suspension was divided i n t o several aliquots and to each was added dropwise, d i f f e r e n t amounts of detergents from e i t h e r a 10% (w/v) or 20% (w/v) stock s o l u t i o n i n TED bu f f e r , or i n some instances added without d i l u t i o n . A f t e r incubation f o r 30 min at 0°C, they were centrifuged at 200,000 x g for 2 h and the transhydrogenase and protein l e v e l s determined i n the supernatant and p e l l e t f r a c t i o n s . P u r i f i c a t i o n of Transhydrogenase from St r a i n W6 A l l steps were performed at 0-4°C with 10-15 g of W6 c e l l s as s t a r t i n g material. The c e l l s were suspended i n 40 ml of TED buffer and lysed by passage through a French pressure c e l l at 1400 kg/cm 2. Unbroken c e l l s were removed by ce n t r i f u g a t i o n at 12,000 x g for 10 min. The supernatant was adjusted to f i n a l volume of 200 ml with TED buffer and 24. was centrifuged at 180,000 x g for 2 h. The resulting pellet was suspended in 20 ml of TED buffer and 5 ml of 8 M urea was added to the membrane suspension. After incubation at 0°C for 10 min, the membrane suspension was diluted to 100 ml with TED buffer and centrifuged at 180,000 x g for 2 h. The resulting pellet was washed by resuspension and recentrifugation at 18,000 x g for 1 h twice with 1 mM Tris-HCl, pH 7.8, 0.2 mM dithiothreitol, 0.2 mM EDTA. The washed membranes were suspended in 20 ml of TED buffer and KCI and sodium deoxycholate (0.25 M stock) were added to final concentrations of 1 M and 15 mM, respectively. The membrane suspension was stirred for 10 min at 0°C and then centrifuged at 180,000 x g for 1 h. The amber supernatant was adsorbed batchwise onto a minimal amount of phenyl-Sepharose (Pharmacia). The resin was washed with TED buffer containing (1 mg/ml) Brij 35 to remove KCI. The resin was transferred to a column and the protein eluted with 20 mg/ml Triton X-100 in the same buffer. The eluted material was adsorbed onto a 32 ml (1.5 x 18 cm) DEAE-Bio-Gel A (Bio-Rad) column equilibrated with TED buffer containing 10% (v/v) glycerol and 1 mg/ml Brij 35. Proteins were eluted with a 0-200 mM NaCl gradient (200 ml) in the same buffer used to equilibrate the column. Fractions containing transhydrogenase activity were pooled and concentrated by ultrafiltration under ]S to a volume of 1 ml using an Amicon XM-50 membrane. The buffer in which the enzyme was dissolved was exchanged to 10 mM sodium phosphate pH 7.4, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mg/ml Brij 35 by gel filtration chromatography on Sephadex G-50. The enzyme was then adsorbed onto a 1 x 3 cm agarose/hexane/nicotinamide adenine dinucleotide column equilibrated with the same buffer. The column was washed sequentially with 10 ml of equilibration buffer, 10 ml of buffer containing 20 mM NaCl, 10 ml of 25. buffer, 5 ml of buffer containing 5 mM NADH, 5 ml of buffer, and the enzyme eluted by addition of 4 ml of buffer containing 10 mM NADH. This was followed by more buffer until a l l of the enzyme had been eluted. NADH was removed by desalting on a column of Sephadex G-50 equilibrated with sodium phosphate, pH 7.0, 1 mM dithiothreitol and 0.5 mg/ml Brij 35. Screening of the Clarke and Carbon Collection for Plasmids Carrying the pnt Gene Each of the 2,112 clones from the Clarke and Carbon collection was grown at 37°C for 48 h in 12.5 ml of M9 medium supplemented with 20 Mg each of leucine, threonine, and tryptophan, 1 ug of thiamine, and 1 U of colicin El per ml. Cells were harvested by centrifugation at 12,000 x g for 5 min at 4°C. The supernatant was discarded and the cells suspended in 2.5 ml of 0.1 M sodium phosphate buffer (pH 7.0) containing 0.2 mM dithiothreitol and 0.2 mM EDTA. The cells were broken by passage through an ice-cold French pressure cell at 1,400 kg/cm2. Samples of 450 ul were used to measure transhydrogenase activity. Preparation of Colicin El Colicin El was prepared from strain W3110 pColEl (101). An overnight culture of W3110 pColEl was streaked onto an LB plate and grown overnight at 37°C. Several of the colonies were then replica plated onto two LB plates. One plate was used as a master plate and grown overnight at 37°C. The remaining plate was incubated at 37°C for 2 h and then the colonies irradiated for 10 sec with a Mineralight short-wave lamp held 1 cm above the agar surface. The plate was incubated at 37°C for 1 h, then overlayed with a mixture of 0.5 ml of X1197 cells in 2.5 ml of top agar 26. (LB media with 0.8% agar), and incubated at 37°C overnight. Zones of c l e a r i n g indicated that the colony contained the C o l E l plasmid. A 50 ml culture of W3110 pColEl (LB media) was grown at 37°C with vigorous shaking to an absorbance of 0.5 at 600 nm. To each of eight 2 1 flasks containing 500 ml of LB media was added 5 ml of the W3110 pColEl culture and the f l a s k s were incubated at 37° with shaking (250 rpm) u n t i l the absorbance had reached 0.5 at 600 nm. Mitomycin C was then added to give a f i n a l concentration of 1 ug/ml and the f l a s k s were shaken for 14 h at 37°C. The c e l l s were harvested by c e n t r i f u g a t i o n at 4,500 x g for 15 min and resuspended i n 60 ml of 0.1 M potassium phosphate buffer, pH 7.0, containing 1 M NaCl. C e l l s were broken by passage three times through an i c e - c o l d French pressure c e l l at 1,400 kg/cm2. The broken c e l l s were centrifuged at 18,000 x g for 90 min and the p e l l e t discarded. Saturated ammonium s u l f a t e (40 ml) was added dropwise to the supernatant which was then s t i r r e d f o r 30 min i n an i c e bath. The p r e c i p i t a t e was removed by cen t r i f u g a t i o n at 12,000 x g for 10 min and the p e l l e t was discarded. Saturated ammonium s u l f a t e (35 ml) was added to the supernatant and the supernatant was s t i r r e d for 30 min i n an ice bath. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n at 12,000 x g for 10 min and the supernatant was discarded. The p e l l e t was suspended i n 5 ml of 0.1 M potassium phosphate bu f f e r , pH 7.5, and dialyzed f or 4 h against 6 1 of the same buffer. The volume was now 10 ml. 10 ml of 20% (v/v) g l y c e r o l was added and the crude c o l i c i n E l was stored at -20°C. T i t r a t i o n of the c o l i c i n was performed using s t r a i n X1197. Overnight cultures of XI197 grown i n LB media were added to top agar (0.5 ml of c e l l s to 2.5 ml of LB containing 0.8% (w/v) agar) and layered on LB p l a t e s . A drop of s e r i a l l y d i l u t e d c o l i c i n E l was placed on the plate which was then incubated overnight at 37°C. The number of units of c o l i c i n was ca l c u l a t e d as the r e c i p r i c a l of the most d i l u t e c o l i c i n s olution which caused l y s i s of the c e l l s on the pla t e . The preparations normally gave t i t r a t i o n s of 1 x 10" to 1 x 10 1 1 units of c o l i c i n per ml. Preparation of Plasmid DNA For large-scale preparations, plasmid DNA was amplified by t r e a t i n g the c e l l s with chloramphenicol as described by Maniatis et a l . (102). C e l l s were grown i n LB media to an absorbance of 0.6 and chloramphenicol was added to a f i n a l concentration of 50 ug/ml. C e l l s were shaken a further 12-18 h. Plasmid DNA was extracted from lysozyme-Triton X-100 lysates (103,104) or by a l k a l i n e l y s i s (102). Normally, the a l k a l i n e l y s i s method was used. This was performed as described below. The b a c t e r i a l p e l l e t from a 500 ml culture was resuspended i n 10 ml of 50 mM glucose, 25 mM T r i s - H C l , pH 8.0, and 10 mM EDTA containing 5 mg/ml lysozyme. Af t e r 5 min at room temperature, 20 ml of 0.2 N NaOH/1% SDS was added and the tubes were l e f t on ic e for 10 min. 15 ml of an i c e - c o l d s o l u t i o n of 5 M potassium acetate, pH 4.8, was added. This s o l u t i o n was prepared from 60 ml of 5 M potassium acetate, 11.5 ml of g l a c i a l a c e t i c acid and 28.5 ml of E^O. The tubes were inverted several times and l e f t standing i n i c e f o r 10 min. The c e l l DNA and b a c t e r i a l debris were removed by c e n t r i f u g a t i o n at 40,000 x g for 30 min at 4°C. The supernatant was transferred to a fresh tube and 0.6 volumes of isopropanol was added to each tube. The contents were mixed and l e f t to stand at room temperature f o r 15 min. The plasmid DNA was recovered by c e n t r i f u g a t i o n at 12,000 x g for 30 min at room temperature. The supernatant was 28. discarded and the p e l l e t of nucleic acid dried b r i e f l y i n a vacuum desiccator. The p e l l e t s were dissolved i n 9 ml of TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) and 9 g of cesium chloride was added. The cesium chloride s o l u t i o n was transferred to a 16 x 76 mm sealable tube and the tube was f i l l e d with a so l u t i o n of ethidium bromide (10 mg/ml i n H 2 O ) . The tube was sealed and centrifuged at 250,000 x g for 36 h at 15°C. Two bands of DNA were normally v i s i b l e i n ordinary l i g h t . The upper band consisted of line a r b a c t e r i a l DNA and nicked c i r c u l a r plasmid DNA; the lower band consisted of closed c i r c u l a r plasmid DNA. The lower band of DNA was col l e c t e d using an 18G needle and the DNA transferred to a tube. Ethidium bromide was removed by adding an equal volume of isoamyl alcohol saturated with water and mixing the two phases. The phases were separated by cent r i f u g a t i o n at 1,500 x g for 3 min at room temperature. The upper phase was discarded. The extraction was repeated several times u n t i l the pink color had disappeared. The aqueous phase was dialyzed against several changes of TE buf f e r . For small-scale i s o l a t i o n s , plasmid DNA was prepared from unamplified overnight cultures, using the a l k a l i n e l y s i s method (102). T y p i c a l l y , 1.5 ml of the culture was poured into a 1.5 ml tube and centrifuged for 1 min in a Micro-Centrifuge. The medium was removed by a s p i r a t i o n and the p e l l e t resuspended i n 100 ul of l y s i s buffer (50 mM glucose, 25 mM Tris-HCl, pH 8.0 and 10 mM EDTA containing 4 mg/ml of lysozyme). A f t e r 5 min at room temperature, 200 y l of an ic e - c o l d s o l u t i o n of 0.2 N NaOH i n 1% SDS was added and the contents mixed by i n v e r t i n g the tube two or three times. The tube was kept on ice for 5 min. Then, 150 u l of an i c e - c o l d s o l u t i o n of potassium acetate pH 4.8, made up as described for the large-scale plasmid i s o l a t i o n , was added. Aft e r 5 min at 0°C the s o l u t i o n 29. was centrifuged i n a Micro-Centrifuge for 7 min. The supernatant was added to a fresh tube and extracted with an equal volume of phenol/chloroform. A f t e r c e n t r i f u g i n g for 2 min i n a Micro-Centrifuge, the supernatant was transferred to a fresh tube. Two volumes of ethanol were added. A f t e r standing at room temperature for 2 min, the tubes were centrifuged at room temperature for 5 min i n a Micro-Centrifuge. The supernatant was removed and the p e l l e t dried b r i e f l y i n a vacuum desiccator. 50 ul of TE buffer containing 50 ug/ml DNase-free pancreatic RNase was added and the mixture incubated at 37°C for 5 min. The preparation of plasmid DNA was stored at -20° c e n t r i f u g a t i o n . P u r i f i c a t i o n of Nucleic Acids DNA was p u r i f i e d by e x t r a c t i o n with phenol/chloroform and p r e c i p i t a t i o n with ethanol. L i q u i f i e d phenol was prepared by e x t r a c t i n g several times with buffer (usually 1.0 M Tris-HCl pH 8.0, followed by 0.1 M Tris-HCl pH 8.0 and 0.2% 13-mercaptoethanol), u n t i l the pH of the aqueous phase was 7.6 (102). 8-Hydroxyquinoline was then added to a f i n a l concentration of 0.1%. This preparation i s r e f e r r e d to as phenol. Chloroform as used i n these experiments i s a 24:1 mixture of chloroform and iso-amyl a l c o h o l . The DNA was mixed with an equal volume of phenol or phenol/chloroform i n a polypropylene tube. The contents were mixed u n t i l an emulsion formed. The two phases were separated by c e n t r i f u g a t i o n i n a Micro-Centrifuge for 1 min. The upper aqueous phase was transferred to a fresh tube. The steps were repeated using chloroform alone. One-tenth volume of 0.3 M sodium acetate, pH 5.5, was added along with two volumes of ethanol. The tubes were stored at -20°C for at l e a s t 1 h. The DNA was 30. recovered by ce n t r i f u g a t i o n for 10 min i n a Micro-Centrifuge. The supernatant was discarded and the p e l l e t dried i n a vacuum d e s i c c a t o r . The DNA p e l l e t was suspended i n TE buffer. Digestion with R e s t r i c t i o n Enzymes R e s t r i c t i o n endonuclease digestions were done according to the manufacturer's i n s t r u c t i o n s . Conditions for p a r t i a l d i g e s t i o n of plasmid DNA were established by adding s e r i a l d i l u t i o n s of enzymes to the plasmid DNA and incubating at 37°C for 1 h. Reactions were terminated by the addition of one-sixth volume of concentrated "loading" buffer (0.25% bromophenol blue, 40% sucrose, 75 mM EDTA) and a sample was loaded onto an agarose gel for electr o p h o r e s i s . Once the correct d i l u t i o n of enzyme had been determined, p a r t i a l digestion of plasmid DNA was c a r r i e d out accordingly and the reactions were stopped by the addi t i o n of 0.5 M EDTA to a f i n a l concentration of 12 mM. Ligations with T4-DNA Ligase 200 ng of l i n e a r i z e d plasmid DNA and a threefold molar excess of the fragment to be subcloned were mixed i n a t o t a l volume of 8 Ml. 1 p l of 10-fold concentrated l i g a t i o n buffer (0.66 M Tris-HCl pH 7.5, 50 mM MgCl 2, 50 mM d i t h i o t h r e i t o l and 10 mM ATP) and 1 ul of 0.9 U/ul T4-DNA li g a s e were added. The contents were mixed with a vortex mixer and l e f t at room temperature for 1 to 12 h. Transformations Bacteria were transformed with plasmid DNA by the calcium ch l o r i d e procedure (102). About 40 ml of LB medium i n a 250 f l a s k was incubated with 0.1 ml of an overnight culture of the c e l l s to be transformed. The c e l l s were grown at 37°C with vigorous shaking to an absorbance of 0.2 to 0.6 at 600 nm. The f l a s k was placed on ice for 10 min and the c e l l s c o l l e c t e d by c e n t r i f u g a t i o n at 3000 x g for 5 min at 2°C. The c e l l p e l l e t was suspended i n 20 ml of 10 mM T r i s - H C l , pH 8.0, 50 mM C a C l 2 . The tube was l e f t on i c e for 25 min and then centrifuged as before. The supernatant was discarded and the c e l l s were gently suspended i n 4 ml of 10 mM Tris-HCl pH 8.0, 50 mM C a C l 2 and kept on i c e . For each transformation, 200 ul of the competent c e l l s were placed i n i c e - c o l d tubes and about 50 ng of plasmid DNA was added. The tubes were l e f t on ice for 40 min and then placed i n a 42°C bath for 2 min. The c e l l s were revived by adding 800 ul of LB media to each tube and incubating the c e l l s at 37° for 1 h. Up to 100 ul of the transformed c e l l s were streaked onto plates containing the desired medium and 1.5% agar. In the case of pUC plasmids 50 u l of 5-bromo-4-chloro-3-indolyl-S-D- galactopyranoside (2% i n N,N'-dimethylformamide) was a l s o present. Clones carrying an i n s e r t within the plasmid were i d e n t i f i e d as white co l o n i e s , whereas clones without i n s e r t s gave blue colonies. Electrophoresis of DNA Agarose slab gels (0.4 to 1.2%) were prepared (Bio-Rad, Mini-Gel) and run i n TBE buffer (0.09 M T r i s , 0.09 M boric acid, 2 mM EDTA) at 60 V for 2 h. Gels were stained i n ethidium bromide (0.5 ug/ml of H 20) and DNA-containing bands were observed under UV l i g h t . Photographs were taken using a Polaroid MP-4 camera f i t t e d with a red f i l t e r and using Type 667 f i l m . Extraction of i n d i v i d u a l DNA bands for l i g a t i o n from low-melting point agarose was performed as described by Burns and Beacham (105). The piece of agarose containing the DNA was put i n a volume of TE buffer so that the f i n a l concentration of agarose would be 0.1%. The agarose was melted by incubation at 65°C for 5 min and then cooled. The DNA i n the s o l u t i o n was then l i g a t e d as described previously. Dephosphorylation of DNA The terminal 5' phosphates were removed from DNA by treatment with c a l f i n t e s t i n a l a l k a l i n e phosphatase (CIP)(102). The DNA was digested with the r e s t r i c t i o n enzyme of choice, extracted once with phenol/chloroform, and the DNA p r e c i p i t a t e d with ethanol. The DNA was dissolved i n a minimum volume of 10 mM T r i s - H C l , pH 8.0. Water and 5 u l of ten-fold concentrated b u f f e r (0.5 M Tris-HCl pH 9.0, 10 mM MgCl 2 > 1 mM ZnC^, 10 mM spermidine) were added to bring the t o t a l volume to 50 u l . 0.01 units of C1P was added to remove the terminal phosphates from 1 pmole of DNA. To dephosphorylate protruding 5' termini, the preparation was incubated at 37°C for 30 min, then a second ali q u o t of CIP was added and the incubation was continued for a further 30 min. To dephosphorylate DNA with blunt ends or recessed 5' termini, the preparation was incubated for 15 min at 37°C followed by 15 min at 56°C. A second ali q u o t of CIP was added and the incubations were repeated at both temperatures. The CIP was then inactivated by adding 40 u l of H 20, 10 ul of STE (100 mM Tris-HCl pH 8.0, 1 M NaCl, 10 mM EDTA), 5 ul of 10% SDS, and heating the sample at 68°C for 15 min. The DNA was extracted twice with phenol/chloroform and p r e c i p i t a t e d with ethanol. 3 3 . Nuclease BAL31 Digestion An equal volume of 2-fold concentrated buffer (24 mM CaC^, 24 mM MgCl 2, 0.4 M NaCl, 40 mM Tris-HCl pH 8.0, 2 mM EDTA) was added to the sample of DNA. The samples were incubated at 37°C for 3 min and then a pre-determined amount of BAL31 was added. At appropriate times EGTA (0.2 M pH b.0) was added to a f i n a l concentration of 20 mM and the samples were placed on i c e . The samples of BAL31-digested DNA were extracted with phenol/chloroform and p r e c i p i t a t e d with ethanol. P u r i f i c a t i o n of Transhydrogenase from JM83 pDC21 A l l steps were performed at 0-4°C with 3-5 g of JM83 pDC21 c e l l s as s t a r t i n g m a t e r i a l . The c e l l s , suspended in 40 ml of TED buffer containing 5 mM MgSO^ and a small amount of DNase I, were lysed by passage through an i c e - c o l d French pressure c e l l at 1,400 kg/cm2. Unbroken c e l l s were removed by c e n t r i f u g a t i o n of the lysate at 12,000 x g for 10 min. The supernatant was adjusted to a f i n a l volume of 100 ml with TED buffer and centrifuged at 210,000 x g for 2 h. The membrane p e l l e t s were suspended i n 50 ml of TED buffer and T r i t o n X-100 was added to a f i n a l concentration of 1% (v/v). A f t e r s t i r r i n g at 0°C for 5 min, the membrane suspension was centrifuged at 210,000 x g for 90 min. The p e l l e t s were suspended i n 50 ml of TED buffer, and sodium cholate (0.5 M) was added to a f i n a l concentration of 50 mM. After s t i r r i n g at 0°C for 5 min, the membrane suspension was again centrifuged at 210,000 x g for 90 min. The p e l l e t was suspended i n 15 ml of TED buffer and KCI was added to a f i n a l concentration of 1 M. Sodium deoxycholate (0.25 M) and sodium cholate (0.5 to) were then added to f i n a l concentrations of 15 mM each. Af t e r s t i r r i n g tor 10 min at 0°C, the insoluble material was removed by 3 4 . centrifugation at 210,000 x g for 4 5 min. The r e s u l t i n g supernatant was layered onto a cushion of 1.1 M sucrose i n TED buffer containing 2.3 mM sodium cholate, and centrifuged at 260,000 x g for 16 h. The tube was punctured and ten 1.4 ml f r a c t i o n s were c o l l e c t e d . Fractions containing transhydrogenase a c t i v i t y were pooled and stored at 0-4°C. Peptide Mapping P r o t e o l y t i c d i g e s t i o n was c a r r i e d out using the method described by Cleveland et a l . (106). The f i r s t slab gel (15 x 14 x 0.075 cm) was composed of 10% polyacrylamide with a 4% stacking gel as described by Laemmli (7). Each well received 60 ug of p u r i f i e d transhydrogenase in Laemmli sample buffer. Electrophoresis was c a r r i e d out at 100 mA/slab. The gels were stained i n 0.1% (w/v) Coomassie Blue, 25% (v/v) isopropanol, 10% (v/v) a c e t i c acid for 30 min, and then destained for 60 min in 10% (v/v) a c e t i c a c i d . The desired polypeptides were excised from the g e l , soaiced for 30 min in buffer B (0.125 M Tris-HCl pH 6.8, 1 mM EDTA, 0.1% (w/v) SDS), and then loaded into the wells of a slab gel (15 x 14 x 0.15 cm) composed of 15% acrylamide with a 4% stacking g e l . To each well was added 10 ul of chymotrypsin (0.25 mg/ml) i n buffer B containing 10% (v/v) g l y c e r o l . Electrophoresis was performed at 100 mA/slab u n t i l the proteins had entered the stacking g e l . The power was then shut o f f . After 30 min, electrophoresis was continued as before. The gels were stained overnight in 0.1% (w/v) Coomassie Blue, 25% (v/v) isopropanol, 10% (v/v) a c e t i c acid and destained i n 10% (v/v) a c e t i c a c i d . 35. Polyacrylamide Gel Electrophoresis Polyacrylamide gel electrophoresis was performed as described by Laemmli (107). The separating gel contained 0.375 M T r i s - H C l , pH 8.8, 0.1% bDS and 7-15% acrylamide (prepared from a stock s o l u t i o n of 30% (w/v) acrylamide and 0.8% (w/v) N,N 1-methylenebisacrylamide i n water). For each 15 ml of g e l , polyacrylamide was effected by the ad d i t i o n of 75 y l of freshly prepared 10% (w/v) ammonium persulfate and 15 Ml of TEMED. A 15 x 14 x 0.075 or 0.15 cm gel was poured and approximately 0.5 ml of t e r t i a r y butanol was layered on the gel surface. A f t e r 1 h, the t e r t i a r y butanol was removed. A stacking gel of 0.125 M T r i s - H C l , pH 6.8, 0.1% (w/v) SDS and 4% acrylamide was prepared. Polymerization was i n i t i a t e d by the addition of 10 ul of TEMED and 15 ul of f r e s h l y prepared 10% (w/v) ammonium pers u l f a t e . The gel mixture was layered over the separating gel and allowed to polymerize for 1 h. Samples were prepared by adding an equal volume of SDS sample buffer (0.125 M Tr i s - H C l , pH 6.8, 4% (w/v) SDS, 20% (v/v) g l y c e r o l , 10% ft-mercaptoethanol). The samples were loaded onto the gel which was immersed i n running buffer (25 mM T r i s base, 192 mM glycine, 0.1% (w/v) SDS). Electrophoresis was c a r r i e d out at 100 mA/slab. The gels were stained with Fairbank's s t a i n (108) of 0.1% (w/v) Coomassie b r i l l i a n t blue R, 25% (v/v) isopropanol and 10% (v/v) a c e t i c a c i d . The gels were destained with 10% (v/v) a c e t i c a c i d . Reconstitution of Transhydrogenase P u r i f i e d transhydrogenase was reconstituted with egg yolk phosphatidylcholine by the cholate d i l u t i o n method (65). P u r i f i e d transhydrogenase (0.5 mg/ml, 26.4 U/mg of protein) was passed through a Sephadex G-50 column to exchange the buffer to 10 mM Hepes-KOH, pH 7.5, 36. 300 mM KCI, 0.1 mM d i t h i o t h r e i t o l , 1.5% (w/v) sodium cholate. One ml of egg phosphatidylchol ine (10 mg/ml) was dried under nitrogen and the residue was suspended i n 5 ml of the same buffer. The l i p i d suspension was c l a r i f i e d by passage through a French pressure c e l l at 1,400 kg/cm2 twice. Ten ug of p u r i f i e d transhydrogenase was then added to 0.2 ml of l i p i d suspension and d i l u t e d into the appropriate assay medium. In v i t r o Protein Synthesis In v i t r o protein synthesis was carried out with a procaryotic DNA-directed t r a n s l a t i o n k i t obtained from Amersham International Corp. Proteins were translated i n the presence of [ 1 sSJmethionine according to the i n s t r u c t i o n s of the s u p p l i e r . A premix for each r e a c t i o n was prepared by mixing together 7.5 ul of supplement s o l u t i o n , 3 u l of amino acid mixture and 2 Ul of [ 1 sS]methionine (1460 Ci/nmole, 15.8 Ci/ml). Reaction mixtures were prepared adding 12.5 ul of DNA (3-5 ug) i n d i l u t i o n buffer to 12.5 ul of the premix. The re a c t i o n mixture was placed in a 37°C water bath and the reaction was started by adding 5 ul of the S-30 c e l l e x t r a c t . A f t e r incubating the reaction at 37°C for 60 min, 5 ul of methionine chase so l u t i o n was added and incubation was continued for a further 5 min. An equal amount of SDS sample buffer was added and the proteins were separated by SDS/polyacrylamide gel electrophoresis. The g e l was stained with Fairbank's s t a i n for 30 min, destainea with 10% (v/v) a c e t i c acid, treated with Amplify (Amersham) for 15 min, and then d r i e d . Autoradiography was performed overnight using Kodak XAR-5 f i l m . In some cases an S-30 extract was used that was prepared from s t r a i n A19 as follows (109). The growth medium was composed of 2.6% (w/v) 37. K 2HP0 4, 0.5% (w/v) KH 2P0 4, 0.9% (w/v) yeast extract, 1% (w/v) glucose, 13.5 ul/ml thiamine and 50 ug/ml of methionine. S t r a i n A19 was grown overnight in 200 ml of medium in a 1 1 f l a s k at 37°C with shaking. One ml of the overnight culture was used to inoculate each of twelve 2 1 f l a s k s containing 400 ml of medium. The c e l l s were grown to an absorbance of 0.3 at 600 nm with shaking (250 rpm) at 30°C. The c e l l s were harvested by c e n t r i f u g a t i o n at 4,500 x g for 15 min. They were suspended in i c e - c o l d buffer S (0.01 M T r i s - a c e t a t e , pH 8.2, 0.014 M MgOAc, 0.06 M KOAc, 1 mM d i t h i o t h r e i t o l ) and centrifuged at 8,000 x g for 5 min. The c e l l s were resuspended in 40 ml of buffer S and recentrifuged. The y i e l d of c e l l s was 2.5 g. The washed c e l l s were suspended i n 10 ml of buffer S and broken in an i c e - c o l d French pressure c e l l at 5b0 kg/cm2. D i t h i o t h r e i t o l was then added to give a f i n a l concentration of 1 mM. The extract was centrifuged twice at 30,000 x g for 30 min, keeping the supernatant each time. Equal parts of mix (1 M Tris-acetate pH 7.8, 5 ml; 0.14 MgOAc, 1.0 ml; 1 M d i t h i o t h r e i t o l , 15 u l ; 20 mM amino acids mixture lacking methionine, 25 ul) and mix 2 (0.2 h ATP, pH 7.0, 0.2 ml; 75 mM sodium phosphoenolphosphate, 6 ml; pyruvate kinase, 0.5 mg) were mixed and 2 ml was added to 8 ml of the c e l l e xtract. A f t e r incubation at 37°C for 90 min i n the dark, the extract was dialyzed against 200 volumes of buffer S for 14 h at 4°, changing the buffer once. The extract was divided into aliquots and stored at -70°C. DNA Sequence Determination DNA sequence determination was performed using the chain termination procedure (110,111). The procedure consists of two parts; preparation of single-stranded phage and DNA sequence determination. Phages M13mpl8 and M13mpl9 were used. 3 8 . I s o l a t i o n of M13 Phage 0.1 ml of an overnight culture of JM103 c e l l s grown i n M9 media was added to 5 ml of YT medium and the culture incubated at 37°C for 2 h with shaking. This culture was added to 45 ml of YT medium and 2 ml was d i s t r i b u t e d to separate culture tubes. Each phage plaque was cored from a plate with a 50 u l disposable micropipet and the agar plug was blown into the medium i n a culture tube. The tubes were incubated at 37°C for 5 h with vigorous shaking. The contents of each tube were poured into a 1.5 ml centrifuge tube and the c e l l s p e l l e t e d by c e n t r i f u g a t i o n i n a Micro-Centrifuge for 1 min. The supernatant (0.8 ml) was added to 200 Ul of 20% (w/v) PEG-6000/2.5 M NaCl, mixed, and l e t stand at room temperature for 15 min. The phage were c o l l e c t e d by c e n t r i f u g a t i o n i n a Micro-Centrifuge for 5 min. The supernatant was removed and the phage p e l l e t was suspended i n 100 y l of TE buffer. The phage p e l l e t was extracted with 50 Ml of phenol by vortexing for 10 sec followed by centrifuging i n the Micro-Cenrifuge for 1 min. The aqueous layer was then extracted three times with 500 ul of water-saturated d i e t h y l ether. The single-stranded DNA was p r e c i p i t a t e d by adding 10 ul of 3 M sodium acetate, pH 5.5, and 300 y l of ethanol and c h i l l i n g the tube at -20°C overnight. The DNA was c o l l e c t e d by c e n t r i f u g a t i o n i n the Micro-Centrifuge for 5 min at 0-4°C. The p e l l e t was dr i e d i n a vacuum desiccator and the DNA resuspended i n 50 y l of TE b u f f e r . Sequence Reaction The f i r s t step of the sequencing reaction was to anneal the primer to the template. To 5 y l of single-stranded M13 DNA was added 1 y l of 17 base deoxynucleotide primer (2.4 yg/ml) and 1 y l of 10-fold 39. concentrated annealing buffer (0.1 M Tris-HCl, pH 8.5, 0.1 M MgCl 2) and 1 u l of ti^O. The tube was placed in 70°C water i n a heating block. The heating block was allowed to cool to about 40°C and 1 u l of 12 uM dATP and 1 ul of deoxyadenosine 5 ' -[<*-3 *P J triphosphate (3000 Ci/mmole, 10 mCi/ml) was added to the DNA. The contents of the tube were mixed and 2 y l was d i s t r i b u t e d to each of the four nucleotide r e a c t i o n tubes each of which contained 2 ul of the appropriate terminator mix. The terminator mixes of the following compositions in 50 mM T r i s - H C l , pH 7.5, 1 mM EDTA: A, 0.5 mM ddTTP, 5.4 uM dTTP, 109 uM dCTP, 109 uM d&lP; B, 50 uM ddCTP, 109 uM dTTP, 5.4 uM dCTP, 109, uM dGTP; C, 0.5 mM ddGTP, 109 uM dTTP, 109 uM dCTP, 5.4 uM dGTP; D, 50 uM ddATP, 77 UM dTTP, 77 UM dCTP, 77 UM dGTP. The r e a c t i o n was started by adding 0.5 units of the Klenow fragment of DNA polymerase i n 2 u l of Klenow d i l u t i o n buffer (10 mM Tris-HCl pH 8.0, 1 mM d i t h i o t h r e i t o l , 50 ug/ml of nuclease-free BSA, 10% (v/v) g l y c e r o l ) to each of the reaction tubes. The tubes were incubated at 37°C for 15 min and then 2 ul of 0.5 mh dATP was added to each tube and incubation was continued for a further 15 min. The reactions were stopped by adding 4 ul of formamide stop mix (90% (v/v) formamide, 20 mM EDTA, 0.03% (w/v) xylene cyanol, 0.03% bromophenol blue) to each reaction mixture. The reaction tubes were placed i n a b o i l i n g water bath for 3 min and then cooled on i c e . Polyacrylamide gels were prepared by f i r s t mixing together 25 g of urea, 7.5 ml of 38% (w/v) acrylamide/2% (w/v) N,N'-methylenebisacrylamide, 5 ml of 10-fold concentrated TBE buffer (1.78 M T r i s , 1.78 M boric a c i d , 20 mM EDTA), 0.5 ml of 10% (w/v) ammonium persulfate i n a t o t a l volume of 50 ml. The gel was degased and polymerization was i n i t i a t e d by adding 20 u l of TEMED. The gel was poured to form a 17 x 30 x 0.05 cm g e l . A f t e r the gel 40. had polymerized, i t was attached to the elect r o p h o r e s i s apparatus and pre-run at 28 amps/gel f o r 10 min using TBE b u f f e r . About 2 u l of each sequencing reaction was loaded i n each well and the gels were run at 28 mA/gel for e i t h e r 2 or 5 h. The gels were then placed on 0.3 mm Whatman paper, covered with Saran Wrap and drie d f o r one hour on a gel-dryer at 80°C. Autoradiography was performed overnight using Kodak XRP-1 f i l m . The f i l m was treated for 5 min with Kodak GBX developer and replenisher and then treated for 5 min with Kodak f i x e r . The developed f i l m was rinsed with water for 30 min and d r i e d . I s o l a t i o n of Transhydrogenase ot and 13 Subunits A 15 x 14 x 0.15 cm 10% polyacrylamide gel with a 4% stacking gel was prepared as described f o r Laemmli gels except that the TEMED and ammonium per s u l f a t e concentrations were reduced by one-half. The s l o t in the stocking gel was 12 cm wide. P u r i f i e d transhydrogenase (450 ug) i n SDS-sample buffer was layered onto the g e l . Electrophoresis was c a r r i e d out at 100 mA per slab u n t i l the bromophenol blue dye front reached the end of the g e l . The gel was placed i n a glass d i s h , washed three times with water and cooled on i c e . Ice-cold 0.2 M KC1 was added to the gel (112). A f t e r 5 min the white protein bands could e a s i l y be seen against a black background. The protein bands were excised from the g e l , cut i n t o very small pieces and then placed i n a screw-cap tube with 4-5 volumes of 0.1% (w/v) SDS. The tube was incubated with shaking for four hours at room temperature. The contents of the tube were placed i n a 10 ml syringe and f i l t e r e d through a 45 um Acrodisc f i l t e r . Cold 100% t r i c h l o r o a c e t i c a c i d was added to the f i l t r a t e to give a f i n a l concentration of 12%. Af t e r incubation on i c e for 2 h, the p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n for 15 min i n a Micro-Centrifuge at 0-4°C. The protein p e l l e t was washed three times with i c e - c o l d 10% t r i c h l o r o a c e t i c acid and three times with i c e - c o l d acetone. The protein p e l l e t was dried i n a vacuum desiccator. The sample was then sent to the Un i v e r s i t y of V i c t o r i a for protein sequencing. The amount of each subunit recovered was estimated to be between 50 and 100 ug on the basis of the i n t e n s i t y of s t a i n i n g when a sample was run on a Laemmli g e l . I s o l a t i o n of Transhydrogenase Subunits using the Prep-Gel Apparatus The BRL Prep-Gel apparatus was used according to the manufacturer's i n s t r u c t i o n s . A 6 cm 10% Laemmli g e l with a 1 cm 4% stocking gel was cast i n the apparatus. Laemmli running buffer containing 0.1 mM sodium mercaptoacetate was used. A sample containing 50-200 ug of p u r i f i e d transhydrogenase i n SDS-sample buffer was layered on the g e l . Electrophoresis was c a r r i e d out at 150 V, c o l l e c t i n g 10 min fract i o n s at a flow rate of 10 ml/h at room temperature. The transhydrogenase subunits eluted a f t e r 5-6 h. Protein Assay Protein concentration was determined according to the method of Lowry et a l . (114) with the exception that 1% (w/v) SDS was incubated i n the assays. Assay of Energy-Independent Transhydrogenase A c t i v i t y The assay of transhydrogenase a c t i v i t y was based on the method of Kaplan (113). The reaction was c a r r i e d out at 25°C i n 50 mM sodium phosphate buffer, pH 7.0, containing 1 mM KCN, 1 mM d i t h i o t h r e i t o l , 0.5 mM EDTA, 1 mM AcNAD and 0.5 mM NADPH i n a f i n a l volume of 1 ml. The reduction of AcNAD by NADPH was measured as an increase i n the absorbance at 375 nm using a Coleman 124 spectrophotometer attached to a chart recorder. 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 5.1 1/mmol/cm (113). One unit of enzyme a c t i v i t y represents the conversion of 1 umol of AcNAD to AcNADH per min. The assay was modified when assaying soluble transhydrogenase by including 0.025% (w/v) B r i j 35 in the assay medium. Assay of Energy-Dependent Transhydrogenase A c t i v i t y This was measured by a modification of the method of Fisher and Sanadi (25). Membrane-bound transhydrogenase was incubated i n an assay medium of 50 mM Tr i s - H C l , pH 7.8, 10 mM MgSO^, 1 mM d i t h i o t h r e i t o l and 0.16 M sucrose. The cuvette was then transferred to a Perkin-Elmer model 124 spectrophotometer, maintained at 37°C by means of a c i r c u l a t i n g water bath. Af t e r 5 min, 10 y l of ethanol, 50 ul of yeast alcohol dehydrogenase (4 mg/ml) and 25 ul of 2.7 mM NAD were added. The absorbance of the reaction mixture at 340 nm was monitored. Af t e r 1 min, 50 ul of 15.7 mM NADP was added and the reduction NADP measured (aerobic-driven transhydrogenase). When the oxygen i n the cuvette was exhausted, the formation of NADPH was now due to the energy-independent transhydrogenase. 10 ul of 65 mM ATP, pH 7.8, was added and the new rate of NADP reduction was measured (ATP-driven transhydrogenase). The rate of the energy driven transhydrogenase was corrected by subtracting the rate of energy-independent transhydrogenation. The e x t i n c t i o n c o e f f i c i e n t was 6.22 1/mmol/cm. One unit of enzyme a c t i v i t y represents the conversion of 1 umole of NADP to NADPH. 43. Fluorescence Assays I n t r a v e s i c u l a r pH changes were followed f l u o r o m e t r i c a l l y with 9-aminoacridine with a Turner Spectrofluorometer (model 420) using the indicated wavelength pair ( e x c i t a t i o n , 420 nm; emission, 500 nm). Assays were conducted i n 10 mM Hepes-KOH buffer, pH 7.5, containing 300 mM KCI, 5 mM h g C l 2 , 0.5-5 tiM 9-aminoacridine, 250 uM NADPH and reactions were i n i t i a t e d by addition of 500 \xM AcNAD. Preparation of RNase that i s Free of DNase A c t i v i t y Pancreatic RNase (RNase A) was dissolved i n 10 mM Tris-HCl pH 7.5/15 mM NaCl at a concentration of 10 mg/ml. The enzyme preparation was boiled for 15 min and then allowed to cool slowly to room temperature. The RNase was dispensed into aliquots and stored at -20°C. Glutamate Dehydrogenase Assay Glutamate dehydrogenase was assayed using the method described by Sakamoto et a l . (115). The following solutions were added to a 1 ml quartz cuvette: 0.5 ml of 0.1 M potassium phosphate buffer, pH 8.0, containing 1 mM EDTA, 0.05 ml of 0.2 M 2-oxoglutarate, 0.05 ml of 0.4 M ammonium ch l o r i d e , 0.01 ml of 10 mM NADPH, 0.39 ml of water and sample. The absorbance of the reaction mixture was followed at 340 nm using a Perkin-Elmer model 124 spectrophotometer maintained at 30°C by means of a c i r c u l a t i n g water bath. The reaction was started by addition of the sample. The same assay was used for glutamate synthase except that 40 mM glutamine was included i n the assay instead of NH.Cl. 4 4 . Pl Transduction Bacteriophage P l v i r was obtained from Dr. P. Dennis of th i s department. The preparation of lysates and transductions were c a r r i e d out as described by M i l l e r (116). The lysate was prepared as follows. An overnight b a c t e r i a l culture from which the lysate was to be prepared was used to inoculate (100 y l ) 10 ml of LB media containing 0.1% (w/v) glucose and 5 mM CaCl^ i n a 50 ml f l a s k . This culture was incubated with shaking u n t i l an absorbance reading of 0.2 at 600 nm was obtained. One ml of this culture was placed i n a test tube containing about 1 x 10 6 phage and l e f t for 20 min at 37°C. To each tube was added 2.5 ml of R-top agar (1% (.w/v) Bacto-tryptone, 0.1% (w/v) yeast extract, 0.8% (w/v) NaCl, 0.1% (w/v) glucose, 2 mM C a C l 2 > 0.8% (w/v) agar) and the e n t i r e contents were poured onto plates containing R-medium (1% (w/v) Bacto-tryptone, 0.1% (w/v) yeast extract, 0.8% (w/v) NaCl, 0.1% (w/v) glucose, 2 mM CaC^, 1.2% (w/v) agar). The plates were incubated at 37°C for 5-6 h. The top agar was scrapped o f f and placed i n a screw-top tube with 4 ml of LB media, 50 ul of 1 M sodium c i t r a t e and 10 drops of chloroform. The contents of the tube were mixed vigorously on a vortex mixer for 1 min and the tube was l e f t at 0-4°C overnight. The agar was removed by c e n t r i f u g a t i o n at the maximum speed i n an International desk-top centrifuge for 30 min. The supernatant was removed and stored i n screw-top tubes i n the presence of a few drops of chloroform. The bacteriophage were t i t r a t e d using s t r a i n W1485. The above procedures were repeated for the t i t r a t i o n using d i f f e r e n t d i l u t i o n s of the phage. The plates were incubated overnight at 37°C and the number of cl e a r plaques were counted. T i t e r s ranged from 1 x 1 0 s - l x 10l 1 phage/ml. 45. Transductions were c a r r i e d out as follows. Overnight cultures of the desired s t r a i n of b a c t e r i a i n LB medium containing 10 mM CaC^ were added to d i f f e r e n t d i l u t i o n s of the phage stock i n a t o t a l volume of 400 l i l . The tubes were incubated at 37°C for 20 min. An equal volume of IM sodium c i t r a t e was added to the c e l l s and 100 ul of samples of the transduction mix were streaked out onto the s e l e c t i o n media. The plates were incubated overnight at 37°C. La b e l l i n g of Membrane Vesicles with [l,tC]DCCD ['"CJDCCD (50 mCi/mMole) was purchased i n ether i n a sealed v i a l . The ether was evaporated at room temperature under nitrogen and the dried contents were taken up i n absolute ethanol (5 mM [ll*C]DCCD). Membranes were prepared and suspended i n 10 mM Tr i s - H C l , pH 7.8, containing 0.25 M sucrose, 5 mM MgSO^ and 0.2 mM d i t h i o t h r e i t o l at a protein concentration of 5 mg/ml. To 0.5 ml of the membrane suspension was added 10 ul of 5 mM [ 1 < ,C]DCCD i n absolute ethanol and the suspension s t i r r e d at 4°C for 12 h. The l a b e l l e d membranes were washed f i v e times i n buffer by resuspension and c e n t r i f u g a t i o n at 250,000 x g for 1 h. Crossed Immunoelectrophoresis This was based on the methods of Nowotny (117), Bjerrum and Lundhal (118) and Mayer and Walker (119). 2.4 ml of 1% (w/v) agarose i n Bjerrum buffer, pH 8.8, containing 100 mM glycine, 38 mM T r i s and 1% (w/v) T r i t o n X-100 at 55°C was poured onto a 50 x 50 mm glass plate surrounded by a p l a s t i c mold. The agarose was allowed to cool to 20 °C and the mold c a r e f u l l y removed such that a 40 x 40 x 1.5 mm gel was obtained. Agarose 46. i n the same buffer was layered around the periphery of the cast gel to the edges of the plate (agarose bridges) to give a more uniform conduction of the current. A row of four wells (3 mm diameter), about 1 cm apart, was cut ouc with a Bio-Rad gel-puncher. The wells were f i l l e d with various levels of antigen and the gel placed in a Pharmacia flat-bed electrophoresis unit at 4°C. Wicks, 4 cm wide (Ultrawicks, Bio-Rad) were placed onto the agarose bridges i n such a manner that they did not overlap onto the wells. The chambers were f i l l e d with Bjerrum buffer, pH 8.8, without any detergent, and electrophoresis was c a r r i e d out for 1.5-2 h at 4°C and 100 V. A f t e r electrophoresis, the gel was cut into s t r i p s (5 mm width) such that each contained a sample well at one end. The i n d i v i d u a l s t r i p s were stained immediately or run i n the second dimension. 1% (w/v) agarose i n Bjerrum buffer pH 8.8 and various l e v e l s of antiserum ( f i n a l volume, 2.4 ml) were mixed at 55°C and cast onto a 40 x 40 x 1.5 mm gel as above. A s t r i p of the gel from the first-dimension was placed at the cathodic end of the gel and agarose bridges constructed around the g e l . The electrode buffer was the same as i n the f i r s t dimension. Electrophoresis was c a r r i e d out perpendicular to the d i r e c t i o n of the f i r s t dimension for 16 to 18 h at 10 V and 4°C. The proteins were stained with Fairbank's s t a i n (108). Reaction of E. c o l i Transhydrogenase with Mitochondrial Anti-Transhydrogenase Membranes were prepared from 3 g each of JM83 pUC13 and JM83 pDCll. Tne proteins (70 ug) were separated on a 0.15 cm 10% SDS/polyacrylamide gel with a 4% stacking g e l . The gel was e q u i l i b r a t e d i n 500 ml of 25 mM T r i s , 192 mM glycine and 20% (v/v) methanol for 30 min. The gel was placed i n a transfer apparatus (Bio-Rad) next to wetted n i t r o c e l l u l o s e i n e q u i l i b r a t i o n buffer and the proteins were transferred to the n i t r o c e l l u l o s e at 100 V for 12 h. The apparatus was cooled by c i r c u l a t i n g tap water- Staining of the gel indicated that the two transhydrogenase subunits had been transferred to the n i t r o c e l l u l o s e . The membrane was washed twice for 30 min i n TBS (20 mM Tris-HCl pH 7.5, 500 mM NaCl, 3% (w/v) BSA) and placed i n a sealed bag with 1:25 d i l u t e d antibody ( g i f t from R.R. Fisher) and incubated overnight at room temperature. Excess antibody was removed by washing the membrane twice with 100 ml of TBS containing 0.05% (w/v) Tween 20. [ l 2 s I ] P r o t e i n A was added and the membrane incubated for 1 h i n a sealed p l a s t i c bag. The excess protein A was washed three times with TBS containing 0.05% (w/v) Tween 20. The n i t r o c e l l u l o s e was a i r dried and developed by autoradiography. No bands were observed. 48. RESULTS I. P h y s i o l o g i c a l Role of Transhydrogenase The p h y s i o l o g i c a l functions of the pyridine nucleotide transhydrogenase of E_. c o l i have not been e s t a b l i s h e d . To gain i n s i g h t into the ro l e o f t h i s enzyme, mutants which lack transhydrogenase a c t i v i t y were i s o l a t e d by Hanson's group (82,84). As shown i n Table 2, one of these mutants, RH-5, has normal growth rates when grown a e r o b i c a l l y on LB, or on a synthetic medium with glucose, g l y c e r o l or fructose as carbon source when compared to the growth rates of the parental s t r a i n . No di f f e r e n c e was observed between anaerobic growth rates of the mutant and parent on synthetic media with glucose or g l y c e r o l plus fumarate as carbon source. These r e s u l t s confirm the observations of Hanson's group (82,84) and support h i s conclusion that under normal growth conditions an a c t i v e transhydrogenase i s not e s s e n t i a l to c e l l v i a b i l i t y . In E. c o l i , the energy-linked transhydrogenase has often been postulated to act as a source of NADPH for the biosynthesis of amino acids since the presence of the l a t t e r i n the growth medium represses the l e v e l of the enzyme i n the c e l l s (57,120). An i n t e r r e l a t i o n s h i p between amino ac i d biosynthesis and transhydrogenase a c t i v i t y may occur during the a s s i m i l a t i o n of nitrogen. Recent work on the E_. c o l i transhydrogenase has implicated t h i s enzyme as a source of NADPH for glutamate dehydrogenase (81). As shown i n F i g . 2, glutamate dehydrogenase i s one of the pathways f o r a s s i m i l a t i o n of ammonia i n E. c o l i . Liang and Houghton (81) reported that glutamate dehydrogenase and transhydrogenase are coregulated during nitrogen l i m i t a t i o n . S i m i l a r r e s u l t s were obtained when E. c o l i s t r a i n W6 was grown on glucose at various concentrations of NH.Cl ( F i g . 3). In Table 2. E f f e c t of transhydrogenase a c t i v i t y on aerobic growth r a t e s . Doubling Time (min) S t r a i n Glucose Glycerol Fructose LB GMS 343 (pnt + ) 58 139 97 36 RH5 (pnt::Tn5) 64 131 95 38 C e l l s were grown in 200 ml of M9 medium containing 0.2% (w/v) of the indicated carbon sources or in LB medium in 2 1 f l a s k s at 37°C with shaking (250 rpm). RH-5 was grown i n the presence of 25 ug/ml kanamycin. C e l l growth was measured from the absorbance of the cul t u r e at 600 nm. 50. F i g . 2 Pathways of nitrogen a s s i m i l a t i o n i n E_. c o l i . a-oxoglutarate HOOC-CO-CH 2 -CHi -COOH HOOC-CH-CH 2 - C H 2 -COOH NHi glutamate CH-CH-COOH 3' J_ NH1 valine (a) CH-CO-COOH a-oxoisovalerate NH 3 + ATP glutamate glutamate glutamine synthetase ADP + P| glutamine NADP [ glutamate synthase - NADPH? a-oxoglutarate (b) v the range of 0.5 to 20 mM NH^Cl the a c t i v i t i e s of both glutamate dehydrogenase and transhydrogenase increased two- to t h r e e f o l d . Higher exogenous NH^Cl concentrations led to a decline i n the s p e c i f i c a c t i v i t i e s of both enzymes. Coordinate changes in the l e v e l s of transhydrogenase and glutamate dehydrogenase could indicate that the enzyme i s involved i n the supply of NADPH, s p e c i f i c a l l y for glutamate dehydrogenase when i t functions i n the synthesis of glutamate. This hypothesis was tested by i n a c t i v a t i n g the other pathway for nitrogen a s s i m i l a t i o n . This pathway includes glutamate synthase. The s t r a i n AB1450 (gltB13), lacking glutamate synthase a c t i v i t y , i s dependent on glutamate dehydrogenase for a s s i m i l a t i o n of ammonia. The transhydrogenase a c t i v i t y of AB1450 was inac t i v a t e d by Pl transduction from the pnt s t r a i n RH-5. The gltB13 pnt mutant obtained d i d not require glutamate for growth on a minimal s a l t s medium. Its growth rate was i d e n t i c a l to that of the parent (Table 3,4). These r e s u l t s i n d i c a t e that transhydrogenase i s not the sole source of NADPH for nitrogen a s s i m i l a t i o n by glutamate dehydrogenase i n E. c o l i . I I . P u r i f i c a t i o n of transhydrogenase from S t r a i n W6 Growth of C e l l s E. c o l i s t r a i n W6 was used as a source of enzyme. The transhydrogenase a c t i v i t y of t h i s s t r a i n i s c o n s i s t e n t l y higher than that of s t r a i n K-12 (81). Transhydrogenase a c t i v i t y i s repressed when E. c o l i i s grown on complex media containing high l e v e l s of amino acids (81,120). Transferring of such c e l l s into a glucose minimal medium r e s u l t s i n a 5- to 10-fold increase i n transhydrogenase a c t i v i t y (55). Therefore, i n F i g . 3. E f f e c t of exogenous NH^Cl on glutamate dehydrogenase and transhydrogenase a c t i v i t i e s i n E. c o l i W6. C e l l s were grown on M9 medium containing various concentrations of NH^Cl. The c e l l s were harvested i n late-exponential phase (as determined by the absorbance of cultures at 600 nm) and washed twice with TED buffer (50 mM T r i s - H C l , pH 7.8, 1 mM d i t h i o t h r e i t o l ) . Membrane and cytoplasmic f r a c t i o n s were prepared from the c e l l s , and the glutamate dehydrogenase (GDH) and transhydrogenase (PNT) a c t i v i t i e s measured as described in Materials and Methods. Measurements were obtained at least from two separate experiments. 53. Table 3. Role of transhydrogenase i n the a s s i m i l a t i o n of ammonia. Enzyme A c t i v i t i e s [umoles min" 1(mg p r o t e i n ) " 1 ] Doubling time Trans- Glutamate Glutamate St r a i n (min) hydrogenase dehydrogenase synthase GMS 343 68 0.36 0.28 0.38 RH-5(pnt::Tn5) 65 0 0.24 0.38 AB1450(gltB13) 78 0.18 0.42 0 AB1450(pnt::Tn5,gltB13) 80 0 0.37 0 C e l l s were grown i n 200 ml of M9 media (containing 25 ug/ml o f kanamycin for transhydrogenase mutants) i n 2 1 flasks at 37°C with shaking. The growth of the c e l l s was measured using the absorbance at 600 nm. The c e l l s were harves ted i n late-exponential phase and washed with TED b u f f e r . Enzyme assays and the preparation of membrane and cytoplasmic f r a c t i o n s are described i n Materials and Methods. 54. Table 4. E f f e c t of transhydrogenase mutation on the growth of glutamate synthase mutants. Doubling Time (min) S t r a i n Glucose Fructose Glycerol LB AB 1450 (gltB13) 78 106 146 31 AB 1450 (gltB13, pnt::Tn5) 80 98 144 35 C e l l s were grown i n 200 ml of M9 medium containing 0.2% (w/v) of the indicated carbon source or i n LB medium i n 2 1 flasks at 37°C with shaking (250 rpm). AB 1450 (gltB13, pnt::Tn5) was grown i n the presence of 25 ul/ml kanamycin. C e l l growth was measured from the absorbance at 600 nm. order to maximize the expression of transhydrogenase, c e l l s were grown i n a glucose minimal medium. The s p e c i f i c a c t i v i t y of transhydrogenase in membranes prepared from c e l l s grown under these conditions ranged from 0.9 to 1.3 pmoles/min per mg of protein. Selection of Detergent Transhydrogenase i s an i n t e g r a l component of the cytoplasmic membrane. In contrast to water-soluble proteins, membrane-bound proteins must be released from the membranes pr i o r to p u r i f i c a t i o n . An e f f e c t i v e detergent should s a t i s f y c e r t a i n conditions. F i r s t , the detergent should be able to release a s i g n i f i c a n t percentage of the enzyme from the membrane. Second, i t should not i n a c t i v a t e the enzyme under study. Third, i t would greatly a i d the p u r i f i c a t i o n scheme i f the detergent could s e l e c t i v e l y s o l u b i l i z e a s i g n i f i c a n t amount of the enzyme without s o l u b i l i z i n g other membrane-bound proteins. On the basis of these c r i t e r i a a number of detergents were tested to select a suitable detergent for the extraction of the transhydrogenase from the cytoplasmic membrane. Stra i n ML 308-225 was used as s t a r t i n g material for these preliminary experiments. Membrane v e s i c l e s were prepared and treated with various l e v e l s of detergents. Transhydrogenase a c t i v i t y was considered as being s o l u b i l i z e d i f i t was not sedimented following c e n t r i f u g a t i o n at 200,000 x g for 2 h. The following detergents s o l u b i l i z e d less than 25% of the membrane-bound transhydrogenase and were considered i n e f f e c t i v e : cholate, taurodeoxycholate, deoxycholate, T r i t o n X-100, B r i j 35, B r i j 96, Lubrol WX, Aminoxide WS 35 and n-octyl-fi-D-glucoside when used at concentrations up to 2%. The r e s u l t s of the treatment with various detergents which s o l u b i l i z e d more than 25% of the a c t i v i t y are shown i n F i g . 4. Optimal s o l u b i l i z a t i o n of the transhydrogenase a c t i v i t y was obtained with the detergent sodium deoxycholate i n the presence of 1 M KCI. At a detergent to protein r a t i o of 0.8, 100% of the a c t i v i t y was s o l u b i l i z e d . Some stimulation of transhydrogenase occurs i n the presence of deoxycholate as 12% of the o r i g i n a l a c t i v i t y was detected i n the p e l l e t f r a c t i o n . Deoxycholate exhibited some s e l e c t i v i t y , i n that only 34% of the membrane protein was s o l u b i l i z e d at a detergent to protein r a t i o of 0.8. Taurodexoycholate i n the presence of 1 M KCI also s o l u b i l i z e d s i g n i f i c a n t amounts of transhydrogenase a c t i v i t y . At a detergent to protein r a t i o of 0.4, taurodeoxycholate s o l u b i l i z e d 73% of the a c t i v i t y but only 37% of the membrane protein. Dodecyl-6-D-maltoside and N-lauroyl sarcosine s o l u b i l i z e d over 50% of the transhydrogenase a c t i v i t y but with less s e l e c t i v i t y when compared to the r e s u l t s obtained with deoxycholate and taurodeoxycholate. At a detergent to protein r a t i o of 1, dodecyl-IJ-D-maltoside s o l u b i l i z e d 64% of the transhydrogenase a c t i v i t y and 49% of the membrane protein. N-lauroyl sarcosine exhibited the least s e l e c t i v i t y of the detergents tested. At a r a t i o of 0.6, 65% of the membrane protein was s o l u b i l i z e d , but only 50% of the transhydrogenase was detected i n the supernatant. Since only 5% of the a c t i v i t y was found i n the p e l l e t , 45% of the a c t i v i t y was in a c t i v a t e d . It i s clear from these studies that the most suitable detergent for the s o l u b i l i z a t i o n of transhydrogenase i s deoxycholate i n the presence of KCI. F i g . 4. S o l u b i l i z a t i o n of membrane-bound transhydrogenase with various detergents. Membrane v e s i c l e s i n TED buffer at a protein concentration of 5 mg/ml were treated with various levels of detergent as described in Materials and Methods. N-lauroyl sarcosine (•-•); sodium deoxycholate in the presence of 1 M KC1 (o-o); taurodeoxycholate i n the presence of IM KC1 (u-u); dodecyl-S-D-maltoside (•-«). Transhydrogenase Activity Protein Detergent-mg Protein-mg 58. P u r i f i c a t i o n of Transhydrogenase The p u r i f i c a t i o n procedure outlined under 'Methods and Materials' i s represented i n Table 5. The pu r i t y of the transhydrogenase at various stages was assessed by SDS-polyacrylamide gel electrophoresis ( F i g . 5). To remove peripheral proteins l i k e l y to contaminate the s o l u b i l i z e d transhydrogenase, membrane v e s i c l e s were extracted sequentially with 2 M urea and low s a l t b u f fer. Over 90% of the transhydrogenase a c t i v i t y was retained i n the membrane v e s i c l e s a f t e r these washing procedures. The extracted membrane v e s i c l e s were incubated with 0.6% deoxycholate i n the presence of 1 M KCI. Cholate was added to prevent the deoxycholate from forming a g e l . A f t e r c l a r i f i c a t i o n by centr i f u g a t i o n , 24% of the t o t a l membrane protein and 75% of the transhydrogenase a c t i v i t y remained i n s o l u t i o n . The high concentration of s a l t had to be removed p r i o r to ion-exchange chromatography. A rapid desalting procedure was developed using hydrophobic chromatography. The s o l u b i l i z e d transhydrogenase was adsorbed onto a minimal amount of phenyl-Sepharose. KCI was removed by washing the r e s i n with a column volume of buffer and the transhydrogenase then eluted with T r i t o n X-100. Although ion-exchange chromatography was performed i n the presence of the detergent B r i j 35, t h i s detergent was found to be i n e f f e c t i v e i n e l u t i n g the bound transhydrogenase from phenyl-Sepharose. Recoveries of about 90% were obtained during t h i s step when T r i t o n X-100 was used. The material from phenyl-Sepharose was adsorbed onto a DEAE-Bio Gel A column and then eluted using a 0-200 mM NaCl l i n e a r gradient containing 0.05% (w/v) B r i j 35. As shown i n F i g . 6, considerable amount of the applied protein did not bind to the ion-exchange r e s i n . Transhydrogenase Table 5. P a r t i a l p u r i f i c a t i o n of transhydrogenase from E. c o l i s t r a i n W6. Transhydrogenase A c t i v i t y F r a c t i o n Protein Total S p e c i f i c % U/mg protein Membranes 311 100 1.3 Extracted Enzyme 74 75 4.1 Af t e r phenyl-Sepharose chromatography 52 63 4.9 Combined f r a c t i o n s a f t e r chromatography on DEAE-Bio-Gel A 7.8 25 13 Combined fr a c t i o n s a f t e r chromatography on NAD-Agarose 1.4 3.9 22 Membranes were prepared from 12.1 g of c e l l s . A f t e r t r e a t i n g the membranes with 2 M urea, the transhydrogenase was s o l u b i l i z e d by the detergent deoxycholate i n the presence of KC1. After d e s a l t i n g the extract using phenyl-Sepharose, the enzyme was p a r t i a l l y p u r i f i e d using ion-exchange and a f f i n i t y chromatography. Experimental d e t a i l s of the p u r i f i c a t i o n are described i n Materials and Methods. 60. F i g . 5. SDS-polyacrylamide gel electrophoresis of f r a c t i o n s at various stages of the transhydrogenase p u r i f i c a t i o n from E. c o l i s t r a i n W6. A sample was removed at each of the various stages of the p u r i f i c a t i o n scheme outlined i n Table 5 and analyzed by SDS/polyacrylamide gel electrophoresis as described i n Materials and Methods. AG-NAD+; + NAD -agarose. F i g . 6. Separation of transhydrogenase by ion-exchange chromatography. S o l u b i l i z e d transhydrogenase (50 mg of p r o t e i n ) , which had been desalted by hydrophobic chromatography, was subjected to chromatography on a 1.5 x 18 cm DEAE Bio Gel A column as described i n Materials and Methods. Fractions of 4.2 ml were c o l l e c t e d and assayed f o r transhydrogenase a c t i v i t y («-a). Protein was detected from the absorbance at 280 nm (u-u) and NaCl concentration by conductivity (o-o). Fractions 28 to 32 were saved. Fraction Number a c t i v i t y was detected i n fractio n s 24-35 with the peak fr a c t i o n s e l u t i n g at 100 mM NaCl. Most of the applied protein was eluted o f f the column p r i o r to the appearance of transhydrogenase a c t i v i t y i n the eluant. Fractions 28-32 were pooled and the buffer was exchanged to 10 mM sodium phosphate pH 7.4, 1 mM EDTA, 1 mM DTT and 0.05 mg/ml B r i j 35 using a Sephadex G-25 column. The desalted transhydrogenase from the ion exchange column was applied to a column of NAD agarose. The column was washed successively with e q u i l i b r a t i o n buffer, buffer containing 20 mM NaCl, buffer containing 5 mM NADH, and buffer with 10 mM NADH. As shown i n F i g . 7, most of the transhydrogenase a c t i v i t y was eluted with 10 mM NADH, although s i g n i f i c a n t amounts were eluted by the 20 mM NaCl wash. The t o t a l number of units of transhydrogenase recovered a f t e r t h i s step represented 1-4% of the s t a r t i n g m a t e r i a l . Analysis of the p u r i f i e d transhydrogenase using SDS-PAGE showed the presence of three major protein bands of molecular weights 100,000, 52,000 and 47,000, as well as several minor protein bands. The p u r i f i c a t i o n procedure discussed above was not e n t i r e l y s a t i s f a c t o r y . Yields of transhydrogenase were low and the pur i t y of the material varied g r e a t l y from p u r i f i c a t i o n to p u r i f i c a t i o n . The greatest v a r i a b i l i t y occurred during the ion-exchange or a f f i n i t y chromatography steps. During ion-exchange chromatography, the best p u r i f i c a t i o n occurred when the transhydrogenase eluted l a t e r than the bulk of the applied protein. Unfortunately, i n many cases the two peaks would overlap s l i g h t l y r e s u l t i n g i n a less pure transhydrogenase preparation. The y i e l d and p u r i t y of the transhydrogenase following a f f i n i t y chromatography varied from run to run. Another problem with the p u r i f i c a t i o n procedure was that the transhydrogenase preparation was contaminated with several other proteins. F i g . 7. P u r i f i c a t i o n of transhydrogenase by a f f i n i t y chromatography. P a r t i a l l y p u r i f i e d transhydrogenase (7.8 mg) was subjected to chromatography on a 1 x 3 cm AG-NAD Type I column (P-L Biochemicals) as described i n Materials and Methods. The column was washed successively with buffer ( f r a c t i o n s 1-3), buffer containing 20 mM NaCl ( f r a c t i o n 4), buffer containing 5 mM NADH ( f r a c t i o n 8), and transhydrogenase eluted with 10 mM NADH ( f r a c t i o n 11). Each f r a c t i o n was assayed for transhydrogenase a c t i v i t y (•-•). Fraction Number 64. Transhydrogenase i s found i n r e l a t i v e l y minor amounts i n the E_. c o l i c e l l membrane. This increases the d i f f i c u l t y of p u r i f i c a t i o n . This d i f f i c u l t y was overcome by increasing the expression of transhydrogenase by cloning the pnt gene onto a multi-copy plasmid. I I I . Cloning of the pnt Gene I d e n t i f i c a t i o n of the pnt plasmids Mutants de f e c t i v e i n the expression of transhydrogenase do not e x h i b i t a r e a d i l y detectable phenotype. Hanson and Rose (84) reported that E_. c o l i s t r a i n s d e f e c t i v e i n both glucose-6-phosphate dehydrogenase and transhydrogenase produced much smaller colonies when grown ana e r o b i c a l l y on minimal media plates when compared to the parent which was defective i n glucose-6-phosphate dehydrogenase alone. However, I found the d i f f e r e n c e i n colony s i z e between the two s t r a i n s to be n e g l i g i b l e . This property r u l e d out any screening of an E. c o l i plasmid bank based on a recognizable phenotypic change when seeking the pnt locus. Therefore, the Clarke and Carbon colony bank was screened based on the r a t i o n a l e that E_. c o l i c e l l s harboring multicopy plasmids containing the pnt gene would contain elevated l e v e l s of the enzyme. Among the approximately 2,200 clones of the Clarke and Carbon c o l l e c t i o n , clones pLC 10-19, pLC 26-24, and pLC 27-35 contained 8- to 10-fold more transhydrogenase a c t i v i t y when compared with the other clones of the bank (Table 6). Over 90% of the enzyme a c t i v i t y was associated with the membranes of these c e l l s . Expression of E_. c o l i transhydrogenase a c t i v i t y i s maximal when c e l l s are grown i n minimal media free of high l e v e l s of 65. Table 6. Transhydrogenase a c t i v i t y i n membranes of selected s t r a i n s from the Clarke-Carbon colony bank Transhydrogenase A c t i v i t y (umoles/min/mg protein) S t r a i n LB medium M9 medium MV12(pLC 14-12) a 0.04 0.18 MV12(pLC 10-19) 0.32 1.33 MV12(pLC 26-24) 0.38 1.52 MV12(pLC 27-35) 0.42 1.80 aThe l e v e l of transhydrogenase a c t i v i t y i n t h i s s t r a i n i s t y p i c a l of those st r a i n s not carrying the pnt gene on a Col EI plasmid. amino acids (57). Increased l e v e l s of transhydrogenase were observed when each of the pnt bearing clones was grown i n minimal M9 medium. Membranes prepared from c e l l s grown i n minimal medium exhibited a f o u r f o l d increase i n transhydrogenase a c t i v i t y as compared with the transhydrogenase a c t i v i t y of membranes prepared from c e l l s grown i n LB medium (Table 6). R e s t r i c t i o n Endonuclease Analysis of the pnt Plasmids R e s t r i c t i o n endonuclease analysis was performed on the three recombinant plasmids from the Clarke and Carbon colony bank to i d e n t i f y the regions of DNA containing the pnt gene. An 8.7-kilobase region was found to be common to the i n s e r t s of a l l three plasmids ( F i g . 8). The region of the E_. c o l i genome bearing the transhydrogenase gene has been p h y s i c a l l y mapped by Bouche1 (121). Comparison of the r e s t r i c t i o n endonuclease maps of the plasmids with that of the genome revealed that the plasmid i n s e r t s overlap a region of the genome between 35.2 and 35.7 min ( F i g . 8). The overlap region common to a l l three plasmids included the 35.4 min region which i s the p o s i t i o n mapped by Hanson and Rose (84) for the pnt gene. Subcloning of the pnt Gene into pUC13 Plasmid pLC 26-24 was digested by r e s t r i c t i o n nuclease P s t l to give four fragments (10.4, 5.9, 1.2 and 0.1 k i l o b a s e s ) . The fragments were separated by gel electrophoresis i n low-melting-point agarose, and the 10.4 kilobase fragment was excised from the g e l . Pstl-digested pUC13, which had been dephosphorylated with c a l f i n t e s t i n a l phosphatase, was l i g a t e d with the 10.4-kilobase fragment, and the l i g a t e d DNA was used to transform s t r a i n JM83. White a m p i c i l l i n - r e s i s t a n t transformants were 67. F i g . 8. Comparison of r e s t r i c t i o n endonuclease maps of Col EI plasmid in s e r t s with a region of the E. c o l i genome. The r e s t r i c t i o n map of the E. c o l i genome was determined by Bouche (121). —1 I I ? T i T pLC 10-19 -, 1 I I T T—L pLC 26-24 1 I I T T pLC 27-35 , , 1 u—1—1—i 1 i T Genome 35.2 min 3 5 7 m i n Eco Rl _L Hind III 1 H 1kb Pst I -r 68. selected and screened for overproduction of transhydrogenase. Plasmid pDCl, containing the 10.4-kilobase P s t l fragment inserted into pUC13, amplified transhydrogenase a c t i v i t y 20-fold i n JM83. A 4.8-kilobase H i n d l l l fragment of pDCl was subcloned into the H i n d l l l s i t e of pUC13 as shown i n F i g . 9 to y i e l d plasmid pDC3. Transhydrogenase a c t i v i t y of JM83 carrying pDC3 was 50-fold greater when compared with JM83 harboring pUC13. L o c a l i z a t i o n of the pnt Gene i n pDC3 Plasmid pDC3 was subjected to r e s t r i c t i o n endonuclease a n a l y s i s , using the r e s t r i c t i o n endonucleases Hpal, BstEII, Xhol, Smal, and S a i l . A f t e r the locations of the r e s t r i c t i o n endonuclease s i t e s had been established, various segments of the 4.8-kilobase H i n d l l l inserts of pDC3 or pDC4 were removed. Plasmids pDC3 and pDC4 d i f f e r only i n the or i e n t a t i o n of the i n s e r t i n the pUC13 vector. Plasmid pUC13 contains a sin g l e EcoRI s i t e . Plasmids i n F i g . 10 are drawn so that t h i s s i t e i s closest to one end of the i n s e r t . The constructed plasmids were used to transform JM83. The c e l l s were grown and t h e i r transhydrogenase l e v e l s were determined ( F i g . 10). Deletion of a 0.55-kilobase Hpal-Smal fragment from pDC3 or a 1.6-kilobase Hpal-Smal fragment from pDC4 to give plasmids pDC8 and pDC9, re s p e c t i v e l y , resulted i n loss of enhanced expression of transhydrogenase a c t i v i t y . These r e s u l t s demonstrated that at least the 2.65-kilobase fragment bounded by the Hpal r e s t r i c t i o n s i t e s was e s s e n t i a l for the expression of enzyme a c t i v i t y . During the construction of pDC9 from pDC4, one clone was i s o l a t e d which contained plasmid pDCll. This plasmid was missing 0.75-kilobases of DNA between the Hpal and H i n d l l l s i t e s ( F i g . 10). There was no Hpal s i t e at t h i s point i n pDC4 so the 69. F i g . 9. Subcloning of DNA carrying the pnt gene. The plasmids and r e l a t i v e positions of the r e s t r i c t i o n s i t e s are drawn approximately to scale. Pstl 70. F i g . 10. R e s t r i c t i o n endonuclease maps of plasmids containing the pnt gene and transhydrogenase a c t i v i t i e s of membranes prepared from c e l l s harboring each of the plasmids. Plasmids were constructed and transformed into JM83. Membranes were prepared from transformants grown i n LB medium and assayed for transhydrogenase (PNT) a c t i v i t y . The transhydrogenase a c t i v i t y of membranes prepared from JM83 pUC 13 was 0.035 U/mg protein. Symbols: P, Pat I; H, Hind I I I ; B, Bst EII; Hp, Hp_a I; E, Ec£ R l ; S, Sal I; X, Xho I; T, Sst I; M, Sma I; s o l i d l i n e s are inserted DNA. PNT P L A S M I D A C T I V i T Y f U m g ) „ n r 1 P H Hp E S E Hp H P P ° C 1 ' 1 L I i, LZ 1 i 0 . 7 0 2 n n r T ? H H P E S E Hp H E M P J, 1 1 1.82 - n r . H Hp E S E Hp H S T P D t - 4 i i— i j J — 1 | iZ i r i 1 J 1 1.73 X B PME PDC 11 H ^ E S E Hp T 2 3 9 X B E PDC 8 T E ? E H P - H 0.032 E X B ~ R Q H Hp E S E T „ „ o o PDC 9 i d. —ij i , 0.038 X B E pDC 2 3 H H p E S H p T 0.042 X B E P D C 21 H P E S E H P T H 2.41 M E P H 1 kb - p U C 13 cleavage may have been made by another enzyme contained i n the Hpal preparation. This plasmid conferred to 70-fold a m p l i f i c a t i o n of transhydrogenase a c t i v i t y i n the membranes of JM83 ( F i g . 10). The gene was further l o c a l i z e d by treatment with the exonuclease BAL31• Plasmid pDCll contains a single H i n d l l l s i t e at one junction of the pUC13 and in s e r t DNA ( F i g . 10) and a si n g l e SstI s i t e within vector pUC13 at the other junction. The plasmid was f i r s t cleaved with SstI and then treated with BAL31 for d i f f e r e n t lengths of time. The BAL31-treated i n s e r t s were cleaved with H i n d l l l to release the fragments and then l i g a t e d into the H i n d l l l and H i n d i s i t e s of pUC13. The r e s u l t i n g plasmids were used to transform JM83. The smallest plasmid pDC15 s t i l l r e t a i n i n g the transhydrogenase gene was then i s o l a t e d , and the process described above was repeated for the other end of the i n s e r t i n th i s plasmid by f i r s t cleaving with H i n d l l l followed by digestion f o r various lengths of time with BAL31. The digested i n s e r t s ware released from the plasmid by cleavage with SstI and then l i g a t e d into the SstI and H i n d i s i t e s of pUC13. One of the r e s u l t i n g plasmids, pDC21, contained a 3.05-kilobase i n s e r t . Inserts 50 to 100 base pairs smaller at e i t h e r end of the i n s e r t of pDC21 did not e x h i b i t transhydrogenase a c t i v i t y . Plasmid pDC21 conferred a 70-fold a m p l i f i c a t i o n of transhydrogenase a c t i v i t y i n the membranes of JM83 ( F i g . 10). I d e n t i f i c a t i o n of the pnt Gene Products During the course of t h i s study, we found that the pnt gene products i r r e v e r s i b l y aggregated when s o l u b i l i z e d i n SDS gel electrophoresis sample buffer at 100°C and did not enter SDS-polyacrylamide gels during electrophoresis. However, two protein products of molecular weight 52,000 72. and 48,000 were observed i n the gels of membranes from JM83 (pDCll), but not i n the gels of membranes from JM83 (pUC13), when s o l u b i l i z a t i o n i n SDS sample buffer was c a r r i e d out at 37°C ( F i g . 11). To e s t a b l i s h that the two protein products of molecular weights 52,000 and 48,000 were plasmid encoded, an i n v i t r o t r a n s c r i p t i o n / t r a n s l a t i o n system was used with plasmid pDCll as template. The proteins, l a b e l l e d with [ 3 5SJmethionine, were separated by SDS-polyacrylamide gel electrophoresis and v i s u a l i z e d by autoradiography of the drie d gels. Two radioactive polypeptides of molecular weights 52,000 and 48,000 were observed to be products of the in v i t r o t r a n s l a t i o n of pDCll but not of pUC13 ( F i g . 12). These two products correspond to the two polypeptides amplified i n membranes of JM83 (pDCll). The lower-molecular-weight radioactive polypeptides seen on the gel are products of the pUC13 vector DNA. Two polypeptides of a combined molecular weight of 100,000 would require the coding capacity of about 2.7 kilobases of DNA. This agrees with the observation that at l e a s t a 3.05-kilobase i n s e r t i s required for the expression of enzyme a c t i v i t y . To e s t a b l i s h whether or not both polypeptides were needed f or transhydrogenase a c t i v i t y , the products of the various plasmids ( F i g . 10) were examined by SDS-polyacrylamide gel electrophoresis ( F i g . 13). The expression of both polypeptides was observed i n JM83 membranes containing e i t h e r pDCll or pDC21, and a m p l i f i c a t i o n of transhydrogenase a c t i v i t y occurred i n both cases. Similar r e s u l t s were obtained with plasmids pDCl, pDC3, and pDC4 (data not shown). Neither of the two polypeptides was observed i n the membranes of c e l l s containing pUCl3 or pDC8. Only one membrane-bound protein was observed as a product of plasmid pDC9. No increase i n transhydrogenase 73 F i g . 11. SDS polyacrylamide gel electrophoresis of membranes of JM83 containing e i t h e r pUC 13 or pDC 11. Membranes were s o l u b i l i z e d i n SDS sample buffer at e i t h e r 37°C for 10 min or at 100°C for 3 min p r i o r to electrophoresis. Lane 1, molecular weight markers; lane 2, JM83 (pUC 13) membranes s o l u b i l i z e d at 100°C; lane 3, JM83 (pDC 11) membranes s o l u b i l i z e d at 100°C; lane 4, JM83 (pUC 13) membranes s o l u b i l i z e d at 37°C lane 5, JM83 (pDC 11) membranes s o l u b i l i z e d at 37°C. 2 3 4 5 94K« 67K< 43K< 74. F i g . 12. Autoradiograph of SDS polyacrylamide electrophoresis gel of [ 3 sSjmethionine-labeled products using plasmids pUC 13 (lane 3) and pDC 11 (lane 4) as templates i n an i n v i t r o t r a n s c r i p t i o n / t r a n s l a t i o n system. Plasmid DNA was added to f i n a l concentration of 100 pg DNA per ml. The reaction mixture was incubated at 37°C for 40 min. The r e a c t i o n was terminated by cooling to 0°C. Samples of the r e a c t i o n mixture were mixed with an equal volume of electrophoresis sample buffer and 10 u l was used for electrophoresis. The polypeptides of lane 1 (molecular weight markers) and lane 2 (membrane polypeptides of JM83 pDC 11) were stained with Coomassie blue. 1 2 3 4 94K — 67K 43K mm ii 30K*» 75. Fig 13. SDS polyacrylamide gel electrophoresis of membrane fr a c t i o n s of JM83 carrying hybrid plasmids. Lanes 1,9, molecular weight markers; lanes 2,8, JM83 (pUC 13); lane 3, JM83 (pDC 9); lane 4, JM83 (pDC 23); lane 5, JM83 (pDC 21); lane 6, JM83 (pDC 8); lane 7, JM83 (pDCll). 1 2 3 4 5 6 7 8 9 94K — 67K«» 43K mm — 94K ••67K • 43K 30K' - *» 30K a c t i v i t y was detected i n these membranes, i n d i c a t i n g that the 52,OOO-molecular-weight polypeptide alone i s not capable of transhydrogenation. The p o s s i b i l i t y that the 48,OOO-molecular-weight polypeptide alone i s responsible for transhydrogenase a c t i v i t y was examined by constructing plasmid pDC23 from pDCll. Plasmid pDCll was digested with S a i l and BstEII, and the recessed 3' ends of the DNA were f i l l e d i n before l i g a t i o n , using the Klenow fragment of E. c o l i DNA polymerase. The r e s u l t i n g plasmid, pDC23, was used to transform s t r a i n JM83. Membranes prepared from the transformed s t r a i n contained the 48,OOO-molecular-weight polypeptide and a polypeptide of molecular weight 42,000 ( F i g . 13). The l a t t e r may be a t r a n s l a t i o n product of the r e s i d u a l DNA r e s u l t i n g from the 0.45-kilobase d e l e t i o n . No increase i n transhydrogenase a c t i v i t y was detected i n these membranes ( F i g . 13), i n d i c a t i n g that the 48,OOO-molecular-weight polypeptide alone i s not capable of transhydrogenation. Complementation of Transhydrogenase A c t i v i t y Further evidence that the 52,000- and 48,OOO-molecular-weight polypeptides compose the transhydrogenase enzyme and were not merely stimulating expression of the chromosomally encoded transhydrogenase gene comes from complementation tests with s t r a i n RH-5. In th i s s t r a i n , transposon Tn5 i s inserted i n the pnt locus with the r e s u l t that there i s complete loss of transhydrogenase a c t i v i t y (84). Since RH-5 transformed poorly, the defective pnt locus was transduced into AB1450, using bacteriophage Pl and s e l e c t i n g for kanamycin resistance. This s t r a i n was then transformed with plasmids pUC13, pDC9, pDC23, and pDC21. Transhydrogenase a c t i v i t y was restored to the r e c i p i e n t by pDC21 only (Table 7), i n d i c a t i n g that both polypeptides are part of the enzyme. Transhydrogenase a c t i v i t y could also be restored by having replicons containing the pDC9 and pDC23 i n s e r t s transformed into the same c e l l s . Plasmid pDC23 was cleaved with H i n d l l l and BamHI and l i g a t e d into pACYC184 which had been cleaved with H i n d l l l and BamHI to give plasmid pDC50. Plasmids pDC9 and pDC50 were transformed into AB1450 (pnt::Tn5). Transformants r e s i s t a n t to both a m p i c i l l i n and chloramphenicol were selected. As shown i n F i g . 14, these transformants contained both pDC9 and pDC50. Plasmid pDC50 i s a low-copy-number plasmid and was found i n lesser amounts than the high-copy number plasmid pDC9. Membranes prepared from clones containing both plasmids had an enhanced l e v e l of transhydrogenase a c t i v i t y (0.33 U/mg pr o t e i n ) . No transhydrogenase a c t i v i t y was found i n the supernatant prepared from AB1450 (pnt;:Tn5). Membranes prepared from AB1450 had a s p e c i f i c transhydrogenase a c t i v i t y of 0.04 U/mg protein. Both the 52,000 and 48,000 proteins were found i n membranes of the c e l l s containing both plasmids i n amounts corresponding to the plasmid copy number from which they were encoded ( F i g . 15). These re s u l t s confirm that the transhydrogenase consist of two subunits of molecular weights 52,000 and 48,000. These w i l l be ref e r r e d to as the a and IE subunits, r e s p e c t i v e l y . Morphological E f f e c t s of pnt Overproduction The three plasmids, pDC9, pDC23 and pDC21, carrying i n t a c t a, 13 and u/ft subunits of transhydrogenase, and pUC13 were transferred into the transhydrogenase inac t i v a t e d s t r a i n AB1450 pnt::Tn5. High le v e l s of the transhydrogenase subunits were found i n the membranes ( F i g . 13) from c e l l s 78. Table 7. Complementation of chromosomal pnt::Tn5 by various pnt a l l e l e s on plasmids. transhydrogenase a c t i v i t y i n membrane Str a i n (umoles/min/mg protein) AB 1450 0.044 AB 1450 pnt::Tn5 0 AB 1450 pnt: :Tn5 pUC13 0 AB 1450 pnt: :Tn5 pDC9 0 AB 1450 pnt: :Tn5 pDC23 0 AB 1450 pnt: :Tn5 pDC21 0.94 E. c o l i s t r a i n AB 1450 pnt::Tn5 was transformed with plasmids pUC13, pDC9, pDC23 and pDC21 and then grown i n LB media supplemented with 25 ug/ml kanamycin and 50 ug/ml a m p i c i l l i n . Membranes were prepared and assayed for transhydrogenase a c t i v i t y as described i n Materials and Methods. 79. Fi g . 14. Agarose gel electrophoresis of plasmids prepared from E. c o l i AB 1450 containing transhydrogenase subunits on separate r e p l i c o n s . Plasmids were prepared from AB 1450 pDC50 (lane 2), AB 1450 pDC50 and pDC9 (lane 3) and AB 1450 pDC9 (lane 4) c e l l s grown i n LB supplemented with 30 ug/ml chloramphenicol (pDC50) and/or 50 ug/ml a m p i c i l l i n as described i n Materials and Methods. The plasmids were digested with endonucleases Xhol and BamHI and the fragments separated by electrophoresis i n an 0.8% agarose g e l . Lane 1: A D N A cleaved with H i n d l l l and EcoRI. Size (bp) 12 3 4 80. F i g . 15. SDS-polyacrylamide gel electrophoresis of membranes prepared from E. c o l i AB 1450 pnt::Tn5 and AB 1450 pnt::Tn5 pDC9, pDC50. C e l l s were grown i n LB media supplemented with 30 ug/ml of kanamycin. A m p i c i l l i n (50 Ug/ml) and chloramphenicol (30 ug/ml) were included i n the medium for the plasmid-containing s t r a i n . Membranes were prepared and electrophoresed i n a 10% SDS/polyacrylamide gel as described i n Materials and Methods. Lane 1: molecular weight markers; lane 2, p u r i f i e d transhydrogenase; lane 3, AB 1450 pnt::Tn5 pDC9 and pDC50 membranes; lane 4, AB 1450 pnt::Tn5 membranes. M r x10 - 3 94 67 ? mm 43 81. carrying pDC21. There was an almost 80-fold greater expression of transhydrogenase a c t i v i t y when compared to the transhydrogenase l e v e l s of AB1450. No growth was observed when c e l l s containing the plasmids pDC9, pDC23 or pDC21 were inoculated into M9 medium and shaken at 37°C for 18 h. C e l l s containing pUC13 grew normally. The presence of the plasmids containing transhydrogenase subunits also i n h i b i t e d growth i n LB medium and decreased the y i e l d of c e l l s (Table 8). The i n h i b i t i o n was most pronounced i n c e l l s containing plasmid pDC21. The y i e l d of c e l l s was reduced by almost 50% when compared to the y i e l d of c e l l s which contained the plasmid pUC13. Observation of the c e l l s using phase contrast microscopy revealed that the st r a i n s with a high l e v e l of transhydrogenase subunits had a heterogeneous s i z e d i s t r i b u t i o n ( F i g . 16). The c e l l s i n thi n section preparation revealed the presence of tu b u l a r - l i k e structures that were sometimes observed i n c e l l s overproducing the transhydrogenase subunits but not observed i n c e l l s containing the plasmid pUC13 ( F i g . 17). These t u b u l a r - l i k e structures were normally observed near the poles of the c e l l s . Another i n t e r e s t i n g e f f e c t of overproduction of transhydrogenase was the loss of aerobic-driven transhydrogenation. In membranes prepared from JM83, energy-independent, aerobic-dependent and ATP-dependent a c t i v i t i e s are 0.009, 0.036 and 0.064 U/mg protein r e s p e c t i v e l y . The corresponding values were 0.14, 0, and 0.44 U/mg protein f o r JM83 pDC21. IV. P u r i f i c a t i o n of Transhydrogenase from S t r a i n JM83 pDC21 Cloning of the pnt gene to form the multicopy plasmid pDC21 resu l t e d 82. Table 8. Growth c h a r a c t e r i s t i c of JM83 carrying various plasmids. Plasmid S p e c i f i c A c t i v i t y of transhydrogenase U/mg protein Relative Growth of Single Colonies Growth Rate (%) Growth Y i e l d (%) pUC13 0 1.0 100 100 pDC21 3.4 0.6 78 52 pDC9 0 0.6 80 64 pDC22 0 0.7 86 81 none 0 1.0 100 100 C e l l s were grown i n LB medium supplemented with 100 ug/ml of a m p i c i l l i n when the c e l l s contained plasmids. The sizes of colonies were measured a f t e r growth on plates containing LB medium containing 100 ug/ml of a m p i c i l l i n at 37°C overnight. Growth rate was measured from the absorbance at 600 nm. The growth y i e l d was measured by adding an equal amount of c e l l s to 200 ml of LB media and growing the c e l l s for 14 h at 37° with shaking (250 rpm). 83. F i g . 16. Microphotographs (phase contrast, 40X ob j e c t i v e ) of E. c o l i JM83 c e l l s containing the plasmids pUC13 (A) or pDC21 (B). Magnification - 1000X. 84. F i g . 17. Thin section micrographs of JM83 pDC21 c e l l s . C e l l s were grown to stationary phase i n LB medium. The c e l l s were c o l l e c t e d by centr i f u g a t i o n , washed with 0.2 M sodium phosphate (pH 7.0), and fi x e d with 2% glutaraldehyde. The c e l l s were washed with phosphate-buffered sucrose, postfixed i n 2% OsO^, dehydrated i n ethanol and embedded i n Epon 812. Sections were stained with lead c i t r a t e and saturated uranyl acetate, and mounted on grids for electron microscopy. 85. i n greater than 70-fold overproduction of transhydrogenase i n c e l l s harboring the plasmid. These c e l l s served as an excellent s t a r t i n g material for the p u r i f i c a t i o n of transhydrogenase as the two subunits, a (M^ 52,000) and fi (M r 48,000), were the two major proteins i n the cytoplasmic membrane. Ext r a c t i o n of membrane v e s i c l e s of E. c o l i s t r a i n JM83 pDC21 sequentially with 1% T r i t o n X-100 and 2% sodium cholate resulted i n s o l u b i l i z a t i o n of 80% of the membrane protein, while approximately 55% of the transhydrogenase remained i n the p a r t i c u l a t e material a f t e r c e n t r i f u g a t i o n (Table 9). Some s e l e c t i v i t y was observed i n the proteins extracted by the detergents. Several of the proteins remaining a f t e r T r i t o n X-100 ext r a c t i o n were e f f i c i e n t l y s o l u b i l i z e d by subsequent treatment with cholate. However, only T r i t o n X-100 was e f f e c t i v e i n s o l u b i l i z i n g some flavoproteins and b-type cytochromes, as well as a number of other proteins. The transhydrogenase could be s o l u b i l i z e d from the extracted v e s i c l e s by 0.5% sodium deoxycholate i n the presence of 1 M KC1. Although cholate (0.5%) alone was i n e f f e c t i v e i n s o l u b i l i z i n g the transhydrogenase, i t was necessary to include t h i s detergent i n order to prevent the s o l u b i l i z e d material from forming a gel when stored for any length of time. The deoxycholate extract was loaded over a 20% sucrose s o l u t i o n and centrifuged overnight at 260,000 x g. The transhydrogenase a c t i v i t y was found i n the sucrose s o l u t i o n . Pooled f r a c t i o n s containing transhydrogenase had a s p e c i f i c a c t i v i t y of 29.9 limol.min" 1 .mg"1 of protein and represented 16% of the o r i g i n a l a c t i v i t y . Recoveries of a c t i v i t y ranged from 10-18%. The p u r i f i e d enzyme retained over 90% of i t s a c t i v i t y when stored for a week 86. Table 9. P u r i f i c a t i o n of transhydrogenase from E_. c o l i s t r a i n JM83 pDC21. Transhydrogenase A c t i v i t y F r a c t i o n Protein Total S p e c i f i c mg % U/mg protein Membranes 188 100 5.2 Af t e r T r i t o n X-100 treatment 61 77 12.1 Af t e r cholate treatment 33 55 16.2 Extracted enzyme 15 38 24.4 Combined fr a c t i o n s a f t e r c e n t r i f u g a t i o n 5 16 29.9 Membranes were prepared from 4.7 g of c e l l s ; a f t e r t r e a t i n g the membranes sequentially with the detergents T r i t o n X-100 and sodium cholate, the transhydrogenase was s o l u b i l i z e d by the detergent sodium deoxycholate i n the presence of KCI. The enzyme was p u r i f i e d from the extract by cent r i f u g a t i o n through a sucrose s o l u t i o n . Experimental d e t a i l s of the p u r i f i c a t i o n are described i n Materials and Methods. 87. at 4°C. A high degree of p u r i f i c a t i o n was achieved as shown by SDS-polyacrylamide gel electrophoresis of the p u r i f i e d material ( F i g . 18). Two bands, a and 8, of apparent molecular weights 52,000 and 48,000 were observed. The absorption spectrum of the p u r i f i e d enzyme indicated that the preparation was devoid of f l a v i n . Both Liang and Houghton (55) and Voordouw et a l . (122) have suggested that the 100,000-molecular-weight protein i s a component of the E_. c o l i transhydrogenase. A 100,000-molecular-weight protein was observed when transhydrogenase was p u r i f i e d from W6 ( F i g . 18). A minor 100,000-molecular-weight protein was observed as a d i f f u s e band i n SDS-polyacrylamide gels of the enzyme preparation p u r i f i e d from JM83 pDC21. This polypeptide became more prominent i n the gels when the samples were stored for several days at 4°C or when t h i o l reducing agents were omitted from the buf f e r s . In order to determine i f the 100,000-molecular weight protein represented an aggregate of the transhydrogenase a. and 6 subunits, the stained bands of the polypeptides were excised from the gels and p a r t i a l l y digested with chymotrypsin using the technique described by Cleveland et a l . (106). The patterns of chymotryptic fragments are shown i n F i g . 19. The pattern of chymotryptic fragments for the 100,000 molecular-weight protein c l o s e l y resembled the pattern of fragments obtained from the a and 8 subunits of the transhydrogenase. This indicates that the 100,000-molecular-weight protein was an aggregate of the a and IE subunits. 88. F i g . 18. SDS-polyacrylamide gel electrophoresis of various f r a c t i o n s obtained during the p u r i f i c a t i o n of transhydrogenase from E. c o l i s t r a i n s JM83 pDC 21 (lanes 1-5) and W6 (lane 6). P u r i f i c a t i o n of the enzyme, gel electrophoresis, and s t a i n i n g were car r i e d out as described i n Materials and Methods. Lane 1, membranes, 36 ug; lane 2, T r i t o n X-100 extracted membranes, 16 ug; lane 3, cholate-extracted membranes, 12 Ug; lane 4, deoxycholate extract, 8 ug; lane 5, pooled sucrose gradient f r a c t i o n s , 6 ug; lane 6, a f f i n i t y column eluent, 10 ug. The posit i o n s of migration of the molecular weight standards are indicated. M r X 10 -3 Mrx10 1 2 3 4 5 6 94- 6 7 - 94- 67- 43- 43- 30- 30- 20.1 14.4 20.1- 14.4- 89. F i g . 19. P a r t i a l p r o t e o l y s i s of the 97,000-molecular-weight protein, and the u and fi subunits of the transhydrogenase. The 100,000-molecular-weight protein and the a and 13 subunits of the transhydrogenase were subjected to Cleveland digestion using chymotrypsin as described i n Materials and Methods. Lane 1, chymotrypsin alone; lane 2, 100,000-molecular-weight protein; lane 3, mixed a and 6 transhydrogenase subunits; lane 4, transhydrogenase 6 subunit; lane 5, transhydrogenase a subunit. The positions of migration of the molecular mass standards are in d i c a t e d . 90. V. Properties of Transhydrogenase K i n e t i c Parameters The steady state k i n e t i c s for reduction of AcNAD by NADPH were determined ( F i g . 20). The apparent Michaelis constants, Km, for NADPH and AcNAD were 23 uM and 33 uM, r e s p e c t i v e l y , for the p u r i f i e d enzyme. The corresponding values for the membrane-bound enzyme were 29 uM and 41 uM, r e s p e c t i v e l y . I n a c t i v a t i o n by Trypsin Studies of 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 mitochondrial transhydrogenase by t r y p s i n provided evidence that conformational changes i n the transhydrogenase molecule are induced by the binding of NADPH (123). As shown in Table 10, E. c o l i transhydrogenase was also i n a c t i v a t e d by t r y p s i n . P r o t e o l y t i c cleavage took place within the a subunit ( F i g . 21). NADPH increased the degree of i n a c t i v a t i o n by tr y p s i n whereas NADP, NADH and NAD did not have any s i g n i f i c a n t e f f e c t on p r o t e o l y s i s ( F i g . 22). These findings are s i m i l a r to those reported f o r the mitochondrial transhydrogenase. VI. Transhydrogenase as a Proton Pump Proteoliposome Energization During the ea r l y stage of p u r i f i c a t i o n when the transhydrogenase was s t i l l membrane-bound, maximum a c t i v i t y was observed i n the presence of an uncoupler. Addition of the uncoupler FCCP to nonenergized, everted membrane v e s i c l e s enhanced the reduction of AcNAD by NADPH and the reduction of NADP by NADH by 3.4-fold and 3.6-fold, r e s p e c t i v e l y F i g . 20. K i n e t i c parameters of transhydrogenase. The a c t i v i t i e s of p u r i f i e d (A and B) and membrane-bound transhydrogenases (C and D) were assayed i n the presence of a fixed concentration of 1.98 mM AcNAD (B and D) or 0.99 mM NADPH (A and C) and varying the concentration of the other nucleotide as described i n Materials and Methods. The assays were ca r r i e d out i n the presence of 1 uM FCCP. V i s expressed i n umoles min" 1 mg - I ® ® Table 10. Treatment of membrane v e s i c l e s prepared from E. c o l i JM83 pDC21 with various l e v e l s of TPCK-trypsin. Trypsin added (ng) % Control A c t i v i t y 0 100 50 86.3 100 65.3 200 25.6 400 1.4 Membranes i n TED at a protein concentration of 2.3 mg/ml were treated with various amounts of t r y p s i n for 5 min at 37°C. Reactions were stopped by the addition of 10 ug of t r y p s i n i n h i b i t o r . Transhydrogenase a c t i v i t y was then measured as described in Materials and Methods. 93. F i g . 21. SDS-polyacrylamide gel electrophoresis of membrane v e s i c l e s prepared from E. c o l i JM83 pDC21 and treated with 0 ng (lane 3), 50 ng (lane 4), 100 ng (lane 5), 200 ng (lane 6) or 400 ng (lane 7) of TPCK-trypsin as described i n Table 10. The proteins were separated on a 10% SDS-polyacrylamide gel as described i n Materials and Methods. Lane 1: molecular weight markers; lane 2: p u r i f i e d transhydrogenase. 1 2 3 4 5 6 7 94. F i g . 22. Inac t i v a t i o n of membrane-bound transhydrogenase by TPCK-trypsin i n the presence of various l e v e l s of nucleotides. Membrane v e s i c l e s were prepared and suspended i n TED buffer at a protein concentration of 2.3 mg/ml. The membrane v e s i c l e s (0.5 ml) were treated with 75 mg of TPCK-trypsin for 5 min at 37°C i n the presence of various amounts of NADPH (•-•), NADP (o-o), NADH (•-•) or NAD (•-•). The reactions were stopped by the addit i o n of 10 Ug of soybean t r y p s i n i n h i b i t o r and the tubes placed on i c e . The transhydrogenase a c t i v i t y was measured as described i n Materials and Methods. I t i s expressed as a percentage of the transhydrogenase a c t i v i t y remaining i n a control with no added nucleotide. Treatment of the cont r o l with 75 ng of TPCK-trypsin for 5 min at 37°C resulted i n a 34% decrease i n transhydrogenase a c t i v i t y . C o O c CD O d> a . o 50 100 200 [Pyridine Dinucleotide] pM (Fig. 23). Uncoupler did not a f f e c t the a c t i v i t i e s of s o l u b i l i z e d or p u r i f i e d transhydrogenase. These data suggest that the i n h i b i t i o n of transhydrogenation i n both d i r e c t i o n s derives from the establishment of a pH gradient across the membrane. Uncouplers would collapse the pH gradient e i t h e r by providing a continual supply of protons to the v e s i c l e i n t e r i o r to be pumped out during the reduction of NADP by NADH or by r e l i e v i n g the b u i l d up of protons i n the v e s i c l e i n t e r i o r during the reduction of AcNAD by NADPH. Various pH probes, such as 9-aminoacridine, pyramine or neutral red may be used to monitor the i n t e r n a l pH of the v e s i c l e s (124-126). Proton t r a n s l o c a t i o n during the reduction of AcNAD by NADPH was followed by measuring the fluorescence of 9-aminoacridine ( F i g . 24). Following the additi o n of both substrates, the fluorescence was s u b s t a n t i a l l y quenched i n d i c a t i n g an uptake of protons into the v e s i c l e s during the rea c t i o n . Quenching was subsequently r e l i e v e d by the addition of FCCP. Measurement of the electrogenic a c t i v i t y of p u r i f i e d transhydrogenase may be c a r r i e d out by r e c o n s t i t u t i n g the enzyme into synthetic liposomes. When p u r i f i e d transhydrogenase was reconstituted into egg phosphatidylcholine v e s i c l e s , the rate of reduction of AcNAD by NADPH was increased threefold by the addition of the uncoupler FCCP. Proton pumping a c t i v i t y was also observed during the reduction of AcNAD by NADPH catalyzed by the reconstituted transhydrogenase as indicated by the quenching of the fluorescence of 9-aminoacridine ( F i g . 24). Interaction of Transhydrogenase with a pH Gradient or Membrane P o t e n t i a l Uncouplers stimulate transhydrogenase i n both d i r e c t i o n s ( F i g . 23) suggesting that r e s p i r a t o r y control seen i n transhydrogenase containing F i g . 23. E f f e c t of FCCP on reverse and forward transhydrogenase a c t i v i t i e s . The influence of uncoupler on the a c t i v i t i e s of the soluble and membrane-bound transhydrogenase a c t i v i t i e s were assayed as described i n Materials and Methods. Experiment A compares the rates of AcNAD reduction catalyzed by e i t h e r (a) s o l u b i l i z e d transhydrogenase or (b) membrane-bound transhydrogenase. Experiment B compares the rate of NADP reduction catalyzed by e i t h e r (a) s o l u b i l i z e d transhydrogenase or (b) membrane-bound transhydrogenase. FCCP (1 yM) was added where indicated. F i g . 24. Quenching of the fluorescence of 9-aminoacridine during the reduction of AcNAD by NADPH catalyzed by e i t h e r membrane-bound or reconstituted transhydrogenase. The fluorescence of 9-aminoacridine was monitored during the reduction of AcNAD by NADPH. The e x c i t a t i o n wavelength was 420 nm and emission was determined at 500 nm. Membranes were prepared from E_. c o l i s t r a i n JM83 pDC21 as described i n Materials and Methods with the exception that 5 mm MgSO^ was included i n a l l steps. The reaction mixtures (2.1 ml) at pH 7.5 contained 10 mM Hepes, 300 mM KCI, 5 mM MgCl 2 and 250 uM NADPH. In addition, experiment A contained 15.8 yg of washed membranes and 8.7 uM 9-aminoacridine. Experiment B contained 2.4 ug of p u r i f i e d transhydrogenase reconstituted into v e s i c l e s with egg yolk phosphatidylcholine, and 1.1 uM 9-aminoacridine. Reactions were started by the a d d i t i o n of 500 UM AcNAD. Where indicated 1.3 uM FCCP was added i n experiment A and 0.3 uM FCCP was added i n experiment B. ® A c N A D A c N A D Q u e n c h i n g 1 m in F C C P 98. v e s i c l e s could r e s u l t from a balancing of the substrate oxidation-reduction p o t e n t i a l against the electrochemical hydrogen ion gradient formed across the membrane. The rate of reduction of NADP by NADH would decrease because of a depletion of i n t e r n a l protons whereas the rate of reduction of NAD by NADPH would be i n h i b i t e d because of an increase i n i n t e r n a l protons i n everted membrane v e s i c l e s . A l t e r n a t i v e l y , transhydrogenation might be con t r o l l e d p r i m a r i l y by the membrane p o t e n t i a l An a r t i f i c i a l l y imposed pH gradient was generated i n membrane v e s i c l e s by carrying out the reduction of AcNAD by NADPH i n a buffer at pH 6.0 using v e s i c l e s which had been e q u i l i b r a t e d with buffer to give an in t e r n a l pH of 8.0. As shown i n F i g . 25A, there was a transient phase of high i n i t i a l a c t i v i t y which gradually declined. Addition of uncoupler caused the expected enhancement of a c t i v i t y . No decrease i n a c t i v i t y was observed when the reaction was c a r r i e d out at an external pH of 7.4 (Fig. 25B). Interactions between transhydrogenase and imposed membrane potentials were investigated with potassium gradients i n the presence of valinomycin. A high external concentration of KC1 (150 mM) and a low in t e r n a l concentration of KC1 ( 5 mM) would generate a minimal membrane po t e n t i a l of 100 mV p o s i t i v e i n s i d e the v e s i c l e s . A membrane p o t e n t i a l of approximately the same size but negative inside the v e s i c l e s was generated by the presence of valinomycin and the same concentration of KCl inside the v e s i c l e s . No s i g n i f i c a n t e f f e c t was observed on the rate of transhydrogenation under ei t h e r of these conditions ( F i g . 26). The same r e s u l t s were observed when everted v e s i c l e s were prepared i n 0.1 M potassium phosphate or sodium phosphate and assayed i n 0.1 M sodium phosphate or potassium phosphate, r e s p e c t i v e l y . F i g . 25. Influence of a transmembrane pH gradient on the rate of reduction of AcNAD by NADPH catalyzed by membrane v e s i c l e s . Membrane vesi c l e s were prepared from E. c o l i JM83 pDC21 i n 0.1 M T r i c i n e buffer, pH 8.0 containing 5 mM MgCl^, 0.2 mM d i t h i o t h r e i t o l . The reduction of AcNAD by NADPH at 375 nm was measured i n 0.1 M MES buffer, pH 6.0, containing 5 mM MgCl^ and 0.2 mM transhydrogenase (A) or i n 0.1 M T r i c i n e , pH 7.4 containing 5 mM, MgCl^ and 0.2 mM d i t h i o t h r e i t o l . 35 ug of protein was added to each assay mixture. FCCP was added to a f i n a l concentration of 1.3 uM. 2 min 100. F i g . 26. Influence of a membrane p o t e n t i a l p o s i t i v e inside (A) or outside (B; on the rate of reduction of AcNAD by NADPH catalyzed by membrane v e s i c l e s . Membrane v e s i c l e s of JM83 pDC21 were prepared i n 0.1 M T r i c i n e pH 7.4 containing 5 mM MgCl^ and 0.2 mM d i t h i o t h r e i t o l and assayed for transhydrogenase a c t i v i t y i n the same buffer containing 150 mM KC1 (A).or membrane v e s i c l e s were prepared i n buffer with KC1 and assayed i n the absence of external KC1 (B). A volume of 2 ul membrane v e s i c l e s containing 12 ug of pro t e i n was used i n the assay. Valinomycin was added to a f i n a l concentration of 1 uM and FCCP to 1.5 uM. The transhydrogenase assay procedure i s described i n Materials and Methods. A B A c N A D F C C P 2 min * 101. N i g e r i c i n promotes the e l e c t r o n e u t r a l exchange of K for protons across membranes. Thus, i t w i l l change the i n t r a v e s i c u l a r pH without a l t e r i n g the av (127). As shown i n F i g . 27, n i g e r i c i n stimulated the reduction of AcNAD regardless of the o r i e n t a t i o n of the membrane pot e n t i a l i n the everted membrane v e s i c l e s . When the external l e v e l of KC1 i s high, n i g e r i c i n , which couples the e f f l u x of protons to the uptake of potassium, would be expected to stimulate the reduction of AcNAD i f the reac t i o n were influenced by apH. In the experiment with potassium loaded v e s i c l e s , n i g e r i c i n again stimulated the reaction by coupling the e f f l u x of protons to the uptake of potassium even though t h i s required the movement of potassium ions against a potassium ion gradient. Thus, these r e s u l t s indicate that the reduction of AcNAD by NADPH i s associated with an inward transport of protons and the rea c t i o n i s c o n t r o l l e d p r i m a r i l y by a pH gradient i n everted membrane v e s i c l e s . I n h i b i t i o n by DCCD Previous studies have shown that the a c t i v i t i e s of the mitochondrial transhydrogenase are affected by DCCD (74,128). This compound i n h i b i t e d proton pump a c t i v i t y without a f f e c t i n g transhydrogenation (50). In addition to the mitochondrial transhydrogenase, DCCD also i n h i b i t s proton-linked ATP synthase (129), ubiquinol-cytochrome c reductase from mammalian and yeast mitochondria (130,131) and cytochrome oxidase (132). In these systems DCCD i n h i b i t s proton t r a n s l o c a t i o n p r i m a r i l y rather than h y d r o l y t i c or redox reactions catalyzed by these enzymes. Treatment of both p u r i f i e d and membrane-bound transhydrogenases of E. c o l i with DCCD inacti v a t e d the enzyme. F i g . 28 shows the k i n e t i c s of i n a c t i v a t i o n of both p u r i f i e d and membrane-bound transhydrogenases by 102, F i g . 27. E f f e c t of ionophores on the reduction of AcNAD by NADPH catalyzed by membrane-bound transhydrogenase i n the presence of a transmembrane p o t e n t i a l p o s i t i v e inside (A) or p o s i t i v e outside (B). Experimental d e t a i l s are outlined i n F i g . 26 except that 10 mM KCI was included i n the external assay medium i n B. N i g e r i c i n , valinomycin and FCCP were added to give f i n a l concentrations of 0.1 uM, 1 uM and 1.5 mM, r e s p e c t i v e l y . 103. F i g . 28. Kin e t i c s of i n h i b i t i o n of membrane-bound and p u r i f i e d transhydrogenase by DCCD. Washed membranes (380 ug) were incubated at 22°C with constant s t i r r i n g i n a 1 ml reac t i o n mixture containing 40 uM Tris-HCl, pH 7.8, 0.8 mM d i t h i o t h r e i t o l , 2% (v/v) ethanol, and 25 uM (•), 50 UM (o), 75 UM (a), 100 UM ("), or 150 UM ( A ) DCCD (experiment B). P u r i f i e d transhydrogenase (76 ug) was incubated at 22°C i n a 1 ml reac t i o n mixture containing 40 mM Tr i s - H C l , pH 7.8, 0.8 mM d i t h i o t h r e i t o l , 2% (v/v) ethanol, 0.02% (w/v) B r i j 35 detergent and 10 UM (•), 20 uM (o), 30 uM (•), 40 uM (u), or 50 uM U) DCCD (experiment A). Samples were removed at the times indicated and assayed for the reduction of AcNAD by NADPH. In both cases the transhydrogenase a c t i v i t y of the cont r o l remained constant. In the insets the log of the slope of the l i n e obtained by p l o t t i n g log control a c t i v i t y (%) against time i s plo t t e d against the log of the concentration of DCCD during preincubation. T i m e (min) 104. DCCD. Plots of the logarithm of the a c t i v i t y , expressed as a percentage of the c o n t r o l value versus time, were l i n e a r , i n d i c a t i n g that the modification of the enzyme was pseudo-first order. The insets show plots of the logarithm of the pseudo-first-order rate constants versus the logarithm of the corresponding concentrations of DCCD. The slopes of the l i n e s , 1.16 for membrane-bound transhydrogenase, 1.03 for p u r i f i e d transhydrogenase, indicate that i n h i b i t i o n r e s u l t s from the i n t e r a c t i o n of approximately one molecule of DCCD per reactive enzyme complex. As shown i n F i g . 29, both the c a t a l y t i c and proton-pumping a c t i v i t i e s of transhydrogenase were i n h i b i t e d at the same rate with 250 uM DCCD. The modification of transhydrogenase by DCCD exhibited some s p e c i f i c i t y as transhydrogenation was not affected a f t e r treatment with the water-soluble carbodiimide EDC (Table 11). Since the enzyme consists of two subunits i t was of i n t e r e s t to see whether i n h i b i t i o n of transhydrogenase a c t i v i t y by DCCD involved covalent modification by the i n h i b i t o r of only one of the subunits. Membranes from JM83 pDC21 were incubated with [ll*C]DCCD, washed several times to remove excess reagent, and then the polypeptides separated by electrophoresis on an SDS-polyacrylamide g e l . As can be seen i n F i g . 30, the o subunit was p r e f e r e n t i a l l y modified. No l a b e l l i n g of the 6 subunit was observed even when the gel was autoradiographed f o r a much longer period of time. As shown i n Table 12, NADH protected the enzyme against i n h i b i t i o n by DCCD, while NADP, and to a lesser extent NADPH, increased the rate of i n h i b i t i o n . NAD, AcNAD and AcNADH had l i t t l e e f f e c t on the rate of i n h i b i t i o n by DCCD. Although both NADH and AcNADH can act as substrates f o r the enzyme, only NADH affected the rate of i n h i b i t i o n by DCCD. A further d i f f e r e n c e between NADH and AcNADH i s shown i n F i g . 31. NADH 105. Fi g . 29. E f f e c t of DCCD on proton tr a n s l o c a t i o n and c a t a l y t i c a c t i v i t i e s of membrane-bound transhydrogenase. Washed membranes were prepared from E. c o l i s t r a i n JM83 pDC21 as described i n Materials and Methods with the exception that 5 mM MgSO^ was included i n a l l steps. The membranes (1.26 mg) were incubated at 22°C with constant s t i r r i n g i n a 1 ml reaction mixture containing 7.5 mM Hepes/KOH (pH 7.5), 1 mM KCN, 0.15 mM d i t h i o t h r e i t o l , 4 mM MgCl 3, 225 mM KC1 and 2% (v/v) ethanol. Treatment was carried out with 250 pM U , a) or zero (n , Q ) DCCD and samples were removed at the times indicated to assay the reaction of AcNAD by NADPH (O, A) i n the presence of 1 uM FCCP or to measure the quenching of fluorescence of 9-aminoacridine (•, a) during the reduction of AcNAD by NADPH. Control values at zero time were 30% quenching of 9-aminoacridine and 6 umol of AcNAD reduced x min" 1 x mg ot protein" 1 . Time(min) 106. Table 11. Treatment of E. c o l i W6 pDC21 membrane with EDC tl-ethyl-3(3-dimethyl-amino-propyl)carbodiimide]. Concentration of EDC (uM) Transhydrogenase A c t i v i t y (U/mg of protein) 0 4.32 10 4.26 50 4.26 100 4.34 200 4.27 500 4.23 1000 4.26 Membranes (5 mg/ml of protein) prepared from E_. c o l i W6 pDC21 were treated with various l e v e l s of EDC for 60 min. Transhydrogenase a c t i v i t y was measured as described i n Materials and Methods. 107. F i g . 30. [l"C]DCCD l a b e l l i n g of membrane-bound transhydrogenase. Washed membranes from E. c o l i s t r a i n JM83 pDC21 (5 mg protein/ml) were incubated f o r 10 h at 4°C i n the presence of 40 mM Tris-HCl (pH 7.8), 0.2 mM d i t h i o t h r e i t o l and 160 uM DCCD (5 uC i ) . The membranes were then sedimented by c e n t r i f u g a t i o n at 175,000 x g for 1 h, and resuspended i n buffer without DCCD. This step was repeated u n t i l a l l of the free l a b e l was removed. Samples were submitted to electrophoresis, stained and fluorographed as described i n Materials and Methods. Lane 1, molecular mass standards; lane 2, [1'*C]DCCD l a b e l l e d membranes stained with Coomassie blue; lane 3, fluorograph of ['''CjDCCD l a b e l l e d membranes. 108. Table 12. E f f e c t of substrates on the i n h i b i t i o n of transhydrogenase a c t i v i t y by DCCD. Nucleotide added k min" 1 None 0.071 NADH 0.019 AcNADH 0.068 AcNAD 0.064 NAD 0.066 NADP 0.26 NADPH 0.15 Preincubation and assay conditions were the same as i n F i g . 26, experiment B, except that the p u r i f i e d transhydrogenase was incubated i n the presence of 75 uM DCCD. The nucleotides indicated were added to the preincubation mixture immediately before the addition of DCCD. A l l data were plotted as i n F i g . 26 and i n h i b i t i o n rate constants were calculated from the slopes of the pseudo-first-order p l o t s , k i s the pseudo-first-order i n h i b i t i o n rate constant. 109. F i g . 31. E f f e c t of NADH and AcNADH on the reduction of AcNAD by NADPH catalyzed by p u r i f i e d transhydrogenase. The reduction of AcNAD by NADPH catalyzed by p u r i f i e d transhydrogenase was assayed i n the presence of d i f f e r e n t concentrations of NADH (•) or AcNADH (•). 30r [ N u c l e o t i d e ] ( m M ) 110. stimulated the enzyme-catalyzed reduction of AcNAD by NADPH at concentrations up to 0.75 mM. Maximum stimulation occurred at 0.1-0.2 mM NADH. At concentrations higher than 0.75 mM, NADH i n h i b i t e d t h i s r e a c t i o n by acting as a competitive i n h i b i t o r at the NAD s i t e . In contrast, low concentrations of AcNADH did not a f f e c t the reaction rate, but at concentrations higher than 25 uM, i n h i b i t e d the re a c t i o n . VII. I s o l a t i o n of Transhydrogenase Subunits for Amino Acid Sequence Analysis P u r i f i c a t i o n of Subunits Using Polyacrylamide Slab Gels An important body of information that i s needed to understand the mechanism and structure of transhydrogenase i s the amino acid sequence of the transhydrogenase subunits. The amino acid sequence of the transhydrogenase subunits was elucidated by sequencing the DNA of the pnt gene (discussed l a t e r ) . The amino acid sequence of the amino-terminal ends of the transhydrogenase a and B subunits was determined to a i d i n the i d e n t i f i c a t i o n of t h e i r respective DNA coding regions. The a and 6 subunits of transhydrogenase must be i n d i v i d u a l l y i s o l a t e d for amino acid sequence a n a l y s i s . This was achieved by SDS-polyacrylamide gel electrophoresis. Two methods of gel electrophoresis were used. I s o l a t i o n of the Transhydrogenase Subunits Using a Commercial Preparative Gel Electrophoresis System The BRL preparative gel electrophoresis system was used to separate the transhydrogenase subunits. In th i s system, the proteins are separated 111. by electrophoresis i n a tube gel and the components c o l l e c t e d by continuous sampling from the base of the g e l . The system was used according to the manufacturer's i n s t r u c t i o n s . ' A 6 cm 10% polyacrylamide gel with a 1 cm 4% stacking gel was poured and allowed to stand overnight at room temperature. A sample of 200 u l containing 50 to 400 ug of p u r i f i e d transhydrogenase i n sample buffer was layered on top of the gel and electrophoresis was c a r r i e d out at 150 V. Fractions were c o l l e c t e d every 10 min at a flow rate of 10 ml/h. Sodium mercaptoacetate (0.1 mM) was included i n the cathode b u f f e r r e s e r v o i r to minimize the destruction of tryptophan, h i s t i d i n e and methionine side chains by free r a d i c a l s or oxidants trapped i n the gel matrix (133). The mercaptoacetate t r a v e l s at the dye front during electrophoresis and scavenges the destructive species i n the gel before the proteins reach them. A l l of the buffers and the apparatus were thoroughly de-gassed to prevent accumulation of bubbles under the e l u t i o n f r i t which would cause a poor separation. A portion of each f r a c t i o n was l y o p h i l i z e d , suspended i n SDS sample buffer and applied to a polyacrylamide slab g e l . As shown i n F i g . 32, some separation of the transhydrogenase subunits was obtained but there was s t i l l some overlap. The separation of the subunits was much worse when larger amounts of protein were applied to the system ( F i g . 33). Therefore only small amounts of the transhydrogenase subunits « 1 0 ug of each subunit) could be recovered i n a p u r i f i e d form using the apparatus. Attempts were made to i s o l a t e larger amounts of each subunit by pooling the p a r t i a l l y p u r i f i e d subunits and re-running them through the electrophoretic system. Again the y i e l d s of recovered p u r i f i e d subunits were low and degradation was observed. Therefore i t was concluded that the system could not be used to resolve the two subunits i n quantities s u f f i c i e n t for sequencing. 112. F i g . 32. SDS-polyacrylamide gel electrophoresis of samples from f r a c t i o n s obtained during the separation of transhydrogenase subunits using the preparative gel electrophoresis system (BRL). 160 ug of p u r i f i e d transhydrogenase was applied to a 6 cm 10% polyacrylamide gel with a 5% stacking gel and electrophoresis was c a r r i e d out at 150 V as described i n Materials and Methods. 200 ul of each 1.6 ml f r a c t i o n was l y o p h i l i z e d and the residue suspended i n sample buffer. The l y o p h i l i z e d portions of fractions 30-45 were applied to a 10% polyacrylamide gel (lanes 2-17, r e s p e c t i v e l y ) along with i n t a c t transhydrogenase (lane 1). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 F i g . 33. SDS-polyacrylamide gel electrophoresis of samples of f r a c t i o n s obtained during the separation of transhydrogenase subunits using the preparative gel electrophoresis system (BRL). The conditions are the same as outlined i n F i g . 32 except that 400 ug of p u r i f i e d transhydrogenase was applied to the g e l . Lanes 1-18 contain samples from f r a c t i o n s 30-49. A 1 2 3 4 5 6 7 8 9 1011 12 1314 114. I s o l a t i o n of Transhydrogenase Subunits by Excision from Polyacrylamide Gels A better method of separating the two subunits was the e x c i s i o n of the protein bands from the g e l . The two transhydrogenase subunits had f i r s t to be located i n the g e l . The protein bands were too c l o s e l y spaced to use stained s t r i p s of gel as a guide. Furthermore the use of Coomassie B r i l l i a n t Blue to v i s u a l i z e the protein i s time-consuming and some proteins, p a r t i c u l a r l y those containing Asp-Pro bands, can be p a r t i a l l y fragmented by b r i e f exposure to low pH. The protein bands can be v i s u a l i z e d without s t a i n i n g using the formation of protein-SDS complexes with potassium (134,135) or by p r e c i p i t a t i o n of non-protein-bound SDS by 4M sodium acetate (136). A s t r i p of gel containing the protein i s cut out and the protein extracted from i t . Approximately 450 ug of p u r i f i e d transhydrogenase was separated on a 15 x 14 x 0.15 cm 10% polyacrylamide g e l . The protein bands were v i s u a l i z e d by t r e a t i n g the gel with cold 0.1 M KC1. The opaque pr o t e i n bands were v i s i b l e a f t e r 5 min when viewed against a black background. The s t r i p s of gel containing the proteins were cut out, diced i n t o small pieces with a sharp razor blade and placed i n a sealable tube with four volumes of 0.1% SDS/1 mM d i t h i o t h r e i t o l . The tube containing the gel was incubated at room temperature with gentle shaking for 4 h. The gel pieces were removed by f i l t r a t i o n through a 0.45 pM pore f i l t e r . The proteins were p r e c i p i t a t e d by adding t r i c h l o r o a c e t i c a c i d to a f i n a l concentration of 12% and allowing the samples to stand on ice for 1 h. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and the protein p e l l e t was washed three times with cold 10% t r i c h l o r o a c e t i c acid and three times with col d acetone. The samples were l y o p h i l i z e d f o r sequencing. As shown i n F i g . 34 the two subunits were i s o l a t e d i n a highly p u r i f i e d form using 115. F i g . 34. SDS-polyacrylamide gel electrophoresis of transhydrogenase subunits p u r i f i e d by e x c i s i o n of the protein band from a g e l . 450 ug of p u r i f i e d transhydrogenase was applied to a 10% polyacrylamide gel to purify the subunits as described i n Materials and Methods. Samples representing 5% or 2.5% of the i s o l a t e d a and S subunits, r e s p e c t i v e l y , were subjected to electrophoresis i n a 10% polyacrylamide g e l . Lane A, u subunit; lane B, fi subunit. 1 2 116. this technique. The a subunit did undergo some degradation but the multiple bands probably represent d i f f e r e n t oxidized forms of the enzyme. The r e s u l t s of the protein sequencing of each of the transhydrogenase subunits are shown i n F i g . 35. The i d e n t i t y of 26 of the f i r s t 28 amino acid residues was obtained for the a subunit and 8 residues were i d e n t i f i e d for the 6 subunit. It was reported that the B subunit was susceptible to the a c i d i c conditions used to suspend the protein f o r sequencing. This caused the protein to fragment. VIII. Nucleotide Sequencing of the pnt Gene The DNA sequence described i n F i g . 36 were determined by the dideoxy chain termination procedure (110) coupled with cloning into bacteriophage M13 (137). The phage M13 has been g e n e t i c a l l y engineered to contain part of the E. c o l i gene for 8-galactosidase and expresses t h i s gene when grown on lac" E. c o l i . This lac region serves as a marker system for d i s t i n g u i s h i n g vector phage (blue plaque-formers) and recombinant phage (white plaque formers) on plates containing X-Gal. The phages used for sequencing the transhydrogenase gene were M13mpl8 and M13mpl9. These contain an i n s e r t of 57 bases with an array of r e s t r i c t i o n s i t e s within the lac gene as shown i n F i g . 37. Fragments can be inserted into t h i s region and recombinant phages distinguished as white plaques. The plaques were grown i n small cultures (2 ml), the phage i s o l a t e d , and the single-stranded DNA uncoated using phenol. The fragments were then sequenced using a synthetic primer. Two d i f f e r e n t strategies were applied at various stages of t h i s work to sequence the transhydrogenase gene. I n i t i a l l y , various r e s t r i c t i o n Subunit Residue Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 ALA or a MET ARG ILE GLY ILE PRO ARG GLU ARG LEU THR ASN GLU THR ARG VAL ALA VAL THR PRO LYS THR GLY GLU GLN - - LYS 13 MET SER GLY THR LEU VAL THR ALA F i g . 35. Amino acid sequence of the transhydrogenase a and 13 subunits. The transhydrogenase subunits were i s o l a t e d as described i n Materials and Methods and sequenced on a gas-phase sequenator at the University of V i c t o r i a . 118. F i g . 36. Nucleotide sequence of the pnt gene region. Each gene i s marked above the proposed points for i n i t i a t i o n of t r a n s l a t i o n . Proposed ribosome-binding s i t e s are underlined. Promoter sequences are boxed and l a b e l l e d -10 and -35. 1 5 3 0 4 5 6 0 7 5 9 0 1 0 5 G A T G T C T C G T T T A T G C G G C G T T C T A A G G T G T T T A T C C C A C T A T C A C G G C T G A A T C G T ^ 1 3 2 T35_- 1 4 7 1 6 2 -10 1 " 1 9 2 2 0 7 2 2 2 T T C A G T A C G T T A T A G G G C G T j r r G ^ J j C T A A T T T A T T T T A A C G G A G J T ^ 2 4 9 2 6 4 2 7 9 2 9 4 3 0 9 3 2 4 3 3 9 M R I G I P R E R L T N E T R V A A T P K T V E Q L L K L G F T V A V G A A G G G A A T A T C A T G C ^ A A T T G G C A T A C C A A G A G A A C G G T T A A C C A A T C A A A C C C G T G T T G C ^ 3 6 6 3 8 1 3 9 6 4 1 1 4 2 6 4 4 1 4 5 6 E S G A V N W Q V L T I K R L C S G R E 1 V E G N S V W Q S E 1 1 L K V N A P G A G A G C G G C G C G G T C A A C T G G C A A G T T T T G A C G A T A A A G C G T T T G T G C A G C G G G C G T G A A A T T G T A G A A G G G A A T A G C G T C T G G C A G T C A G A G A T C A 4 8 3 4 9 8 5 1 3 5 2 8 5 4 3 5 5 8 5 7 3 L D D E I A L L N P G T T L V S F I W P A Q n P E L M Q K L A E R N V T V M A T T A G A T G A T G A A A T T G C G T T A C T G A A T C C T G G G A C A A C G C T G G T G A G T T T T A T C T G G C C T G C G C A G A A T C C G G A A T T A A T G C A A A A A C T T G 6 0 0 6 1 5 6 3 0 6 4 5 6 6 0 6 7 5 6 9 0 M D S V P R 1 S R A Q S L D A L S S M A N I A G Y R A I V E A A H E F G R F F A T G G A C T C T G T G C C G C G T A T C T C A C G C G C A C A A T C G C T G G A C G C A C T A A G C T C G A T G G C G A A C A T C G C C G G T T A T C G C G C C A T T G T T G A A G C G G C A C A T G A A 7 1 7 7 3 2 7 4 7 7 6 2 7 7 7 7 9 2 8 0 7 T G Q I T A A G K V P P A K V M V I G A G V A G L A A 1 G A A N S L G A 1 V R A C C G G G C A A A T T A C T G C G G C C G G G A A A G T G C C A C C G G C A A A A G T G A T G G T G A T T G G T G C G G G T G T T G C A G G T C T G G C C G C C A T T G G C G C A G C A A A C A G T C T C G G C G C G A T T G 8 3 4 8 4 9 8 6 4 8 7 9 8 9 4 9 0 9 9 2 4 A F D T R P E V K E Q V Q S M G A E F L E L D F K E E A G S G D G Y A K V M S G C A T T C G A C ^ C C C G C C C G G A A G T G A A A G A A C A A G T T C A A A G T A T G G G C G C G G A A T T C C T C G A G C T G G A T r T T A A A G A G G A A G C T G G C A G C G G C G A T G G C T A T G C 9 5 1 9 6 6 9 8 1 9 9 6 1 0 1 1 1 0 2 6 1 0 4 1 D A F I K A E M E L F A A Q A K E V D I 1 V T T A L I P G K P A P K L I T R E G A C G C G T T C A T C A A A G C G G A A A T G G A A C T C T T T G C C G C C C A G G C A A A A G A G G T C G A T A T C A T T G T C A C C A C C G C G C T T A T T C C A G G C A A A C C A G C G C C G A A G C T A A T T A C C C G T G A A 1 0 6 8 1 0 8 3 1 0 9 8 1 1 1 3 1 1 2 8 1 1 4 3 1 1 5 8 M V D S M K A G S V 1 V D L A A Q N G G N C E Y T V P G E I F T T E N G V K V A T G G T T G A C T C C A T G A A G G C G G G C A G T G T G A T T G T C G A C C T G G C A G C C C A A A A C G G C G G C A A C T G T G A A T A C A C C G T G C C G G G T G A A A T C T T C A C T A C G G A 1 1 8 5 1 2 0 0 1 2 1 5 1 2 3 0 1 2 4 5 1 2 6 0 1 2 7 5 I G Y T D L P G R L P T Q S S Q L Y G R N L V N L L K L L C K E K D G N I T V A T T G G T T A T A C C G A T C T T C C G G G C C G T C T C C C G A C G C A A T C C T C A C A G C T T T A C G G C A G A A A C C T O 1 3 0 2 1 3 1 7 1 3 3 2 1 3 4 7 1 3 6 2 1 3 7 7 1 3 9 2 D F D D V V I R G V T V I R A G E I T W P A P P I Q V S A Q P Q A A Q K A A P G A T T T T G A T G A T G T G G T G A T T C G C G G C G T G A C C G T G A T C C G T G C G G G C G A A A T T A C C T G G C C G G C ^ 1 4 1 9 1 4 3 4 1 4 4 9 1 4 6 4 1 4 7 9 1 4 9 4 1 5 0 9 E V K T E E K C T C S P W R K Y A L M A L A I I L F G W M A S V A P K E F L G G A A G T G A A A A C T G A G G A A A A A T G T A C C T G C T C A C C G T G G C G T A A A T A C G C G T T G A T G G C G C T G G C A A T C A T T C T T T T T G G C T C 1 5 3 6 1 5 5 1 1 5 6 6 1 5 8 1 1 5 9 6 1 6 1 1 1 6 2 6 H F T V F A L A C V V G Y Y V V W N V S H A L H T P L M S V T N A I S G I 1 V C A C T T C A C C G T T T T C G C G C T G G C C T G C G T T G T C G G T T A r T A C G T G G T G T G G A A T G T A T C G C A C G C G 1 6 5 3 1 6 6 8 1 6 8 3 1 6 9 8 1 7 1 3 1 7 2 8 1 7 4 3 V G A L L Q 1 G Q G G W V S F L S F 1 A V L I A S 1 N 1 F G G F T V T Q R M G T C G G A G C A C T G T T G C A G A T T G G C C A G G G C G G C T G G G T T A G C T T C C T T A G T 1 7 7 0 1 7 8 5 1 8 0 0 1 8 1 5 1 8 3 0 1 8 4 5 1 8 6 0 M S G G L V T A A Y I V A A 1 L F 1 F S L A G L S K H E T A A A T G T T C C G C A A A A A T T A A G G G G T A A C A T A T G T C T C G A G G A T T A G T T A C A G C T C pntB »» 1 8 8 7 1 9 0 2 1 9 1 7 1 9 3 2 1 9 4 7 1 9 6 2 1 9 7 7 S R Q G N N F G I A G M A I A L I A T I F G P D T G N V G W I L L A M V I G G T C T C G C C A G G G T A A C A A C T T C G G T A T C G C C G G G A T G G C G A T T G C G T T A A T C C C A A C C A 2 0 0 4 2 0 1 9 2 0 3 4 2 0 4 9 2 0 6 4 2 0 7 9 2 0 9 4 A 1 G 1 R L A K K V E M T E M P E L V A I L H S F V G L A A V L V G F N S Y L G C A A T T G G T A T C C G T C T G G C G A A G A A A G T T G A A A T G A C C G A A A T G C C A G A A C T G G T G G C G A T C C T G C A T A G C T T C G T G G G T C T G G C G G C A G T G C T G G T T G G C T T T A A C A G C T A T C T G 2 1 2 1 2 1 3 6 2 1 5 1 2 1 6 6 2 1 8 1 2 1 9 6 2 2 1 1 H H D A G M A P 1 L V N 1 H L T E V F L G . I F I G A V T F T G S V V A F G K L C A T C A T G A C G C G G G A A T G G C A C C G A T T C T G G T C A A T A T T C A C C T G A C G G A A G T G T T C C T C G G T A T C T T C A T C G G G G C G G T A A C G T T C A C G G G T T C G G T G G T G G C G T T C G G C A A A C T G 2 2 3 8 2 2 5 3 2 2 6 8 2 2 8 3 2 2 9 8 2 3 1 3 2 3 2 8 C G K I S S K P L M L P N R H K M N L A A L V V S F L L L I V F V R T D S V G T G T G G C A A G A T T T C G T C T A A A C C A T T G A T G C T G C C A A A C C G T C A C A A A A T G A A C C T G G C G G C T C T G G T C G T T T C C T T C C T G C T G C T G A T T G T A T ^ 2 3 5 5 2 3 7 0 2 3 8 5 2 4 0 0 2 4 1 5 2 4 3 0 2 4 4 5 L Q V L A L L I M T A I A L V F G W H L V A S I G G A D M P V V V S M A E L V C T G C A A G T C C T G G C A T T G C T G A T A A T G A C C G C A A T T G C G C T G G T A T T C G G C T G G C A T T T A G T C G C C T C C A T C G G T G G T G C A G A T A T G C C A G T G G T G G T G T C G A 2 4 7 2 2 4 8 7 2 5 0 2 2 5 1 7 2 5 3 2 2 5 4 7 2 5 6 2 L R L G G C G C G L Y A Q Q . R P V I V T G A L V G S S G A 1 L S Y I M C K A M C T C C C G C T G G G C G G C T G C G G C T G C G G G C T T T A T G C T C A G C A A C G A C C T G T C 2 5 8 9 2 6 0 4 2 6 1 9 2 6 3 4 2 6 4 9 2 6 6 4 2 6 7 9 N R S F I S V I A G G F G T D G S S T G D D Q E V G E H R E 1 T A E E T A E L A A C C G T T C C T T T A T C A G C G T T A T T G C G G G T G G T T T C G G C A C C G A C G G C T C T T C T A C T G G C G A T G A T C A G G A A G T C 2 7 0 6 2 7 2 1 2 7 3 6 2 7 5 1 2 7 6 6 2 7 8 1 2 7 9 6 L K N S H S V I I T P G Y G M A V A Q A Q Y P V A E I T E K L R A R G 1 N V R C T G A A A A A C T C C C A T T C A G T G A T C A T T A C T C C G G G G T A C G G C A T G G C A G T C G C G C A G G C G C A A T A T C C T G T C G C T G A A A T T A C T G A G A A A T T G C G C G 2 8 2 3 2 8 3 8 2 8 5 3 2 8 6 8 2 8 8 3 2 8 9 8 2 9 1 3 F G 1 H P V A G R L P G H M N V L L A E A K V P Y D I V L E M D E I N D D F A T T C G G T A T C C A C C C G G T C G C G G G G C G T T T G C C T G G A C A T A T G A A C G T A T T G C T G G C T G A A 2 9 4 0 2 9 5 5 2 9 7 0 2 9 8 5 3 0 0 0 3 0 1 5 3 0 3 0 D T D T V L V I G A N D T V N P A A Q D D P K S P I A G M P V L E V W K A Q N G A T A C C G A T A C C G T A C T G G T C A T T G G T G C T A A C G A T A C G G T T A A C C C G G C G G 3 0 5 7 3 0 7 2 3 0 8 7 3 1 0 2 3 1 1 7 3 1 3 2 3 1 4 7 V I V F K R S M N T G Y A G V O . N P L F F K E N T H M L F G D A K A S V D A I G T G A T T G T C T r r A A A C G T T C G A T G A A C A C T G G C T A T G C T G G T G T G C A A 3 1 6 9 3 1 7 9 3 1 8 9 3 1 9 9 3 2 0 9 3 2 1 9 3 2 2 9 3 2 3 9 L K A L C T G A A A G C T C T G T A A C C C T C G A C T C T G C T G A G G C C G T C A C T C T T T A T T G A G A T C G C T T A A C A G A A C G G C G A T G C G A C T C T A [ REVERSE SEQUENCING 1 PRIMER (+| STRAND SYNTHESIS |-> STRAND SYNTHESIS C A O O A A A C A Q C I A I S A C - - - l l l l l l l l l l l l l l l l l e i « < ) A A A C i o c i i f « 4 C C A i e * i t « c c A A T i c c A C c r c c c t A c c c c c G C A i c c i c i A a A G i c c A C C i G C A G G C A i Q C A A C c n c t i C * c i a o c c o i c a i i i i A c i i c o i c a i a * c i « o a 15-BASE SEQUENCING PRlMEfl i i a c A O c * c r « * c c c III III I I IMI I I 11 I M I I I I 11 I I I I I II I I I M I I I I ,1 I I 111 I I I 11 I I I I I i i t i i i i i t i u t II II HYBRIDIZATION PROBE PRIMER I I I J L I I I I I I I c o M , S t l l A t w t l l » M i n t X 0 < l S W I * * M i ton I M a M H I M i Met i Ml3mplfi 11111 ,11111 11111111111 l i l l l l l l l l i 111111111 IIIIIIIIIIIIIUII 17-BASE SEQUENCING PRIMER C A O O A A A C A Q C I A f a A C C A i a A I I A CCCCAAGCI lGCAIGCCIGCAGGICGACIC IAGAGGAICCCCGCCTACCGAGCICGAAnCA C I O O C C O I C O I l l l A C A A C a l c A | i A 6 f « a « ( , | _ ! I I J I I I I Htnau sp«i tut s * t i « « t JAAIIIII ) « M i s u i tsotn ACS I U l I f * m • SAM I MI3mpl9 Co 0 T3 I—' CO (U 9 a- B xi o a H -3 OP 1 (D OQ H* O 3 ca o 121. fragments (mostly EcoRI/HincII fragments) of pDC21 were cloned into M13 for sequencing. However, i t proved d i f f i c u l t to i s o l a t e s u f f i c i e n t clones to cover the whole gene. Therefore, the majority of the clones f or sequencing were generated using exonuclease BAL31. Three sets of fragments were generated using BAL31. The f i r s t set of fragments were generated by cleaving the unique Smal s i t e of pDC21, t r e a t i n g the l i n e a r i z e d plasmid DNA with BAL31 for various lengths of time and then r e l e a s i n g the shortened pnt fragment with P s t l and H i n d l l l . The BAL31 fragments were cloned into Pstl/Smal cleaved M13mpl9. The second set of fragments were generated by cleaving the unique P s t l s i t e of pDC21, trea t i n g the l i n e a r i z e d plasmid with BAL31 for various lengths of time and then r e l e a s i n g the shortened fragments by cleavage with BamHI. The fragments were cloned into the BamHI/SmaI s i t e of M13mpl9. Clones containing fragments of the opposite strand of pDC21 were generated by cleaving pDC21 with BstEII, t r e a t i n g with BAL31 for various lengths of time and then re l e a s i n g two fragments containing opposite ends of the pnt gene by cleaving with H i n d l l l and BamHI. These fragments were cloned into e i t h e r BamHI/HincII-treated M13mpl8 or H i n d l l l / H i n c I I - t r e a t e d Ml3mpl9. The M13 phage containing the fragments were transformed into JM103 and the sequencing protocols c a r r i e d out as described i n Materials and Methods. Thus, the sequence was b u i l t up i n an orderly and rapid manner i n both o r i e n t a t i o n s , as summarized i n F i g . 38. The predicted amino acid sequences for the a and 6 transhydrogenase subunits are shown i n F i g . 36. The amino acid composition of the two subunits i s shown i n Table 13. The predicted molecular weight for the a and S subunits are 53,906 and 48,667 r e s p e c t i v e l y . 122 Fig. 38. Summary of clones used to e s t a b l i s h the nucleotide sequence. Horizontal arrows represent extent of sequences determined and t h e i r o r i e n t a t i o n s . Symbols: Hi, H i n d l l l ; Ps, P s t l ; H, Hpal; P, P v u l l ; He, H i n d i ; E, EcoRI; X, Xhol; Sa, S a i l ; Bs, BstEII; B, BamHI; Sm, Smal; S, SstI. Boxed l i n e s are inserted DNA and dotted l i n e s are pUC13 vector DNA. Hi pnt A- HPHc K n— E X HcSa ""r Ps E Bs P _i i i_ • pntB Sm B S E S E pDC 11 pDC 21 0.5 kb Table 13. Amino acid composition of the transhydrogenase subunits. No. of residues/subunit Content (mole %) a 13 a 13 GLY 41 48 8.17 10.39 ALA 59 55 11.75 11.90 LEU 42 47 8.37 10.17 ILE 38 38 7.57 8.22 VAL 51 48 10.16 10.39 PRO 24 17 4.78 3.68 GLU 31 19 6.18 4.11 GLN 20 10 3.98 2.16 ASP 16 18 3.19 3.90 ASN 18 18 3.59 3.90 THR 30 22 5.98 4.76 SER 27 25 5.38 5.41 MET 15 19 2.99 4.11 CYS 6 4 1.20 0.87 ARG 20 13 3.98 2.81 LYS 24 17 4.78 3.68 HIS 4 12 0.80 2.60 TYR 8 8 1.59 1.73 PHE 20 21 3.98 4.55 TRP 8 3 1.59 0.65 502 462 124. DISCUSSION P h y s i o l o g i c a l Role Mutants have been i s o l a t e d which lack transhydrogenase a c t i v i t y (82,84). One of the mutants, RH-5, had normal growth rates when grown a e r o b i c a l l y on a synthetic medium with glucose, g l y c e r o l or fructose as carbon source, when compared to the growth rates of the parental s t r a i n . The mutant also grew normally under anaerobic conditions on synthetic media with glucose or g l y c e r o l and fumarate as carbon source (Table 2). These r e s u l t s agree with those of Hanson (82,84). Thus, transhydrogenase i s not an e s s e n t i a l source of NADPH for the c e l l . In agreement with the findings of Liang and Houghton (85) glutamate dehydrogenase and transhydrogenase a c t i v i t i e s were found to be coregulated during nitrogen l i m i t a t i o n . Coordinate changes i n the l e v e l s of transhydrogenase and glutamate dehydrogenase suggest that transhydrogenase may be a d i r e c t source of NADPH for the glutamate dehydrogenase r e a c t i o n . This hypothesis was tested by i n a c t i v a t i n g transhydrogenase i n a s t r a i n of E. c o l i dependent s o l e l y on the glutamate dehydrogenase pathway for a s s i m i l a t i o n of ammonia. The mutant d i d not require glutamate f o r growth. This rules out the hypothesis that transhydrogenase i s the sole source of NADPH for ammonia a s s i m i l a t i o n by glutamate dehydrogenase i n E. c o l i . These r e s u l t s suggest that the transhydrogenase of E. c o l i may not function as a component i n a s p e c i f i c pathway. Since the catabolism of E_. c o l i i s almost e n t i r e l y NAD-linked, the function of the energy-linked transhydrogenase may be to channel reduction equivalents from the NADH pool to the NADPH pool under thermodynamically unfavourable conditions. A 125. number of other enzymes including glucose 6-phosphate dehydrogenase and NADP-specific malic enzyme also contribute to the NADPH pool. Therefore, i t i s not s u r p r i s i n g that transhydrogenase mutants grow normally. It i s i n t e r e s t i n g to note that mutants defective i n both glucose 6-phosphate dehydrogenase and transhydrogenase grow much slower than mutants defective i n only one of the enzymes (84). Cloning and Expression of Transhydrogenase A great deal of time and e f f o r t was devoted to the p u r i f i c a t i o n of transhydrogenase from E. c o l i s t r a i n W6 during the i n i t i a l stage of th i s project. The transhydrogenase was p u r i f i e d to near homogeneity by a combination of hydrophobic, ion-exchange and a f f i n i t y chromatography. Analysis of the p u r i f i e d transhydrogenase using SDS-PAGE showed the presence of three major protein bands of molecular weights 100,000, 52,000 and 47,000 as well as several minor protein bands. The SDS-PAGE p r o f i l e c l o s e l y resembled the r e s u l t s reported by Liang and Houghton (55) although t h e i r gels showed only one protein band i n the molecular weight 50,000 region. The p u r i f i c a t i o n procedure from s t r a i n W6 was not e n t i r e l y s a t i s f a c t o r y . The r e p r o d u c i b i l i t y of the procedure was poor. Much of the v a r i a b i l i t y occurred during the a f f i n i t y chromatography step. Persson et a l . (50) also reported that y i e l d s and pur i t y of mitochondrial transhydrogenase varied greatly when p u r i f i e d using a f f i n i t y chromatography with AG-NAD r e s i n s . Another problem with the p u r i f i c a t i o n procedure was that the transhydrogenase preparation was contaminated with several other proteins. Therefore i t was decided to amplify the l e v e l s of transhydrogenase i n the c e l l s by cloning the pnt gene onto a multi-copy plasmid. 126. Based on the r a t i o n a l e that E. c o l i c e l l s harbouring plasmids containing the pnt gene would contain elevated l e v e l s of enzyme, three clones were i s o l a t e d from the Clarke and Carbon colony bank which contained the transhydrogenase gene. That the pnt gene had been cloned was shown by the following. ( i ) An 8.7-kilobase fragment common to a l l three plasmids was included i n the 35.5-min region of the E. c o l i genome previously mapped as the locus for the pnt gene ( F i g . 8). ( i i ) Transhydrogenase a c t i v i t y was repressed when the plasmid-bearing c e l l s were grown i n a r i c h medium (Table 6). ( i i i ) Analysis of membranes of the plasmid-bearing s t r a i n s showed the amplified expression of the two polypeptides of molecular weights 52,000 and 48,000 ( F i g . 11) which were observed i n the p a r t i a l l y p u r i f i e d preparation of the transhydrogenase of E. c o l i s t r a i n W6 ( F i g . 5). Similar polypeptides were formed during i n v i t r o t r a n s c r i p t i o n / t r a n s l a t i o n of pDCll DNA ( F i g . 12). (i v ) Transhydrogenase a c t i v i t y was restored to a transhydrogenase-defective mutant when transformed with plasmid pDC21 (Table 7). Subcloning of the pnt gene in t o pUC13 resu l t e d i n up to 70-fold a m p l i f i c a t i o n of transhydrogenase a c t i v i t y . I n i t i a l attempts to i d e n t i f y the protein products by SDS-PAGE f a i l e d because heating at 100°C i n the presence of SDS gel electrophoresis sample buffer caused aggregation of the proteins so that they did not enter the gel ( F i g . 11). Liang and Houghton demonstrated that the r a t i o of 100K to 50K protein could be a l t e r e d upon a l k y l a t i o n . Therefore, the 100K protein probably represents an unusually stable dimer of one or both of the lower molecular weight 127. components. The rodA gene product (138), lactose permease (139), and g l y c e r o l phosphate permease (140) are other proteins which aggregate when boiled i n SDS. A l l four proteins which behave i n t h i s manner are cytoplasmic membrane proteins. Both polypeptides are needed for the expression of transhydrogenase a c t i v i t y . Deletion of a 1.6-kilobase H p a l - H i n d l l l fragment from pDC4 to give pDC9 resulted i n the loss of the 48,000-molecular weight polypeptide from the membranes of plasmid-bearing c e l l s ( F i g . 12). Deletion of the 0.55-kilobase H p a l - H i n d l l l fragment from the 4.8-kilobase H i n d l l l i n s e r t of pDC3 to give pDC8 resulted i n the loss of both polypeptides. The 52,000-molecular weight polypeptide was l o s t when a 0.45-kilobase S a l l - B s t E I I fragment was deleted from pDCll ( F i g . 12). No transhydrogenase a c t i v i t y was associated with any of these plasmids. These r e s u l t s suggest that a l l or part of the promoter region i s found i n the 0.60-kilobase H p a l - H i n d l l l region followed by the regions coding for the 52,000 and 48,000-molecular weight polypeptides, r e s p e c t i v e l y . Compatible plasmids which would express e i t h e r the 50,000 or 48,000-molecular-weight polypeptides were constructed and transformed together into J.. c o l i AB1450 (pnt::Tn5) lacking transhydrogenase a c t i v i t y . In such c e l l s , active transhydrogenase was formed i n d i c a t i n g that the transhydrogenase of E. c o l i i s composed of two d i f f e r e n t subunits of molecular weights 52,000 and 48,000 ( F i g . 15). The two subunits of molecular weights 52,000 and 48,000 were designated as the a and ft subunits r e s p e c t i v e l y of transhydrogenase. Expression of the pnt gene of the multicopy plasmid pDC21 resulted i n greater than 70-fold overproduction of transhydrogenase i n c e l l s harbouring the plasmids. The a and B subunits were the two most 128. abundant polypeptides i n the cytoplasmic membranes of these c e l l s . Biosynthesis of transhydrogenase to amounts greater than those of wild-type s t r a i n s had e f f e c t s on morphology, growth rate, growth y i e l d and c e l l d i v i s i o n of these c e l l s (Table 8, F i g . 16). Deletion of portions of the genes coding for the a or fi subunits so that only one i n t a c t subunit was synthesized i n the c e l l s i n large amounts also caused the abnormal p h y s i o l o g i c a l and morphological e f f e c t s (Table 8). Therefore, the changes are not the r e s u l t of a pyridine nucleotide imbalance which may be produced by excess le v e l s of transhydrogenase. The p h y s i o l o g i c a l and morphological phenomena observed i n the E. c o l i s t r a i n s , which synthesize high l e v e l s of transhydrogenase, are thus to be regarded as a sole consequence of the abnormally high amount of protein being inserted i n the membrane. Tubular-like structures were observed i n the c e l l s containing excess l e v e l s of the transhydrogenase subunits ( F i g . 17). Lemire et a l . (141) found unusual tubular v e s i c l e s i n t h e i r membrane preparations of an E. c o l i s t r a i n overproducing the membrane-bound fumarate reductase. The same group did a thorough study on the morphological changes caused by overproduction of fumarate reductase under growth conditions where the enzyme accounted for more than 50% of the inner-membrane protein (142). They found that the membrane accommodated t h i s excess fumarate reductase without reducing the l e v e l s of other membrane-associated enzymes. At the same time, the amount of membrane l i p i d increased such that the l i p i d / p r o t e i n r a t i o remained constant, i n d i c a t i n g that the t o t a l amount of membrane had doubled. The excess membrane was l o c a l i z e d i n tubular structures which branched from the cytoplasmic membrane and were composed of an aggregate of fumarate reductase and l i p i d . The tubules only appeared a f t e r the cytoplasmic membrane became highly enriched i n fumarate 129. reductase. Changes i n l i p i d composition were also observed. The major change i n phospholipid composition upon a m p l i f i c a t i o n of fumarate reductase was the disappearance of the acyl phosphatidylglycerol and the appearance of c a r d i o l i p i n . Similar t u b u l a r - l i k e structures could be observed i n some of the c e l l s containing excess transhydrogenase. However, no transhydrogenase a c t i v i t y was detected i n the supernatant f r a c t i o n a f t e r c e n t r i f u g a t i o n at 50,000 x g for 90 min of envelopes prepared by French press l y s i s . Under these conditions, Weiner et a l . (141) reported that small fragments of the fumarate reductase-enriched tubules remained i n the supernatant f r a c t i o n . Abnormal p h y s i o l o g i c a l and morphological e f f e c t s were also reported by von Meyenburg et a l . (143) on overproduction of membrane-bound ATP synthase i n E. c o l i . They observed that 10- to 12-fold overproduction of ATP synthase resulted i n pronounced i n h i b i t i o n of c e l l d i v i s i o n and growth and i n formation of membrane c i s t e r n ( s ) and v e s i c l e s within the c e l l s . Inclusion bodies, probably representing deposits of excess ATP synthase, were also observed i n these c e l l s . P u r i f i c a t i o n and Characterization of Transhydrogenase Cloning of the pnt gene to form the multicopy plasmid pDC21 resulted i n greater than 70-fold overproduction of transhydrogenase i n c e l l s harbouring the plasmid. These c e l l s served as excellent s t a r t i n g material for the p u r i f i c a t i o n of transhydrogenase as the a and & subunits were the two major proteins i n the cytoplasmic membrane ( F i g . 11). The transhydrogenase was p u r i f i e d from the amplified membranes by a simple procedure employing d i f f e r e n t i a l s o l u b i l i z a t i o n of proteins by detergents followed by ce n t r i f u g a t i o n through a 1.1 M sucrose so l u t i o n ( F i g . 18). 130. The presence of the two subunits (a and IS) i n the p u r i f i e d enzyme confirmed that both gene products of the pnt gene are needed for a functional transhydrogenase. The two subunits are present i n equimolar amounts. In s o l u b i l i z e d preparations these two subunits i r r e v e r s i b l y aggregate over a period of time to form a species of molecular weight 95,000-100,000. The extent of aggregation i s increased when the subunits are s o l u b i l i z e d i n the absence of disulphide-reducing agents. The structure of the E. c o l i transhydrogenase d i f f e r s markedly from the well-studied transhydrogenase of the bovine heart mitochondrion and of R. rubrum. The bovine heart mitochondrial transhydrogenase has been p u r i f i e d to homogeneity and consists of a sin g l e polypeptide chain of molecular weight 97,000-120,000 (47-50). In contrast, the transhydrogenase of R. rubrum consists of a soluble peripheral p r o t e i n factor having a molecular weight of 70,000 and an i n t e g r a l membrane-bound component of unknown molecular weight (59,60). Neither component alone exhibits transhydrogenase a c t i v i t y . The E_. c o l i transhydrogenase d i f f e r s from the R. rubrum enzyme i n that both subunits are t i g h t l y bound to the cytoplasmic membrane and are not released even i n the presence of M 6 urea. The two components can be released only by detergents such as deoxycholate i n the presence of high concentrations of s a l t s . Reconstitution of p u r i f i e d transhydrogenase into egg phosphatidylcholine v e i s c l e s resulted i n a 70-80% decrease i n enzymatic a c t i v i t y . Addition of the uncoupler, FCCP, enhanced enzymatic a c t i v i t y . Similar r e s u l t s were obtained with membrane-bound transhydrogenase, c a t a l y z i n g the reaction i n e i t h e r d i r e c t i o n ( F i g . 23). These data suggest that the i n h i b i t i o n of transhydrogenase i n both d i r e c t i o n s derives from a rapid estalishment of a pH gradient across the membrane. Translocation of protons can also be measured d i r e c t l y using pH-sensitive fluorescent probes such as 9-aminoacridine (124-126). During the reduction of AcNAD by NADPH by transhydrogenase reconstituted into v e s i c l e s , 9-aminoacridine fluorescence was s u b s t a n t i a l l y quenched, i n d i c a t i n g that protons were taken into the v e s i c l e s ( F i g . 24). A d d i t i o n a l support for a proton-translocating function of transhydrogenase was provided by the ATP-dependent stimulation of the reduction of NADP by NADH, catalyzed by membrane v e s i c l e s containing both proton-translocating ATPase and transhydrogenase. The a v a i l a b l e information thus seems to favour a chemiosmotic type of coupling mechanisms for energy-linked transhydrogenation. However, caution i s warranted i n i n t e r p r e t i n g the present data with regard to a coupling mechanism. Transmembrane proton tr a n s l o c a t i o n catalyzed by transhydrogenase remains to be shown d i r e c t l y , although the fluorescent probe used here c l e a r l y indicates an NAD plus NADPH-dependent a c i d i f i c a t i o n of the v e s i c l e s . Fluorescent probes may not probe the in t e r n a l pH of the v e s i c l e s e x c l u s i v e l y , but also i n t e r a c t with the surface or the i n t e r i o r of the membrane (144). Nevertheless, 9-aminoacridine has been reported to behave as an id e a l monoamine which d i s t r i b u t e s across the liposomal membrane i n response to a transmembrane pH gradient (145,146). Experiments with n i g e r i c i n and valinomycin suggest a p r e f e r e n t i a l regulation of the enzyme by a proton gradient rather than a membrane po t e n t i a l ( F i g . 25-27). Persson et a l . (50) reported that the mitochondrial transhydrogenase from bovine heart was p r e f e r e n t i a l l y regulated by a proton gradient rather than a membrane p o t e n t i a l . 132. Both transhydrogenation and proton pump a c t i v i t y of the enzyme were i n h i b i t e d by covalent modification of one act i v e enzyme unit by one molecule of DCCD ( F i g . 29). L a b e l l i n g of the transhydrogenase with [ l HC]DCCD indicated that the a subunit was p r e f e r e n t i a l l y modified (.Fig. 30). Mitochondrial transhydrogenase i s also i n h i b i t e d by DCCD (74, 128). Pennington and Fisher (74) postulated that DCCD may modify the mitochondrial transhydrogenase i n a putative proton binding domain outside the active s i t e . By contrast Phelps and Hat e f i (128) have suggested that DCCD reacts near the NAD(H) binding s i t e of beef heart mitochondrial transhydrogenase. Persson et a l . (50) observed an i n h i b i t i o n of proton pump a c t i v i t y without an e f f e c t on hydride transfer suggesting that proton translocation and hydride transfer are not o b l i g a t o r i l y l i n k e d . However, my r e s u l t s with E. c o l i transhydrogenase suggest that proton translocation and c a t a l y t i c a c t i v i t i e s are t i g h t l y coupled ( F i g . 29). This does not imply that DCCD reacts with the proton pump. It has become evident that t h i s reagent i s not a s p e c i f i c i n h i b i t o r of proton pumps (147). Homyk and Bragg (88) concluded that NADH may also bind to an a l l o s t e r i c s i t e by studying the k i n e t i c s of modification by 2,3-butanedione of a r g i n y l residues of the membrane-bound E. c o l i transhydrogenase. The present r e s u l t s are consistent with t h i s conclusion ( F i g . 31). Furthermore, the NADH analogue AcNADH appears not to bind to th i s s i t e since i t does not stimulate the enzyme-catalyzed reduction of AcNAD by NADPH or protect the enzyme from modification by DCCD (Table 12). The protection by NADH might mean that DCCD binds to the enzyme at or near the a l l o s t e r i c NADH-binding s i t e that stimulates the reduction of AcNAD by NADPH. An a l t e r n a t i v e explanation i s that the binding of NADH to 133. the enzyme induces a conformational change that makes the DCCD-binding residues less a c c e s s i b l e . By contrast, the binding of NADP appears to induce a conformational change which causes the DCCD-binding residue to become more accessible to th i s reagent (Table 12). Substrate-induced conformers of transhydrogenase are l i k e l y to play an important r o l e i n the function of the enzyme. Further evidence that binding of low concentrations of substrates cause a conformational change i s seen from the i n a c t i v a t i o n of the enzyme by t r y p s i n (Table 10). NADPH increased the degree of i n a c t i v a t i o n by tr y p s i n whereas NADP, NADH and NAD did not have any s i g n i f i c a n t e f f e c t on pr o t e o l y s i s ( F i g . 20). It i s i n t e r e s t i n g to note that s i m i l a r r e s u l t s were obtained for the rat l i v e r mitochondrial transhydrogenase (87). These r e s u l t s suggest that the NADP-enzyme complex has a d i f f e r e n t conformation from the NADPH-enzyme complex. This hypothesis i s supported by the find i n g that s u l f h y d r y l group modification by N-ethyl maleimide with e i t h e r the mitochondrial enzyme or the enzyme from E. c o l i was enhanced by NADPH, whereas NADP afforded protection against modification (46,86). A working hypothesis for a mechanism of the E. c o l i transhydrogenase can be based on the findings that transhydrogenase acts as a proton pump and can e x i s t i n three d i f f e r e n t conformations as shown i n F i g . 39. The res t i n g conformation (E^) i s transformed to by binding NADP. The conformation of the enzyme changes to E^ upon hydride ion tr a n s f e r . At the same time a proton i s translocated from the outer surface of the membrane to the inner surface. The d i r e c t i o n of transhydrogenation would be determined by the r a t i o of substrates and products, and also by the pH gradient. 134. F i g . 39. Proposed mechanism of transhydrogenase i n in t a c t E_. c o l i c e l l s . , E^ and E^ are d i f f e r e n t conformations of the enzyme. De t a i l s of the model are found i n the Discussion. NADH.. nH + NADP + NADH + E, ̂  E -nH out 1 N A D p ^ 2 out H transfer N A D \ nH. + NAD + NADPH E -nH. NADPH^ 3 i n 135. Nucleotide Sequence of the pnt Gene In order to predict the amino acid sequence of the a and 6 subunits °f J[« c o l i transhydrogenase, the nucleotide sequence of the i n s e r t of plasmid pDC21 was determined ( F i g . 36). Two open reading frames of 1406 and 1386 nucleotides were found i n the nucleotide sequence. The predicted amino acid sequences were compared with the N-terminal amino acid sequences of the u and B subunits ( F i g . 35). It was found that the 1406 and 1386 nucleotide open reading frames corresponded to the a and B subunits r e s p e c t i v e l y . However, only 45 nucleotides remained upstream of the a subunit and 69 nucleotides downstream of the B subunit i n the i n s e r t of plasmid pDC21. Therefore, promoter and t r a n s c r i p t i o n termination signals for pnt gene t r a n s c r i p t i o n i n plasmid pDC21 were l i k e l y to be found i n the vector DNA as expression of transhydrogenase was very high i n c e l l s containing plasmid pDC21. The missing nucleotide sequences of the pnt gene promoter region were derived from plasmid pDCll. Promoter sequence elements, determining the p o s i t i o n of t r a n s c r i p t i o n a l i n i t i a t i o n i n E_. c o l i , contain two regions of conserved DNA sequence located at about 10 and 35 nucleotides upstream from the t r a n s c r i p t i o n s t a r t s i t e (the '-10' and '-35' sequences [148]). The 246-base p a i r nucleotide sequence 5' to the a ATG codon was searched for a '-10' (TATAAT) and a 1-35'(TTGACA) consensus sequence (149). A promoter-like sequence was found at positions 138-143 (TTGTTA) and posi t i o n s 163-168 (TAACAT)(Fig. 36). The nucleotide sequence between the termination codon of the a subunit and the i n i t i a t i o n codon of the B subunit i s too short (30 nucleotides) to contain a promoter. Therefore, the a and B subunits are probably transcribed together. 136. Four-nucleotide sequences at posit i o n s 238-241 (AGGG) and p o s i t i o n s 1775-1778 (AGGG) show some homology to the ribosome binding s i t e consensus sequence (AGGAGGT)(150) and are located at the correc t distance from the a and (3 i n i t i a t i o n points of t r a n s l a t i o n r e s p e c t i v e l y ( F i g . 36). No obvious ter m i n a t i o n - l i k e structure was observed i n the sequence of the inserted DNA following the coding region. The non-random usage of codons i n the pnt gene coding regions further supports the assignment of the open reading frames. The codons CTA, ATA, AGA and AGG are r a r e l y used i n E. c o l i (151). The codons are used sparingly i n the pntA and B genes ( F i g . 36). Grojean and F i e r s (151) have analyzed codon usage i n JS. c o l i genes. They found that an e f f i c i e n t in-phase t r a n s l a t i o n i s f a c i l i t a t e d by proper choice of degenerate codewords ending with a T or C promoting a codon-anticodon i n t e r a c t i o n with intermediate strength (optimal energy) over those with very strong or very weak i n t e r a c t i o n energy. Generally, e f f i c i e n t l y expressed genes show a c l e a r preference for a C i n the t h i r d base p o s i t i o n of codons having A and/or T and a preference f o r a T i n the t h i r d base p o s i t i o n of codons having C and/or G. Conversely, codon usage i n weakly expressed genes such as repressor genes follows exactly the opposite r u l e s . Codon usage i n both the a and 13 subunit genes does not c l e a r l y resemble codon usage i n e i t h e r weakly or strongly expressed genes (Table 14). The codon usage r e f l e c t s a moderately e f f i c i e n t t r a n s l a t i o n of transhydrogenase mRNA. In normal E. c o l i c e l l s , transhydrogenase only represents between 0.1% to 0.5% of the cytoplasmic membrane pr o t e i n . This i s much more expression than weakly expressed genes such as repressor proteins but much less than e f f i c i e n t l y expressed genes such as RNA polymerase and ribosomal proteins. Table 14. Codon usage i n the E. c o l i pnt genes. TTT F 9 8 TTC F 11 13 TTA L 4 3 TTG L 6 6 CTT L 8 3 CTC L 4 3 CTA L 2 0 CTG L 18 34 ATT I 24 19 ATC I 11 18 ATA I 3 1 ATG M 15 19 GTT V 13 9 GTC V 11 10 GTA V 4 7 GTG V 23 22 a B TCT S 1 7 TCC S 2 4 TCA S 6 1 TCG S 5 6 CCT P 2 4 CCC P 0 0 CCA P 5 4 CCG P 17 9 ACT T 5 4 ACC T 17 9 ACA T 3 2 ACG T 5 7 GCT A 2 13 GCC A 11 6 GCA A 18 12 GCG A 28 22 a B TAT Y 4 5 TAC Y 4 3 TAA 0 0 TAG 0 0 CAT H 2 7 CAC H 2 5 CAA Q 9 4 CAG Q 11 6 ATT N 10 4 AAC N 8 14 AAA K 20 12 AAG K 4 5 GAT D 9 11 GAC D 7 7 GAA E 24 15 GAG E 7 4 a_ 6 TGT C 2 2 TGC C 4 2 TGA 0 0 TGG W 8 3 CGT R 10 7 CGC R 6 4 CGA R 1 1 CGG R 1 1 AGT S 5 2 AGC S 8 5 AGA R 2 0 AGG R 0 0 GGT G 10 22 GGC G 22 14 GGA G 1 5 GGG G 8 7 138. The amino acid sequences of the transhydrogenase o and fl subunits were predicted from the nucleotide sequences. Both subunits are rather hydrophobic. Analysis of the amino acid sequence of both subunits for l o c a l hydropathy and predicted secondary structure suggests that presence of at least f i v e transmembrane segments i n the a subunit and seven transmembrane segments i n the fl subunit ( F i g . 40,41). The r e l i a b i l i t y of such predictions are unknown because of an absence of a database of membrane proteins of known structure. The a subunit of E. c o l i transhydrogenase reacts quite s p e c i f i c a l l y and covalently with DCCD. Other pro t e i n - t r a n s l o c a t i n g enzymes react with DCCD and the s i t e of i n t e r a c t i o n with DCCD has been i d e n t i f i e d i n some cases (152). They e x h i b i t s t r i k i n g s i m i l a r i t y : i n a l l cases DCCD reacts with a carboxyl residue located i n an otherwise hydrophobic and highly conserved region of these polypeptides. This residue may be important to proton-translocation. However, no homologous sequence was found i n the amino acid sequence of e i t h e r subunit of E. c o l i transhydrogenase. This observation supports the supposition that DCCD i s not a s p e c i f i c i n h i b i t o r of proton pumps (147). 139. F i g . 40. Hydropathy plot of the transhydrogenase a subunit based on the procedure of Kyte and D o o l i t t l e (153). The positions of basic residues (A ) , a c i d i c residues (A) and regions of predicted alpha h e l i x (-) and beta sheet (niltl) are indicated. Possible transmembrane segments are shown (I-V). Secondary structure was determined by the method of Gamier et a l . (154). -3\ x: AA A A A A A A \ ^ A i A i f um- i _l_ A A ^A Ct A A A A A /, A | II I I I _l l_ A A A A - L tS.±A A A A A,-^ A A - - | | II - i _ - I as o T 3 > . X. 5 0 100 150 200 250 V? IV ~ i 1 r . 'i -3 L A £. A a A A i i IIIIII in in i _i_ — I I I I I H I U - 250 300 350 400 Residue number 450 500 L40. F i g . 41. Hydropathy plot of the transhydrogenase 13 subunit based on the procedure of Kyte and D o o l i t t l e (153). The positions of basic residues (*), a c i d i c residues (A) and regions of predicted alpha h e l i x (-) and beta sheet (inn) are ind i c a t e d . Possible transmembrane segments are shown (I-VTI). Secondary structure was determined by the method of Garnier et a l . (154). I II III IV V VI VII i — ~ — i i 1 i — = — i i ' — i i 1 i i — : — 3 -3 L x: O -a A£* A A * \ & A A A A A A A A A A A A II I llll I I II I I I I - I tl SI || 0 5 0 1 0 0 1 5 0 2 0 0 2 5 0 a. A A A ^ A A « ^ A A A ^ A A A A AA A A A ^ A A A A A / £ A A A A^ A^ AA A -3L »—«i i it »i i i M i i i II* ill - I - II ii 250 300 350 v 400 450 Residue number 141. REFERENCES 1. Colowick, S.P., N.O. Kaplan, E.F. Neufeld and M.M. C i o t t i (1952) J . B i o l . Chem. 195, 95-105. 2. Kaplan, N.O., S.P. Colowick and E.F. Neufeld (1953) J . B i o l . Chem. 205, 1-15. 3. Fisher, R.R. and S.R. Earle (1982) i n The pyridine nucleotide coenyzmes ( J . Everse, B. Anderson and K.-S. You, eds.) pp.279-324, Academic Press, New York. 4. Rydstrom, J . (1977) Biochim. Biophys. Acta 463, 155-184. 5. Rydstrom, J . , J.B. Hoek and L. Ernster (1976) i n The enzymes (P.D. Boyer, ed.) Vol. 13, pp. 51-88, Academic Press, New York. 6. Louie, D.D. and N.O. Kaplan (1970) J . B i o l . Chem. 245, 5691-5698. 7. Chung, A.E. (1970) J . B a c t e r i o l . 102, 437-438. 8. Kaplan, N.O., S.P. Colowick, E.F. Neufeld and M.M. C i o t t i (1953) J . B i o l . Chem. 205, 17-29. 9. Cohen, P.T. and N.O. Kaplan (1970) J . B i o l . Chem. ̂ 45, 2825-2836. 10. van den Brock, H.W.J., J.S. Santema, J.H. Wassink and C. Veeger (1971) Eur. J . Biochem. 24, 31-45. 11. Middleditch, L.E., R.W. Atchison and A.E. Chung (1972) J . B i o l . Chem. 247, 6802-6809. 12. Louie, D.D., N.O. Kaplan and J.D. Lean (1972) J . Mol. B i o l . 70, 651-664. 13. Murthy, P.S. and A.E. Brodie (1964) J . B i o l . Chem. 239, 4292-4297. 14. Asano, A, K. Imai and R. Sato (1967) Biochim. Biophys. Acta 143, 477-486. 15. Downs, A.J. and C.W. Jones (1975) Arch. M i c r o b i o l . 105, 159-167. 16. Kay, W.W. and P.D. Bragg (1975) Biochem. J . 150, 21-29. 17. C o l l i n s , P.J. and C.J. Knowles (1977) Biochim. Biophys. Acta 480, 77-82. 18. Keist e r , D.L. and N.J. Yike (1967) Biochemistry 6_, 3847-3857. 19. Orlando, J.A., D. Sabo and C. Curnym (1966) Plant P h y s i o l . 41, 937-945. 142. 20. Kaplan, N.O. , M.N. Swartz, M.E. Freeh and M.M. C i o t t i (1956) Proc. Natl. Acad. S c i . kl, 481-487. 21. Danielson, L. and L. Ernster (1963) Biochem. Biophys. Res. Commun. 10, 91-96. 22. Danielson, L. and L. Ernster (1963) Biochem. Z. 338, 188-205. 23. Houghton, R.L., R.J. Fisher and D.R. Sanadi (1975) Biochim. Biophys. Acta 396, 17-23. 24. Keist e r , D.L. and N.J. Yike (1966) Biochem. Biophys. Res. Commun. 24, 519-525. 25. Fisher, R.J. and D.R. Sanadi (1971) Biochim. Biophys. Acta 245, 34-41. 26. Bragg, P.D. and C. Hou (1968) Can. J . Biochem. 46_, 631-641. 27. Bragg, P.D. and C. Hou (1972) FEBS L e t t . 28, 309-312. 28. Lee, C.P. and L. Ernster (1964) Biochim. Biophys. Acta 81, 187-190. 29. Rydstrom, J . , A. T e i x e i r a da Cruz and L. Ernster (1970) Eur. J . Biochem. 17_, 56-62. 30. Cox, G.B., N.A. Newton, J.D. B u t l i n and F. Gibson (1971) Biochem. J 125, 489-493. 31. Kanner, B.J. and D.C. Gutnick (1972) FEBS L e t t . _22, 197-199. 32. Hanson, R.L. and E.P. Kennedy (1973) J . B a c t e r i o l . 114, 772-781. 33. Cox, G.B., F. Gibson, L.M. McCann, J.D. B u t l i n and F.L. Crane (1973) Biochem. J . 132, 689-695. 34. Montal, M., B. Chance, C.P. Lee and A. Az z i (1969) Biochem. Biophys. Res. Commun. 34, 104-110. 35. Kawasaki, T., K. Satoh and N.O. Kaplan (1964) Biochem. Biophys. Res. Commun. 17, 648-654. 36. Dontsov, A.E., L.L. Grinius, A.A. J a s a i t i s , I.I. Severina and V.P. Skulachev (1972) Bioenergetics 3_» 277-303. 37. Chetkausstaite, A.V. and L.L. Grinius (1979) Biokhimiya 44, 1101-1110. 38. Van de Stadt, R.J., F.J.R.M. Nieuwenhuis and K. Van Dam (1971) Biochim. Biophys. Acta 234, 173-176. 39. M i t c h e l l , P. and J . Moyle (1965) Nature 208, 1205-1206. 40. Skulachev, V.P. (1970) FEBS L e t t . 11, 301-308. 143. 41. M i t c h e l l , P. (1972) Bioenergetics 3_, 5-24. 42. Skulachev, V.P. (1974) Ann. N.Y. Acad. S c i . 227, 188-202. 43. M i t c h e l l , P. (1966) B i o l . Rev. 41_, 445-502. 44. M i t c h e l l , P. (1977) Ann. Rev. Biochem. 46, 996-1005. 45. Rydstrom, J . , J.B. Hoek, B.G. Ericson, and T. Hundal (1976) Biochim. Biophys. Acta 430, 419-425. 46. Houghton, R.L., R.J. Fisher and D.R. Sanadi (1976) Biochem. Biophys. Res. Commun. 73, 751-757. 47. Hojeberg, B. and J . Rydstrom (1977) Biochem. Biophys. Res. Commun. 78_, 1183-1190. 48. Anderson, W.M. and R.R. Fisher (1978) Arch. Biochem. Biophys. 187, 180-190. 49. Wu, L.N.Y., R.M. Pennington, J.D. Everett and R.R. Fisher (1982) J. B i o l . Chem. 257, 4052-4055. 50. Persson, B. K. Enander, H.L. Tong and J . Rydstrom (1984) J . B i o l . Chem. 259, 8626-8632. 51. Capaldi, R.A. and G. Vanderkooi (1972) Proc. Natl. Acad. S c i . 69_, 930-932. 52. Anderson, W.M. and R.R. Fisher (1981) Biochim. Biophys. Acta 635, 194-199. 53. Wu, L.N.Y. and R.R. Fisher (1983) J . B i o l . Chem. 258, 7847-7851. 54. Hanson, R.L. (1979) J . B i o l . Chem. 254, 888-893. 55. Liang, A. and R.L. Houghton (1980) FEBS L e t t . K)9, 185-188. 56. Homyk, M. (1981) Ph.D. th e s i s , University of B r i t i s h Columbia, Vancouver. 57. Bragg, P.D., P.L. Davies and C. Hou (1972) Biochem. Biophys. Res. Commun. 4_7, 1248-1255. 58. Fisher, R.R. and R.J. G u i l l o r y (1969) J . B i o l . Chem. 244, 1078-1079. 59. Fisher, R.R. and R.J. G u i l l o r y (1969) FEBS L e t t . 3, 27-30. 60. Fisher, R.R. and R.J. G u i l l o r y (1971) J . B i o l . Chem. 246, 4687-4693. 61. Konings, A.W.T. and R.J. G u i l l o r y (1973) J . B i o l . Chem. 248, 1045-1050. 144. 62. Jacobs, E., K. Heriot and R.R. Fisher (1977) Arch. M i c r o b i o l . 115, 151-156. 63. Fisher, R.R. and R.J. G u i l l o r y (1971) J . B i o l . Chem. 2A6, 4679-4686. 64. Ear l e , S.R., W.M. Anderson and R.R. Fisher (1978) FEBS L e t t . 9J,, 21-24. 65. Ear l e , S.R. and R.R. Fisher, (1980) Biochemistry lj), 561-569. 66. Rydstrom, J . (1979) J . B i o l . Chem. 254, 8611-8619. 67. Pennington, R.M. and R.R. Fisher (1983) FEBS L e t t . JJ4, 345-349. 68. Deamer, D.W., R.C. Prince and A.R. Crafts (1972) Biochim. Biophys. Acta 274, 323-335. 69. Ear l e , S.R. and R.R. Fisher (1980) J . B i o l . Chem. 155, 827-830. 70. Senior, A.E. and J.G. Wise (1983) J . Membr. B i o l . 73_, 105-124. 71. Clejan, L. and D.S. Beattie (1983) J . B i o l . Chem. 258, 14271-14275. 72. Esposti, M.D., E.M.M. Meier, J . Timoneda and G. Lenaz (1983) Biochim. Biophys. Acta 725, 349-360. 73. Casey, R.P., M. Thelen and A. Az z i (1980) J . B i o l . Chem. 255, 3994-4000. 74. Pennington, R.M. and R.R. Fisher (1981) J . B i o l . Chem. ̂ 56, 8963-8969. 75. Phelps, D.C. and Y. Hate f i (1984) Biochemistry 23_, 4475-4480. 76. Phelps, D.C. and Y. Hate f i (1984) Biochemistry 23, 6340-6344. 77. Jocelyn, P.C. and J . Dickson (1980) Biochim. Biophys. Acta 590, 1-12. 78. Bellamo, G., A. Martino, P. Richelmi, G.A. Moore, S.A. Jewell and S. Orrenius (1984) Eur. J . Biochem. 140, 1-6. 79. Houghton, R.L., R.J. Fisher and D.R. Sanadi (1976) Arch. Biochem. Biophys. 176, 747-752. 80. Gerolimatos, B. and R.L. Hanson (1978) J . B a c t e r i o l . 134, 394-400. 81. Liang, A. and R.L. Houghton (1981) J . B a c t e r i o l . 146, 997-1002. 82. Zahl, K.J., C. Rose and R.L. Hanson (1978) Arch. Biochem. Biophys. 190, 598-602. 83. Hanson, R.L. and C. Rose (1979) J . B a c t e r i o l . L38, 783-787. 84. Hanson, R.L. and C. Rose (1980) J . B a c t e r i o l . 141, 401-404. 145. 85. Skulachev, V.P. (1974) Ann. N.Y. Acad. S c i 227, 188-202. 86. O'Neal, S.G. and R.R. Fisher (1977) J . B i o l . Chem. _252, 4552-4556. 87. Blazyk, J.F., D. Zam and R.R. Fisher (1976) Biochemistry _15, 2843-2848. 88. Homyk, M. and P.D. Bragg (1979) Biochim. Biophys. Acta _571, 201-217. 89. T e i x e i r a da Cruz, A., J . Rydstrom and L. Ernster (1971) Eur. J . Biochem. 23, 203-211. 90. T e i x e i r a da Cruz, A., J . Rydstrom and L. Ernster (1971) Eur. J . Biochem. 23_, 212-219. 91. Rydstrom, J . (1972) Eur. J . Biochem. _31, 496-504. 92. Chen, S. and R.J. G u i l l o r y (1984) J . B i o l . Chem. 259, 5945-5953. 93. Wu, L.N.W., S.R. Earle and R.R. Fisher (1981) J . B i o l . Chem. 256, 7401-7408. 94. H a t e f i , Y., D.C. Phelps and Y.M. Galante (1980) J . B i o l . Chem. 255, 9526-9529. 95. Wu, L.N.Y. and R.R. Fisher (1982) J . B i o l . Chem. 257, 11680-11683. 96. Kozlov, I.A., Y.M. Milgrom, L.A. Saburova and A.Y. Sobolev (1984) Eur. J . Biochem. JA5, 413-416. 97. Enander, K. and J . Rydstrom (1982) J . B i o l . Chem. 2^6, 14760-14766. 98. Lee, CP., N. Simard-Duquesne, L. Ernster and H.D. Hoberman (1965) Biochim. Biophys. Acta 105, 397-409. 99. Lazdunski, M. (1972) Curr. Top. Bioenerg. 6, 267-310. 100. Klingenberg, M. (1981) Nature 290, 449-454. 101. Swartz, S.A. and D.R. H e l i n s k i (1971) J . B i o l . Chem. 246, 6318-6327. 102. Maniatis, J . , E.F. F r i t s c h and J . Sambrook (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold spring Harbor, N.Y. 103. Katz, L., O.T. Kingsbury and D.R. H e l i n s k i (1973) J . B a c t e r i o l . 114, 577-591. 104. Katz, L. P.H. Williams, S. Sato, R.W. L e a v i t t and D.R. H e l i n s k i (1977) Biochemistry 16_, 1677-1683. 105. Burns, D.M. and I.R. Beacham (1983) Anal. Biochem. 135, 48-51. 146. 106. Cleveland, D.W., S.G. Fischer, M.W. Kirscher and U.K. Laemmli (1977) J . B i o l . Chem. _252, 1102-1106. 107. Laemmli, U.K. (1970) Nature 227, 680-685. 108. Fairbanks, G. T.L. Steck and D.F.H. Wallach (1971) Biochemistry 10, 2606-2617. 109. Brusilow, W.S.A., R.P. Gunsalus and R.D. Simoni (1983) Methods Enzymol. 9_7, 189-195. 110. Sanger, F., S. Nicklen and A.R. Coulson (1977) Proc. Natl. Acad. S c i . 74, 5463-5467. 111.. Sanger, F.and A.R. Coulson (1978) FEBS L e t t . 87, 107-110. 112. Oker-Blom, C. (1984) Ph.D. th e s i s , Univ. of H e l s i n k i , H e l s i n k i . 113. Kaplan, N.O. (1967) Methods Enzymol. 10, 317-322. 114. Lowry, O.H., N.J. Rosebrough, A.L. Farr and A.J. Randall (1951) J . B i o l . Chem. 193, 265-275. 115. Sakamoto, N., A.M. Kotre and M.A. Savageau (1975) J . B a c t e r i o l . 124, 775-783. 116. M i l l e r J.H. (1972) Experiments i n molecular genetics. Cold Spring Harbor Laboratory, New York. 117. Nowotny, A. (1979) Basic Exercises i n Immunochemistry. Springer-Verlag, New York. 118. Bjerrum, O.J. and P. Lundhal (1974) Biochim. Biophys. Acta 342, 69-80. 119. Mayer, R.J. and J.H. Walker (1980) Immunochemical Methods i n B i o l o g i c a l Sciences: Enzymes and Proteins, Academic Press, Inc., New York. 120. Houghton, R.L., R.J. Fisher and D.R. Sanadi (1976) Arch. Biochem. Biophys. 176, 747-752. 121. Bouchg, J.P. (1982) J . Mol. B i o l . _154, 1-20. 122. Voordouw, G., S.M. van der Vies and A.P.N. Themmen (1983) Eur. J . Biochem. 131, 527-533. 123. Blazyk, J.F. and R.R. Fisher (1975) FEBS Le t t . 50, 227-230. 124. Deamer, D.W., R.C. Prince and A.R. Crafts (1972) Biochim. Biophys. Acta 274, 323-335. 125. Rottenberg, H. and C P . Lee (1975) Biochemistry 14, 2675-2680. 126. Casadio, R. and Melandri, B.A. (1977) J . Bioenerget. Biomembr. 9, 17-29. 147. 127. Pressman, B.C. (1976) Annu. Rev. Biochem. 4_5, 501-530. 128. Phelps, D.C. and Y. Hate f i (1981) J . B i o l . Chem. ̂ 56, 8217-8221. 129. Senior, A.E. and J.G. Wise (1983) J . Membr. B i o l . 73,, 105-124. 130. Clejan, L. and D.S. Beattie (1983) J . B i o l . Chem. 258, 14271-14275. 131. E s p o s t i , M.D., E.M.M. Meier, J . Timoneda and G. Lenaz (1983) Biochim. Biophys. Acta 725, 349-360. 132. Casey, R.P., M. Thelen and A. Azzi (1980) J . B i o l . Chem. 255, 3994-4000. 133. Hunkapiller, M.W., E. Lujan, F. Ostrander and L.E. Hood (1983) Meth. Enzymol. 91_, 227-236. 134. Nelles, L.P. and J.R. Bambury (1976) Anal. Biochem 7_3, 522-531. 135. Hager, D.A. and R. Burgess (1980) Anal. Biochem. 97_, 76-86. 136. Higgins, R.C. and M.E. Dahmus (1979) Anal. Biochem. 9J3, 257-260. 137. Messing, J . and J . V i e i r a (1982) Gene 19_, 269-276. 138. Stocker, N.G., J.M. Pratt and B.G. Spratt (1983) J . B a c t e r i o l . 155, 854-859. 139. Teather, R.M., B. M u l l e r - H i l l , U. Abrutsch, G. Aichele and P. Overath (1978) Mol. Gen. Genet. 159, 239-248. 140. Larson, T.J., G. Schumaker and W. Boos (1983) J . B a c t e r i o l . 152, 1008-1021. 141. Lemire, B.D., J . J . Robinson, R.D. Bradley, D.G. Scraba and J.H. Weiner (1983) J . B a c t e r i o l . 155, 391-397. 142. Weiner, J.H.., B.D. Lemire, M.L. Elmes, R.D. Bradley and D.G. Scraba (1984) J . B a c t e r i o l . 158, 590-596. 143. von Meyenburg, K., B.B. Jorgensen and B. van Deurs (1984) EMBO J . 3, 1791-1797. 144. F i o l e t , J.W.T., E.P. Bakker and K. van Dam (1974) Biochim. Biophys. Acta 368, 432-445. 145. Lee, H.C. and J.G. Forte (1978) Biochim. Biophys. Acta 508, 339-356. 146. Rogan, C.I. and W.R. Widger (1975) Biochem. Biophys. Res. Commun. 62, 744-749. 147. A z z i , A., R.P. Casey and M.J. Nalecy (1984) Biochim. Biophys. Acta 768, 209-226. 148. 148. Rosenberg, M. and D. Court (1979) Annu. Rev. Genet. 13., 319-353. 149. Hawley, D.K. and W.R. McClure (1983) Nucleic Acids Res. 8, 2237-2255. 150. Gold, L., D. Pribnow, T. Schneider, S. Shinedling, B.S. Singer and G. Stormo (1981) Annu. Rev. M i c r o b i o l . 35, 365-403. 151. Grosjean, H. and W. F i e r s (1982) Gene 18_, 199-209. 152. S o l i o z , M. (1984) TIBS 9, 309-312. 153. Kyte, J . and R.F. D o o l i t t l e (1982) J . Mol. B i o l . 157, 105-132. 154. Gamier, J . , D.J. Osguthorpe and B. Robson (1978) J . Mol. B i o l . 120, 97-120.

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