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A Study of an aldehyde dehydrogenase from Pseudomonas aeruginosa Von Tigerstrom, Richard G. C. 1967

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A STUDY OF AN ALDEHYDE DEHYDROGENASE FROM PSEUDOMONAS AERUGINOSA by RICHARD G. C. VON TIGERSTROM B. S. A. Un i v e r s i t y of B r i t i s h Columbia, 1964 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of MICROBIOLOGY We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1967 In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced deg ree at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I ag ree 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 s t u d y . I f u r t h e r ag ree 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 pu rposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n -t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Mi c-rohi o l ogy  The U n i v e r s i t y o f B r i t i s h Co lumb ia Vancouve r 8, Canada D a t e January, 1968 i i ABSTRACT An aldehyde dehydrogenase was found in c e l l extracts of Pseudomonas aeruginosa ATCC 9027 grown on several carbon sources. It was present in highest concentration in c e l l extracts after growth of the organism on ethylene glycol or ethanol . The enzyme from ethanol-grown cells was purified by protamine sulfate, ammonium sulfate, acetone, and isoelectric precipitation, ion exchange chromatography and gel f i l t r a t i o n . After an eighteen-to twenty-fold purification with a twenty-three per cent yield of activity a homogeneous preparation was obtained, as evidenced by ultracentrifugation, electrophoresis, and other c r i t e r i a . The enzyme was found to be unstable in crude preparations. This in s t a b i l i t y was overcome by the use of bi s u l f i t e buffer. The enzyme oxidizes a wide variety of aldehydes. The products of glycolaldehyde and glyceraldehyde oxidation were identified as the free acids. The pH optimum for f:he reaction was found to be between pH 8.0 and 8.6. The enzyme is more active with NAD+ as the hydrogen acceptor than with NADP+. Potassium or ammonium was found to be essential for activity. Less activity was obtained in the presence of rubidium. Aldehyde dehydrogenases from five other species of Pseudomonas were also activated by potassium. Michaelis constants for aldehyde substrates, NAD+, NADP+, and the activating ions were determined. In addition to the activating ion, a reducing agent was required for enzymatic activity. It could be replaced, in part, by EDTA or o-phenanthroline. No inhibition was observed with EDTA3 but o_-phen-anthroline inhibited the enzyme reaction in the presence of a reducing i i i agent. However, zinc was not found to be present i n the p u r i f i e d aldehyde dehydrogenase. Aldehyde dehydrogenase also was i n h i b i t e d by iodoacetamide, I | iodoacetate, arsenite,.Cu , and p_-chloromercuribenzoate. Enzymatic a c t i v i t y also was l o s t when tryp s i n was added to the enzyme preparation. This loss of a c t i v i t y and the i n h i b i t i o n by the a l k y l a t i n g agents vere s p e c i f i c a l l y prevented by the addition of the a c t i v a t i n g ion and NAD* to the enzyme preparation. Some protection from digestion by t r y p s i n was afforded by potassium alone. However, i n the absence of NAD"1" potassium accelerated the rate of i n h i b i t i o n by a l k y l a t i n g agents. A molecular weight of 200,000 was determined f o r aldehyde dehydrogenase by several methods. At low i o n i c strength the enzyme underwent a p a r t i a l d i s s o c i a t i o n with loss of enzymatic a c t i v i t y . This d i s s o c i a t i o n could be reversed by increasing the s a l t concentration. D i s s o c i a t i o n and as s o c i a t i o n of the enzyme into subunits of approximately equal size could be followed i n the u l t r a c e n t r i f u g e and on starch gel electrophoresis. The d i s s o c i a t e d form of the enzyme was i s o l a t e d a f t e r starch gel electrophoresis and found to be completely i n a c t i v e . F u l l enzymatic a c t i v i t y was obtained only when the associated enzyme was protected from oxidation. The enzyme was soluble below i t s i s o e l e c t r i c point (pH 4.8) but denatured s as evidenced by sedimentation, d i f f u s i o n , and v i s c o s i t y studies. The molecular weight of the enzyme preparation at pH 3.0 was estimated to be approximately one-half of that found at pH 7.0. i v Aldehyde dehydrogenase contained r e l a t i v e l y large amounts of a l l oommon amino a c i d s . The lowest amount was obtained for c y s t e i c a c i d : 23 to 24 residues per mole. Studies with ^C-iodoacetamide showed that the enzyme was completely i n h i b i t e d when approximately three moles of iodoacetamide were taken up per mole of enzyme. Fo l lowing chymotryptic d i g e s t i o n of l a b e l l e d aldehyde dehydrogenase, a f r a c t i o n conta in ing a large percentage of the r a d i o a c t i v i t y was p a r t i a l l y p u r i f i e d by ion exchange chromatography and gel f i l t r a t i o n . This f r a c t i o n contained one peptide spec ies , or severa l very s i m i l a r peptide spec ies , probably der ived from t h e ' a c t i v e s i t e of t<he' enzyme. TABLE OF CONTENTS PAGE INTRODUCTION . 1 LITERATURE REVIEW 3 I. The r o l e of Aldehyde Dehydrogenase i n Metabolism . . . 3 I I . Aldehyde Oxidation 5 I I I . Aldehyde Dehydrogenases 6 IV. D i s s o c i a t i o n of Enzymes 9 MATERIALS AND METHODS . .. . ' 11 I. Materials . . • . . . .•• • . .. . . . . . • • • . • • .... 11 I I . Organisms 11 I I I . Growth of Organisms 12 Growth of P. aeruginosa f o r Enzyme Production . . . 12 Growth of P. aeruginosa on D i f f e r e n t Carbon Sources. 12 IV. Preparation of C e l l Extracts . .- 13 V. Enzyme Assays 13 Aldehyde Dehydrogenase • 13 Catalase 14 Yeast Alcohol Dehydrogenase .14 Alcohol Dehydrogenase i n C e l l Extracts of P 14 aeruginosa VI. Preparation of Columns for Chromatography 15 Cellex-T .. . .' 15 Gel F i l t r a t i o n .16 v i PAGE Bio-Rad AG 50W-X2 for Chromatography of Peptides . . . 17 VII. Electrophoresis of Proteins 17 Starch Gel Electrophoresis 17 Polyacrylamide Disc Electrophoresis 18 Detection of Aldehyde Dehydrogenase Activity in Gels . 18 VIII. Sucrose Gradient Centrifugation 18 IX. Studies with the Analytical Ultracentrifuge 19 Sedimentation . . . . . . . 19 Diffusion 19 Approach to Sedimentation Equilibrium . . . 20 X. Determination of the Isoelectric Point 20 XI., Viscosity Determinations 21 XII. Determination of Amino Acid Composition 21 Acid Hydrolysis • 21 Performic Acid Oxidation 21 Amino Acid Analysis 22 Estimation of Tyrosine and Tryptophan 22 XIII. Studies with ^C-Iodoacetamide . . . . . 22 XIV. Enzymatic Hydrolysis of Aldehyde Dehydrogenase 23 ..XV. Analytical Methods 24 Protein . . ....... . . . . . . . . . . ............. 24 Dry Weight . . . 24 Nitrogen . 24 v i i PAGE Estimation of Peptides with Ninhydrin . . . ... . . . 24 Zinc . . . . . . . . . . . . . . . . . . . . . .25 XVI. Paper Chromatography . . .25 Products of the Reaction of the Enzyme with Glyceraldehyde and Glycolaldehyde 25 EXPERIMENTAL RESULTS . . . ... . ......... . . ... 26 I. Induction of Aldehyde Dehydrogenase 26 I I . P u r i f i c a t i o n of Aldehyde Dehydrogenase . . . 28 Step 1. Preparation of C e l l Extract 28 Step 2. Protamine Sulfate Treatment 29 Step 3. Ammonium Sulfate F r a c t i o n a t i o n . . . . . . . 29 Step 4. Acetone F r a c t i o n a t i o n 29 Step 5. I s o e l e c t r i c F r a c t i o n a t i o n . . 30 Step 6. Ion Exchange Chromatography . 30 Step 7. Gel F i l t r a t i o n 31 I I I . P u r i f y of Aldehyde Dehydrogenase . . . . . . . . 36 Electrophoresis i n Starch Gel . . . . . . ....... . . 36 Polyacrylamide Disc Electrophoresis . . '. . . . . . . .36 Aldehyde Dehydrogenase i n the A n a l y t i c a l U l t r a c e n t r i f u g e 39 IV. Physical Properties of Aldehyde Dehydrogenase 43 S o l u b i l i t y . . . .. . . ... 43 I s o e l e c t r i c Point 43 v i i i PAGE Molecular Weight of Aldehyde Dehydrogenase . . . ... .43 Physical Properties of Aldehyde Dehydrogenase below the I s o e l e c t r i c Point 48 Absorbance 51 Amino Acid Composition of Aldehyde Dehydrogenase .. . . 53 V. Requirements f or Enzyme A c t i v i t y 56 Assay of Maximum A c t i v i t y . . . . . . .. . .56 Ionic Requirements 58 Enzyme:Specificity . . . . . . . . . . . 62 Michaelis Constants 65 Reducing Agents . . . . . . . . . 70 E f f e c t of pH 70 VI. S t a b i l i t y 73 S t a b i l i t y of the Enzyme i n C e l l Extracts 73 S t a b i l i t y of the P u r i f i e d Enzyme . . . . . . . . . . . . . . 75 E f f e c t of pH . 76 Inactivation by Heat . . . . . . . 76 VII. I n h i b i t i o n and Protection 79 VIII. Analysis of the Enzyme for Zinc 83 IX. D i s s o c i a t i o n and Deactivation of Aldehyde Dehydrogenase. 84 Pattern of Dissociated and Reassociated Aldehyde Dehydrogenase i n the Ul t r a c e n t r i f u g e . . . . . . . . . . 85 Ele c t r o p h o r e t i c M o b i l i t y of Dissociated and Reassociated Aldehyde Dehydrogenase .85 i x PAGE .Deactivated State of Dissociated Enzyme i n Protein Bands Isolated by Starch Gel Electrophoresis . . . 85 Conditions for D i s s o c i a t i o n and Deactivation . . . . 88 Conditions f o r Reassociation ... . . . . . . ... . . 90 Conditions for Reactivation . . . . . 90 Size of the Dissociated Enzyme 92 Influence of Other Reagents 93 X. L a b e l l i n g of Aldehyde Dehydrogenase with ^ C -Iodoacetamide and P a r t i a l P u r i f i c a t i o n of an Active Peptide . . . . . . . . . . . . . . . 95 DISCUSSION 100 SUMMARY . 114 BIBLIOGRAPHY . . 116 X LIST OF FIGURES FIGURE PAGE 1. Chromatography of Aldehyde Dehydrogenase on Cellex-T . . . . 32 2. Gel F i l t r a t i o n of Aldehyde Dehydrogenase on Sephadex G-200 . 34 3. Starch Gel Electrophoresis of AldehydeDehydrogenase at Two Stages of the P u r i f i c a t i o n 37 4. Polyacrylamide Disc Electrophoresis of Aldehyde Dehydrogenase at D i f f e r e n t Stages of the P u r i f i c a t i o n . . 38 5. Polyacrylamide Disc Electrophoresis of D i f f e r e n t Quantities of P u r i f i e d Aldehyde Dehydrogenase . . . 40 6. Sedimentation of Aldehyde Dehydrogenase at pH 7.0 41 7. Determination of Sedimentation C o e f f i c i e n t of Aldehyde Dehydrogenase 42 8. I s o e l e c t r i c Point of Aldehyde Dehydrogenase . . . . 44 9. Gel F i l t r a t i o n of Aldehyde Dehydrogenase and Reference Enzymes . . . . . . . . 46 10. Sucrose Gradient Centrifugation of Aldehyde Dehydrogenase and Reference Enzymes 47 11. Determination of the D i f f u s i o n C o e f f i c i e n t f o r Aldehyde Dehydrogenase 49 12. Schlieren Pattern of Aldehyde Dehydrogenase at 5227 r.p.m. . 50 13. Sedimentation of Aldehyde Dehydrogenase at pH 3.0 52 14. E f f e c t of Hydrolysis Time on Amino Acid Composition . . . . . 55 15. E f f e c t of Sequence of Addition of Components to the Reaction Mixture . . . . . . . . . . . . . . 57 x i PAGE 16. Double Reciprocal Plots of V e l o c i t y against NAD+ or NADP+ Concentration 66 17. Double Reciprocal Plot of V e l o c i t y against Glyceraldehyde Concentration 67 18. Double Reciprocal Plot of V e l o c i t y against Glycolaldehyde Concentration 68 19. Double Reciprocal Plot of V e l o c i t y against Ammonium or Potassium Ion Concentration . . . . . . . . . . . . 69 20. The E f f e c t of pH on Glycolaldehyde Oxidation 72 21. Heat Ina c t i v a t i o n of Aldehyde Dehydrogenase 78 22. E f f e c t of the Presence of K+ or Na+ on the I n h i b i t i o n of Aldehyde Dehydrogenase by Iodoacetamide 80 23. ' Sedimentation of Dissociated and Reassociated Aldehyde Dehydrogenase i n the Ultr a c e n t r i f u g e . . . 86 24. Starch Gel Electrophoresis of Dissociated and Reassociated Aldehyde Dehydrogenase . . . . ......... . . . . . 87 25. Enzymatic A c t i v i t y , of Isolated Dissociated and Associated Aldehyde Dehydrogenase . . . . . . . . . . . . . 89 26. Sucrose Gradient Centrifugation of Dissociated Aldehyde Dehydrogenase . . . . . . . . . . • . . .; 94 27. Ion Exchange Chromatography of Chymotryptic Digest of •^C-Labelled Aldehyde Dehydrogenase . . . . . . . 97 28. .Gel F i l t r a t i o n of 1 4 C - L a b e l l e d Chymotryptic Peptide . . . . . . . 99 x i i LIST OF TABLES TABLE PAGE I. Aldehyde Dehydrogenase A c t i v i t y i n C e l l Extracts of P. aeruginosa Grown on D i f f e r e n t Substrates 27 I I . Summary, of the P u r i f i c a t i o n of Aldehyde Dehydrogenase from P. aeruginosa 35 I I I . Amino Acid Composition of Aldehyde Dehydrogenase 54 IV. Ionic Requirements f or Aldehyde Dehydrogenase A c t i v i t y . . . 59 V. E f f e c t of Monovalent Cations of Potassium-Activated Aldehyde Oxidation 60 -VI. Aldehyde Dehydrogenase A c t i v i t y i n C e l l Extracts of Six Di f f e r e n t Strains of Pseudmonas 61 VII. S p e c i f i c A c t i v i t y of Crude and P u r i f i e d Enzyme Preparations with D i f f e r e n t Substrates and Hydrogen Acceptors 63 VI I I . Substrate S p e c i f i c i t y • 64 IX. A c t i v i t y of Aldehyde Dehydrogenase i n the Presence of EDTA and D i f f e r e n t Reducing Agents . 71 X. E f f e c t of Reducing Agents, B i s u l f i t e , and Phosphate on S t a b i l i t y of Aldehyde Dehydrogenase i n C e l l Extracts . . . 74 XI. E f f e c t of B i s u l f i t e on the S t a b i l i t y of P u r i f i e d Aldehyde Dehydrogenase . . . . . . . . . 77 XII. I n h i b i t i o n of Aldehyde Dehydrogenase by Iodoacetate and Protection by the A c t i v a t i n g Ion and Coenzyme 81 X l l l PAGE XIII. Digestion of Aldehyde Dehydrogenase by Trypsin and Protection by the A c t i v a t i n g Ion and Coenzyme . . . . 82 XIV. A. Reactivation a f t e r D i s s o c i a t i o n by D i a l y s i s against 1 mM EDTA B. Reactivation a f t e r D i s s o c i a t i o n by D i a l y s i s against 1 mM T r i s . . . . . . . . . . . . . . 91 x i v ACKNOWLEDGEMENTS I would l i k e to express my sincere appreciation to Dr. W. E. Ra z z e l l for his h e l p f u l suggestions and c r i t i c i s m s during the course of t h i s study. I also am g r a t e f u l to Dr. L. B. S m i l l i e and Dr. C. M. Kay of the Department of Biochemistry, U n i v e r s i t y of Alb e r t a , f o r t h e i r advice and permission to use t h e i r equipment. I would l i k e to thank Miss Maxine H i l b e r t for t e c h n i c a l assistance with some of the experiments and Mrs. Marilyn Vantour for typing t h i s t h e s i s . INTRODUCTION The i n t e r e s t i n aldehyde dehydrogenase evolved from studies on t r i o s e and t r i o s e phosphate metabolism i n Pseudomonas aeruginosa. The p o s s i b i l i t y of a non-phosphorylative pathway for g l y c e r o l oxidation, such as that reported i n an Arthrobacter species (Shethna and Bhat, 1962), was investigated. Although a g l y c e r o l dehydrogenase could not be demonstrated, the presence of an aldehyde dehydrogenase and aldehyde reductase f o r glyceraldehyde (Gadd, 1962; von Tigerstrom, 1964) and a hydroxypyruvate reductase i n glucose- or glycerol-grown c e l l s suggested that t h i s pathway of g l y c e r o l degradation might occur. However, further studies on the oxidation of ^ C g l y c e r o l by r e s t i n g c e l l s of g l y c e r o l -grown P. aeruginosa revealed a large percentage of phosphorylated intermediates i n the e a r l y stages of oxidation. F i f t y per cent of t h i s was i d e n t i f i e d as CC-glycerophosphate. This finding.and the demonstration of a glycerokinase i n c e l l extracts of glycerol-grown c e l l s , but not i n glucose-grown c e l l s , led to the conclusion that most, i f not a l l , g l y c e r o l i s metabolized v i a Q;-glycerophosphate:and not by a non-phosphorylative pathway (von Tigerstrom, unpublished r e s u l t s ) . Growth of P. aeruginosa on alcohols such as ethanol, ethylene g l y c o l , n-propanol and n-butanol r e s u l t e d i n a s i g n i f i c a n t increase of the aldehyde dehydrogenase a c t i v i t y i n c e l l extracts of t h i s organism, compared to the l e v e l s detected i n c e l l extracts of g l y c e r o l - or glucose-grown c e l l s . I t was found that t h i s enzyme could be obtained i n very high y i e l d from - 2 -ethanol-grown c e l l s and conditions were discovered f o r maintaining.the enzyme.in a stable state. I t seemed possible , therefore,.that the enzyme could be obtained i n a highly p u r i f i e d form which would allow studies of the requirements, for enzymatic a c t i v i t y of a pure preparation and studies of some of the molecular properties of an aldehyde dehydro-genase. Such studies had not been possible so f a r with any other aldehyde.dehydrogenase. In add i t i o n , further i n v e s t i g a t i o n of the enzyme seemed warranted since i t appeared to d i f f e r from other aldehyde dehydrogenases reported i n Pseudomonas species; s p e c i f i c a l l y the phosphate-requiring enzyme from Pseudomonas fluorescens (Jakoby, 1958a) and the. enzyme from P. aeruginosa requiring. Ca"*"1" or F e ^ and f l a v i n f o r a c t i v i t y (Heydeman and Azoulay, 1963). - 3 -LITERATURE REVIEW I. The Role of Aldehyde Dehydrogenase i n Metabolism Aldehyde dehydrogenases have been reported from a great v a r i e t y of sources. Their r o l e i n the metabolism of animal tissues i s a general one, that of degradation of toxic aldehydes occurring from the breakdown of other metabolites. This may also be true f o r many microorganisms. In addition,.aldehyde dehydrogenases have a s p e c i f i c place i n metabolic sequences f o r the degradation of alcohols or more reduced substrates as i t occurs i n the a c e t i c acid b a c t e r i a and i n pseudomonads. Oxidation of alcohols has been investigated p r i m a r i l y i n Acetobacter species. The i n i t i a l step i n ethanol oxidation i s c a r r i e d out by a NADP+ -dependent alcohol dehydrogenase i n Acetobacter peroxidans (Tanenbaum, 1956). A NAD+ - dependent alcohol dehydrogenase also has been reported f o r t h i s organism (Atkinson, 1956). The r e s u l t i n g aldehyde i s oxidized to ac e t i c ac i d by an aldehyde dehydrogenase which i s NADP+ s p e c i f i c (Tanenbaum, 1956). The aldehyde dehydrogenase.from Acetobacter  suboxydans has been p a r t i a l l y p u r i f i e d and studied by King and Chelde l i n (1956). The metabolism of the a c e t i c a c i d b a c t e r i a has been reviewed by Wood (1957) and the oxidation of a l i p h a t i c g l y c o l s by these organisms was summarized by DeLey and Kersters (1964). Although a c e t i c a c i d b a c t e r i a are regarded as potent alcohol o x i d i z e r s , ; t h i s i s also true.of Pseudomonas species. Stanier (1947), - 4 -i n v e s t i g a t i n g the acid production from ethanol by pseudomonads i n buffered media, found that nine out of th i r t e e n species thrived on ethanol as the sole carbon source. S e v e n , a l l fluorescent pseudomonads, produced a c e t i c a c i d to varying degrees, some i n good y i e l d . P. aeruginosa was the only organism which did not produce a c i d , although u t i l i z i n g ethanol as the sole carbon source. Several alcohol dehydrogenases have been reported i n pseudomonads. Shaw (1956) reported a po l y o l dehydrogenase from a Pseudomonas species which was NAD+ - l i n k e d . Azoulay and Heydeman (1963) describe a NAD+ -linked heptanol dehydrogenase from P. aeruginosa. Ethanol was also oxidized by t h i s enzyme but at a reduced rate. For most alcohol dehydrogenases, pyridine nucleotides are the primary hydrogen acceptors. However, Anthony and Zatman (1964) reported the i s o l a t i o n of a methanol-grown Pseudomonas sp. M 27, and i n a subsequent report (1965) they describe the.alcohol dehydrogenase and p a r t i a l p u r i f i c a t i o n of the enzyme. This alcohol dehydrogenase uses phenazine methosulfate as the primary hydrogen acceptor and no natural hydrogen acceptor could be found. Maximum enzymatic a c t i v i t y was obtained with methanol or ethanol as the substrate. Approximately eighty per cent of the maximum a c t i v i t y was obtained with propanol, butanol,.and ethylene g l y c o l . Two aldehyde dehydrogenases from Pseudomonas species have been reported i n the l i t e r a t u r e . The aldehyde dehydrogenase from ethylene glycol-grown P. fluorescens oxidizes a wide v a r i e t y of a l i p h a t i c and aromatic aldehydes, i n c l u d i n g acetaldehyde (Jakoby, 1958a). The enzyme reported from P. aeruginosa grown on p a r a f f i n hydrocarbons i s also active with acetaldehyde although higher aldehydes are oxidized at a f a s t e r rate - 5 -(Heydeman and Azoulay,. 1963) . Unless the oxidation of ethanol stops at a c e t i c a c i d as i n some .Acetobacter species, acetate becomes a key metabolic intermediate, leading to l i p i d synthesis on the one hand and.into the t r i c a r b o x y l i c a c i d cycle and the glyoxylate cycle on the other (Kornberg, 1966). IIV Aldehyde Oxidation The concept of an aldehyde mutase as proposed by Dixon and Lutwak-Mann (1937) could no longer be accepted as the only mechanism for aldehyde oxidation a f t e r the i s o l a t i o n of an aldehyde dehydrogenase from bovine l i v e r by Racker (1949) and of an.alcohol dehydrogenase from l i v e r by Bonnichsen (1950). Aldehyde oxidation i s now considered to be the reaction of an aldehyde, the hydrogen donor, with a hydrogen acceptor, generally a pyridine nucleotide. If water i s the t h i r d reactant, the product i s an a c i d . If water i s replaced by phosphate, glutathione, or coenzyme A, the product i s the respective ester of the free a c i d . For some time i t was thought that the aldehyde hydrate was the reacting species. However, F r i d o v i c h (1966), using xanthine oxidase, has shown that the aldehyde i s the reactant and not the aldehyde hydrate and he points out that t h i s also may be true for the dehydrogenation reaction of aldehyde dehydrogenases. The change of the free energy (AF) for the oxidation of acet-aldehyde to a c e t i c a c i d was c a l c u l a t e d by Kaplan (1951) to be -12.5 kc. The formation of a c e t y l coenzyme A from acetaldehyde and reduced coenzyme A has AF =-4.2 kc. This l a t t e r r e a c t i o n i s r e v e r s i b l e , whereas - 6 -the oxidation of acetaldehyde to a c e t i c acid has not been reversed experimentally. I I I . Aldehyde Dehydrogenases A number of aldehyde dehydrogenases have been reported and p a r t i a l l y p u r i f i e d from mammalian tissues and microorganisms. A review of these enzymes was prepared by Jakoby i n 1963 (Jakoby, 1963). Reports of an aldehyde dehydrogenase from P. aeruginosa grown on hydro-carbons (Heydeman and Azoulay, 1963) and aldehyde dehydrogenases from r a t , monkey, and bovine brain (Erwin and D e i t r i c h , 1966) have appeared since then. A l l enzymes studied except those acting on semialdehydes are r e l a t i v e l y nonspecific and oxidize a l i p h a t i c , or a l i p h a t i c and aromatic, aldehydes. As a r u l e , NAD"1".or NADP+ i s the hydrogen acceptor and some enzymes are more s p e c i f i c than others i n t h i s respect. S p e c i f i c i o n i c requirements for enzymatic a c t i v i t y have been reported f o r several aldehyde dehydrogenases. The NAD+ -linked aldehyde dehydrogenase from yeast requires K + or Rb + (Black, 1951). The NADP+ -linked aldehyde dehydrogenase from yeast i s active i n the presence of Mg (Seegmiller, 1953). An enzyme from P. fluorescens i s reported to require phosphate or arsenate (Jakoby, 1958a) while the aldehyde dehydrogenase from P. aeruginosa grown on hydrocarbons uses Fe or Ca (Heydeman and Azoulay, 1963). These d i f f e r e n t s p e c i f i c i t i e s might suggest that these enzymes d i f f e r i n t h e i r properties although a l l oxidize the same f u n c t i o n a l group. On the other hand, there are some properties which are shared by most, i f not a l l , aldehyde dehydrogenases. - 7 -One general property of aldehyde dehydrogenases i s that of i n s t a b i l i t y . This was the main factor which, u n t i l now, had prevented extensive p u r i f i c a t i o n and studies of the physical and molecular properties of aldehyde dehydrogenases, such as have been possible with a l c o h o l , l a c t i c ,.• glyceraldehyde,3-phosphate and other dehydrogenases. Another common property of aldehyde dehydrogenases i s the involvement of s u l f h y d r y l groups i n the active s i t e . The enzymes reported by Jakoby (1958a) and by Burton and Stadtman (1953) show an absolute requirement for mercaptans f o r enzymatic a c t i v i t y . The yeast enzymes and the l i v e r aldehyde dehydrogenase are stimulated by 2-mercapto-ethanol. Jakoby (1958b) investigated these enzymes and found that arsenite would cause : i n h i b i t i o n of enzymatic a c t i v i t y . i n the presence of a mono-mercaptan. Other aldehyde dehydrogenases were also found to be i n h i b i t e d by arsenite (Jakoby, 1963). The i n h i b i t i o n was reversed by 2,3-dimercaptopropanol (BAL), a dimercaptan. In p a r a l l e l to the i n v e s t i g a t i o n which led to the i s o l a t i o n of l i p o i c a c i d and the e l u c i d a t i o n of i t s r o l e i n <*--keto acid oxidation (Reed ejt a l , 1953) , two c l o s e l y linked s u l f h y d r y l groups on the enzyme were proposed to be involved i n aldehyde oxidation (Jakoby, 1958b). That, i n f a c t , s u l f y d r y l groups are involved i n aldehyde oxidation was substantiated by the use of a number of s u l f h y d r y l reagents and by the work of Stoppani and M i l s t e i n (1957) which demonstrated the s p e c i f i c protection obtained with pyridine nucleotides against the i n h i b i t i o n of the two yeast and the l i v e r aldehyde dehydrogenases by s u l f h y d r y l reagents. Protection was also afforded by the aldehyde substrate and the a c t i v a t i n g ion, suggesting that the aldehyde and pyridine nucleotide may be attached to the s u l f h y d r y l groups on the enzyme while c a t a l y s i s i s taking place. - 8 -However, Nirenberg and Jakoby (1960), working with succinic semi-aldehyde dehydrogenase found that arsenite i n h i b i t i o n was competitive with the aldehyde substrate but not with pyridine nucleotide. Furthermore, Nirenberg and Jakoby showed that s u c c i n i c semialdehyde dehydrogenase was more susceptible to t r y p s i n digestion i n the presence of the cofactor and that t h i s s u s c e p t i b i l i t y was not changed i n the presence of £-chloromercuribenzoate or arsenite. This, and the f a c t that these i n h i b i t o r s did not replace the cofactor from the enzyme, led to the conclusion that NADP+ was attached to a s i t e other than a s u l f h y d r y l group. Such data as well as the c l o s e l y -positioned s u l f h y d r y l groups on the enzyme w i l l have to be considered i n proposing a mechanism f o r aldehyde oxidation. Some of the p o s s i b i l i t i e s are discussed by Jakoby (1963). I n h i b i t i o n of the potassium-activated yeast enzyme and the l i v e r aldehyde dehydrogenase with o-phenanthroline led to further i n v e s t i -gation of these enzymes by Stoppani, Schwarcz, and Freda (1966). I | K i n e t i c data indicated that Zn i s an i n t r i n s i c constituent of both enzymes and a s i g n i f i c a n t l e v e l of Zh"^" was found i n yeast aldehyde dehydrogenase preparations. A binding of NAD+ to the dival e n t metal was suggested. The f i n d i n g of Nirenberg and Jakoby (1960) that the presence of pyridine nucleotide increased the s e n s i t i v i t y of su c c i n i c semialdehyde dehydrogenase to t r y p s i n digestion suggested that a change of conformation of the enzyme molecule occurs i n the presence of the cofactor. A change i n the phy s i c a l arrangement of the yeast aldehyde dehydrogenase i n the presence of monovalent a c t i v a t i n g cations, was suggested by the work of Sorger - 9 -and Evans (1966). They observed that K + and Rb + were more e f f e c t i v e i n preventing the loss of enzyme a c t i v i t y due to heat treatment than other non-activating ions. In ad d i t i o n , K +.and Rb + were found to be more e f f e c t i v e i n . r e s t o r i n g enzyme a c t i v i t y a f t e r d i a l y s i s of the. enzyme against T r i s phosphate buffer than non-activating ions. Although a conformational change of the enzyme appears l i k e l y , d i r e c t studies must await extensive p u r i f i c a t i o n of.the proteins. IV. D i s s o c i a t i o n of Enzymes As more and more enzymes are p u r i f i e d and as t h e i r p h y s i c a l properties are examined, i t i s found that most large enzymes consist of several polypeptide chains held together by physical and, or,.chemical bonds to form the enzymatically active u n i t . Under c e r t a i n conditions t h i s active unit w i l l separate into subunits. This d i s s o c i a t i o n of proteins into smaller parts and the reverse process, the ass o c i a t i o n of the subunits,.has been the f i e l d of intensive i n v e s t i g a t i o n s . This subject,.and studies of conformational changes i n proteins, :.are very extensive ones and several.reviews have been published. A s s o c i a t i o n of proteins i s discussed from the point of protein-protein i n t e r a c t i o n s by Waugh (1954). R e i t h e l (1963) reviewed the subject of d i s s o c i a t i o n and a s s o c i a t i o n extensively, i n c l u d i n g a discussion on the techniques used i n these studies. The subunit structure of proteins, and the genetic aspects of t h i s f i e l d was the subject of a Brookhaven symposium (1964). The i s o l a t i o n of protein subunits was discussed by Porter (1966) and the l a t e s t work on conformation was reviewed by Timasheff and Garbunoff (1967). - 10 -The i n v e s t i g a t i o n of d i s s o c i a t i o n and a s s o c i a t i o n of proteins i s l a r g e l y empirical. It i s based on exposure of these macromolecules to denaturing conditions, such as high concentrations of urea or guanidinium s a l t s , high or low hydrogen ion concentration and i o n i c strength, detergents, and o x i d i z i n g or reducing agents. Some chelating agents,.metabolites or cofactors have proved to be very s p e c i f i c i n producing d i s s o c i a t i o n and a s s o c i a t i o n i n some enzymes. These d i s s o c i a t i o n s are often thought of as being s i g n i f i c a n t i n the regulation of enzyme a c t i v i t y . The p r i n c i p a l methods of detecting d i s s o c i a t i o n and.association have been u l t r a c e n t r i f u g a l a n a l y s i s , measurement of d i f f u s i o n constants and v i s c o s i t y , electrophoresis, chromatography and gel f i l t r a t i o n . Due to the i n s t a b i l i t y and lack of p u r i t y of aldehyde dehydro-genases, d i s s o c i a t i o n of these enzymes had not been previously investigated. - 11 -MATERIALS AND METHODS I. Materials A l l chemicals were obtained from commercial sources and were of a high standard of p u r i t y . Cellex-T (eriethylaminoethyl-cellulose), Bio-Gel P-60, and Bio-Rad AG 50W-X2 were purchased from Bio-Rad Laboratories; Sephadex G-200 and Sephadex G-25, from Pharmacia. Starch (hydrolyzed) f o r electrophoresis was from Connaught Medical Research Laboratories and the reagents for polyacrylamide disc electrophoresisv/were^friom' Eas.tman Organic Chemicals. Yeast alcohol dehydrogenase and tr y p s i n were products of Worthington Biochemical Corporation and <*--chymotrypsin was from C a l -biochem. Gatalase was prepared from horse blood^-. The pyridine used as the solvent f o r column chromatography of peptides was obtained from Fisher S c i e n t i f i c Company. One l i t e r was refluxed with two grams of ninhydrin for one hour p r i o r to d i s t i l l a t i o n . The i n i t i a l 800 ml. of the d i s t i l l a t e were c o l l e c t e d and used. . I I . Organisms P. aeruginosa ATCC 9027 and P. fluorescens A312 were obtained from the Department of Microbiology, U n i v e r s i t y of B r i t i s h Columbia. P. ^"Catalase was the g i f t of Dr. G. R. Schonbaum, Department of Biochemistry, U n i v e r s i t y of Alberta. - 12 -fluorescens s t r a i n s 36', 22' and 26 and P. o v a l i s were obtained from the Department of Microbiology, U n i v e r s i t y of Alberta. I I I . Growth of Organisms Growth of P. aeruginosa for Enzyme Production - P. aeruginosa ATCC 9027 was grown i n Roux f l a s k s containing 100 ml. of medium c o n s i s t i n g of NH4H2PO4, 0.3%; K2HPO4, 0.4%; FeS04.5H20, 5 p.p.m.; yeast extract, 0.1%; tryptone, 0.1%,; adjusted to pH 7.4 before s t e r i l i z a t i o n . Separate s t e r i l e solutions of MgS04.7H20 and 95% ethanol were prepared and added at the time of i n o c u l a t i o n to y i e l d concentrations of 0.05% and 0.3% r e s p e c t i v e l y . A 1%, inoculum was added and the culture was incubated for 24 hours at 30°, with a further addition of ethanol equal to 0.47» of the medium a f t e r 10 hours of growth. c The organism was also grown for 48 hours at 25° i n some cases. The second ad d i t i o n of ethanol (0.47o) was then made a f t e r 24 hours of growth. This method was used when P. aeruginosa, P. fluorescens A312, 6 P. fluorescens 36 1, _P. fluorescens 22', P. fluorescens 26 , and P. o v a l i s were grown i n the presence of ethanol to compare the l e v e l and i o n i c requirements of aldehyde dehydrogenase, i n these organisms. Stock cultures of P..aeruginosa were stored.at 4° i n the l i q u i d medium used for enzyme production a f t e r growth at 30° for 24 hours. The culture was checked for p u r i t y p e r i o d i c a l l y by p l a t i n g on Plate Count agar and i t was tested f o r pyocyanin production. Growth of P. aeruginosa on D i f f e r e n t Carbon Sources - The media used for growth of the organism on d i f f e r e n t carbon sources had the same - 13 -composition. as the medium used f o r enzyme production with the exception of the carbon source. The cultures were incubated f o r 48 hours at 25°. Glucose, g l y c e r o l , ethylene g l y c o l , , and ethanol were added to 0.27, concentration at the time of ino c u l a t i o n and again a f t e r 24 hours. Potassium acetate, pH 4.0, was added at 0.17o concentration at the time of i n o c u l a t i o n and a further 0.157o was added at 15 and 30 hours. Inoculum from a culture grown on the respective carbon source was used. IV. Preparation of C e l l Extracts The c e l l s were harvested by c e n t r i f u g a t i o n , washed once i n the buffer described i n the text,.and suspended i n the buffer to a con-ce n t r a t i o n of 200 mg. wet weight per ml. The c e l l suspension was immersed i n an ice-water bath and subjected to sonication f o r 7 minutes (for 50 ml. quantities) or 15 minutes ( f o r 100 ml. quantities) using a Bronwill Biosonik (Bronwill S c i e n t i f i c , Rochester, N.Y.) at maximum power output. The temperature of the c e l l suspension during t h i s operation was kept below 10°. V. Enzyme Assays Aldehyde Dehydrogenase - The enzyme re a c t i o n was followed by measuring the rate of appearance of NADH at 340 mu. i n a 1.0 cm. l i g h t path at 35° with a G i l f o r d Automatic Spectrophotometer Model 2000. Unless otherwise s p e c i f i e d , the incubation mixture contained the following constituents i n a final.volume of 1.0 ml.: potassium phosphate buffer, - 14 -pH 7.2, 100 mM; 2-mercaptoethanol, 10 mM; NAD+, 2 mM; an appropriate amount of enzyme; and glycolaldehyde, 1 mM. The reac t i o n was started by addition of the aldehyde af t e r preincubation f or f i v e minutes at 35° of a l l other components. D i l u t i o n s of the enzyme were made with 100 mM potassium phosphate buffer-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0. A measurable reaction rate i s obtained with approximately 1.0 ug of p u r i f i e d aldehyde dehydrogenase. One unit of enzyme a c t i v i t y , i s defined as the amount c a t a l y z i n g the formation of 1 umole of NADH per minute and s p e c i f i c a c t i v i t y as units per mg. of protein. Catalase - Catalase a c t i v i t y was measured according to the method of Chance and Maehly (1955) in. which the decrease i n absorbance at 230 mu due to the decomposition of hydrogen peroxide i s followed. The react i o n mixture contained 10 mM potassium phosphate b u f f e r , pH 7.0, 0.03% hydrogen peroxide and approximately 0.03 ug. of catalase from horse blood. The reaction was c a r r i e d out at 25° using a G i l f o r d Automatic Spectrophotometer Model 2000. Yeast Alcohol Dehydrogenase - Alcohol dehydrogenase a c t i v i t y was measured i n a reac t i o n mixture containing 20 mM sodium pyrophosphate, pH 8.8, 2 mM NAD+, 100 mM ethanol and approximately 0.02 ug of yeast alcohol dehydrogenase (Vallee and Hoch,1955). The increase i n absorbance at 340 mu due to reduction of NAD"1" was measured at 25° i n a 1.0 cm. l i g h t path using a G i l f o r d Automatic Spectrophotometer Model 2000. Alcohol Dehydrogenase i n C e l l Extracts of P. aeruginosa - Alcohol dehydrogenase a c t i v i t y i n c e l l extracts of ethanol-grown c e l l s was measured by the method of Va l l e e and Hoch (1955) as used for yeast alcohol - 15 -dehydrogenase. It was also measured with 100 mM T r i s Chloride, pH 7.8, replacing the sodium pyrophosphate buffer and i n the presence and absence of 10 mM 2-mercaptoethanol. The ethanol employed was tested f o r possible aldehyde contaminants by using i t as the substrate i n the r e a c t i o n mixture f o r aldehyde dehydrogenase with p u r i f i e d aldehyde dehydrogenase. Alcohol dehydrogenase a c t i v i t y was also measured according to the method of Anthony and Zatman (1965). The r e a c t i o n mixture contained: 100 mM T r i s c h l o r i d e , pH 9.0, 5 mM ethanol, 0.11 mM phenazine metho-s u l f a t e , 0.04 mM 2,6-dichlorophenol indophenol, and 15 mM ammonium chloride i n a t o t a l volume of 1.0 ml. The decrease i n absorbance at 600 mn due to reduction of 2,6-dichlorophenol indophenol and dependent on the presence of phenazine methosulfate, ethanol and c e l l extract was measured. VI. Preparation of Columns f o r Chromatography Cellex^T - Cellex-T ( t r i e t h y l a m i n o e t h y l - c e l l u l o s e ) was suspended i n approximately ten volumes of d i s t i l l e d water and f i n e material, was decanted three times. The c e l l u l o s e was then suspended i n M NaCl-0.1 M NaOH and packed into a. chromatographic column and washed with the a l k a l i n e s a l t s o l u t i o n u n t i l the absorbance at 260 mu of the e f f l u e n t had decreased to a value less than 0.05. Washing with f i v e column volumes of 0.1 M NaCl-0.01 M EDTA, pH 8.0, followed. The c e l l u l o s e was f i n a l l y washed free of chloride with d i s t i l l e d water and stored at 4°. For ion exchange chromatography, washed Cellex-T i n 0.1 M NaCl was packed under s l i g h t pressure i n a glass column. The Cellex-T was - 16 - . converted from the chloride to the phosphate form by washing with 0.1 M potassium phosphate, pH 7.0, u n t i l the e f f l u e n t was free of chloride ions. The column was e q u i l i b r a t e d with the s t a r t i n g buffer before a p p l i c a t i o n of the enzyme preparation. Regeneration of the ion exchanger was c a r r i e d out by washing successively with M NaCl, 0.1 M potassium phosphate, pH 7.0 and the s t a r t i n g b u f f e r . Gel F i l t r a t i o n - Sephadex G-200 was suspended i n approximately ten volumes of d i s t i l l e d water and swelling of the gel was allowed to proceed f o r at least 24 hours. During t h i s time f i n e material was decanted f i v e to s i x times. The gel was then washed three times with 50 mM potassium phosphate-10 mM 2-mercaptoethanol-l mM EDTA, pH 7.0. For packing of the column, a wide-stem funnel was attached to the top of a b u f f e r - f i l l e d column. The Sephadex s l u r r y was poured into the funnel and allowed to s e t t l e with overhead s t i r r i n g . A f t e r a column bed of 3 to 5 cm. had s e t t l e d , packing;was continued with a slow flow rate and a pressure head of no more than 10X of the column height by adjusting the p o s i t i o n of the o u t l e t . A f t e r packing was complete, the column was washed with at least two column volumes before the enzyme preparation was applied. D i l u t e protein solutions were applied i n a 10% sucrose s o l u t i o n by layering the s o l u t i o n on top of the gel bed. Concentrated protein solutions were applied without sucrose. During the operation of gel f i l t r a t i o n , , t h e • flow rate was adjusted by p o s i t i o n i n g the buffer r e s e r v o i r at a height of 10% of the column height above the o u t l e t . - 17 -Bio-Gel P-60 and Sephadex G-25 were treated and packed into columns i n a s i m i l a r way but with less consideration given to the height of the pressure head for these columns. Bio-Rad AG 50W-X2 for Chromatography of Peptides - Bio-Rad AG 50W-X2 was washed according to the method of Moore and Stein (1951). In t h i s procedure the r e s i n i s obtained i n the sodium form. I t was converted to the pyridinium form and e q u i l i b r a t e d with 0.2 N pyridine acetate, pH 3.1, and used for chromatography as described by Schroeder e_t a l (1962). A column, 1.4 by 34.0 cm. was packed with ion exchange r e s i n under s l i g h t a i r pressure. The column was washed with two column volumes of pyridine acetate b u f f e r , pH 3.1, at 38° before the protein digest was applied. The digest was eluted at 38° with a l i n e a r gradient c o n s i s t i n g of 350 ml. 0.2 N pyridine acetate, pH 3.1, and 350 ml. 2.0 N pyridine acetate, pH 5.0. The flow rate of 28 ml. per hour was c o n t r o l l e d with a Buchler P o l y s t a l t i c pump. VII. Electrophoresis of Proteins Starch Gel Electrophoresis - Starch gel electrophoresis was performed according to a modification of the method of Smithies (1955, 1959a) on gel s l i c e s 3 cm. wide, 20 cm. long and approximately 0.3 cm. thick. A p o t e n t i a l of 150 v o l t s was applied for two hours at 20-22° or f o r three and one-half to four hours at 4°. The starch blocks were prepared with 12.57= starch i n 30 mM T r i s borate b u f f e r , pH 8.5, 8.0 and 7.6, or i n 10 mM potassium phosphate b u f f e r , pH 7.0. The electrode compartments contained the same b u f f e r s , r e s p e c t i v e l y , at ten times the above concentrations. Protein.was detected with a 0.27. amido black s o l u t i o n i n methanol-water-acetic acid (5:5:1). - 18 -Polyacrylamide Disc Electrophoresis - Polyacrylamide disc e l e c t r o -phoresis was performed according to the method of Davis (1964) i n which the separation gel i s buffered at approximately pH 9.0. The enzyme was applied i n a large-pore sample gel or else layered on the prepared gels in,a: 20%, sucrose s o l u t i o n . Electrophoresis was performed at 20-22° with a current of 2 ma. per gel tube f o r 110 minutes. The protein was detected by s t a i n i n g with 1% amido black i n 7%, a c e t i c a c i d . The excess s t a i n was removed by electrophcteesis inlth&IMetaloglass rapid gel destaining apparatus. Detection of Aldehyde Dehydrogenase A c t i v i t y i n Gels - A modi-f i c a t i o n of the method of Fine and C o s t e l l o (1963) was used to detect aldehyde dehydrogenase a c t i v i t y i n starch or acrylamide gels. The gels were incubated f o r 3 minutes at 20-22° i n a reaction mixture containing: potassium phosphate, 100 mM; NAD+, 1 mM; and 2-mercaptoethanol, 5 mM. To i n i t i a t e the r e a c t i o n 0.1 ml. of 20 mM glycolaldehyde, 0.1 ml of 10 mg. per ml. p_-nitroblue tetrazolium and 0.02 ml. of 5 mg. per ml. phenazine methosulfate were added per 10 ml. reaction mixture. Formation of a dark band indicated a region of aldehyde dehydrogenase a c t i v i t y . V I I I . Sucrose Gradient Centrifugation Sucrose was d i s s o l v e d i n the desired buffer to a concentration of 5% and 20% w/v. The gradient, c o n s i s t i n g of 2.5 ml. 20%, sucrose s o l u t i o n and 2.5 ml. 5%, sucrose s o l u t i o n , was prepared i n n i t r o c e l l u l o s e centrifuge tubes using a Buchler gradient maker. The enzyme s o l u t i o n , 0.1 ml. or 0.3 ml., was layered onto the gradient and c e n t r i f u g a t i o n was c a r r i e d out f o r 13 hours at 5° i n the Beckman Model L-2 u l t r a c e n t r i f u g e with the SW 39L - 19 -swinging bucket rotor at 35,000 r.p.m. After c e n t r i f u g a t i o n approximately 0.2 ml. f r a c t i o n s were c o l l e c t e d , s t a r t i n g from the bottom of the tube. IX. Studies with the A n a l y t i c a l U l t r a c e n t r i f u g e A Beckman Model E a n a l y t i c a l u l t r a c e n t r i f u g e was used f o r a l l 2 experiments . Sedimentation - The observed sedimentation c o e f f i c i e n t , S ^ g , was obtained by c e n t r i f u g a t i o n at 59,780,r.p.mv^cand the sedimentation co-e f f i c i e n t , S20 ws w a s c a l c u l a t e d according to Schachman (1957). Protein solutions of approximately 1 mg. per ml. were used when the progress of sedimentation was viewed by u l t r a v i o l e t optics and approximately 5 mg. per ml. when the s c h l i e r e n optics were employed. Sedimentation c o e f f i c i e n t s of aldehyde dehydrogenase were obtained i n the following solutions: 100 mM potassium phosphate-10 mM or 1 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0; 1 mM EDTA, pH 7.2; and 5 mM KC1, pH 3.0. D i a l y s i s against the respective solutions for 16 to 24 hours preceeded each u l t r a c e n t r i f u g a l a n a l y s i s . D i f f u s i o n - The d i f f u s i o n constant f o r aldehyde dehydrogenase was obtained according to the method of Moller (1964). The enzyme was dissolved at a concentration of approximately. 1 mg. per ml. i n eit h e r 80 mM potassium phosphate-8 mM d i t h i o t h r e i t o l - 0 . 8 mM EDTA, pH 7.0, or 5 mM KC1, pH 3.0 and centrifuged at 59,780 r.p.m. f o r 40 minutes and 180 minutes r e s p e c t i v e l y . The speed was then reduced at 16,200 r.p.m. The progress of sedimentation at high speed and that of d i f f u s i o n at low speed was observed ^ Studies with the u l t r a c e n t r i f u g e were c a r r i e d out with the kind co-operation of Dr. C. M. Kay of the Department of Biochemistry, U n i v e r s i t y of Al b e r t a , and the t e c h n i c a l assistance of Mr. M. R. Aarbo. - 20 -with u l t r a v i o l e t o p t i c s . From the change of the slope of the boundary 1 — 9 9 ~r^Z r1 (1-sw^t) was c a l c u l a t e d . The d i f f u s i o n constant was obtained from the graph of t h i s value plotted against time, and corrected f o r temperature and v i s c o s i t y of the solvent to obtain the value T>2Q w-Approach to Sedimentation Equilibrium - To obtain a value for the molecular weight of aldehyde dehydrogenase, the method of Archibald, which i s described by Schachman (1957), was used. Aldehyde dehydrogenase, 5.4 mg. per ml. i n 100 mM potassium phosphate-1 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0, was centrifuged at 5227 r.p.m. at 20°. Pictures were taken at an angle of 65° at 32 minute i n t e r v a l s . Calculations were made from the pattern obtained at the meniscus. X. Determination of the I s o e l e c t r i c Point i Ten u l of a 4.8 mg. per ml..solution of aldehyde dehydrogenase i n 10 mM potassium phosphate, pH 7.0, were added to tubes containing 1 ml. of cold 100 mM potassium phosphate adjusted to the desired hydrogen ion concentrations with HC1. Tubes covering the pH range from pH 3.0 to pH 7.0 were prepared and incubated f or 9 hours at 5°. They were then centrifuged at 27,000 x g for 15 minutes. Protein determinations according to the method of Lowry et a l (1951) were performed on the p r e c i p i t a t e d protein. The i s o e l e c t r i c point was determined from a graph showing the protein p r e c i p i t a t e d at the d i f f e r e n t hydrogen ion concentrations. ci - 21 -XI. V i s c o s i t y Determinations V i s c o s i t y measurements were c a r r i e d out i n a c a p i l l a r y v i s -cometer tube immersed i n a constant temperature water bath at 5°. A volume of 1.75 ml. was required f o r these determinations. The temp-erature was c o n t r o l l e d by means of a Sargent Heater and C i r c u l a t o r for thermostatic baths and a Sargent:Thermonitor. Cooling was effected by c i r c u l a t i n g a 0° ethylene glycol-water mixture through the cooling c o i l . The i n t r i n s i c v i s c o s i t y , [nj , was calculated from the s p e c i f i c v i s c o s i t y , . r^gp, by taking the protein concentration into account since [n^] •= t| Sp/c, where c = grams of protein per cm . Values were not extrapolated to zero protein concentration. XII. Determination of Amino Acid Composition Acid Hydrolysis - Samples of aldehyde dehydrogenase were prepared f o r a c i d hydrolysis according to the method of Moore and Stein (1963). To a serie s of 18 mm. test tubes containing .1 ml. of aldehyde dehydrogenase (0.1 mg.) i n 1 mM potassium phosphate, pH 7.0, was added 1 ml. of r e d i s t i l l e d 12 N HC1. The samples were frozen and evacuated f o r 20 minutes. They were then sealed under vacuum and hydrolyzed at 110° fo r 12, 24, 48, or 72 hours. Duplicate samples were prepared f o r each hydrolysis time. Performic Acid Oxidation - Duplicate samples, each c o n s i s t i n g of one ml. of aldehyde dehydrogenase containing 0.1 mg. protein per ml.,, i n 1 mM potassium phosphate, pH 7.0, were taken to dryness by l y o p h i l i z a t i o n i n 18 - 22 -mm. test tubes. Two ml. of performic acid s o l u t i o n were added and allowed to react f o r 12 hours at 0°. Hydrogen bromide, 0.3 ml. of a 48% s o l u t i o n , was then added and the combined s o l u t i o n was taken to dryness by f l a s h evaporation at 45° (Moore, 1963). Samples were prepared i n duplicate and hydrolyzed i n 2 ml. of 6 N HCl'for 24 hours at 110°. Amino Acid Analysis - The hydrolyzed samples of aldehyde dehydrogenase and performic acid oxidized-aldehyde dehydrogenase were taken to dryness by f l a s h evaporation and dissolved i n 1 ml. of sodium c i t r a t e b u f f e r , pH 2.2. An a l i q u o t , u sually 200 u l . , of these solutions wasc subjected to amino acid a nalysis using the Beckman Automatic Amino Acid Analyzer Model 120B . The r e s u l t s were expressed as grams of amino acid per 100 grams of protein and as number of residues per 200,000 grams of protein. Estimation of Tyrosine and Tryptophan - The tyrosine and tryptophan content of aldehyde dehydrogenase was estimated by the method of Beaven and Holiday (1952). The absorbance of the enzyme i n 0.1 N NaOH was measured at 280 mu and 294.4 mu. The absorbance at 320 mu and 360 mu was also determined and extrapolated to 280 mu and 294.4 mu to correct for cloudiness of the so l u t i o n as proposed by these authors. 14 XIII. . Studies with C-Iodoacetamide Aldehyde dehydrogenase, 32.5 mg. i n 5 ml. of 100 mM potassium phosphate-1 mM d i t h i o t h r e i t o l , pH 7.0, was reacted with iodoacetamide-1-I am indebted to Dr. L. B. S m i l l i e of the Department of Biochemistry, U n i v e r s i t y of A l b e r t a , f o r the use of the amino acid analyzer and to Mr. E. Paradowski for performing the analyses. - 23 -•^C at 25°. Three successive additions of 0.05 ml. of 8.1 mM iodoacet-amide-l-^C (1.24 c./mole) were made to the enzyme s o l u t i o n at zero, 30, and 60 minutes. Aliquots of 10 u l each were removed and d i l u t e d for determination of enzymatic a c t i v i t y a f t e r 30, 90 and 120 minutes. After 120 minutes the protein was separated from the unreacted i n h i b i t o r by gel f i l t r a t i o n using a 2.1 by 23 cm. Bio-Gel P-60 column which was eq u i l i b r a t e d with 0.05 M NH4HCO3. Absorbance at 280 mu. and radioact-i v i t y of the effluents-fractions were determined. R a d i o a c t i v i t y was measured by adding samples to 5 ml. of Bray's s o l u t i o n (Bray, 1960) and counting the v i a l s i n a Nuclear Chicago L i q u i d S c i n t i l l a t i o n Counter Mark 1. The e f f i c i e n c y of counting was approximately 657,. Reaction of aldehyde dehydrogenase with iodoacetamide-l-^C was also c a r r i e d out i n the presence of 1 mM NAD+ under otherwise i d e n t i c a l conditions. XIV. Enzymatic Hydrolysis of Aldehyde Dehydrogenase Approximately 30 mg. of aldehyde dehydrogenase was suspended i n 5 ml. of d i s t i l l e d water and heated to 100° for 3 minutes. Ammonium bicarbonate was added to a f i n a l concentration of 50 mM. The denatured protein suspension was incubated at 37° f o r 20 hours with 2 mg. of chymotrypsin and a second addition of 1.3 mg. of <*• -chymotrypsin was made a f t e r 12 hours. The digest was l y o p h i l i z e d over c i t r i c a c i d . P a r t i a l l y p u r i f i e d i odoacetamide-l-^C-labelled peptide, approximately 0.15 umoles i n 0.05 M T r i s c h l o r i d e pH 8.5, was incubated i n the presence of 10 ug. of t r y p s i n at 37° for 2 hours. ( - 24 -XV. A n a l y t i c a l Methods Protein - Protein was determined by the method of Lowry et_ a l (1951). C r y s t a l l i n e bovine serum albumen was used as the standard. The protein concentration of p u r i f i e d aldehyde dehydrogenase also was determined by absorbance at 280 mu a f t e r the r e l a t i o n of absorbance at 280 mu to dry weight had been established. Dry Weight - Dry weights were determined on 1 ml. samples of approximately 3 mg. per ml. aldehyde dehydrogenase which had been dialysed against 1 mM potassium phosphate b u f f e r , pH 7.0, f o r 48 hours at 0°. Duplicate samples of protein s o l u t i o n and phosphate buffer were placed i n aluminum f o i l cups, 2 cm. diameter, and dried to constant weight at 80° and 90°. The weighings were c a r r i e d out with a Mettler Micro Gram-Atic Balance. Duplicate weighings of 2.8 mg. d i f f e r e d by less than 2%, those of 0.2 mg. by approximately 6%. The dry weight obtained for aldehyde dehydrogenase was r e l a t e d to the absorbance at 280 mu of the same s o l u t i o n . Nitrogen - Nitrogen was determined by the method of Minari.aarid Z i l v e r s m i t (1963). Protein samples (approximately 100 ug.) were digested by r e f l u x i n g f o r 90 minutes i n 16 mm. test tubes i n the presence of SeOC^ i n IL^SO^ and subjected to N e s s l e r i z a t i o n . Ammonium su l f a t e dried to constant weight was used as the standard. Estimation of Peptides with Ninhydrin - The photometric determination of peptides was made according to the method of Moore.and Stein (1954). Samples of 50 u l . to 200 u l . were reacted with 0.5 ml. of ninhydrin reagent at 100° for 15 minutes. Af t e r cooling of the reaction mixture, - 25 -0.5 ml. of 50% ethanol was added as the d i l u e n t . Absorbance was determined at 570 mu. Zinc - Aldehyde dehydrogenase was dialysed f o r 40 hours at 0° against 10 mM KC1 adjusted to pH 7.2 with T r i s . This enzyme so l u t i o n (4.8 mg. per ml.) was analyzed for Zn"*"1" using a Perkin Elmer 303 4 . Atomic Absorption Spectrophotometer . Standard s o l u t i o n s , containing 0.5 to 4.0 ug. per ml. zinc ywere prepared with ZnS04 using the same buffer s o l u t i o n as f o r the d i a l y s i s of the enzyme. A preparation of yeast alcohol dehydrogenase (5.0 mg. per ml.) was dialysed i n p a r a l l e l I | with the aldehyde dehydrogenase and analyzed f o r Zn content. XVI. Paper Chromatography Products of the Reaction of the Enzyme with Glyceraldehyde and Glycolaldehyde - The products of the rea c t i o n of aldehyde dehydrogenase with glyceraldehyde and glycolaldehyde were i d e n t i f i e d by comparison with known compounds using descending chromatography on Whatman No. 40 paper. The solvent systems butanol-acetic acid-water (12:3:5) and et h y l acetate-pyridine-water (12:5:4) were employed. The compounds were detected by spraying with bromcresol green reagent or dipping i n s i l v e r n i t r a t e reagent (Smith, 1960). The use of th i s equipment was possible through the kind permission of Dr. S. Pawluk, Department of S o i l Science, U n i v e r s i t y of Alberta. - 26 -EXPERIMENTAL.RE SULTS I. Induction of Aldehyde Dehydrogenase The s p e c i f i c a c t i v i t y of aldehyde dehydrogenase i n c e l l extracts prepared from P. aeruginosa grown on f i v e d i f f e r e n t substrates i s shown i n Table I. When ethanol was used as the carbon source, the s p e c i f i c a c t i v i t y of the enzyme was s i x t y - f i v e times greater than when glucose was used. Ethanol was selected, therefore, as the substrate f o r growth of t h i s organism to obtain a high y i e l d of aldehyde dehydrogenase f or p u r i f i c a t i o n . The organism was r o u t i n e l y grown i n s t i l l c ulture i n a medium containing s a l t s , ethanol, yeast e x t r a c t , and tryptone. A high s p e c i f i c a c t i v i t y of aldehyde dehydrogenase also was obtained i n extracts of c e l l s grown i n the absence of yeast extract and tryptone, however the t o t a l y i e l d of c e l l s was reduced s i g n i f i c a n t l y . Employ-ment of a r o t a r y shaker, compared to growth i n s t i l l c u l t u r e , reduced the enzyme y i e l d by approximately f i f t y per cent. Growth of the organism i n bulk culture with forced aeration necessitated the use of extremely large amounts of anti-foaming.agent. P. aeruginosa grown i n bulk culture with forced aeration i n an inorganic salts-glucose medium could be adapted to growth on ethanol a f t e r the depletion of glucose, but the s p e c i f i c a c t i v i t y of the enzyme increased only te n - f o l d i n a two hour period. Therefore growth with ethanol i n s t i l l c u lture was the preferred method for production of enzyme. - 27 -TABLE I ALDEHYDE DEHYDROGENASE ACTIVITY IN CELL EXTRACTS OF P. AERUGINOSA GROWN ON DIFFERENT SUBSTRATES Carbon Source S p e c i f i c A c t i v i t y umoles/min./mg. protein Glucose 0.012 Acetate 0.051 Gl y c e r o l 0.071 Ethylene g l y c o l 0.364 Ethanol 0.780 The organism was grown f o r 48 hours at 25° i n media containing the respective carbon sources as described i n "Materials and Methods". Inoculum from a culture grown on the same carbon source was used. The c e l l s were harvested, washed once i n 10 mM potassium phosphate containing 10 mM 2-mercaptoethanol, pH 7.0, and suspended i n the same buffer. C e l l extracts were prepared by sonication and c e n t r i f u g a t i o n at 31,700 x g for 45 minutes. - 28 -The induction of aldehyde dehyrogenase i n ethanol-grown c e l l s indicates that the enzyme has an important r o l e i n the metabolism of this substrate. Anthony and Zatman (1965) have reported an alcohol dehydrogenase i n methanol-grown c e l l s of Pseudomonas sp. M27, using phenazine methosulfate as the primary hydrogen acceptor. Both t h i s enzyme and an NAD + -dependent alcohol dehydrogenase were found i n c e l l extracts of P. aeruginosa grown on ethanol. Therefore during growth on th i s substrate acetaldehyde can be considered to be the natural substrate f o r aldehyde dehydrogenase. However, since acetaldehyde shows considerable substrate i n h i b i t i o n even at very low concentrations, as w i l l be reported, glycolaldehyde was used as the substrate i n the routine assay of the enzyme. I I . P u r i f i c a t i o n of Aldehyde Dehydrogenase A l l operations were performed at 0-3° unless otherwise stated. Step 1. Preparation of C e l l Extract - C e l l s from s i x l i t e r s of culture medium were harvested by ce n t r i f u g a t i o n - at 13,200 x g, washed once with 50 mM potassium phosphate-10 mM mercaptoethanol, pH 7.0, and suspended i n 336 ml. of 100 mM sodium b i s u l f i t e - 1 0 mM mercaptoethanol, pH 7.0. The c e l l s were disrupted by sonicating 50 ml. quant i t i e s of the suspension for 7 minutes, using a Bronwill Biosonik at maximum output. The temperature of the suspension during this operation was kept below 10°. The sonic extract was d i l u t e d to 560 ml. and the f i n a l buffer concentration was adjusted to 50 mM potassium phosphate-100 mM sodium b i s u l f i t e - 1 0 mM mercaptoethanol, pH 7.0. After - 29 -centrifugation at 31,700 x g for 60 minutes, 527 ml. of c e l l extract were obtained. Step 2. Protamine Sulfate Treatment - Twenty per cent by volume of protamine sulfate solution (20 mg. per ml., pH 5.0) was added with stir r i n g to 527 ml. of c e l l extract. Stirring was continued for 20 minutes and the precipitate was seddmented by centrif-ugation at 31,700 x g for 30 minutes. The volume of the supernatant solution was 608 ml. Step 3. Ammonium Sulfate Fractionation - Solid ammonium sulfate, 127 g.was added, with s t i r r i n g , to 608 ml. of the solution obtained in step 2. Stirring was continued for an additional 30 minutes and the precipitate was removed by centrifugation at 31,700 :;x g for 30 minutes. The volume of the supernatant solution was 655 ml. To this a further 61.5 g. of ammonium sulfate were added as above. After centrifugation.the precipitate, which contained the enzyme activity, was dissolved in 5 mM potassium phosphate-10 mM sodium bisulfite-10 mM mercaptoethanol-0.5% ethylene glycol, pH 7.0, to give 100 ml. total volume. Step 4. Acetone Fractionation - This operation was performed in a salt-ice bath at -15°. During the addition of acetone the temp-erature of the preparation was kept below 3°. Sixty ml. of acetone, chilled to -15°, were added, with s t i r r i n g , to 100 ml. of the ammonium sulfate fraction. The precipitate was removed by centrifugation at 23,300 x g for 10 minutes at -5° and set aside (PI). This procedure was repeated with the addition of 25 ml. (PII) and then 40 ml. of cold acetone (PHI) . Precipitates I, II, and III, thus obtained, were each - 30 -suspended i n 100 mM potassium phosphate-100 mM sodium b i s u l f i t e - 1 0 mM mercaptoethanol-1.07, ethylene g l y c o l , pH 7.0, to a f i n a l volume of 100 ml. F r a c t i o n III alone u s u a l l y contained 7370 of the enzyme a c t i v i t y . To t h i s f r a c t i o n 43 g. of s o l i d ammonium su l f a t e were added and, a f t e r being s t i r r e d f o r 2 hours, the mixture was centrifuged at 27,000 x g for 20 minutes. The p r e c i p i t a t e was recovered, suspended i n 50 ml. of 100 mM potassium phosphate-10 mM mercaptoethanol-5 mM EDTA, pH 7.0, and the suspension was centrifuged as before to remove any denatured protein. The volume was then adjusted to 78 ml. with the same bu f f e r . Step 5. I s o e l e c t r i c F r a c t i o n a t i o n - To 78 ml. of the acetone f r a c t i o n 12.9 g. of ammonium sul f a t e were added. The s l i g h t amount of p r e c i p i t a t e which formed a f t e r 30 minutes s t i r r i n g was removed by c e n t r i f u g a t i o n at 27,000 x g f o r 10 minutes. Molar a c e t i c a c i d (containing:16.5 g. ammonium su l f a t e per 100 ml.) was used to lower the pH of the preparation. The pH was brought from 6.5. to 5.4 by the ad d i t i o n of 5.1 ml. of the acid mixture with s t i r r i n g . A f t e r c e n t r i -fugation at 27,000 x g for 10 minutes, 5.2 ml. were added to adjust the pH to 4.8. The f r a c t i o n p r e c i p i t a t i n g between pH 5.4 and pH 4.8 was c o l l e c t e d , a f t e r a further 10 minutes of s t i r r i n g , by c e n t r i f u g a t i o n as above. I t was dissolved with 100 mM potassium phosphate-10 mM mercaptoethanol-5 mMEDTA-1.07, ethylene g l y c o l , pH 7.0, to y i e l d 15 ml. of s o l u t i o n . Step 6. Ion Exchange Chromatography - This step was performed at room temperature (20-22°). Cellex-T ( t r i e t h y l a m i n o e t h y l - c e l l u l o s e ) i n the ch l o r i d e form was packed i n a 2.1 cm. diameter column to a height - 31 -of 49 cm., converted to the phosphate form with 0.1 M potassium phos-phate, pH 7.0, and e q u i l i b r a t e d with the s t a r t i n g buffer (5 mM potassium phosphate-10 mM mercaptoethanol-1 mM EDTA-1.07> ethylene g l y c o l , pH 7.0). The enzyme preparation obtained i n step 5 was d i a l -yzed against 500 ml. of the s t a r t i n g buffer f or 4 hours, with one change of buffer at 2 hours, and applied to the column. Twenty ml. of s t a r t i n g buffer were added to wash the enzyme into the column. A l i n e a r gradient was used to elute the enzyme. This consisted of 450 ml. s t a r t i n g buffer and 450 ml. of 300 mM potassium phosphate-10 mM mercaptoethanol-1 mM.EDTA-1.0% ethylene g l y c o l , pH 7.0. Fractions of 8.9 ml. were c o l l e c t e d at a flow rate of 3 ml. per minute. The protein concentration and aldehyde dehydrogenase a c t i v i t y of the f r a c t i o n s were determined, y i e l d i n g the e l u t i o n curve shown i n Figure 1. Fractions 50 to 63 were pooled (volume- 124 ml.) and to th i s was added, with s t i r r i n g , 6.2 ml. of 1.0 M potassium phosphate, pH 7.0, and 56 g. of ammonium s u l f a t e . A f t e r the suspension had been kept at 4° for 15 hours the p r e c i p i t a t e d protein 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 , d i s s o l v e d and made to a volume of 4.0 ml. using 50 mM potassium phosphate-10 mM mercaptoethanol-1 mM EDTA, pH 7.0. Step 7. Gel F i l t r a t i o n - This step was performed at room temperature. A column,.2.5 cm. diameter was packed with Sephadex G-200 i n 50 mM potassium phosphate-10 mM mercaptoethanol-1 mM EDTA, pH 7.0, to a height of 38 cm. and washed with two volumes of the same buffer. The enzyme preparation obtained i n step 6 was applied to the column and eluted with the above buffer at a flow rate of 0.6 ml. per minute. - 32 -FIGURE 1 CHROMATOGRAPHY OF ALDEHYDE DEHYDROGENASE ON CELLEX-T Protein (315 mg.) was applied to a column 2.1 x 49 cm. and eluted with a l i n e a r gradient of phosphate as described i n the text. The enzyme a c t i v i t y was determined using the standard assay procedure except that the assay was c a r r i e d out at 20-22°. The re s u l t s are expressed as units per 1.0 ml. of the f r a c t i o n . Absorbance at 280 mu Enzyme a c t i v i t y - 33 -Fractions of 2.4 ml. were c o l l e c t e d a f t e r 23 ml. of the eluent had passed through the column. Protein concentration and aldehyde dehydrogenase a c t i v i t y were determined, y i e l d i n g the r e s u l t s shown i n Figure 2. Fractions 38 to 56 (44 ml.) were pooled and to t h i s was added, with s t i r r i n g , 4.4 ml. of 1.0 M potassium phosphate, pH 7.0, and 21 g. of ammonium s u l f a t e . A f t e r being kept at 4° for 15 hours the suspension was centrifuged and the p r e c i p i t a t e was dissolved, made to 4.0 ml. with 100 mM potassium phosphate-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0, and stored at -22°. A summary of the p u r i f i c a t i o n i s shown i n Table I I . This procedure has been c a r r i e d out many times with s i m i l a r r e s u l t s and almost i d e n t i c a l f i n a l s p e c i f i c a c t i v i t i e s and y i e l d s . A d d i t i o n a l steps, performed on a p u r i f i e d preparation, such as e l u t i o n from calcium phosphate g e l , ammonium su l f a t e p r e c i p i t a t i o n , ion exchange chromatography at pH 6.0 or pH 8.0, and p r e c i p i t a t i o n of the enzyme by d i a l y s i s at low i o n i c strength did not r e s u l t i n any increase of s p e c i f i c a c t i v i t y . The c e l l extract of P. aeruginosa has a strong reddish-brown c o l o r . This i s gradually removed during the p u r i f i c a t i o n . The l a s t traces of c o l o r , s t i l l observed a f t e r the ion exchange chromatography, are removed i n the f i n a l step. A s o l u t i o n of 20 mg. per ml. protein of the f i n a l product i s c o l o r l e s s . T f r T U B E NO. -34 -FIGURE 2 . GEL FILTRATION OF ALDEHYDE DEHYDROGENASE ON SEPHADEX G-200 Protein (116 mg.) i n 4.0 ml. volume was applied to a column of Sephadex G-200 (2.5 x 38 cm.) and eluted i n 50 mM potassium phosphate-10 mM 2-mercaptoethanol-l mM EDTA, pH 7.0. The enzyme a c t i v i t y was determined as described i n "Materials and Methods" using 10 u l . of a one to ten d i l u t i o n of each f r a c t i o n . The r e s u l t s are expressed as units per 1.0 ml. of the f r a c t i o n . Absorbance at 280 mu Enzyme a c t i v i t y - 35 -TABLE II SUMMARY OF THE PURIFICATION OF ALDEHYDE DEHYDROGENASE FROM P. AERUGINOSA Tota l T o t a l S p e c i f i c Step Volume Recovery protein a c t i v i t y a c t i v i t y ml. mg. units units/mg. protein 7o 1. C e l l extract 527 8450 5500 0.65 100 2. Protamine s u l f a t e 608 6400 5380 0.84 98 3. Ammonium s u l f a t e 100 2300 4430 :.i;9 81 4. Acetone p r e c i p i t a t i o n 78 675 3520 5.2 64 5. I s o e l e c t r i c p r e c i p i t a t i o n 15 315 2280 7.3 42 6. Cellex-T column 4 116 1690 14.5 30 7. Sephadex G-200 4 86 1250 14.7 23 - 36 -I I I . Purity of Aldehyde Dehydrogenase A high degree of pu r i t y of aldehyde dehydrogenase was suggested by the elutions curves from the ion exchange cellulose (Figure 1) and from Sephadex G-200 (Figure 2). A s i m i l a r correspondence of absorbance at 280 mu and 'enzyme a c t i v i t y was found i n a 5% to 20% sucrose gradient a f t e r c e n t r i f u g a t i o n at 35,000 r.p.m. for 13 hours. Electrophoresis i n Starch Gel - In starch gel electrophoresis the p u r i f i e d protein migrates toward the anode as one major band at pH 6.8, 7.4, and 8.5. The electrophoresis of two preparations of d i f f e r e n t purity, i s shown i n Figure 3. Staining of the gels f o r enzyme a c t i v i t y demonstrated that aldehyde dehydrogenase i s associated with the major protein band under these conditions. The f a i n t band with f a s t e r e l e c t r o p h o r e t i c m o b i l i t y , which can be observed i n the gel electrophoresis of the p u r i f i e d preparation, i s believed to be the dis s o c i a t e d form of the enzyme. Exposure of the protein to low s a l t concentrations (1.0 mM) for prolonged periods of time p r i o r to e l e c t r o -phoresis r e s u l t s i n an increase i n the protein concentration of the f a i n t , faster-moving band. This can be reversed by addition of s a l t s and indicates a d i s s o c i a t i o n - a s s o c i a t i o n of the protein. This property of aldehyde dehydrogenase i s described i n the section on D i s s o c i a t i o n and Deactivation, page 84. Polyacrylamide Disc Electrophoresis - Figure 4 shows the r e s u l t s of polyacrylamide d i s c electrophoresis of three preparations at d i f f e r e n t stages of the p u r i f i c a t i o n procedure. As i n starch g e l electrophoresis, the p u r i f i e d enzyme shows only one major protein band. Enzyme a c t i v i t y - 37 -FIGURE 3 STARCH GEL ELECTROPHORESIS OF ALDEHYDE DEHYDROGENASE AT TWO STAGES OF THE PURIFICATION The gel i n the center shows a sample a f t e r acetone p r e c i p i t a t i o n (step 4) and the gels on l e f t and r i g h t show the enzyme s o l u t i o n a f t e r gel f i l t r a t i o n (step 7). Approximately 100 ug. and 50 ug. of protein were applied to the gel s l i c e s r e s p e c t i v e l y , and electrophoresis was performed with T r i s borate b u f f e r , pH 8.5, as described i n "Materials and Methods". The anode i s at the bottom of the p i c t u r e . The dif f e r e n c e i n m o b i l i t y of the enzyme r e s u l t s merely from v a r i a t i o n s i n the gel bed, and not from any change i n the protein. - 38 -FIGURE 4 POLYACRYLAMIDE DISC ELECTROPHORESIS OF ALDEHYDE DEHYDROGENASE AT DIFFERENT STAGES OF THE PURIFICATION The gel at the l e f t shows c e l l extract (step 1, 240 ug. p r o t e i n ) , the gel i n the center the acetone p r e c i p i t a t e (step 4, 70 ug. p r o t e i n ) , and the gel.at the r i g h t the enzyme s o l u t i o n a f t e r gel f i l t r a t i o n (step 7, 40 ug. p r o t e i n ) . Electrophoresis was c a r r i e d out as described under "Materials and Methods". The anode i s at the bottom of the p i c t u r e . - 39 -could be demonstrated i n t h i s band only i f the enzyme was applied to the prepared g e l immediately p r i o r to the electrophoresis, which was c a r r i e d out for approximately two hours. Incorporation of the protein into the sample gel during the a d d i t i o n a l 90 minutes required for gel preparation r e s u l t e d i n loss of enzyme a c t i v i t y . However, the e l e c t r o p h o r e t i c pattern of the protein was not changed s i g n i f i c a n t l y by t h i s alternate method of a p p l i c a t i o n . To estimate the possible amount of contaminating protein i n the p u r i f i e d enzyme, 2, 4, 8, 20, 40, and 80 ug. of enzyme protein were subjected to electrophoresis. These r e s u l t s are shown i n Figure 5. In the two gels containing 40 ug. and 80 ug. of protein d i f f u s e bands with a f a s t e r electrophoretic m o b i l i t y than the major band were observed. These were estimated to represent less than 2.5% of the t o t a l protein applied. Due to the d i s s o c i a t i o n phenomenon of the enzyme, the f a i n t f a s t bands may not n e c e s s a r i l y be a protein contaminant but could represent, at least i n part, d i s s o c i a t e d aldehyde dehydrogenase. Aldehyde Dehydrogenase i n the A n a l y t i c a l Ultracentrifuge - The s c h l i e r e n patterns of a sample of p u r i f i e d aldehyde dehydrogenase at a concentration of 3.7 mg. per ml. at three d i f f e r e n t time i n t e r v a l s are shown i n Figure 6. A high degree of homogeneity i s indicated. The r e l a t i o n s h i p of the logarithm of the distance sedimented to time i s shown i n Figure 7. The. l i n e a r r e l a t i o n s h i p indicates that the sedimentation constant did not change during the course of the experiment. A sedimentation constant, w » °^ 9.4 S was c a l c u l a t e d from the data. ' It -—' _ - 40 -FIGURE 5 POLYACRYLAMIDE DISC ELECTROPHORESIS OF DIFFERENT QUANTITIES OF PURIFIED ALDEHYDE DEHYDROGENASE The gels from l e f t to r i g h t contained the following quantities of p u r i f i e d enzyme: 2, 4, 8, 20, 40, and 80 ug. of protein. Electrophoresis was c a r r i e d out as described under "Materials and Methods". The anode i s at the bottom of the p i c t u r e . - 41 -FIGURE 6 SEDIMENTATION OF ALDEHYDE DEHYDROGENASE AT pH 7.0 Aldehyde dehydrogenase (3.7 mg. per ml.) i n 100 mM.pot-assium phosphate-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7 .0 , was sedimented at 59,780 r.p.m. i n the u l t r a c e n t r i f u g e at 2 0 ° . The sedimentation proceeds from r i g h t to l e f t . Pictures were taken at 8, 24,. and 40 minutes a f t e r a t t a i n i n g f u l l speed. - 42 -FIGURE 7 DETERMINATION OF SEDIMENTATION COEFFICIENT OF ALDEHYDE DEHYDROGENASE The log of the distance sedimented (x) i s plotted versus time. The conditions of c e n t r i f u g a t i o n are as stated i n the legend to Figure 6. c - 43 -IV. Physical Properties of Aldehyde Dehydrogenase S o l u b i l i t y - Aldehyde dehydrogenase i s soluble at moderately high s a l t concentration above i t s i s o e l e c t r i c point. It was found to be insoluble above 35% saturation with ammonium su l f a t e and i n very d i l u t e s a l t s o l u t i o n s , as for example 0.05 mM EDTA. Almost complete recovery of enzyme a c t i v i t y was possible a f t e r p r e c i p i t a t i o n at high s a l t concentration. However, only 25% of the a c t i v i t y was recovered a f t e r p r e c i p i t a t i o n by d i a l y s i s against a s o l u t i o n of low s a l t concentration. I s o e l e c t r i c Point - As evidenced by the p r e c i p i t a t i o n at low hydrogen ion concentrations, absorption to triethylaminoethyl-c e l l u l o s e , and movement towards the anode i n electrophoresis, aldehyde dehydrogenase i s negatively charged at pH 7.0 or above. The i s o e l e c t r i c point of the enzyme was determined by measuring, q u a n t i t a t i v e l y , the amount of protein p r e c i p i t a t e d i n 0.1-M potassium phosphate adjusted to the desired pH values with hydrochloric a c i d . The r e s u l t s are shown i n .Figure 8. The i s o e l e c t r i c point of the enzyme was found to be pH 4.8. Although a s o l u t i o n of aldehyde dehydrogenase i s obtained below the i s o e l e c t r i c pH,.attempts to neu t r a l i z e an a c i d i f i e d sample led to p r e c i p i t a t i o n of the protein with loss of a l l enzymatic a c t i v i t y . It appears that the enzyme i s denatured i n the course of a c i d i f i c a t i o n , and the denatured form i s insoluble at neutral pH. Molecular Weight of Aldehyde Dehydrogenase - The molecular weight of aldehyde dehydrogenase was compared with that of catalase T - 44 -FIGURE 8 ISOELECTRIC.POINT OF ALDEHYDE DEHYDROGENASE The i s o e l e c t r i c point was determined as described i n "Materials and Methods". The Figure shows the protein p r e c i p i t a t e d at the d i f f e r e n t hydrogen ion concentrations. - 45 -from horse blood, which has. a molecular weight of 225,000 to 250,000 (Nicholls and Schonbaum, 1963), and yeast alcohol dehydrogenase, which has a molecular weight of 151,000 (Kagi and V a l l e e , 1960), by gel f i l t r a t i o n with Sephadex G-200 and by u l t r a c e n t r i f u g a t i o n i n a sucrose density gradient. The molecular weight was also c a l c u l a t e d from sedimentation and d i f f u s i o n c o e f f i c i e n t s and by the method of Archibald (Schachman, 1957). The r e s u l t s obtained from gel f i l t r a t i o n are shown i n Figure 9. A l l three enzymes were eluted from the column a f t e r the void volume, with e l u t i o n volumes for aldehyde dehydrogenase, catalase, and yeast alcohol dehydrogenase being 93 ml., 94 ml., and 103 ml. r e s p e c t i v e l y . The indicated molecular si z e f o r aldehyde dehydrogenase i s , therefore, s i m i l a r to that of catalase (225,000 to 250,000). The r e s u l t s obtained a f t e r c e n t r i f u g a t i o n of aldehyde dehydrogenase and the two reference enzymes i n a 5% to 207» sucrose gradient are presented i n Figure 10. Aldehyde dehydrogenase was located i n the gradient between catalase and yeast alcohol dehydro-genase. The density of aldehyde dehydrogenase i s therefore between the two reference enzymes which suggests that i t has a molecular weight of approximately 200,000. The sedimentation c o e f f i c i e n t , S20 w> f ° r aldehyde dehydrogenase was determined to be 9.4 S (Figure 6 and Figure 7). Under s i m i l a r conditions values of 9.0 and 9.2 were also obtained. The d i f f u s i o n c o e f f i c i e n t was determined by the method of Moller (1964), i n which the d i f f u s i o n of the boundary of a protein i n a c e n t r i f u g a l f i e l d over a 1 i - 46 -FIGURE 9 GEL FILTRATION OF ALDEHYDE DEHYDROGENASE AND REFERENCE ENZYMES A 1 ml. s o l u t i o n of aldehyde dehydrogenase, catalase (M.W. 225,000 to 250,000) and yeast alcohol dehydrogenase (M.W. 151,000) was.-applied to a column of Sephadex G-200, 2.5 by 37.5 cm., e q u i l i b r a t e d with 100 mM.potassium phosphate-1 mM 2-mercaptoethanol, pH 7.0. Fractions of approximately 1.9 ml. were c o l l e c t e d at a flow rate of 0.3 ml. per minute. Enzyme assays were c a r r i e d out as out l i n e d under "Materials and Methods". Aldehyde dehydrogenase Catalase Yeast alcohol dehydrogenase - 47 -FIGURE 10 SUCROSE GRADIENT CENTRIFUGATION OF ALDEHYDE DEHYDROGENASE AND REFERENCE ENZYMES A 0.1 ml. s o l u t i o n of aldehyde dehydrogenase, catalase (M.W. 225,000 to 250,000) and yeast alcohol dehydrogenase (M.W:. 151,000) was applied to a 5% to 207„ sucrose gradient i n 100 mM potassium phosphate-1 mM 2-mercaptoethanol, pH 7.0. Fractions were c o l l e c t e d a f t e r c e n t r i f u g a t i o n at 35,000 r.p.m. for 13 hours at 5°. Enzymatic a c t i v i t y was determined as described under "Materials and Methods". 0 - - - - 0 Aldehyde dehydrogenase • • Catalase A A Yeast alcohol dehydrogenase - 48 -period of time i s measured. The values of 1 -2 ( i - s w 2 t ) were 4y2 ^ cal c u l a t e d from these measurements and Figure 11 shows t h e i r r e l a t i o n -7 2 to time. A value of the d i f f u s i o n c o e f f i c i e n t , D£Q w = 4.4 x 10 cm. s e c , was obtained from the slope of t h i s l i n e a f t e r appropriate cor-rections f o r temperature and buffer concentration. In a l l c a l c u l a t i o n s the p a r t i a l s p e c i f i c volume (V) was assumed to be 0.73 cm.^ per gram (Smith, 1963). From the s 2 n j W = 9.4 S and D 2 U ) W = 4.4 x 10" 7 cm.2 sec. a molecular weight of 191,000 was calculated using the Svedberg equation. The use of S20 w = 9.0 S y i e l d s a molecular weight of 183,000 and the use of S20 w = 9.2 S y i e l d s a molecular weight of 187,000. Molecular weight determinations c a r r i e d out by the method of Archibald (Schachman, 1957) indicated values of 168,000 and 207,000. This method i s based on the p r i n c i p l e of equilibrium at the meniscus of a protein s o l u t i o n i n the u l t r a c e n t r i f u g e . a t r e l a t i v e l y low speeds. Figure 12 shows the. pattern which was obtained at the meniscus at 5227 r.p.m. Although complete agreement was not obtained between the r e s u l t s of d i f f e r e n t methods of molecular weight determination, a weight of 200,000 grams per mole f o r aldehyde dehydrogenase i s probably very close to the actual weight and was used for further c a l c u l a t i o n s . Physical Properties of Aldehyde Dehydrogenase below the  I s o e l e c t r i c Point - Whereas the active molecule at pH 7.0 has an S value of 9.0 to 9.4, aldehyde dehydrogenase i n 5.0 mMKGl, pH 3.0, shows a homogeneous peak i n the u l t r a c e n t r i f u g e with an S20 w of 2.0 S I 2 3 S E C O N D S x i d " 4 - 49 -FIGURE 11 DETERMINATION OF THE DIFFUSION COEFFICIENT FOR ALDEHYDE DEHYDROGENASE Aldehyde dehydrogenase (1.2 mg. per ml.) i n 80 mM potassium phosphate-8 mM d i t h i o t h r e i t o l - 0 . 8 mM EDTA, pH 7.0, was centrifuged at 59,780 r.p.m. for 40 minutes and then at 16,200 r.p.m. for 512 minutes. JJ2(1 -sw^t) was ca l c u l a t e d from the slope of the protein 4y^ boundary and plo t t e d against time. The d i f f u s i o n c o e f f i c i e n t was obtained from the slope of t h i s l i n e . - 50 -FIGURE 12 SCHLIEREN' PATTERN OF ALDEHYDE DEHYDROGENASE AT 5227 R.P.M. Aldehyde dehydrogenase (5.4 mg. per ml.) i n 100 mM potassium phosphate-1 mM d i , t h i o t h r e i t o l - l mM EDTA, pH 7.0, was centrifuged at 5227 r.p.m. i n the a n a l y t i c a l u l t r a c e n t r i f u g e . Two s c h l i e r e n patterns are shown from which molecular weights of 168,000 ( l e f t ) and 207,000 (right) were ca l c u l a t e d . - 51 -(Figure 13). Gel f i l t r a t i o n through Sephadex G-200 i n 5.0 mM KC1, pH 3.0, d i d not indicate that the enzyme has a smaller molecular size than the active enzyme at pH 7.0, since a l l the protein applied to the column emerged i n the e f f l u e n t at the point where the active enzyme would be expected. However, c a l c u l a t i o n of the molecular weight from the sedimentation c o e f f i c i e n t and the d i f f u s i o n co-e f f i c i e n t , which was estimated by the method of Moller (1964), indicated a molecular weight of approximately, half that obtained f o r aldehyde dehydrogenase at pH 7.0. V i s c o s i t y of the enzyme was determined i n a c a p i l l a r y viscometer tube at 5°. The enzyme was dissolved i n three d i f f e r e n t solvent systems at a concentration of approximately 5 mg. per ml. protein and 1.75 ml. were used f o r each determination. Each measurement was done i n dup-l i c a t e . Aldehyde dehydrogenase i n 0.1 M potassium phosphate-1.0 mM d i t h i o t h r e i t o l - 1 . 0 mM EDTA, pH 7.0, had an i n t r i n s i c v i s c o s i t y ([*}] ) of 3.6; i n 1.0 mM EDTA, pH 7.2,[r|]= 5.7; and i n 5.0 mM.KCl, pH 3.0, = 17.6. This large increase i n v i s c o s i t y when the enzyme was dissolved at pH 3.0 indicates an unfolding of the molecule, which would also explain the low sedimentation c o e f f i c i e n t . Absorbance - Aldehyde dehydrogenase i n 50 mM potassium phosphate pH 7.0, has an absorbance maximum at 277 mu and shoulders at 282 mu and 289 mu. From dry weight measurements and determination of the absorbance at 280 mu,.it was c a l c u l a t e d that a p u r i f i e d enzyme so l u t i o n at 1.0 mg. per ml. concentration i n 1.0 mM potassium phosphate, pH 7.0, has an.absorbancy of 1.04 at 280 mu. The r a t i o of absorbance at 280 mu and 260 mu was 1.83. - 52 -FIGURE 13 SEDIMENTATION OF ALDEHYDE DEHYDROGENASE AT pH 3.0 Aldehyde dehydrogenase (5.0,mg. per ml.) i n 5 mM KCl,.pH 3 .0 , was sedimented at 59,780 r.p.m. i n the u l t r a c e n t r i f u g e at 6 ° . The sedimentation proceeds from r i g h t to l e f t . Pictures were taken at 16, 48, and 80 minutes a f t e r a t t a i n i n g f u l l speed. - 53 -Amino Acid Composition of Aldehyde Dehydrogenase - The amino acid analyses were performed with the Beckman automatic amino acid analyser. Samples of p u r i f i e d aldehyde dehydrogenase, 0.1 mg., were hydrolysed under vacuum at 110° i n 6 N HC1, according to the method of Moore and Stein (1963). Samples were prepared i n duplicate and hydrolysis was c a r r i e d out f o r 12, 24, 48, and 72 hours. The r e s u l t s are shown i n Table I I I . The values shown are the mean values c a l c -ulated from the r e s u l t s obtained a f t e r d i f f e r e n t hydrolysis times with the following exceptions: the values f o r v a l i n e and isoleucine are mean values obtained a f t e r 72 hours h y d r o l y s i s , and the values for serine and threonine were obtained by extrapolation to zero hours hy d r o l y s i s . The r e l a t i o n s h i p of time of hydrolysis to the values obtained f o r these four amino acids is. shown i n Figure 14. Cysteic acid and methionine sulfone were determined separately a f t e r performic a c i d oxidation (Moore, 1963) of a sample of aldehyde dehydrogenase and 24 hours h y d r o l y s i s . T o t a l nitrogen content of aldehyde dehydrogenase was determined a f t e r d i g e s t i o n of a protein sample i n s u l f u r i c acid i n the presence of selenium oxychloride as the c a t a l y s t and N e s s l e r i z a t i o n according to the method of Minari and Zi l v e r s m i t (1963). Aldehyde dehydrogenase was found to contain 16.07o nitrogen. Tryptophan, which i s completely destroyed upon acid hydrolysis was estimated by the spectrophotometry.method of Beaven and Holiday (1952). From the absorbance of aldehyde dehydrogenase i n 0.1 M sodium hydroxide at 280 mu and 294.4 mu, i t was calculated that there were 33 moles of tryptophan and 110 moles of tryosine per mole of aldehyde - 54 -TABLE III AMINO ACID COMPOSITION OF ALDEHYDE DEHYDROGENASE Amino acid Moles/lOO g. Moles/mole Grams/lOO g. Lysine .0397 H i s t i d i n e .0181 Arginine .0343 Aspartic + ,asparagine .0727 Threonine .044 Serine .037 Glutamic + glutamine .0949 Proline .0382 Glycine .0818 Alanine .1029 Valine .055 Isoleucine .0535 Leucine .0710 Tyrosine .0300 Phenylalanine .0335 Cysteic acid .0118 Methionine .0151 Tryptophan 79.4 36.2 68.6 145.3 88.0 74.0 189.8 76.4 163.6 205.8 110.0 107.0 142.0 60.0 67.0 23.6 30.1 (33) 5.80 2.81 5.97 9.68 5.24 3.88 13.94 4.39 6.14 9.17 6.44 7.02 9.33 5.43 5.54 1.43 2.25 (3.16) t o t a l 107.61 g. - 55 -FIGURE 14 EFFECT OF HYDROLYSIS TIME ON AMINO ACID COMPOSITION Aldehyde dehydrogenase was hydrolysed and analysed for amino acid content as o u t l i n e d under "Materials and Methods". Values obtained for amino acids which vary with the hydrolysis time were pl o t t e d . Values for threonine and serine were obtained by extra-p o l a t i o n to zero hydrolysis time. The values obtained at 72 hours were used f o r v a l i n e and i s o l e u c i n e . - 56 -dehydrogenase. The value obtained f o r tyrosine i s not i n agreement with the value obtained by the automatic amino acid analysis and, therefore, the value f o r tryptophan can only be accepted as an approximation. Table III also shows the number of amino acid residues per mole of enzyme. Each amino acid i s present i n r e l a t i v e l y large quantities and the composition does not lend i t s e l f to speculation or c a l c u l a t i o n of the molecular weight or number of subunits. V. Requirements for Enzyme A c t i v i t y Assay of Maximum A c t i v i t y - Aldehyde dehydrogenase was assayed with a reaction mixture containing b u f f e r , reducing agent, NAD+, K +, enzyme and aldehyde as outlined i n "Materials and Methods". The aldehyde substrate was added l a s t to the otherwise complete re a c t i o n mixture a f t e r 5 minutes incubation at 35°. This resulted i n maximum a c t i v i t y . Figure 15 shows the r e s u l t s obtained when other components of the r e a c t i o n mixture were added to i n i t i a t e the r e a c t i o n . The re a c t i o n rate was markedly reduced i n a l l cases where the enzyme was not preincubated, at least b r i e f l y , with K , NAD , and reducing agent before the aldehyde was introduced. The e f f e c t was greatest when K + and NAD+ were added l a s t . .The r e s u l t s strongly suggest that a reaction between the reduced + enzyme and NAD must occur before the aldehyde can be oxidized and that K + i s e s s e n t i a l for t h i s r e a c t i o n . The potassium ion may p a r t i c i p a t e d i r e c t l y i n the union of coenzyme and enzyme, or i t may acti v a t e the enzyme i n some other manner. The r e s u l t s also suggest that coenzyme M I N U T E S - 57 -FIGURE 15 EFFECT OF SEQUENCE OF ADDITION OF COMPONENTS TO THE REACTION MIXTURE The assay was performed as i n "Materials and Methods" except that 0.1 M potassium phosphate buffer was replaced by 0.1 M T r i s chloride buffer, pH 7.4, and 0.1 M KC1. The following components were added l a s t to an otherwise complete reaction mixture which had been incubated f o r 5 minutes at 35°: 1. Enzyme and aldehyde l a s t 2. Aldehyde l a s t (routine assay) 3. Enzyme l a s t 4. NAD+ l a s t 5. 2-mercaptoethanol and aldehyde l a s t 6. K + and NAD+ l a s t 7. K + omitted. (When two components were added.last, the one mentioned f i r s t was added 20 seconds before the one mentioned second). - 58 -and aldehyde may compete for the same s i t e on the enzyme. The increasing reaction rate when and NAD+ are added l a s t to the reaction mixture, and the substrate i n h i b i t i o n by aldehydes at r e l a t i v e l y low concentrations, which w i l l be described l a t e r , are indications of t h i s . Ionic Requirements - Figure 15 also demonstrates the dependence of the reaction oh the a c t i v a t i n g ion, K +. In a T r i s chloride-buffered assay system 1C7 was e f f e c t i v e l y replaced by NH4 and, to a lesser extent, by Rb +. These r e s u l t s and the e f f e c t of other monovalent and divalent cations are shown i n Table IV. I t should be noted, i n p a r t i c u l a r , that phosphate did not activ a t e the enzyme. The possible i n h i b i t o r y e f f e c t of some monovalent cations on the enzyme reaction:: was tested i n a potassium phosphate-buffered assay system. Of the s a l t s tested, only L i C l may be s l i g h t l y i n h i b i t o r y . The r e s u l t s are shown i n Table V. The e f f e c t of sodium arsenate was also tested i n t h i s system and i t was found to i n h i b i t the reaction by approximately 507». This e f f e c t was ascribed to i n h i b i t i o n by the anion, since sodium chloride was not i n h i b i t o r y . In view of the report of an aldehyde dehydrogenase from Pseudomonas fluorescens which i s dependent on phosphate or arsenate f o r a c t i v i t y (Jakoby, 1958a), a study was made of the i o n i c requirements of aldehyde dehydrogenase from several other members of the genus Pseudomonas. Six d i f f e r e n t organisms were grown with ethanol as the carbon source i n the medium described i n "Materials and Methods", and the c e l l extracts were assayed for t h e i r aldehyde-oxidizing capacity i n a potassium phosphate- and a sodium phosphate-buffered assay system. The r e s u l t s are shown i n Table VI. None of the a c t i v i t i e s i n the sodium - 59 -TABLE IV IONIC REQUIREMENTS FOR ALDEHYDE DEHYDROGENASE ACTIVITY Additions Relative rate None KC1 K phosphate NH4CI RbCl L i C l NaCl Na phosphate CsCl MgCl 2 MnCl2 < 6 100* 100 110 65 < 6 < 6 < 6 < 6 < 6 < 6 a-The r e a c t i o n i n the presence of KC1 i s a r b i t r a r i l y assigned a value of 100. The assays were performed as outlined under "Materials and Methods" except 0.1 M potassium phosphate was replaced by.0.1 M T r i s c h l o r i d e , pH 7.4. A l l s a l t s l i s t e d i n the Table were added to give a f i n a l concentration of 0.1 M. The enzyme was dissolved i n 0.1 M T r i s chloride-0.01 M d i t h i o t h r e i t o l , pH 7.4. - 60 -TABLE V EFFECT OF MONOVALENT CATIONS ON POTASSIUM-ACTIVATED ALDEHYDE OXIDATION Additions r Relative Rate None 100 a KC1 91 NH4CI 100 RbCl 91 NaCl 91 L i C l 86 CsCl 91 The reaction i n the presence of K phosphate with no additions i s a r b i t r a r i l y assigned a value of 100. The enzyme was assayed as outlined under "Materials and Methods" with K phosphate as the bu f f e r . The s a l t s l i s t e d were added to give a f i n a l concentration of 0 , 1 M, TABLE VI ALDEHYDE DEHYDROGENASE ACTIVITY IN CELL EXTRACTS OF SIX DIFFERENT STRAINS OF PSEUDOMONAS Source of c e l l extract S p e c i f i c a c t i v i t y + K + + Na + umoles/min, ./mg. -protein P. fluorescens 36' 0.80 0.054 p. fluorescens 22' 0.65 0.035 p. fluorescens 26^ 0.32 0.022 p. fluorescens A 312 0.23 0.018 p. o v a l i s B 8 0.40 0.023 p. aeruginosa ATCC.9027 0.55 0.021 To prepare the inoculum a l l organisms were transferred twice i n the medium described i n "Materials and Methods" with ethanol as the carbon source. The organisms were then grown i n the same medium for 48 hours at 25°. The c e l l s were harvested, washed once i n 10 mM potassium phosphate containing 10 mM 2-mercaptoethanol, pH 7.0, and suspended i n the same bu f f e r . C e l l extracts were prepared by sonication and c e n t r i f u g a t i o n at 31,700 x g for 30 minutes. Aldehyde dehydrogenase a c t i v i t y was determined a f t e r d i a l y s i s and d i l u t i o n of the c e l l extracts with 50 mM T r i s c h l o r i d e buffer containing 10 mM 2-mercaptoethanol, pH 7.0. Enzyme a c t i v i t y was determined as usual except that 0.1M potassium phosphate b u f f e r , pH 7.2, or 0.1 M sodium phosphate b u f f e r , pH 7.2, was used i n the reaction mixture. - 62 -phosphate-buffered assay system were greater than 870 of those measured i n the potassium phosphate assay system. The same requirement f o r potassium was observed i n comparing the a c t i v i t y of c e l l extracts i n a T r i s chloride-buffered assay system with or without the addition of K + as KC1. Therefore, the aldehyde dehydrogenase a c t i v i t y of a l l the Pseudomonas species tested was dependent on K~*" f o r a c t i v i t y and not onpphosphate. The phosphate-requiring aldehyde dehydrogenase was reported by Jakoby (1958a) to be present i n P. fluorescens grown with ethylene g l y c o l as the carbon source. However, c e l l extracts of P. aeruginosa grown on ethylene g l y c o l were found i n the present work to have the same requirement f o r K + as c e l l extracts from ethanol-grown c e l l s . Enzyme S p e c i f i c i t y - Aldehyde dehydrogenase oxidized a v a r i e t y of aldehydes and NAD+ or, to a lesser extent, NADP+ could serve as the hydrogen acceptor. The s p e c i f i c a c t i v i t i e s of crude c e l l extracts and the p u r i f i e d enzyme preparation were determined with several substrates. The constant r a t i o of the s p e c i f i c a c t i v i t y of the p u r i f i e d enzyme preparation to the s p e c i f i c a c t i v i t y of the crude c e l l extract (Table VII) indicates that the a c t i v i t y with the d i f f e r e n t substrates i s due to one enzyme. The s p e c i f i c a c t i v i t y of a p u r i f i e d enzyme preparation determined with several aldehyde substrates at d i f f e r e n t concentrations i s shown in Table VIII. The lower s p e c i f i c a c t i v i t i e s f o r acetaldehyde propionaldehyde, butyraldehyde and isobutyraldehyde at the higher con-centrations in d i c a t e substrate i n h i b i t i o n . This e f f e c t i s not observed - 63 -TABLE VII SPECIFIC ACTIVITY OF CRUDE AND PURIFIED ENZYME PREPARATIONS WITH DIFFERENT SUBSTRATES AND HYDROGEN ACCEPTORS Substrate S p e c i f i c a c t i v i t y Ratio of Crude P u r i f i e d s p e c i f i c a c t i v i t y umoles/min. /mg. protein S p e c i f i c a c t i v i t y purified/crude Glyceraldehyde + NAD+ 0.16 2.8 17.5 Glycolaldehyde + NAD+ 0.73 12.7 17.4 Glycolaldehyde + NADP+ 0.03 0.48 16.0 Acetaldehyde + NAD+ 0.88 16.0 18.2 The s p e c i f i c a c t i v i t y of aldehyde d lehydrogenase i n the c e l l extract and p u r i f i e d enzyme preparations was determined using the usual assay procedure. The following aldehyde and hydrogen acceptor concentrations were present i n the reaction mixture: glyceraldehyde, 5 mM; glycolaldehyde, 1 mM; acetaldehyde, 0.05 mM; NAD+, 2 mM and NADP"^ , 2 mM. The enzyme concentrations were adjusted to obtain a measurable r e a c t i o n r a t e . - 64 -TABLE VIII SUBSTRATE SPECIFICITY S p e c i f i c A c t i v i t y Substrate 0.1 mM 0.2 mM 1.0 mM 5.0 mM Substrate Substrate Substrate Substrate umoles/min./mg. protein Glycolaldehyde 3.2 5.3 10.2 Acetaldehyde 11.4 9.25 4.85 Propionaldehyde 7.6 5.6 4.2 Butyraldehyde 3.9 2.9 1.7 Isobutyraldehyde 7.7 7.3 4.9 Benzaldehyde 0.3 0.5 1.9 Glyceraldehyde 0.4 1.8 The s p e c i f i c a c t i v i t y of p u r i f i e d aldehyde dehydrogenase was determined as usual i n the presence of the final.concentrations of aldehydes indicated i n the table. - 65 -with glycolaldehyde, benzaldehyde and glyceraldehyde at the concentrations used. The products of glycolaldehyde and glyceraldehyde oxidation were i s o l a t e d from separate 10 ml. reaction mixtures. These contained the same components as the routine assay described i n "Materials and Methods", except an excess of p u r i f i e d aldehyde dehydrogenase was added and several additions of NAD+ and aldehyde were made,.to increase the y i e l d of the product. A f t e r concentration of the re a c t i o n mixtures and d e s a l t i n g , the products were i d e n t i f i e d as g l y c o l l i c acid and g l y c e r i c acid r e s p e c t i v e l y , using descending paper chromatography with two d i f f e r e n t solvent systems. Michaelis Constants - The a f f i n i t y constants of the enzyme for several substrates and a c t i v a t i n g ions were determined. A l l . r e s u l t s are presented as the double r e c i p r o c a l plot proposed by Lineweaver and Burk (1934). Figure 16 shows the r e s u l t s obtained with varying NAD+.and NADP+ concentrations. The IC^  f o r NAD+ was 3.7 x 10"^ M and the KJJJ f o r NADP +was 3.0 x 10"3 M. These r e s u l t s are i n agreement with the much greater a c t i v i t y found with NAD+ than NADP+ i n the routine assay mixture where the coenzyme i s present at 2.0.x 10"-^  M. Figure 17 and Figure 18 show the r e s u l t s obtained with varying glyceraldehyde and glycolaldehyde concentrations r e s p e c t i v e l y . The KJJ, f o r glyceraldehyde was 1.4 x 10" M and for glycolaldehyde, 4.0 x 10~ 4 M. As was found with other aldehyde substrates (Table VIII) high glycolaldehyde concentrations i n h i b i t e d the enzyme re a c t i o n . The values f o r K + and NH^+, 6.0 x 10"-^  M and 12.0 x 10 M r e s p e c t i v e l y (Figure 19), were very s i m i l a r . T - 66 -FIGURE 16 DOUBLE RECIPROCAL PLOTS OF VELOCITY AGAINST NAD+ OR NADP+ CONCENTRATION The assays were c a r r i e d out as described i n "Materials and Methods". Approximately 0.02 units of enzyme ( i n 100 mM T r i s chloride-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0) and 0.1 units + + of enzyme were used with varying NAD and NADP concentrations, r e s p e c t i v e l y . - 67 -FIGURE 17 DOUBLE RECIPROCAL PLOT OF VELOCITY-AGAINST GLYCERALDEHYDE CONCENTRATION The assay was c a r r i e d out as described i n "Materials and Methods" and 0.1 units of enzyme were used per rea c t i o n mixture. There i s no apparent substrate i n h i b i t i o n . - 68 -FIGURE 18 DOUBLE RECIPROCAL PLOT OF VELOCITY AGAINST GLYCOLALDEHYDE CONCENTRATION The assays were c a r r i e d out as described under "Materials and Methods" and 0.02 units of enzyme were used per reaction mixture. Substrate i n h i b i t i o n i s apparent, but has been d i s -regarded in. the l i n e plot used f o r c a l c u l a t i o n s . - 69 -FIGURE.19 DOUBLE RECIPROCAL PLOT OF VELOCITY AGAINST AMMONIUM OR POTASSIUM ION CONCENTRATION Both ions were used as the chloride s a l t . NAD was the hydrogen acceptor and glycolaldehyde the substrate. The assays were c a r r i e d out as described i n "Materials and Methods" and the enzyme was dissolved i n 100 mM T r i s chloride-10 mM d i t h i o t h r e i t o l - 1 mM EDTA,.pH 7.0, and 0.02 units were added per reaction mixture. - 70 -Reducing Agents - The enzyme was assayed r o u t i n e l y i n the presence of 10 mM 2-mercaptoethanol. This reagent could be replaced, i n part, by EDTA, or replaced e f f e c t i v e l y by lower concentrations of cysteine, d i t h i o t h r e i t o l , or 2,3-dimercaptopropanol. The a c t i v i t i e s obtained with EDTA and 2-mercaptoethanol were not a d d i t i v e . These r e s u l t s are shown i n Table IX. The observation that EDTA can replace reducing agents i n the reaction mixture to a considerable degree suggests that aldehyde dehydrogenase has maximum a c t i v i t y i n the reduced state and when i t i s protected from heavy metal ion contaminants. A reducing agent can serve both functions, whereas EDTA only w i l l protect the enzyme from heavy metal ion i n h i b i t i o n . At high concentrations of cysteine (5-10 mM) the i n i t i a l f a s t r eaction rate was arrested a f t e r approximately four minutes. This loss of a c t i v i t y was reversed by the addition of glycolaldehyde. This may indicate the formation of a thiohemiacetal due to. the reaction of the aldehyde substrate and th i s reducing agent. This phenomenon was not observed when the other reducing agents or glutathione were employed. E f f e c t of pH - The optimum pH f o r the i n i t i a l r e action rate was determined by assaying aldehyde dehyrogenase a c t i v i t y i n a T r i s c h l o r i d e -potassium phosphate buffer system at pH values between pH 5.7 and pH 9.3. The enzyme was preincubated at 35° for f i v e and ten minutes before i n i t i a t i o n of the r e a c t i o n by addition of the aldehyde. The r e s u l t s are shown i n Figure 20. The pH optimum f o r the i n i t i a l reaction rate i s between pH 8.0 and pH 8.6. The values obtained a f t e r d i f f e r e n t preincubation times are i n close agreement. This indicated that the preincubation i t s e l f , even at hydrogen ion concentrations which r e s u l t - 71 -TABLE IX ACTIVITY OF ALDEHYDE DEHYDROGENASE IN THE PRESENCE OF EDTA AND DIFFERENT REDUCING AGENTS Addition Concentration S p e c i f i c a c t i v i t y mM umoles/min./mg. protein None - 1.7 EDTA 0.1 1.9 1.0 3.8 10.0 4.8 20.0 5.2 2-Mercaptoethanol 0.1 3.1 1.0 6.4 10.0 12.5 20.0 12.8 2-Mercaptoethanol + EDTA 10.0 + 10.0 11.0 Cysteine 0.1 3.5 1.0 11.0 10.0 14.2 D i t h i o t h r e i t o l 0.01 6.3 0.1 11.3 1.0 13.2 • 10.0 14.2 2,3-Dimercaptopropanol 0.01 8.1 (BAL) 0.1 11.3 1.0 12.8 10.0 14.9 P u r i f i e d aldehyde dehydrogenase dissolved i n 0.1 M potassium phosphate, pH 7.0, was assayed as usual except 2-mercaptoethanol was replaced by the additions l i s t e d i n the table. S P E C I F I C ACTIVITY FIGURE 20 THE EFFECT OF pH ON ,GLYCPLALDEHYDE.;:OXIDAT;I;ON The assays were performed as described i n "Materials and Methods"except that the reaction mixture also contained 0.1 M T r i s c h l o r i d e . The pH of the reaction mixture was determined immediately a f t e r the assay was completed. ® Aldehyde added a f t e r 5 minutes preincubation O Aldehyde added a f t e r 10 minutes preincubation - 73 -i n i n s t a b i l i t y of the enzyme, does not a f f e c t s i g n i f i c a n t l y the measurement of i n i t i a l r e action r a t e s . Since a l i n e a r rate was obtained f o r a longer period of time at pH 7.2 than at hydrogen ion concentrations close to the pH optimum, the assay was r o u t i n e l y c a r r i e d out at pH 7.2. VI. S t a b i l i t y S t a b i l i t y of the Enzyme i n C e l l Extracts - I n i t i a l l y , the i n s t a b i l i t y of aldehyde dehydrogenase i n c e l l extracts had been a serious problem. When the c e l l e x t r a c t , was prepared i n phosphate buffer containing 2-mercaptoethanol, losses of 30-507„ of the a c t i v i t y occurred upon storage at 4° f o r 16 hours or during the course of ammonium s u l f a t e f r a c t i o n a t i o n . A c t i v i t y losses were reduced by storage at -22° or a f t e r gel f i l t r a t i o n using Bio-Gel P-60. B i s u l f i t e , used as Na2S2 n5, adjusted to pH 7.0 with KOH, was found to be very e f f e c t i v e i n reducing a c t i v i t y losses. Table X shows the e f f e c t of 2-mercaptoethanol, d i t h i o t h r e i t o l , and b i s u l f i t e on the s t a b i l i t y of aldehyde dehydrogenase i n crude c e l l extracts. When the c e l l extract was prepared i n 100 mM b i s u l f i t e - 1 0 mM 2-mercaptoethanol, pH 7.0, i n s i g n i f i c a n t losses of a c t i v i t y were observed during storage at -22° for several weeks or at 4° or 25° for several days. B i s u l f i t e could not be replaced by hydrazine, hydroxylamine, ascorbate, or EDTA. The exact r o l e of b i s u l f i t e i n s t a b i l i z i n g the enzyme has not been determined as yet. Aldehydes are toxic to the enzyme as evidenced by substrate i n h i b i t i o n and b i s u l f i t e i s known to form a d d i t i o n products with aldehydes. However, th i s property of b i s u l f i t e does not appear to -. 74 -TABLE X EFFECT OF REDUCING AGENTS, BISULFITE, AND PHOSPHATE ON STABILITY OF ALDEHYDE DEHYDROGENASE IN CELL EXTRACTS Conditions Concentration 7o of i n i t i a l a c t i v i t y mM 7o 2-Mercaptoethanol 10 12 20 32 50 55 D i t h i o t h r e i t o l 10 . 12 B i s u l f i t e 30 80 60 83 120 97 Phosphate 3 30 27 Also contained 10 mM 2-mercaptoethanol C e l l extracts were prepared by sonication i n eit h e r 5 mM potassium phosphate containing 10 mM 2-mercaptoethanol, pH 7.0, or 5 mM potassium phosphate containing. 10 mM d i t h i o t h r e i t o l , pH 7.0. 2-Mercaptoethanol, b i s u l f i t e (pH 7.0), and potassium phosphate were also added to the c e l l extract prepared with 2-mercaptoethanol to give .the f i n a l concentrations shown i n the table. A c t i v i t y of aldehyde dehydrogenase was determined i n each sample before and a f t e r storage at 4° for 6 days. - 75 -account f o r the s t a b i l i z i n g e f f e c t since hydroxylamine and hydrazine, which also form addition products with aldehydes, did not show a s t a b i l i z i n g e f f e c t on the enzyme and the addi t i o n of aldehydes to c e l l extracts d i d not increase i n s t a b i l i t y . The enzyme i s s t a b i l i z e d to some degree by gel f i l t r a t i o n which may support the idea that b i s u l f i t e i s combining with, or protecting the enzyme from, some factor i n the c e l l extract. B i s u l f i t e i s a very strong reducing agent and th i s may be the reason f o r i t s e f f e c t . This i s supported by the f a c t that the presence of high concentrations of 2-mercaptoethanol also re s u l t e d i n greater s t a b i l i t y of the enzyme (as shown i n Table X). However, ascorbate, which i s also a strong reducing agent, could not replace b i s u l f i t e . S t a b i l i t y of the P u r i f i e d Enzyme - During the e a r l y stages of p u r i f i c a t i o n the enzyme preparation can be stored i n phosphate buffer containing 2-mercaptoethanol at -22° without s i g n i f i c a n t loss of a c t i v i t y . Preparations c a r r i e d past step 6 i n the p u r i f i c a t i o n were found to denature upon fr e e z i n g and thawing with the loss of 90-1007, of the a c t i v i t y . This was prevented by the addition of 50 mM b i s u l f i t e or 1.0% ethylene g l y c o l to the buffer system, or by s u b s t i t u t i o n of d i t h i o t h r e i t o l f o r 2-mercaptoethanol. The enzyme preparation a f t e r the f i n a l step of p u r i f i c a t i o n , dissolved to a concentration of 15 mg. per ml..in 100 mM potassium phosphate-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0, was found to be stable at -22° f o r several months. D i l u t e enzyme preparations (50 ug. per ml.) at the f i n a l stage of p u r i f i c a t i o n deteriorated more r e a d i l y upon free z i n g and thawing but could be stored - 76 -at -22° without loss of a c t i v i t y , i n the presence of 30% ethylene g l y c o l . D i l u t e p u r i f i e d enzyme preparations stored at 4° i n 100 mM potassium phosphate-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0, were stable f o r several days and 10 mM d i t h i o t h r e i t o l could be replaced by 100 mM 2-mercaptoethanol. At t h i s temperature b i s u l f i t e showed a favorable e f f e c t on the s t a b i l i t y of the p u r i f i e d enzyme preparation. These r e s u l t s are shown i n Table XI. E f f e c t of pH - The s t a b i l i t y of. the enzyme at d i f f e r e n t hydrogen ion concentrations was determined with a d i l u t e p u r i f i e d enzyme preparation (40 ug. per ml.). The pH region from pH 6.8 to 7.2 was found to be the most favorable for storage at 4°. Ina c t i v a t i o n by Heat - The s t a b i l i t y of aldehyde dehydrogenase at temperatures from 40-60° was determined i n two buffer systems: 150 mM potassium phosphate-10 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0, and 100 mM potassium phosphate-50 mM b i s u l f i t e - 1 0 mM d i t h i o t h r e i t o l - 1 mM EDTA, pH 7.0. The r e s u l t s are shown i n Figure 21. They indic a t e a r e l a t i v e l y low resistance of the enzyme to heating and also the s t a b i l i z i n g e f f e c t of b i s u l f i t e on the enzyme which was e s p e c i a l l y noticeable i n the temperature range from 45° to 53°. A d i l u t e , p u r i f i e d enzyme preparation i n a potassium phosphate-buffered system was e f f e c t i v e l y protected from heat i n a c t i v a t i o n at 46° by the presence of 0.4 mM NAD+. In the presence of NAD+, 82%, of the i n i t i a l a c t i v i t y remained a f t e r heating f o r 20 minutes, whereas 44%, remained without NAD+. In a p a r a l l e l experiment using sodium phosphate buffer,. 33%, and 13%, of the i n i t i a l a c t i v i t y remained i n the presence and TABLE XI EFFECT OF BISULFITE ON THE STABILITY OF PURIFIED ALDEHYDE DEHYDROGENASE Addition Concentration 7, of i n i t i a l activity mM % None - 46 Bisulfite 40 110 K phosphate 40 45 Purified aldehyde dehydrogenase (50 ug. per ml.) in 100 mM potassium phosphate-10 mM dithiothreitol-1.0 mM EDTA, pH 7.0, was stored with or without the addition of bis u l f i t e or an equivalent concentration of phosphate. The activity in each preparation was determined before and after storage at 4° for 16 days. - 78 -FIGURE. 21 HEAT INACTIVATION OF ALDEHYDE DEHYDROGENASE A p u r i f i e d enzyme preparation (50 ug. per ml.) i n 0.5 ml. of the buffer systems outlined i n the text was subjected to 10 minutes heat treatment at the temperatures indicated. The a c t i v i t y remaining a f t e r the treatment i s expressed as % of the i n i t i a l a c t i v i t y . O Enzyme i n potassium phosphate buffer Enzyme i n potassium phosphate buffer + b i s u l f i t e - 79 -absence of NAD+ r e s p e c t i v e l y . Therefore, NAD+ i n the presence of the potassium ion i s much more e f f e c t i v e i n protecting the enzyme from heat i n a c t i v a t i o n than.in the presence of the sodium ion. VII. I n h i b i t i o n and Protection Aldehyde dehydrogenase i s i n h i b i t e d by reagents reacting with I | s u l f h y d r y l groups, such as iodoacetate, iodoacetamide, Cu , and p_-chloromercuribenzoate. The i n h i b i t i o n by iodoacetate and iodoacetamide was found to proceed at a f a s t e r rate i n the presence of the a c t i v a t i n g ion, K +, than i n the presence of a non-activating ion, Na +. The inhi b -i t i o n of the enzyme with .0.1 mM iodoacetamide i s shown i n Figure 22. Similar r e s u l t s were obtained when 1.0 mM iodoacetate was used as the i n h i b i t o r . The enzyme was protected from iodoacetate i n h i b i t i o n by 0.4 mM NAD when K +, or NH^+, was also present. The s p e c i f i c i t y of this protection with K + and NAD+ or NADH i s demonstrated i n Table XII. The presence of K + and NAD"1" also gives protection from i n h i b i t i o n by iodo-acetamide. I t appears that K + or NH^ i s required f o r the binding of the coenzyme to the protein at or near the s i t e of a l k y l a t i o n and that when th i s occurs the enzyme, i s protected from i n h i b i t i o n . A s i m i l a r s p e c i f i c i t y was observed when the loss of a c t i v i t y due to t r y p s i n d i g e s t i o n was followed. Potassium plus NAD+, or K + plus NADH, protected the enzyme whereas K4" plus NADP+ did not. These r e s u l t s are shown i n Table XIII. Potassium alone and, to a lesser extent, Na + provided some protection from t r y p s i n d i g e s t i o n , i n d i c a t i n g some con-formational change of the enzyme due to the presence of the a c t i v a t i n g ion. 40 80 120 160 M I N U T E S - 80 -FIGURE 22 EFFECT OF THE PRESENCE OF K + OR Na + ON THE INHIBITION OF ALDEHYDE DEHYDROGENASE BY IODOACETAMIDE Aldehyde dehydrogenase (74 ug. per ml. i n 100 mM T r i s chloride-1 mM d i t h i o t h r e i t o l , pH 7.2) was incubated for 30 minutes at 20-22° i n the presence and absence of 100 mM KC1 or NaCl. Iodoacetamide was then added to a f i n a l concentration of 0.1 mM and i n h i b i t i o n of the enzyme preparation was followed f o r 150 minutes. No addition + KC1 + NaCl - 81 -TABLE XII INHIBITION OF ALDEHYDE DEHYDROGENASE BY IODOACETATE AND PROTECTION BY THE ACTIVATING ION AND COENZYME Additions °/„ of i n i t i a l a c t i v i t y None 11 KC1 8 NaCl 17 NAD+ 10 KC1 + NAD + 77 NaCl + NAD+ 8 KC1 + NADP+ 19 KC1 + NADH+ 81 To a p u r i f i e d enzyme preparation (74 ug. per ml.) i n 100 mM T r i s chloride-1 mM d i t h i o t h r e i t o l , pH 7.2, the additions l i s t e d i n the table were made to give a f i n a l concentration of 100 mM KC1 or NaCl, and 0.4 mM NAD+, NADP+ or NADH. After incubation f o r 30 minutes at 25°, iodoacetate was added to give a f i n a l concentration of 2 mM. Control samples without.iodoacetate were also included i n the experiment. The Table shows the % of the i n i t i a l a c t i v i t y which remained a f t e r a 2 hour incubation at 25°. - 82 -TABLE XIII DIGESTION OF ALDEHYDE DEHYDROGENASE BY TRYPSIN AND PROTECTION BY THE ACTIVATING ION AND COENZYME Additions 7« of i n i t i a l a c t i v i t y None 16 KCl 51 NaCl 28 NAD+ 12 KCl + NAD+ 91 NaCl + NAD+ 25 KCl + NADP+ 48 KCl + NADH+ 89 The experimental d e t a i l s for th i s experiment are the same as those i n the legend to Table XII except that iodoacetate was replaced by t r y p s i n at a f i n a l concentration of 0.1 |_ig. per ml. The 7, of the i n i t i a l a c t i v i t y remaining a f t e r 60 minutes dig e s t i o n at 25° i s shown i n the Table. - 83 -Arsenite, a common i n h i b i t o r of aldehyde dehydrogenases (Jakoby, 1958b) also i n h i b i t s the enzyme i s o l a t e d from P. aeruginosa. Arsenite at 0.5 mM, 1.0 mM and 5.0 mM i n the r e a c t i o n mixture r e s u l t e d i n 38%, 60%,, and 92% i n h i b i t i o n r e s p e c t i v e l y . The i n h i b i t i o n with 5.0 mM arsenite was reversed by 80%, upon the addition of 5.0 mM d i t h i o t h r e i t o l . Arsenite i n h i b i t i o n also i s reversed completely by d i l u t i o n . Ortho-phenanthroline i n h i b i t s aldehyde dehydrogenase by 44% at 0.5 mM and 67%, at 1.0 mM concentration when added to the reaction mixture during the enzyme assay, but showed a stimulatory e f f e c t on the r e a c t i o n i n the absence of reducing agent. An enzyme preparation (2.8 mg. per ml.) i n h i b i t e d to greater than 907° by 2.0 mM o-phenanthro-l i n e at 20-22° during a 3 hour period, was not activated a f t e r gel f i l t r a t i o n , using Bio-Gel P-60, to separate the i n h i b i t o r from the pro t e i n , or by subsequent incubation of the separated protein with 0.01 mM Zn 4 4", Co44", Fe 4 4", or Mg44". VIII . Analysis of the Enzyme f o r Zinc Yeast aldehyde dehydrogenase has been reported to contain zinc (Stoppani, Schwarcz, and Freda, 1966). The i n h i b i t i o n of the P. aeruginosa aldehyde dehydrogenase by o-phenanthroline suggested that zinc; might be present i n t h i s enzyme.as w e l l , although addition of zinc to the i n h i b i t e d enzyme a f t e r separation of the i n h i b i t o r and enzyme by gel f i l t r a t i o n did not restore a c t i v i t y and other chelating agents, such as EDTA, di d not i n h i b i t the enzyme. P u r i f i e d aldehyde dehydrogenase was, therefore, analyzed f o r zinc using an atomic - 84 -absorption spectrophotometer. A s o l u t i o n of 4.8 mg. per ml. of the enzyme, which had retained at least 65% of the o r i g i n a l s p e c i f i c a c t i v i t y a f t e r extensive d i a l y s i s , contained no detectable z i n c . From the r e s u l t s obtained with standard solutions i t was estimated that 0.1 ug. per ml. of zinc could have been detected. A s o l u t i o n of yeast alcohol dehydrogenase of s i m i l a r concentration contained more than 5: ug. per ml. of zinc i n agreement with the r e s u l t s of V a l l e e and Hoch (1955). It was concluded that the aldehyde dehydrogenase of P. aeruginosa does not contain t h i s metal ion. IX. D i s s o c i a t i o n and Deactivation of Aldehyde Dehydrogenase Enzyme preparations dissolved i n 0.1 M potassium phosphate b u f f e r , pH 7.0, i n the presence of reducing agent were found to have maximum s p e c i f i c a c t i v i t y compared to preparations i n buffers without reducing agent. I t was further observed that d i a l y s i s against solutions of low s a l t concentration (1 mM EDTA, pH 7.2) r e s u l t e d i n a rapid loss of enzyme a c t i v i t y ( d eactivation). Such deactivation was accompanied by a change i n the sedimentation pattern observed i n the u l t r a c e n t r i f u g e and i n the electrophoretic m ob il it y of the protein upon starch gel electrophoresis ( d i s s o c i a t i o n ) . Although add i t i o n of potassium phosphate reversed the d i s s o c i a t i o n observed when d i a l y s i s was performed with e i t h e r d i l u t e EDTA or T r i s , r e a c t i v a t i o n was obtained only i n the former case; r e a c t i v a t i o n of enzyme reassociated with phos-phate a f t e r d i a l y s i s against T r i s required a reducing agent. Thus, reassociated enzyme was not f u l l y r e a c t i v a t e d i n the absence of reducing agent, whereas re a c t i v a t e d enzyme was always found to be reassociated. - 85 -The experimental r e s u l t s are presented below. Pattern of Dissociated and Reassociated Aldehyde Dehydrogenase  i n the U l t r a c e n t r i f u g e - Untreated aldehyde dehydrogenase with a s p e c i f i c a c t i v i t y of 13.5 shows only one component with a w of 9.0 to 9.4. Figure 23 shows the pattern of a d i s s o c i a t e d enzyme sample. Two major components with w of 7.1 and 5.0 and a minor component with an w • o f 2.8 are observed. After r e a s s o c i a t i o n one major component with S20 w of 9.0 and a minor component, S2Q)W °f 5.5>.are present. The s p e c i f i c a c t i v i t i e s of the preparations were 4 for the d i s s o c i a t e d enzyme and 10.2 for the reassociated enzyme. Ele c t r o p h o r e t i c M o b i l i t y of Dissociated and Reassociated  Aldehyde Dehydrogenase - The p u r i f i e d aldehyde dehydrogenase i n the a c t i v e state shows one major band i n starch gel electrophoresis. A f t e r d i s s o c i a t i o n by d i a l y s i s against 1 mM EDTA, pH 7.2, a second band with f a s t e r e l e c t r o p h o r e t i c m o b i l i t y was observed. Re-a s s o c i a t i o n , by a d d i t i o n of potassium phosphate and incubation at elevated temperatures, reversed t h i s change and only one major band with the m o b i l i t y of the untreated enzyme was observed (Figure 24). Reducing agents had no e f f e c t on the electrophoretic m o b i l i t y of e i t h e r the f a s t band or the slow band. Deactivated State of Dissociated Enzyme i n Protein Bands  Isolated by Starch Gel Electrophoresis - Staining of the a c t i v e aldehyde dehydrogenase f o r enzymatic a c t i v i t y a f t e r starch gel e l e c t r o -phoresis revealed one band, whereas two bands were observed when the enzyme was applied i n the d i s s o c i a t e d state. However, since the assay conditions would be expected to permit rea s s o c i a t i o n and r e a c t i v a t i o n , B - 86 -FIGURE 23 SEDIMENTATION OF DISSOCIATED AND REASSOCIATED ALDEHYDE DEHYDROGENASE IN THE ULTRACENTRIFUGE The enzyme preparations (5.7 mg. peri ml.) were sedimented at 59,780 r.p.m..at 20°. The sedimentation proceeds from r i g h t to l e f t . Pictures were taken at 16, 40, and 64 minutes a f t e r the rotor attained f u l l speed. The enzyme was di s s o c i a t e d by d i a l y s i s against 1 mM EDTA, pH 7.2, (A) and an aliqu o t of t h i s preparation was reassociated by the addi t i o n of potassium phosphate,. pH 7.0, and d i t h i o t h r e i t o l to f i n a l concentrations of 80 mM and 8 mM r e s p e c t i v e l y , (B). Both preparations were incubated at 30° for 60 minutes p r i o r to c e n t r i f u g a t i o n . A Dissociated aldehyde dehydrogenase B Reassociated aldehyde dehydrogenase I 2 3 4 5 6 - 87 -FIGURE.24 STARCH GEL ELECTROPHORESIS OF DISSOCIATED AND REASSOCIATED ALDEHYDE DEHYROGENASE Electrophoresis was c a r r i e d out at pH 8.5 for 3.5 hours at 4°. Aldehyde dehydrogenase (5.0 mg. per ml.) was di s s o c i a t e d by d i a l y s i s f o r 48 hours at 0° against 1 mM EDTA, pH 7.2. Pri o r to electrophoresis the following additions were made to the enzyme preparation and the preparations were incubated f o r 30 minutes at the temperatures indicated: 1,3 and 5 No additions - incubated at 30° 2 + K phosphate, pH 7.0, to 90 mM - incubated at 0° 4 + K phosphate, pH 7.0, to 90 mM - incubated at 30° 6 + D i t h i o t h r e i t o l to 9 mM - incubated at 30° - 88 -other methods were required to show whether both bands had enzymatic a c t i v i t y under conditions which do not r e a d i l y reassociate the enzyme. A starch g e l prepared by electrophoresis of di s s o c i a t e d aldehyde dehydrogenase was cut into segments perpendicular to the d i r e c t i o n of movement of the prot e i n . The segments were eluted with d i l u t e EDTA i n the cold and assayed f o r enzymatic a c t i v i t y before and a f t e r r e a c t i v a t i o n . The r e s u l t s , shown i n Figure 25 indicate that the slow band (associated enzyme) i s always maximally active whereas the f a s t band (dissociated enzyme) i s completely i n a c t i v e unless r e a c t i v a t e d . Conditions f o r D i s s o c i a t i o n and Deactivation - A loss of enzymatic a c t i v i t y and the appearance of the f a s t band on starch gel electrophoresis (deactivation and d i s s o c i a t i o n ) occurred when the enzyme was dial y s e d against 1 mM EDTA at pH 7.2, 8 .0 , or 8 .5 , or against 1 mM T r i s c h l o r i d e , pH 7.2. D i a l y s i s against 10 mM EDTA, pH 7.2, however, produced o n l y . i n s i g n i f i c a n t changes: that i s , there was no d i s s o c i a t i o n and l i t t l e d e activation. Also, treatment of the enzyme i n 10 mM EDTA with o-phenanthroline at a concentration which completely i n h i b i t s aldehyde dehydrogenase (1.5 mM) did not give r i s e to a f a s t e r band on electr o p h o r e s i s . These r e s u l t s indicated that the d i s s o c i a t i o n and deactivation was not due to the chelating action of EDTA, but rather to the exposure of the enzyme to low s a l t concentrations. Comparison of the deactivation of 10 mg. per:ml. and 0.1 mg. per ml. solutions of aldehyde dehydrogenase showed that t h i s process i s r e l a t i v e l y inde-pendent of protein concentration. o 2 < 3 co ro i r o m I. [ ro ~ r ~ ENZYME ACTIVITY CD CO ~i o ~1~ - 89 -FIGURE 25 ENZYMATIC ACTIVITY OF ISOLATED DISSOCIATED AND ASSOCIATED ALDEHYDE DEHYDROGENASE Aldehyde dehydrogenase (5.8 rag. per ml.) was d i s s o c i a t e d by d i a l y s i s against 1 mM.EDTA, pH 8.0,.and subjeeted to starch gel electrophoresis at pH 8.5 for 2 hours at 20-22°. The starch gel was then cut into segments and these were eluted with 1 mM EDTA i n the col d . Enzymatic a c t i v i t y was determined before and a f t e r incubation of the eluted f r a c t i o n s at 30° f o r 40 minutes i n 80 mM potassium-phosphate, pH 7.0, and 8 mM d i t h i o t h r e i t o l . - - - - Enzyme a c t i v i t y eluted with EDTA at 0° Enzyme a c t i v i t y a f t e r incubation at 30° with 80 mM potassium phosphate-8 mM d i t h i o t h r e i t o l - 90 -Conditions f o r Reassociation - When aldehyde dehydrogenase was dis s o c i a t e d by d i a l y s i s against 1 mM T r i s c h l o r i d e , pH 7.2, addition of potassium phosphate buffer alone caused a reass o c i a t i o n of the f a s t band observed upon electrophoresis to the slower one (as i n Figure 24). S i m i l a r i l y , when enzyme was di s s o c i a t e d by d i a l y s i s against 1 mM EDTA, add i t i o n of phosphate to 0.1 M caused the enzyme to reassociate. The rea s s o c i a t i o n did not proceed at 0°, but occurred over a period of time at 30°. In a d d i t i o n a l experiments of the same kind, r e a s s o c i a t i o n was brought about by incubation a f t e r addition of sodium phosphate, sodium c h l o r i d e , potassium ch l o r i d e and l i t h i u m chloride at neutral pH and 0.1 M concentration. Therefore, r e a s s o c i a t i o n i s brought about by incubation at increased s a l t concentrations and i s not r e l a t e d to the presence of the a c t i v a t i n g ion. Conditions f o r Reactivation - Following d i a l y s i s against 1 mM EDTA, pH 7.2, the enzyme could be rea c t i v a t e d to a considerable degree with phosphate alone, as shown i n Table XIV. Addition of d i t h i o t h r e i t o l i n the presence of phosphate further increased the degree of r e a c t i -v a t i o n . However, d i a l y s i s against T r i s under the same conditions yielded d i s s o c i a t e d enzyme which could not be reactivated i n the presence of phosphate alone, the addition of d i t h i o t h r e i t o l being e s s e n t i a l to the r e a c t i v a t i o n process (Table XXV), However, no increase i n a c t i v i t y was observed a f t e r incubation with d i t h i o t h r e i t o l or EDTA alone. The r e a c t i v a t i o n process i s r e l a t i v e l y independent of the protein concen-t r a t i o n , and i s e s s e n t i a l l y complete a f t e r 20 minutes, with a further s l i g h t increase up to 60 minutes. As expected, the process of r e a c t i -v a t i o n , l i k e r e a s s o c i a t i o n , does not proceed at 0°. - 91 -TABLE XIV A. REACTIVATION AFTER DISSOCIATION BY DIALYSIS AGAINST 1 mM EDTA S p e c i f i c a c t i v i t y Additions At 0 minutes Aft e r 60 minutes at 30c umoles/min./mg. protein None 0.6 0.4 K'phosphate . 0.6 4.8 D i t h i o t h r e i t o l 0.8 0.7 K phosphate + d i t h i o t h r e i t o l .1.2 9.7 B. REACTIVATION AFTER DISSOCIATION BY DIALYSIS AGAINST 1 mM TRIS Additions S p e c i f i c a c t i v i t y At 0 minutes Aft e r 60 minutes at 30c |imoles/min./mg. protein None 0.5 0.3 K phosphate 0.4 0.5 D i t h i o t h r e i t o l 0.6 0.5 K phosphate + d i t h i o t h r e i t o l 0.6 3.7 The enzyme (5.0 mg^/ml., s p e c i f i c a c t i v i t y 13.5) was dial y s e d 48 hours at 0° against (A) 1 mM EDTA, pH 7.2, and (B) 1 mM T r i s c h l o r i d e , pH 7.2. For r e a c t i v a t i o n , 10 p l . dialysed enzyme was d i l u t e d into 0.6 ml. 1 mM EDTA, pH 7.2, containing the additions shown i n the Table. K phosphate was added to a f i n a l concentration of 80 mM and d i t h i o t h r e i t o l to 8 mM. Samples were assayed at the time of d i l u t i o n and a f t e r 60 min-utes at 30°. Assays were performed as usual except the enzyme was added 5 seconds before the aldehyde. Rates obtained a f t e r 1 min. were recorded. - 92 -Thus, reactivated enzyme was always found to be reassociated, but reassociated enzyme was more or less dependent on reducing agent for r e a c t i v a t i o n . It appears that EDTA plays a s p e c i a l r o l e during the d i s s o c i a t i o n - r e a s s o c i a t i o n process, perhaps protecting the c a t a l y t i c a l l y - a c t i v e s i t e s of the enzyme from an i n h i b i t i o n which i s r e v e r s i b l e by d i t h i o t h r e i t o l . Since EDTA was i n e f f e c t i v e i n reversing the deactivation of the enzyme r e s u l t i n g from d i a l y s i s against T r i s , d i t h i o t h r e i t o l i s s p e c i f i c a l l y - r e q u i r e d to restore the a c t i v i t y l o s t i n the absence of EDTA. A s i m i l a r phenomenon was observed, to a lesser extent, under conditions where d i s s o c i a t i o n does not take place. D i a l y s i s of the enzyme against 1 mM T r i s chloride - 1 0 0 mM potassium phosphate, pH 7.0, r e s u l t e d i n a decrease of the s p e c i f i c a c t i v i t y to 117o of the o r i g i n a l a c t i v i t y and the a c t i v i t y was increased to 707» of the o r i g i n a l by a d d i t i o n o f ^ d i t h i o t h r e i t o l to-10 mM concentration. D i a l y s i s against 1 mM EDTA-100 mM potassium phosphate, pH 7.0, decreased the a c t i v i t y only to 387, of the o r i g i n a l and addition of d i t h i o t h r e i t o l restored 847, of the o r i g i n a l a c t i v i t y . Since d i s s o c i a t i o n had not occurred, the loss of a c t i v i t y on d i a l y s i s was e n t i r e l y due to lack of reducing agent, and EDTA could p a r t i a l l y replace the reducing agent. Size of the Dissociated Enzyme - The observed d i s s o c i a t i o n of the enzyme may r e s u l t from the l i b e r a t i o n of two subunits. The 9 S component of the active enzyme and the 7.1 S component of the di s s o c i a t e d prepar-a t i o n are thought to be i d e n t i c a l , since the low s a l t concentration at which the d i s s o c i a t e d preparation was centrifuged may cause p a r t i a l unfolding of the molecule which would r e s u l t i n a lower sedimentation c o e f f i c i e n t . This component corresponds to the slow band i n starch gel - 93 -e l e c t r o p h o r e s i s . S i m i l a r l y , the 5.5 S component i n the reactivated and the 5.0 S component i n the d i s s o c i a t e d preparation are thought to be i d e n t i c a l and to correspond to the f a s t band i n the electrophoresis. Attempts were made to separate the two components by gel f i l t r a t i o n and sucrose gradient c e n t r i f u g a t i o n . Good separation was not obtained. On Sephadex G-200, when the d i s s o c i a t e d preparation was applied, the f r o n t of the. protein p r o f i l e was more active than the t a i l end before r e a c t i v a t i o n and the protein i n the t a i l end could be reactivated to a greater extent than the protein i n the f r o n t . This r e s u l t i s consistent with the conclusion that the active molecule i s larger than the d i s s o c i a t e d molecule. A c t i v i t i e s before and a f t e r r e -a c t i v a t i o n of d i s s o c i a t e d aldehyde dehydrogenase, recovered from sucrose gradient c e n t r i f u g a t i o n , are shown i n Figure 26. These r e s u l t s i n d i c a t e , as expected, that the d i s s o c i a t e d molecule i s less dense than the active molecule. Influence of Other Reagents - Attempts were made to deactivate or d i s s o c i a t e aldehyde dehydrogenase by the a d d i t i o n of urea, sodium l a u r y l s u l f a t e and para-hydroxymercuribenzoate, reagents which have been found to cause d i s s o c i a t i o n of other enzymes. The enzyme was p r e c i p i t a t e d and denatured at approximately 2 M urea. Neither enzymatic a c t i v i t y nor electrophoretic mobility, was changed at lower concentrations of urea. Sodium l a u r y l s u l f a t e and para-hydroxymercuri-benzoate also denatured the enzyme at 2 mM and 0.2 mM concentrations r e s p e c t i v e l y . L i t t l e or no change of the electrophoretic m o b i l i t y was observed at lower.concentrations. ENZYME ACTIVITY - 94 -FIGURE 26 SUCROSE GRADIENT CENTRIFUGATION OF DISSOCIATED ALDEHYDE DEHYDROGENASE Aldehyde dehydrogenase was di s s o c i a t e d by d i a l y s i s against 1 mM EDTA, pH 7.2, and 0.8 mg. (0.3 ml.) was applied to a 5% to 207o sucrose gradient prepared i n 1 mM EDTA, pH 7.2. Fractions were c o l l e c t e d a f t e r c e n t r i f u g a t i o n at 35,000 r.p.m. f o r 13 hours at 5° and assayed before and a f t e r r e a c t i v a t i o n by the addi t i o n of potassium phosphate, pH 7.0, to 80 mM and d i t h i o t h r e i t o l to 8 mM and incubation at 30° f o r 60 minutes. O -— • Enzyme a c t i v i t y before r e a c t i v a t i o n - O Enzyme a c t i v i t y a f t e r r e a c t i v a t i o n - 95 -X. L a b e l l i n g of Aldehyde Dehydrogenase with ^C-Iodoacetamide  and P a r t i a l P u r i f i c a t i o n of an Active Peptide As mentioned previously, iodoacetate and iodoacetamide i n h i b i t aldehyde dehydrogenase arid there i s s p e c i f i c protection of the enzyme from t h i s i n h i b i t i o n i n the presence of the a c t i v a t i n g ion and coenzyme. It appeared, therefore, that these i n h i b i t o r s were combining with aldehyde dehydrogenase at the active s i t e and that t h i s r e a c t i o n was blocked when the a c t i v a t i n g ion and coenzyme were present. It was thought that the use of radioactive iodoacetamide might demonstrate whether s p e c i f i c s u l f h y d r y l groups on the enzyme were reacting with the i n h i b i t o r and that, i f so, i t might be possible to i s o l a t e a peptide, or peptides, derived from the active s i t e . Aldehyde dehydrogenase was reacted with iodoacetamide-l-^C as stated i n "Materials and Methods". Successive additions of i n h i b i t o r were made u n t i l i n h i b i t i o n of enzymatic a c t i v i t y , was 87%. Aft e r separation of the ^ C - l a b e l l e d protein from the unreacted iodoacetamide by gel f i l t r a t i o n i t was. c a l c u l a t e d that 2.55 moles of i n h i b i t o r had been taken up per mole of enzyme (M.W. 200,000). Almost i d e n t i c a l q uantitative r e s u l t s were obtained i n s i m i l a r experiments. They indi c a t e d that approximately 3 moles of iodoacetamide would be taken up per mole of enzyme at 100%, i n h i b i t i o n of enzymatic a c t i v i t y , whereas 23 c y s t e i c a c i d residues were found per mole of enzyme i n the amino acid a n a l y s i s . In a p a r a l l e l experiment the reaction was c a r r i e d out under i d e n t i c a l conditions except for the presence of 1 mM NAD+. A 60%, - 96 -i n h i b i t i o n of enzymatic a c t i v i t y occurred and 1.56 moles of iodoacetamide were taken up per mole of enzyme. The l a b e l l e d protein was heat denatured and digested extensively with CC -chymotrypsin. Approximately 100%, of the radio-a c t i v i t y was s o l u b i l i z e d . In preliminary experiments i t was observed that t r y p s i n digestion s o l u b i l i z e d only 65% of the r a d i o a c t i v i t y . The chymotryptic digest, dissolved i n 0.2 N pyridine acetate,-pH 3.1, was applied to a column of Bio-Rad AG 50W-X2 at 38° e q u i l i b r a t e d with 0.2 N pyridine acetate, pH 3.1, and eluted with a l i n e a r gradient of 0.2 N pyridine acetate, pH 3.1 and 2 N pyridine acetate, pH 5.0. Figure 27A shows the e l u t i o n of the chymotryptic peptides of aldehyde dehydrogenase reacted with iodoacetamide i n the absence of NAD+, and Figure 27B shows the r e s u l t s obtained when the enzyme was reacted i n the presence of NAD+. In both experiments the area around tube number 40 contained 43%, of the r a d i o a c t i v i t y applied to the column. The remaining 57%, was eluted i n several peaks, none of which had high a c t i v i t y . This indicated that one.species of peptide, probably one involved i n the act i v e s i t e of the enzyme, was l a b e l l e d p r e f e r e n t i a l l y . By comparing the two e l u t i o n curves i n Figure 27A and Figure 27B i t i s evident that the a c t i v i t y , i n t h i s main peak was reduced when the enzyme was reacted with the i n h i b i -tor i n the presence of NAD+. The reduction i n the r a d i o a c t i v i t y was proportional to the reduction of i n h i b i t i o n of enzymatic a c t i v i t y observed. The other areas containing r a d i o a c t i v i t y , with the exception of tubes 10 to 20, were also a f f e c t e d by the presence of NAD+. I t may be that some of these radioactive peaks r e s u l t from incomplete digestion - 97 -FIGURE 27 ION EXCHANGE CHROMATOGRAPHY OF CHYMOTRYPTIC DIGEST OF 1 4C-LABELLED ALDEHYDE DEHYDROGENASE A. The chymotryptic digest of 32.5 mg. aldehyde dehydrogenase, i n h i b i t e d with iodoacetamide-1-^C, was applied to a 1.4 by 34 cm-, column of Bio-Rad AG 50W-X2 i n 7.0 ml of 0.2 N pyridine acetate, pH 3.1. The column was eluted at 38° with a l i n e a r gradient of 0.2 N pyridine acetate, pH 3.1 (350 ml.) and 2 N pyridine acetate, pH 5.0 (350 ml.). F r a c t i o n s of.2.35 ml..were c o l l e c t e d at a flow rate of 0.47 ml. per minute. The column was washed with 2 N pyridine a f t e r completion of the gradient. The r a d i o a c t i v i t y and the color produced i n the ninhydrin r e a c t i o n of the f r a c t i o n s were determined. B. The chymotryptic digest of 30.8 mg. aldehyde dehydrogenase, i n h i b i t e d with iodoacetamide-l--'"^C i n the presence of 1 mM NAD+ was chromatographed as i n A. The r a d i o a c t i v i t y of the f r a c t i o n s was determined. R a d i o a c t i v i t y _ _ _ _ _ _ _ Color with Ninhydrin - 98 -with CY-chymotrypsin or they may be derived from d i f f e r e n t sites.on the enzyme which are also affected i n t h e i r r e a c t i v i t y towards iodo-acetamide by the presence of NAD+. As evidenced from the ninhydrin assay, the active f r a c t i o n , tubes 37 to 42 Figure 27A, contained only a r e l a t i v e l y small f r a c t i o n of the t o t a l material applied to the column. This indicated a high p u r i f i c a t i o n of the active peptide or peptides. To obtain more information about the s i z e and, or, number of active peptides, the main peak was pooled and subjected to gel f i l t r a t i o n on Sephadex G-25. Figure 28 shows the e l u t i o n of the active f r a c t i o n . The r a d i o a c t i v i t y was eluted i n a s i n g l e peak 156 ml. a f t e r the void volume. Under i d e n t i c a l conditions S-amidomethylcysteine was eluted at 216 ml. a f t e r the void volume. Unless charge e f f e c t s influenced the e l u t i o n of the peptide, the molecular weight i s of the order of 1000 to 2000. The active f r a c t i o n was applied again to the same Sephadex G-25 column a f t e r t r y p s i n d i g e s t i o n and eluted as before. The a c t i v i t y was eluted i n a s i n g l e peak and i n the same p o s i t i o n as before the t r y p s i n treatment. This indicated that the s i z e of the peptide was not s i g n i f i c a n t l y reduced by t r y p s i n d i g e s t i o n . Therefore, the peptide material of high r a d i o a c t i v i t y obtained a f t e r chymotryptic d i g e s t i o n has one p a r t i c u l a r charge as evidenced: from ion exchange chromatography, has a unique molecular s i z e , and i s not a f f e c t e d by t r y p t i c d i g e s t i o n . Most l i k e l y , the l a b e l l e d material consists of one peptide species or of several very s i m i l a r peptide species. - 99 -FIGURE 28 GEL FILTRATION OF ^C-LABELLED CHYMOTRYPTIC.PEPTIDE ;The r a d i o a c t i v e material i s o l a t e d by. ion exchange chromato-graphy (tubes 37-42 Figure 27A) was applied i n 3.0 ml. to a 2.5 by 94 cm. Sephadex G-25 column.and.eluted with 0.05 M a c e t i c a c i d . Fractions of 3.8 ml. were c o l l e c t e d a f t e r 150. ml of solvent had passed through the column. The flow rate was 1.0 ml. per minute. R a d i o a c t i v i t y is.expressed as counts per minute per f r a c t i o n . The void volume ( V 0 ) and the point of e l u t i o n of S-amidomethylcysteine ( S-AMC ) are i n d i c a t e d . -100 -DISCUSSION The potassium-activated aldehyde dehydrogenase of P. aeruginosa has a major r o l e i n the metabolism of alcohols - as indicated by the induction of t h i s enzyme when the organism i s grown with ethanol or ethylene g l y c o l as the carbon source. Five other s t r a i n s of the genus Pseudomonas also contained a potassium-activated aldehyde dehydrogenase when grown under s i m i l a r conditions and t h i s suggests the general presence of t h i s enzyme i n pseudomonads. However, other aldehyde dehydrogenases have been found i n t h i s genus. Jakoby (1958a) reported a phosphate-requiring enzyme from P. fluorescens grown on ethylene g l y c o l ; and P. aeruginosa grown on p a r a f f i n hydrocarbons, which are metabolized through the alcohol and aldehyde, has an aldehyde dehydro-genase r e q u i r i n g Fe"1-1" or Ca~^~ (Heydeman and Azoulay, 1963). Acetobacter  suboxydans, often regarded as c l o s e l y r e l a t e d to the pseudomonads, has ' an aldehyde dehydrogenase which has no i o n i c requirements. It also prefers NADP+ as the coenzyme whereas the three enzymes from Pseudomonas use NAD+ p r e f e r e n t i a l l y (King and Cheldelin, 1956). P. aeruginosa produces an alcohol dehydrogenase, and acetaldehyde can be regarded as the natural substrate f o r aldehyde dehydrogenase when the organism i s grown on ethanol. Acetaldehyde i s a very v o l a t i l e substrate and, although the enzyme i s strongly i n h i b i t e d by low concentrations of acetaldehyde i n v i t r o , r e l a t i v e l y high concentrations of t h i s substrate i n the culture may be necessary for maximum production of aldehyde dehydrogenase. This i s indicated by the f a c t that the highest s p e c i f i c a c t i v i t y of t h i s enzyme was obtained i n s t i l l cultures as com-pared to shaken or aerated c u l t u r e s . - 101 -The conditions under which the organism was grown made i t possible to obtain large amounts of aldehyde dehydrogenase i n c e l l extracts and the discovery of the protective e f f e c t of b i s u l f i t e made i t possible to p u r i f y the enzyme with a high y i e l d . The p u r i f i c a t i o n procedure f i n a l l y devised consists of r e l a t i v e l y few simple steps and a homogeneous preparation i s obtained a f t e r approximately twenty-f o l d p u r i f i c a t i o n . I t can be cal c u l a t e d that the enzyme constituted about four per cent of the soluble proteins i n the c e l l . The p o s s i b i l i t y of obtaining large amounts of this enzyme i n p u r i f i e d form makes t h i s protein e s p e c i a l l y a t t r a c t i v e f o r studies of physi c a l properties and as an a n a l y t i c a l t o o l for the detection or quantitative measurements of aldehyde. The homogeneity of the f i n a l p u r i f i e d pro-duet i s indicated by u l t r a c e n t r i f u g e data and electrophoresis.6f,the a c t i v e , associated molecule. These two c r i t e r i a for homogeneity are supported by the e l u t i o n p r o f i l e s from ion exchange c e l l u l o s e and gel f i l t r a t i o n as we l l as by the homogeneity of the preparation at pH 3.0 and on i s o e l e c t r i c p r e c i p i t a t i o n . In add i t i o n , other methods for protein f r a c t i o n a t i o n d i d not increase the s p e c i f i c a c t i v i t y of the p u r i f i e d enzyme preparation. The potassium-activated aldehyde dehydrogenase of P. aeruginosa shares several of the properties common to other aldehyde dehydrogenases. I t has a wide substrate s p e c i f i c i t y , and substrate i n h i b i t i o n was observed with a l l aldehydes tested except glyceraldehyde and benz-aldehyde. This i n h i b i t i o n was e s p e c i a l l y noticeable with acetaldehyde and propionaldehyde. The s p e c i f i c i t y and substrate i n h i b i t i o n s are - 102 -si m i l a r to those observed f or the potassium-activated aldehyde dehydro-genase from yeast (Black, 1951) and the phosphate-requiring enzyme from P. fluorescens (Jakoby, 1958a). The s p e c i f i c i t y d i f f e r s from that of the aldehyde dehydrogenase induced i n P. aeruginosa by growth on p a r a f f i n hydrocarbons which i s most active on a l i p h a t i c aldehydes containing s i x to eight carbon atoms and does not oxidize glycer-aldehyde (Heydeman and Azoulay, 1963) and the aldehyde dehydrogenase of Acetobacter suboxydans which i s most active with acetaldehyde, propionaldehyde and butyraldehyde but does not oxidize glycolaldehyde or glyceraldehyde (King and Cheldelin, 1956). In the presence of glycolaldehyde, NAD+ i s reduced by the enzyme from P. aeruginosa at a much higher rate than NADP"*". This i s also r e f l e c t e d i n the great diff e r e n c e i n the Michaelis constants f o r the two hydrogen acceptors. This i s the case with most other aldehyde dehydrogenases except for the Mg - activated yeast enzyme (Seegmiller, 1953), and the Acetobacter enzyme (King and Cheldelin, 1956), and various semialdehyde dehydrogenases. A reducing agent such as 2-mercaptoethanol, cysteine, d i t h i o -t h r e i t o l or 2,3-dimercaptopropanol i s required by the enzyme. Similar requirements were reported f o r other aldehyde dehydrogenases (Black, 1951; Jakoby, 1958a; Heydeman and Azoulay, 1963). In studies of the e f f e c t of mono- and dimercaptans on the aldehyde dehydrogenases from P. fluorescens the dimercaptans were found to be much better reducing agents (Jakoby, 1958a). These r e s u l t s and further studies with other - 103 -aldehyde dehydrogenases and of the e f f e c t of arsenite led to the conclusion that two c l o s e l y - p o s i t i o n e d s u l f h y d r y l groups are involved i n the active s i t e (Jakoby, 1958b). Similar observations were made with the enzyme studied here. Dimercaptans were much more e f f e c t i v e i n the enzyme assay at low concentrations than 2-mercaptoethanol, a l -though cysteine, a monoraercaptan, was very e f f e c t i v e for obtaining maximum a c t i v i t y at r e l a t i v e l y low concentrations. Arsenite i n h i b i t s t h i s enzyme i n the presence of 2-mercaptoethanol and the i n h i b i t i o n i s reversed by d i t h i o t h r e i t o l . Thus, the postulation of c l o s e l y - p o s i t i o n e d s u l f h y d r y l groups at the active s i t e can be extended to the potassium-activated aldehyde dehydrogenase from P. aeruginosa. In view of the c l o s e l y - p o s i t i o n e d s u l f h y d r y l groups at the active s i t e , reducing agents seem to have a s p e c i a l importance i n keeping these f u n c t i o n a l groups i n the reduced state. D i s u l f i d e : ' formation between the two s u l f u r atoms, which would lead to loss of enzymatic a c t i v i t y could e a s i l y be v i s u a l i z e d unless they are positioned on a r i g i d surface of the enzyme, fa r enough apart to prevent t h i s r e a c t i o n . Chelating agents such as EDTA and o-phenanthroline were found to replace the reducing agent to a considerable degree and almost 507« of the maximum enzymatic a c t i v i t y could be obtained with EDTA. The f a c t that the a c t i v i t i e s obtained with EDTA and reducing agent are not additive i n d i c a t e s that t h e i r a c t i o n i s most l i k e l y on the same s i t e . The enzyme i s most ac t i v e i n the reduced state and when protected from metal ion i n h i b i t i o n . Both conditions can be brought about by mercaptans whereas chel a t i n g agents only protect the enzyme from metal ion i n h i b i t i o n . - 104 -I n h i b i t i o n of enzymatic a c t i v i t y by EDTA has not been observed eit h e r i n the presence or absence of reducing agent. Ortho-phenanth-r o l i n e , however, although stimulating enzymatic a c t i v i t y , i n the absence of reducing agents, i s a strong i n h i b i t o r i n the presence of 2-mercaptoethanol. A s i m i l a r observation was made with aldehyde dehydrogenases from yeast and l i v e r by Stoppani, Schwarcz and Freda (1966). Their k i n e t i c data suggested the involvement of zinc i n both enzymes and analysis of a yeast aldehyde dehydrogenase pre-paration showed s i g n i f i c a n t amounts of z i n c , although quantitative data were not presented. The i n h i b i t i o n of enzymatic a c t i v i t y by o-phenanthroline i s often i n d i c a t i v e of the presence of z i n c , as f o r example with alcohol dehydrogenase (Sund and T h e o r e l l , 1963) and l a c t i c dehydrogenase (Schwert and Winer, 1963). However, although o-phenanthroline causes i n h i b i t i o n and d i s s o c i a t i o n of glutamic dehydro-genase as with yeast alcohol dehydrogenase, non-chelating analogues of o-phenanthroline also i n h i b i t the enzyme and, therefore, i n h i b i t i o n by o-phenanthroline may not n e c e s s a r i l y mean, that zinc i s a constituent of the enzyme (Yielding and Tomkins, 1962). In contrast to the potassium-activated aldehyde dehydrogenase of yeast, analysis of the potassium-activated aldehyde dehydrogenase of P. aeruginosa showed the enzyme did not contain z i n c , although the i n h i b i t i o n with o-phenan-th r o l i n e appears s i m i l a r . Since zinc i s believed to be the s i t e of attachment of the coenzyme i n the yeast enzyme, t h i s would indicate a major d i f f e r e n c e i n the a c t i v e s i t e of the two enzymes. - 105 -P. fluorescens grown on ethylene g l y c o l produces a phosphate-or arsenate-requiring aldehyde dehydrogenase (Jakoby, 1958a). At the beginning of the studies on the aldehyde dehydrogenase from P. aeruginosa i t was believed that the enzyme was s i m i l a r or i d e n t i c a l to that of P. fluorescens since potassium phosphate was e s s e n t i a l f o r a c t i v i t y . However, further studies on the i o n i c requirements f o r a c t i v i t y showed that t h i s response was due to an absolute requirement for the c a t i o n , potassium, which could be replaced by ammonium or, less e f f e c t i v e l y , by rubidium. The requirement for potassium .was not unique for the enzyme from t h i s organism, since.aldehyde dehydrogenases from f i v e other pseudomonads showed a s i m i l a r requirement. The potassium s a l t of phosphate was used i n the report on the phosphate-r e q u i r i n g enzyme from P. fluorescens but the author does not spec i f y the e f f e c t of other phosphate s a l t s or report any studies to d i s t i n -guish between the e f f e c t of the c a t i o n and anion. I t would be of i n t e r e s t to know more about the r e l a t i o n s h i p of these two enzymes since the properties of the enzyme from P. aeruginosa are otherwise i n agreement with those which have been reported for the phosphate-r e q u i r i n g enzyme from P. fluorescens. The enzyme from P. aeruginosa seems to be s i m i l a r to the pot-assium-activated yeast enzyme (Black, 1951) i n i o n i c requirements, except that ammonium i s a better a c t i v a t i n g ion than rubidium whereas the opposite was reported for the yeast enzyme. According to Kachmar and Boyer (1953) comparison of the i o n i c r a d i i of hydrated a l k a l i metal and ammonium ions shows that potassium, rubidium and ammonium ions have approximately the same r a d i i i n contrast to sodium and l i t h i u m . - 106 -Stoppani and M i l s t e i n (1957) reported that any one of potassium, NAD+, or acetaldehyde protected the potassium-activated yeast aldehyde dehydrogenase from inhnhLtion by t h i o l reagents. Sorger and Evans (1966) showed that monovalent cations, e s p e c i a l l y the a c t i v a t i n g :ions, had a favorable e f f e c t on the s t a b i l i t y of t h i s enzyme and suggested that the ions may cause a physi c a l rearrangement of the protein. In the present work, i n h i b i t i o n and protection studies with iodoacetate and iodoacetamide conducted with the aldehyde dehydrogenase of P. aeruginosa show that potassium alone does not protect the enzyme but accelerates i n h i b i t i o n . NAD+ alone also had nooeffect, whereas the combination of potassium (or ammonium) and NAD+ or NADH gave good protection from iodoacetate i n h i b i t i o n . This e f f e c t i s as s p e c i f i c as the requirement f o r c a t a l y s i s of aldehyde oxidation. It i s of in t e r e s t i n t h i s respect that maximum enzymatic a c t i v i t y i n the assay i s obtained when the enzyme i s incubated with the a c t i v a t i n g ion, reducing agent and NAD+ before the aldehyde i s added to the reac t i o n mixture. A p a r a l l e l observation was made by Jakoby (1958a) for the assay of the enzyme from P. fluorescens. It appears that potassium activates the enzyme, which on the one. hand may lead to f a s t e r iodo-acetate i n h i b i t i o n , , and on the other f a c i l i t a t e s binding of the co-enzyme to the active s i t e . The l a t t e r reaction d i r e c t l y , or i n d i r e c t l y , blocks the re a c t i o n with the t h i o l reagent. That a ph y s i c a l rearrangement of the enzyme takes place i n the presence of the a c t i v a t i n g ion and coenzyme i s borne out by the f a c t that the presence of potassium and NAD also protects the enzyme from t r y p s i n d i g e s t i o n and heat i n a c t i v a t i o n . A non-activating c a t i o n , such - 107 -as sodium, may bring about a s l i g h t conformational change as evidenced by s l i g h t l y greater resistance to tr y p s i n digestion, but the e f f e c t i s less than with potassium, while a c t i v a t i o n and binding of NAD + are not f a c i l i t a t e d . In contrast, i t i s i n t e r e s t i n g to note that i n the case of su c c i n i c semialdehyde dehydrogenase (Nirenberg and Jakoby, 1960) the presence of the coenzyme leads to a more rapid d i g e s t i o n of the enzyme with t r y p s i n . There are a great number of enzymes of d i f f e r e n t function which are activ a t e d by potassium and, or, ammonium (Dixon and Webb, 1964) but with r e l a t i v e l y few of these could a c t i v a t i o n be r e l a t e d to NAD+ or NADP+ binding. This could be i n l i n e with the r e s u l t s obtained here, namely that the a c t i v a t i o n of the enzyme molecule by the ion i s an independent step and must preceed the binding of the coenzyme. The conformational change caused by the a c t i v a t i n g . i o n may be due to n e u t r a l i z a t i o n of a s p e c i f i c charge on the enzyme molecule. Whether the negatively charged active peptide i s o l a t e d a f t e r treatment with •^C-l a b e l l e d iodoacetamide has any s i g n i f i c a n c e i n view of a c t i v a t i o n by cations, or i s a mere coincidence, remains to be demonstrated. The r o l e of the ion i n a c t i v a t i n g the enzyme has to take into account the s t r i c t l i m i t s of size of the cations which are e f f e c t i v e . As to the points of attachment of the coenzyme one can, at th i s stage, only suggest that they may be near or at the s u l f h y d r y l groups which are i n h i b i t e d by rea c t i o n with iodoacetamide. I n s t a b i l i t y of aldehyde dehydrogenases was a widely observed c h a r a c t e r i s t i c of t h i s group of enzymes. This was e s p e c i a l l y true of the potassium-activated yeast enzyme which deteriorated within a few - 108 -hours a f t e r being brought to a c e r t a i n stage i n the p u r i f i c a t i o n (Black, 1955). At the beginning of the studies on the enzyme from P. aeruginosa i n s t a b i l i t y was also a great problem but was overcome by the use of b i s u l f i t e b u f f e r . B i s u l f i t e was used at pH 7.0 where the two active species, HSO3 and SO-", are present i n approximately equal amounts (Bennet, Swoboda, and Massey, 1966). The b i s u l f i t e ion i s w e l l known fo r the formation of addition products with the aldehyde group. It also forms add i t i o n products with pyridine nucleotides and t h e i r d e r i v a t i v e s and these complex to protein molecules ( P f l e i d e r e r , J e c k e l , and Wieland, 1956; C i a c c i o , 1966). I n h i b i t i o n of enzyme reactions with b i s u l f i t e was reported for several dehydrogenases when r e l a t i v e l y low concentrations of t h i s compound were used (Ciaccio, 1966). Inhi-b i t i o n by b i s u l f i t e , although not studied i n d e t a i l , has not been observed with aldehyde dehydrogenase. This may be due to the large amounts of NAD"1" used i n the assay and to the presence of excess aldehyde substrate. The reducing capacity of a b i s u l f i t e - s u l f i t e mixture i s ascribed to the s u l f i t e ion (Creeth and Nic h o l , 1960; C e c i l and Loening, 1960). Although a s t a b i l i z a t i o n of the aldehyde dehydrogenase by formation of addi t i o n products cannot be ruled out, the reducing capacity of t h i s reagent i s the most l i k e l y reason f o r the s t a b i l i z a t i o n e f f e c t . C e c i l and Wake (1962) studied the reaction of the d i s u l f i d e bond i n a v a r i e t y of proteins. A l l i n t e r c h a i n d i s u l f i d e bonds and some in t r a c h a i n d i -s u l f i d e bonds were broken under conditions s i m i l a r to those where aldehyde dehydrogenase i s s t a b i l i z e d by a b i s u l f i t e - s u l f i t e mixture. - 109 -According to the amino acid a n a l y s i s , there are twenty-three or twenty-four p o t e n t i a l s u l f h y d r y l groups per enzyme molecule. The i n h i b i t i o n studies with ^C-iodoacetamide indicate that there are r e l a t i v e l y few s u l f h y d r y l groups reacting under the conditions tested and t h i s might be taken to i n d i c a t e that most cysteine moieties are involved i n d i s u l f i d e bond formation. The great s t a b i l i t y of the enzyme i n b i s u l f i t e buffer even at elevated temperatures, however, points to the f a c t that there are no i n t e r c h a i n and p o s s i b l y few or no i n t r a c h a i n d i s u l f i d e bonds i n the active molecule. The requirement of reducing agents for a c t i v i t y and the loss of enzymatic a c t i v i t y on d i a l y s i s without reducing agents, which would lead to d i s u l f i d e bridge formation, i s i n accord with t h i s , although more study i s required. Although s p e c i f i c tests for carbohydrate i n the p u r i f i e d aldehyde dehydrogenase have not been. carried', out, the r e s u l t s of the amino acid a n a l y s i s , the absorbance spectrum and r e l a t i o n of absorbance to dry weight and the nitrogen content of the preparation i n d i c a t e that the enzyme i s not associated with carbohydrate or other non-protein material. The general p h y s i c a l c h a r a c t e r i s t i c s of aldehyde dehydrogenase are those of a t y p i c a l globular p r o t e i n , being insoluble at low and high s a l t concentrations. I t i s negatively charged at neutral pH and soluble i n moderately high s a l t concentrations and i t i s soluble below the i s o e l e c t r i c point. The increase i n v i s c o s i t y when the enzyme s o l u t i o n i s d i s s o l v e d below the i s o e l e c t r i c point indicated an unfolding of the globular p r o t e i n . -110 -In r e l a t i v e l y high s a l t concentrations (0.1 M) aldehyde dehydro-genase shows one component i n the u l t r a c e n t r i f u g e and one major band upon starch gel electrophoresis. In low s a l t concentrations, however, a second component with a lower S value i s observed i n the u l t r a -centrifuge and a second band with f a s t e r electrophoretic m o b i l i t y appears upon electrophoresis. A change of the S value alone could have res u l t e d from a d i s s o c i a t i o n of the enzyme or from an unfolding of the molecule, which also would account for a decrease i n density. That these phenomena: are not always c l e a r l y distinguishable unless c a r e f u l studies are made of d i f f u s i o n constants, increase i n v i s c o s i t y and sedimentation c o e f f i c i e n t s was pointed out by Yagi and Ozawa (1962) th e i r studies of D-amino acid oxidase. In the case of aldehyde dehydro genase, an increase of v i s c o s i t y was observed but other physical data were d i f f i c u l t to obtain since the apparent d i s s o c i a t i o n did not go to completion. However, the appearance of a band i n electrophoresis with f a s t e r m o b i l i t y was a good i n d i c a t i o n that ~a smaller molecule was obtained. Starch gel i s known to act as a molecular sieve (Smithies, 1959b) and a smaller molecule with the same charge as a larger one i s expected to have a f a s t e r m o b i l i t y . An unfolded protein on the other hand would be retarded unless the unfolding were outweighed by an increase i n charge. The r e s u l t s obtained therefore indicated a p a r t i a l d i s s o c i a t i o n of the active aldehyde dehydrogenase, probably into two subunits of equal structure. This d i s s o c i a t i o n i s reversed by the addition of s a l t to the protein s o l u t i o n followed by incubation at elevated temperature; - I l l -A d i s s o c i a t i o n and ass o c i a t i o n of thyroglobulin, dependent on i o n i c strength and observable i n starch gel electrophoresis and the u l t r a c e n t r i f u g e with s i m i l a r patterns as those for aldehyde dehydro-genase, was reported by Spiro (1961). Ionic strength- dependent d i s s o c i a t i o n and a s s o c i a t i o n has been observed also with proteins l i k e chymotrypsins, hemoglobins, hexokinase, i n s u l i n and thyroglobulins ( R e i t h e l , 1963). D i s s o c i a t i o n at low i o n i c strength could be explained by e l e c t r o s t a t i c repulsion of i o n i c groups at low s a l t concentration. Tanford (1964) points out that inorganic s a l t s would favor d i s s o c i a t i o n i f most newly exposed groups are h y d r o p h i l i c , but would i n h i b i t i t i f newly exposed groups are hydrophobic. According to t h i s , aldehyde dehydrogenase would be expected to expose hydrophobic groups upon d i s s o c i a t i o n , which may perhaps be responsible f o r the o r i g i n a l binding of the subunits. The binding of the subunits of aldehyde dehydrogenase by hydrophobic i n t e r a c t i o n would be i n agreement with the temperature dependance of the a s s o c i a t i o n (Kauzmann,1959). The low sedimentation c o e f f i c i e n t which was observed when aldehyde dehydrogenase was dissolved at pH 3.0 was accompanied by a very high v i s c o s i t y and a r e l a t i v e l y low d i f f u s i o n c o e f f i c i e n t as compared to the values obtained f or the enzyme at pH 7.0. This indicated an extensive unfolding of the molecule. A molecular weight of approximately one-half that of the active molecule was estimated for this material and therefore i t may represent the unfolded subunits r which are observed upon d i s s o c i a t i o n i n low i o n i c strength buffer. - 112 -Although there was some d i f f i c u l t y i n assessing the s p e c i f i c a c t i v i t y of a d i s s o c i a t e d enzyme preparation since assay conditions permit r e a s s o c i a t i o n , i t could be shown that enzyme a c t i v i t y i s l o s t upon d i s s o c i a t i o n and that i t i s regained a f t e r r e a s s o c i a t i o n . Furthermore i t could be demonstrated that the subunit, i s o l a t e d a f t e r starch gel electrophoresis, has no enzymatic a c t i v i t y unless re-associated. The enzymatic a c t i v i t y of aldehyde dehydrogenase i s therefore dependent on the union of at least two smaller molecules to form the active enzyme. Whether t h i s d i s s o c i a t i o n - a s s o c i a t i o n has any p h y s i o l o g i c a l s i g n i f i c a n c e i n regulating enzymatic a c t i v i t y within the c e l l can not be determined at t h i s stage. I t does not appear l i k e l y unless metabolites are found to have an influence on the d i s s o c i a t i o n and a s s o c i a t i o n of the enzyme. The studies with radioactive iodoacetamide led, a f t e r chymo-t r y p t i c d i g e s t i o n , to the p a r t i a l p u r i f i c a t i o n of a h i g h l y - l a b e l l e d material with a unique charge, si z e and resistance to further digestion by t r y p s i n . Although further tests are required, i t i s believed to be one peptide or several very s i m i l a r peptide species. Jakoby (1958b) postulated that aldehyde dehydrogenases have two c l o s e l y - p o s i t i o n e d s u l f h y d r y l groups at the active s i t e . The r e s u l t s reported here indicate two possible arrangements of these s u l f h y d r y l groups. They could e i t h e r be located very close to each other within one peptide chain or they could be positioned on two s i m i l a r or i d e n t i c a l peptide chains and brought together by the p o s i t i o n i n g of subunits to give the active molecule. This l a t t e r p o s s i b i l i t y seems l i k e l y i n view of the - 113 -apparent d i s s o c i a t i o n of the aldehyde dehydrogenase into two in a c t i v e subunits. The t h i r d p o s s i b i l i t y , that the two s u l f h y d r y l groups are positioned i n the same peptide chain removed from each other and brought together by f o l d i n g of the peptide, seems less l i k e l y unless they are located i n very s i m i l a r amino acid sequences, since two peptides of high r a d i o a c t i v i t y would be expected i n digests of the protein i f t h i s were the case. Although time and f a c i l i t i e s to investigate the ac t i v e peptide of the aldehyde dehydrogenase of P. aeruginosa i n more d e t a i l were l i m i t i n g , the approach used here seems promising and might lead to the i d e n t i f i c a t i o n of the amino acids and t h e i r sequence around the act i v e s i t e as w e l l as to the e l u c i d a t i o n of the p o s i t i o n of the active s u l f h y d r y l groups i n the protein. 114 -SUMMARY Aldehyde dehydrogenase from P. aeruginosa was p u r i f i e d a f t e r growth of the organism on ethanol as the carbon source. The enzyme was found to be present i n c e l l extracts i n large amounts and a homogeneous enzyme preparation was obtained i n high y i e l d a f t e r an eighteen-to twenty-fold p u r i f i c a t i o n . The aldehyde dehydrogenase i s s t a b i l i z e d by b i s u l f i t e buffer at pH 7.0. A wide v a r i e t y of aldehydes are oxidized by the enzyme. NAD+ i s the preferred hydrogen acceptor and the enzyme i s much less active with NADP+. The enzyme requires potassium or ammonium ions f o r cat-a l y t i c a c t i v i t y . Rubidium ions are less e f f e c t i v e . Aldehyde dehydrogenases from f i v e other Pseudomonas species also required potassium ions for enzyme a c t i v i t y . In addition to the a c t i v a t i n g ion, a reducing agent i s required and th i s can be replaced to some extent by EDTA or ortho-phenanthroline. In the presence of 2 -mercaptoethanol ortho-phenanthroline i n h i b i t s aldehyde dehyrogenase. However, zinc was not found to be present i n p u r i f i e d aldehyde dehydrogenase. The enzyme was i n h i b i t e d by iodoacetate, iodoacetamide, a r s e n i t e , copper ions and para-chloromercuribenzoate... The i n h i b i t i o n . by iodoacetate and the loss of enzymatic a c t i v i t y due to tr y p s i n d i g e s t i o n was prevented, s p e c i f i c a l l y , by the addition of the a c t i v a t i n g ion and NAD+ or NADH. Potassium alone' seemed to accelerate a l k y l a t i o n and give some protection from t r y p s i n d i g e s t i o n . An a c t i v a t i o n of the enzyme molecule by potassium, which must preceed NAD+ binding, was - 115 -implicated. The i n h i b i t i o n by arsenite was reversed by d i t h i o t h r e i t o l . The molecular weight of aldehyde dehydrogenase was determined to be approximately 200,000. In low i o n i c strength buffer the molecule was observed to undergo a p a r t i a l d i s s o c i a t i o n , perhaps into two subunits, with loss of enzymatic a c t i v i t y . The d i s s o c i a t i o n was r e v e r s i b l e by ad d i t i o n of s a l t and incubation at elevated temperatures with recovery of most of the i n i t i a l a c t i v i t y . Below the i s o e l e c t r i c point the enzyme i s soluble but denatured - as evidenced by sedimentation, diffusion,.and v i s c o s i t y studies. The molecular weight of t h i s material appears to be approximately one-half that of the active aldehyde dehydrogenase. The amino acid composition of aldehyde dehydrogenase revealed the presence of r e l a t i v e l y large amounts of a l l common amino acids. The one present i n lowest amount was c y s t e i c acid with twenty-three to twenty-four residues per mole of enzyme. I n h i b i t i o n with iodoacetamide-l-^C indicated the uptake of approximately three moles of i n h i b i t o r per mole of enzyme for 100% i n h i b i t i o n of enzymatic a c t i v i t y . A chymotryptic peptide containing a large percentage of the r a d i o a c t i v i t y was p a r t i a l l y p u r i f i e d . - 11.6 -BIBLIOGRAPHY Anthony, C., and L. J . Zatman. 1964. The microbial oxidation of methanol. 1. 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