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Amine metabolism in mycobacteria isolated from poikilothermic animals. Evelyn, Trevor Patrick Todd 1963

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AMINE METABOLISM I I MYCOBACTERIA ISOLATED FROM POIKILOTHERMIC ANIMALS by TREVOR P.T. EVELYN B.S.A., Ontario A g r i c u l t u r a l College, 1958 M.S.A., Ontario A g r i c u l t u r a l College, I960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department o f Bacteriology and Immunology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1963 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that 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 reference and study. I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n p e r mission. Department of The U n i v e r s i t y of B r i t i s h Columbia,. Vancouver 8, Canada. Date f ^ A f - / ^ g . PUBLICATIONS B a c t e r i o l o g i c a l Studies of Fresh-water Fish, I: I s o l a t i o n of aerobic bacteria from several species of Ontario f i s h . Evelyn, T.P.T. and McDermott, L.A. Canad. J . Microbiol., ]_, 375-382 (1961). The U n i v e r s i t y of B r i t i s h Columbia FACULTY OF GRADUATE STUDIES PROGRAMME OF THE FINAL ORAL EXAMINATION FOR THE DEGREE OF DOCTOR OF PHILOSOPHY of TREVOR PATRICK TODD EVELYN B.S.A., Ontario A g r i c u l t u r a l College, 1958 M.S.A., Ontario A g r i c u l t u r a l College, 1960 (Un i v e r s i t y of Toronto) . TUESDAY, SEPTEMBER 24, 1963, at 3:30 P.M. IN ROOM 201, WESBROOK BUILDING COMMITTEE IN CHARGE Chairman: F.H. Soward C. E. Dolman p.A. Larkin D. C.B. Duff W.J. Polglase W.S. Hoar H.L.A. Tarr External Examiner: R.Y. Stanier U n i v e r s i t y of C a l i f o r n i a Berkeley AMINE METABOLISM IN MYCOBACTERIA ISOLATED FROM POIKILOTHERMIC ANIMALS ABSTRACT Three mycobacterial strains i s o l a t e d from f i s h were studied to determine possible roles that amines might play i n the physiology of these microorganisms, An examination of c e l l s of one mycobacterial s t r a i n which possessed some a b i l i t y to metabolize putrescine when not previously exposed to this com-pound showed that no d i - or polyamines were present. Amines, therefore, may not be involved i n the meta-b o l i c processes occurring in such c e l l s . A number of amines were tested as substrates for mycobacteria. Only putrescine was metabolized by the s t r a i n s tested. The a b i l i t y to u t i l i z e putrescine was enhanced by growing the c e l l s i n the presence of the compound. Studies with i n t a c t c e l l s showed that putrescine was approximately 457, oxidized. During the reaction, the only products excreted were ammonia and carbon dioxide. The former accounted f or 69-767,, of the amino-nitrogen of putrescine and the l a t t e r for at least 37.5% of the putrescine-carbon under CO2- free conditions. Radioactive studies with 1,4-C^ putrescine i n d i -cated that approximately 257, of the was assimilated at a point corresponding to 43-447, of the maximum l e v e l of oxidation. During the reaction, nucleic acid, l i p i d s , and protein were found to be l a b e l l e d . An examination of c e l l - f r e e extracts obtained from putrescine-adapted c e l l s revealed the presence of four enzymes involved i n the u t i l i z a t i o n of putrescine. GRADUATE STUDIES F i e l d of Studys Bacteriology j Pathogenic Microbiology f C.E. Dolman i Directed Studies i n B a c t e r i o l o g i c a l L i t e r a t u r e D.C.B. Duff Topics i n Intermediary Metabolism Enzymology . W.J. Polglase Biochemistry of Proteins and , Carbohydrates W.J. Polglase Other Studies: Biology of Fishes C.C. Lindsey ABSTRACT Three mycobacterial strains i s o l a t e d from f i s h were tested f o r ' t h e i r a b i l i t y to metabolize various amines, supplied i n d i v i d u a l l y as sources of carbon. A series of s i x a l i p h a t i c and two c y c l i c amines was used. Growth occurred only i n putreseine. The a b i l i t y to oxidize putrescine was enhanced by growing the c e l l s i n the presence of t h i s compound. One mycobacterial s t r a i n appeared to possess some a b i l i t y to oxidize putrescine without pre-exposure to the compound. However, when c e l l s of t h i s s t r a i n were examined f o r the presence of putrescine and r e l a t e d polyamines, no such compounds were detected. In the mycobacterial s t r a i n s tested, putrescine did not appear to a f f e c t the rate of endogenous r e s p i r a t i o n . Therefore, i n reporting oxygen consumption due to putrescine, the endogenous oxygen consumption has been subtracted from the t o t a l oxygen uptake. During the oxidation of putrescine, the oxygen uptake curves "broke" at a point corresponding to approximately 43$ of the maximum l e v e l of oxidation. Thereafter, very small increases i n the l e v e l of oxidation occurred, so that by the end of the experiment, over 50$ of the molecule appeared to be assimilated. Up to 69-76$ of the amino-nitrogen of the putrescine molecule was released as ammonia during the reaction, the rest of the nitrogen being assimilated. Measurement of carbon dioxide production i i 14 using 1,4-C putrescine revealed that under CO^-free conditions, at l e a s t 1.5 juM of carbon dioxide were released per of putrescine. 14 When c e l l s were incubated with 1,4-C putrescine, 25$ of the r a d i o a c t i v i t y was assimilated. The remaining 14 r a d i o a c t i v i t y was l i b e r a t e d as C 0 2« Chemical f r a c t i o n a t i o n of the c e l l s indicated that f r a c t i o n s containing l i p i d , n u cleic a c i d and protein were l a b e l l e d . C e l l - f r e e extracts obtained from putrescine-adapted c e l l s contained four enzymes responsible f o r the conversion of putrescine to succinic a c i d . The enzymes occurred i n the 10,000 X G^supernatants of the c e l l - f r e e extracts and were a l l active at pH 9*0. Putrescine was oxidatively deaminated to y i e l d - p y r r o l i n e . The l a t t e r compound was then oxidized to y-aminobutyric acid by a dehydrogenase enzyme requiring IAD or HADP. Gamma-aminobutyric acid underwent a transamination witho<-ketoglutaric acid to y i e l d succinic semialdehyde and glutamic acid. Succinic semialdehyde was oxidized to succinic acid by an NAD- or NADP- requiring dehydrogenase. The s p e c i f i c i t y f o r putrescine, shown by the tested mycobacterial str a i n s , i s discussed. x i ACCTOWXEDGEMEHTS The a u t h o r w o u l d l i k e t o t h a n k D r . D . C . B . D u f f , D e p a r t m e n t o f B a c t e r i o l o g y a n d Immunology, f o r s u p e r v i s i n g t h i s s t u d y . The a u t h o r i s a l s o d e e p l y i n d e b t e d t o D r . C . E . D o l m a n , Head o f D e p a r t m e n t o f B a c t e r i o l o g y a n d Immunology, f o r a l l o w i n g t h i s p r o j e c t t o be c o n d u c t e d i n h i s D e p a r t m e n t , and f o r h i s v a l u a b l e c r i t i c i s m s i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t . G r a t e f u l t h a n k s a r e a l s o e x t e n d e d t o D r . W . J , P o l g l a s e , D e p a r t m e n t o f B i o c h e m i s t r y , f o r h i s h e l p f u l s u g g e s t i o n s , a n d t o D r . J . J . R . C a m p b e l l , D e p a r t m e n t o f D a i r y i n g , f o r h i s g e n e r o u s p e r m i s s i o n t o c o n d u c t t h e r a d i o a c t i v e s t u d i e s i n h i s D e p a r t m e n t . F i n a n c i a l a i d r e c e i v e d f r o m t h e F i s h e r i e s R e s e a r c h B o a r d o f C a n a d a , i s a l s o g r a t e f u l l y a c k n o w l e d g e d . i i i TABLE OF CONTENTS INTRODUCTION 1 REVIEW OF LITERATURE 2 MATERIALS AND METHODS 17 I Organisms 17 II Growth conditions... 18 III Media 18 IV. Preparation of washed c e l l suspensions 19 V Standardization of washed c e l l suspensions..20 VI Preparation of c e l l - f r e e extracts 20 VII Manometric measurements 22 1. Intact c e l l s 22 2. C e l l - f r e e extracts 23 VIII Studies with i n t a c t c e l l s 2 3 1. A b i l i t y of washed c e l l s to oxidize putrescine 23 2. Influence of pre-exposure to putrescine on the oxidation of putrescine 23 3. A b i l i t y of washed c e l l s to oxidize a l i p h a t i c amines 24 4. Amines as sole carbon sources 2 5 5. Occurrence of amines i n mycobacteria....26 6. Influence of putrescine on endogenous r e s p i r a t i o n 27 7. Oxygen, ammonia and carbon dioxide relationships during putrescine oxidation 28 8. I n h i b i t o r studies with i n t a c t c e l l s 31 i v IX: Studies with c e l l - f r e e extracts .33 1. Diamine oxidase enzyme 33 2 . A 1 -pyrroline dehydrogenase 34 3 . y-aminohutyric a c i d - ©C-ketoglutarie acid transaminase 36 4 . Succinic semialdehyde dehydrogenase . . 3 8 X" A n a l y t i c a l Methods 40 1 • Ammonia 40 2. -pyrroline 40 3 . Glutamic acid 41 4 . Succinic a c i d 42 5* 2 , 4-dinitrophenylhydrazones 44 6. Paper chromatography 46 XI Isotopic studies with i n t a c t c e l l s 40, 1. Incubation with substrate 49 2 . Sampling and c e l l f r a c t i o n a t i o n procedure 51 EXPERIMENTAL RESULTS AND DISCUSSION 53 I Studies with i n t a c t c e l l s 53 1. A b i l i t y of washed c e l l s to oxidize putrescine 53 2 . Influence of pre-exposure to putrescine on the oxidation of putrescine 53 3 . A b i l i t y of washed c e l l s to oxidize a l i p h a t i c amines . . . . 5 8 4 . Amines as sole carbon sources 63 5» Occurrence of amines i n mycobacteria. .64 V 6. Influence of putrescine on endogenous r e s p i r a t i o n 68 7. Oxygen, ammonia and carbon dioxide relationships during putrescine oxidation 71 8. Inh i b i t o r studies with i n t a c t c e l l s 80 II Studies with c e l l - f r e e extracts 98 1. Diamine oxidase (D.O.) 98 2. A'-pyrroline dehydrogenase (P.D.) 107 3. y-aminobutyric acid - ©C-ketoglutarie acid transaminase 114 4» Succinic semialdehyde dehydrogenase (S.S.D.) 118 II I Isotopic studies with i n t a c t c e l l s 122 GENERAL CONCLUSIONS AND DISCUSSION 132 APPENDIX 137 BIBLIOGRAPHY 139 v i LIST OF TABLES Table 1 L i s t of cultures obtained for study 17 Table 2 Oxygen uptake by washed c e l l s acting on 0.01 M putrescine at pH 7.2 at 4.5 hours 53 Table 3 E f f e c t of adaptation to amines on t h e i r subsequent oxidation by s t r a i n C.C. cells...5 9 Table 4 Amines as sole carbon sources f o r grovrth... .63 Table 5 E f f e c t of putrescine on endogenous r e s p i r a t i o n of s t r a i n S.C. and N.T.H. c e l l s : one concentration of c e l l s acting on various concentrations of putrescine 69 Table 6 E f f e c t of putrescine on endogenous r e s p i r a t i o n of s t r a i n S.C. c e l l s : three concentrations of c e l l s acting on one concentration of putrescine 70 Table 7 The e f f e c t of various i n h i b i t o r s on the oxygen consumption by s t r a i n S.C. c e l l s acting on 3 . 0 ^ of putrescine at 4 hours 81 Table 8 The e f f e c t of various i n h i b i t o r s on the oxygen consumption by s t r a i n S.C. c e l l s acting on 3.0 nM of putrescine. 84 Table 9 The e f f e c t of arsenite on s t r a i n S.C. c e l l s : accumulation of DPNH-reacting materials 85 Table 10 Ninhydrin-positive areas on chromatograms obtained from HC1 extracts of variously treated s t r a i n C.C. c e l l s . . . . . 92 Table 11 Ninhydrin-positive areas on chromatograms obtained from HC1 extracts of variously treated S.C. c e l l s 94 Table 12 The e f f e c t of heat, on D.O. a c t i v i t y i n c e l l - f r e e extracts 101 v i i T a b l e 13 The e f f e c t s o f v a r i o u s i n h i b i t o r s on D . O . a c t i v i t y i n c e l l - f r e e e x t r a c t s 102 T a b l e 14 A l i p h a t i c a m i n e s a s s u b s t r a t e s f o r D . O . i n c e l l - f r e e e x t r a c t s o b t a i n e d f r o m p u t r e s c i n e - a d a p t e d c e l l s 105 T a b l e 15 The a b i l i t y o f s t r a i n C O . c e l l - f r e e e x t r a c t s t o c a t a l y z e t h e t r a n s a m i n a t i o n b e t w e e n v a r i o u s 6/-amino a c i d s a n d «4 - k e t o g l u t a r i c a c i d 116 T a b l e 16 D i s t r i b u t i o n o f C 1 4 " o c c u r r i n g d u r i n g t h e o x i d a t i o n o f 1,4-C p u t r e s c i n e (3 . 0 ;uM) b y p u t r e s c i n e - a d a p t e d s t r a i n I I . T . N . c e l l s 123 T a b l e 17 I n c o r p o r a t i o n o f C 1 4 " i n t o v a r i o u s N . T . N , c e l l f r a c t i o n s d u r i n g t h e o x i d a t i o n o f 1,4-C"*"4- p u t r e s c i n e (3 . 0 yjM) 124 T a b l e 18 D i s t r i b u t i o n o f i n c o r p o r a t e d C 1 4 " i n v a r i o u s c e l l f r a c t i o n s o b t a i n e d f r o m s t r a i n N . T . N , c e l l s i n c u b a t e d w i t h 1,4-C, p u t r e s c i n e (3 . 0 jM) .....127 v i i i LIST OF FIGURES F i g . 1 Oxidation of putrescine by putrescine-adapted (curve l ) and unadapted (cu.rve 2) s t r a i n N.T.I, c e l l s 54 F i g . 2 Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n CO. c e l l s 55 F i g . 3 Oxidation of putrescine by putreseine-adapted (curve l ) and unadapted (curve 2) s t r a i n S.O. c e l l s ....56 F i g . 4 Diamine oxidase a c t i v i t y i n c e l l - f r e e extracts obtained from putrescine-adapted (curve l ) and unadapted (curve 2) s t r a i n S.C. c e l l s . Reaction mixtures contained i n a volume of 3.0 ml.: C e l l - f r e e extract, 1.0ml., equivalent to 3.0 mg. protein; putrescine, 3.0 pE; t r i s buffer, pH 9.0, 100 uM; HgO 57 F i g . 5 E f f e c t of pre-exposure to spermidine on the subsequent oxidation by s t r a i n C.C. c e l l s of 7.5 ;uM of putrescine (curve 1), 3.25;uM of each of putrescine and 1,3-diaminopropane (curve 2), 7«5 joM of 1,3-diaminopropane (curve 3), and 7.5 >uM of spermidine (curve 4) 61 F i g . 6 Separation of amines by i o n exchange. Samples were eluted from a column of Dowex 50 H+ r e s i n with a l i n e a r l y increasing concentration of HCl(0-1.5 H) 65 F i g . 7 Oxygen consumption and ammonia production during putrescine oxidation by s t r a i n N.T.N, c e l l s . Curve 1: Og uptake with 3.0 jaM putrescine (1.0 jjM/ml.). Curve 2: NH, production with putrescine (l.OjuM/ml.) 72 F i g . 8 Oxygen consumption and ammonia production during putrescine qxidation by s t r a i n S.C. c e l l s . Curve 1: 0 2 uptake with 6.0 jM putrescine (2.0 uM/ml.). Curve 2: NH^ production with putrescine (2.0 jM/wl.)..•.75 i x Fig* 9 Oxygen consumption during the oxidation of 6.0 ;uM of putrescine by s t r a i n C.C. c e l l s 78 F i g . 10 E f f e c t of axsenite on the metabolism of putrescine by s t r a i n S.C. c e l l s . Curve 1: On uptake with 3.0 ;uM putrescine (1.0;uM/ml7) Durve 2: Same as curve 1, but arsenite (5.0 juM/ml.) present. Curve 3s Leakage of pyruvate with putrescine (1.0 >aM/ml.). Curve 4s Same as curve 3, but arsenite (3.0 ^ uM/ml.) present 87 F i g . 11 E f f e c t of arsenite on oxygen consumption and ammonia output by s t r a i n S.C. c e l l s acting on putrescine. Curve Is 0 2 uptake with 3.0 juM putrescine (1.0 .nM/ml.). Curve 2: Same as curve 1, but arsenite (9.0jaM/ml.) present. Curve 3$ NH output with putrescine (1.0, jiM/ml.). Curve 4s Same as curve 3, but arsenite (9.0 JM/TBI.) present 89 F i g . 12 E f f e c t of arsenite on oxygen consumption and ammonia output by s t r a i n C.C. c e l l s acting on putrescine. Curve 1: 0 2 uptake with 3.0 jiM putrescine (l.O /iM/ml.). Curve 2: Same as curve 1, but arsenite (3.0 jiM/ml.) present. Curve 3$ NH* production with putrescine (1.0 yM/ml.) present. Curve 4s Same as curve 3» but arsenite (3.0;aM/ml.) present 90 F i g . 13 The optimum pH f o r diamine oxidase a c t i v i t y . Reactions conducted i n c i t r a t e (pH 4.5-6.0) and phosphate (pH 6.0-8.0) buffers (curve 1) and i n t r i s buffer (pH 7.5-9.0) (curve 2) 103 F i g . 14 Str a i n C.C. d'-pyrroline dehydrogenase a c t i v i t y (pH 9.0;. Curve Is Mercaptoethanol + NAD. Curve 2: Mercaptoethanol + NADP. Curve 3 s NAD. Curve 4s NADP 108 F i g . 15 Str a i n S.C. d ' - p y r r o l i n e dehydrogenase a c t i v i t y (pH 9.0).. Curve Is Mercaptoethanol + NAD. Curve 2: Mercaptoethanol + NADP 108 F i g . 16>.Manometric assay of s t r a i n C.C. t^-pyrroline dehydrogenase a c t i v i t y pH 7.2) 110 Pig. 17 Manometric assay of s t r a i n C.C. -pyrroline dehydrogenase a c t i v i t y (pH 9 . 0 ) 113 Pig. 18 ^-aminobutyric a c i d - oC-ketoglutaric acid optimum pH curve. Glutamic acid synthesized at 30 mins. (curve 1) and at 60 mins. (curve 2) 117 Pig. 19 E f f e c t of pH and reaction time on y-aminobutyric a c i d - oc-ketoglutaric acid transaminase a c t i v i t y 117 Pig. 20 S t r a i n C.C. succinic semialdehyde dehydrogenase a c t i v i t y (pH 9 . 0 ) . Curve 1: NAD. Curve 2: NADP 119 f i g , 21 S t r a i n S.C. succinic semialdehyde dehydrogenase a c t i v i t y (pH 9»0). Curve 1: NAD. Curve 2: NADP 119 Pig. 22 D i s t r i b u t i o n of r a d i o a c t i v i t y during the oxidation of 1,4-C 4 putrescine by putreseine-adapted s t r a i n N.T.N, c e l l s . Reaction mixture supernatant (curve l ) ; combined c e l l f r a c t i o n s (curve 2); c e l l s , calculated as the difference between c e l l s + supernatant and supernatant (curve 3 ) ; c 1 4o 2 ( curve 4 ) ; 0 2 uptake occurring during the reaction (curve 5) "'126 INTRODUCTION A m i n e s a p p e a r t o be n a t u r a l c o n s t i t u e n t s o f b i o l o g i c a l m a t e r i a l s . They a r e w i d e l y d i s t r i b u t e d i n n a t u r e e i t h e r i n f r e e o r c o n j u g a t e d f o r m s . T h i s f a c t , t o g e t h e r w i t h a n i n c r e a s i n g a w a r e n e s s as t o t h e m u l t i p l i c i t y o f e f f e c t s t h a t a m i n e s h a v e on b i o l o g i c a l s y s t e m s , h a s r e c e n t l y s t i m u l a t e d a s u b s t a n t i a l amount o f r e s e a r c h on t h e p o s s i b l e r o l e s t h a t amines may p l a y i n b i o l o g y . A r a p i d l y g r o w i n g l i t e r a t u r e i n d i c a t e s t h a t a m i n e s a r e r e q u i r e d f o r , o r s t i m u l a t o r y f o r , t h e g r o w t h o f some m i c r o o r g a n i s m s ; t h a t amines may i n some manner be i n v o l v e d w i t h n u c l e i c a c i d s a n d t h a t t h e y may p l a y a p a r t i n m a i n t a i n i n g t h e s t r u c t u r a l i n t e g r i t y of . f r a g i l e b i o l o g i c a l u n i t s . The compounds a r e a l s o known t o h a v e a number o f o t h e r p h y s i o l o g i c a l and p h a r m a c o l o g i c a l e f f e c t s b u t i f a s p e c i f i c p h y s i o l o g i c a l r o l e e x i s t s f o r t h e s e s u b s t a n c e s , i t s t i l l r e m a i n s t o be d i s c o v e r e d . T h i s s t u d y was t h e r e f o r e u n d e r t a k e n i n a n a t t e m p t t o d i s c o v e r t h e p o s s i b l e r o l e s t h a t a m i n e s may p l a y i n t h e p h y s i o l o g y o f m y c o b a c t e r i a i s o l a t e d f r o m p o i k i l o t h e r m i c a n i m a l s . C e l l s were t e s t e d i n i t i a l l y f o r t h e i r a b i l i t y t o m e t a b o l i z e p u t r e s c i n e a n d c e r t a i n o t h e r n a t u r a l l y - o c c u r r i n g a m i n e s . I n a d d i t i o n , c e l l s were e x a m i n e d f o r t h e p r e s e n c e o f a m i n e s . 2 REVIEW OF LITERATURE  Distribution of amines Normal animal tissues contain significant concentrations of the polyamines spermine and spermidine (1,23,24,35,42,82, 116) i n addition to smaller.amounts of the aliphatic diamines 1,3-diaminopropane, 1,4-diaminobutane (putrescine) and 1,5-diaminopentane (cadaverine) (1,116). In these tissues the amines appear to be free although Kosaki et a l (53) have reported the presence of a possible spermine conjugate isolated from malignant tissues. Furthermore, the enzyme, transglutaminase, exists i n many.animal tissues and i s capable of catalyzing the exchange of the amide group of some proteins with a number of amines. It i s therefore possible that protein-amine conjugates are more widespread i n animal tissues than i s now realized (14,15,66,67,69,70,86). In microorganisms, substantial amounts of spermine, spermidine, 1,3-diaminopropane and putrescine have been reported i n the free form (8,17,31,32,37,38,107,116). Gram-negative bacteria contain, i n general, much higher concen-trations of amines than Gram-positive bacteria and yeasts (37,38,107). Conjugated amines, are more frequently encountered i n microorganisms than i n animal tissues. Acetylated derivatives of putrescine, spermidine and spermine have been reported for Escherichia c o l i and Staphylococcus aureus (22,79,80). The former organism i s also reported to produce a peptide containing spermidine ( 2 1 ) . Among the viruses, signi-ficaht amounts of putrescine and spermidine, have been reported for certain phages of E. c o l i (2,3»49) while the apparent lack of amines i n other bacteriophages and plant viruses (2) may have been due to the release of the amines to the suspending medium. Plant materials also contain amines. Herbst and Snell (36) have reported the presence of putrescine i n orange juice. Amines as growth factors and stabilizers Putrescine serves as a growth factor for a mutant of Aspergillus nidulans (95.) and for the bacterium Haemophilus  parainfluenzae (36). Amine requirements have also been reported for Neisseria perflava (64), Pasteurella tularensis (112) and Achromobac.ter fi s c h e r i ( 2 2 ) . Spermine, spermidine and putrescine stimulate the growth of Lactobacillus casei (5l)« With P. tularensis. N. perflava and A. fischeri the growth-furthering effect of the added polyamine appears not to be due to specific growth factor activity, but rather to a decrease i n the sensitivity of the microorganism to osmotic l y s i s conferred by the polyamine (55,56,57,59)• The stabilizing effect of amines has been demonstrated for protoplasts of E. c o l i , Micrococcus lysodeikticus and Pseudomonas aeruginosa (33,56,99,100,101). Mitochondrial swelling induced by hypotonic media and various chemicals has i n many cases been inhibited by various poly- and diamines 4 (39,100,101). The d i s s o c i a t i o n of ribosomes in t o t h e i r various components has been i n h i b i t e d by spermidine (17). S t a b i l i z a t i o n of phage preparations against shock has also been accomplished with 1,5-diaminopentane (27) and spermine and spermidine (105). A s i m i l a r resistance to shock i s conferred on the transforming UNA5 preparation of B a c i l l u s  s u b t i l i s by spermine, spermidine and putrescine (104). Although the exact mechanism of the s t a b i l i z a t i o n e f f e c t i s not known, i t i s probable that the high concentrations of phospholipids i n b a c t e r i a l protoplasts and mitochondria allow the formation of stable phospholipid-amine complexes. Similar complexing of the nucle i c acids of phages, transforming p r i n c i p l e s and ribosomal ribonucleic a c i d with amines could also conceivably produce more s t a b i l i z e d structures. Such complexes are not d i f f i c u l t to conceive of since the ca t i o n i c amines should be attracted to anionic materials l i k e phospholipids and nucleic acids. Indeed, such complexes have r e a d i l y been demonstrated (2 ,25,27,50,60,61,77). Other e f f e c t s of amines - Apart from t h e i r growth-promoting properties* amines have been shown to protect against the adverse e f f e c t s of drugs and chemicals i n bacteria, yeast c e l l s and phage (13, 49,65 ,92,96). On the other hand, amines or t h e i r degradation products exert toxic e f f e c t s on a number of b i o l o g i c a l systems. In bacteria, yeasts and phage tested, spermine 5 was found to be more toxic than spermidine, while putrescine exhibited l i t t l e or no t o x i c i t y (49,75,77,85,102). In cer t a i n bacteriophages a decrease i s noted i n the burst size when the systems are exposed to spermine and spermidine (49). Among the mycobacteria, the oxidation product(s) of spermine appear(s) to exert a greater t o x i c i t y than the parent polyamine (40,41,42). Similar observations have been made with E. c o l i and S.. aureus (102) and with malignant mammalian c e l l s (1). A precise mechanism which explains the t o x i c i t y of polyamines i s not immediately available, but there i s growing support f o r the idea that amines may be involved i n the cation balance i n c e r t a i n organisms (22,77). The displacement of non-toxic metal cations by polyamines may p a r t i a l l y explain t o x i c i t y (34,58). In E. c o l i . S. aureus and yeasts (22,75,85) amines exert a more tox i c e f f e c t under a l k a l i n e conditions. Under these conditions, a c e t y l a t i o n of the d i - and polyamines i s accelerated i n E. c o l i (22). Acetylation could serve to reduce a r i s e i n cation concentration. In addition, acetylated spermine i s reported to be l e s s toxic than free spermine and a s i g n i f i c a n t reduction i n the synthesis of putrescine and spermidine i n E. c o l i occurs when the organism i s exposed to exogenous spermidine (22). The plant studies of Smith and Richards (94) also lend support to the cation balance theory. These workers discovered that under conditions of potassium 6 deficiency, barley and red clover plants contain greater concentrations of putrescine and agmatine. These data would suggest that the lack of an inorganic cation l e d the plants to synthesize a substitute organic cation. In animals, amines, p a r t i c u l a r l y spermine, are reported to cause necrosis of the renal tubules (81,102) while i n plants, abnormal chromosomal behaviour was observed when the tissues were exposed to spermine, cadaverine and putrescine (19,63). For a more detailed discussion of the b i o l o g i c a l s i g n i f i c a n c e of amines, the reader i s referred to the excellent review by Tabor et a l (110). Aminest t h e i r synthesis, and degradation Apart from, the general phenomena e l i c i t e d by b i o l o g i c a l materials when exposed to various amines, the synthesis and degradation of t h i s group of compounds i s also of i n t e r e s t . Synthesis of amines The r e l a t i v e l y simpler amines usually a r i s e from the corresponding amino acids by decarboxylation. Gale (30a, 30b,30c,30d) showed that the n a t u r a l l y occurring amines histamine, tyramine, putrescine, cadaverine and agmatine were produced from the. analogous amino acids by a number of b a c t e r i a . Information bearing on t h i s topic has been adequately reviewed by Gale (28). 7 The more complex polyamines, spermidine and spermine are probably derived from simpler compounds. Most studies in v o l v i n g polyamine synthesis have-employed microorganisms, although minced r a t prostate showed some a b i l i t y to synthesize spermidine (109). Studies with growing E. c o l i and Aspe r g i l l u s nidulans (109) and with Neurospora crassa (31, 32) employing l a b e l l e d substrates, indicated C -U 5 14 putrescine and 2-0 methionine to be precursors of spermidine and spermine. When other microorganisms and T A t h e i r extracts, were studied using 2-C methionine, l a b e l l e d spermidine was also i s o l a t e d (8). Prom such studies i t was concluded that putrescine was incorporated into spermidine as an i n t a c t u n i t , while on the other hand, methionine served by donating the three-carbon moiety of the spermine carbon chain. Enzymes i s o l a t e d from E. c o l i (98,108,110) were capable of catalyzing, the following reactions: Mg** (1) ATP + L-methionine > S-adenosylmethionine + H j ^ 4 Mg*4" (2) S-adenosylmethionine »C0p + "decarboxylated adenosylmethionine"* (3) "Decarboxylated methionine" + putrescine methylthioadenosine + spermidine The synthesis of spermine has not yet been elaborated, but i t i s reasonable to assume that spermidine may accept a propylamine group from anpther molecule of "decarboxylated * 5* deoxyadenosyl-(5*), 3-aminopropyl-(l) methyl sulfonium s a l t . 8 a d e n o s y l m e t h i o n i n e w t o f o r m s p e r m i n e . The d e g r a d a t i o n o f a m i n e s The i n i t i a l s t e p i n t h e d e g r a d a t i o n o f monoamines h a s "been t h e s u b j e c t o f a r e v i e w b y Z e l l e r (117). The d e g r a d a t i o n o f o t h e r a m i n e s , c h i e f l y d i a m i n e s , h a s b e e n r e v i e w e d b y Z e l l e r (117) a n d T a b o r (106).' I t i s g e n e r a l l y a g r e e d t h a t mono- and d i a m i n e s a r e f i r s t o x i d a t i v e l y d e a m i n a t e d t o y i e l d ammbnia a n d h y d r o g e n p e r o x i d e : (1) BCHgHHg + 0 2 + H 20 * ECHO + IH3 + HgO I n t h e p r e s e n c e o f c a t a l a s e , t h e n e t r e a c t i o n becomes: (2) R0H2HH2 + 0.5 0 2 y SCHO + HH3 The p r o d u c t i o n o f HgOg d u r i n g t h e r e a c t i o n h a s b e e n d e m o n s t r a t e d by c o u p l e d o x i d a t i o n u s i n g s u b s t r a t e s n o t a t t a c k e d b y t h e enzyme b u t w h i c h a r e o x i d i z e d b y t h e H 20 2 (48,97) o r b y d e s t r u c t i o n o f t h e H 2 0 2 f o r m e d , w i t h c a t a l a s e (106). U n l i k e d i a m i n e o x i d a s e s , monoamine o x i d a s e s a r e n o t s e n s i t i v e t o c a r b o n y l r e a g e n t s and t h e r e f o r e i t h a s b e e n p o s s i b l e t o d e m o n s t r a t e t h a t a l d e h y d e s a r e p r o d u c t s o f t h e r e a c t i o n b y t r a p p i n g t h e l a t t e r ^ w i t h c a r b o n y l r e a g e n t s a n d i d e n t i f y i n g t h e d e r i v a t i v e s so f o r m e d (117). I n t h e c a s e o f d i a m i n e o x i d a s e s , t h i s t e c h n i q u e i s n o t g e n e r a l l y a p p l i c a b l e 9 as these enzymes are usually i n h i b i t e d by carbonyl reagents. The same d i f f i c u l t y e xists f o r the enzymes which oxidize the polyamines, spermine and spermidine (76,115). Since the polyamines are s t r u c t u r a l l y more complex than the diamines, products derived from the former have only recently been p a r t i a l l y characterized. In the case of the diamines putrescine and possibly cadaverine, the aminoaldehyde formed1 by the action of diamine oxidase undergoes a spontaneous c y c l i z a t i o n with the elimination of water to form d' - p y r r o l i n e and 4«-piperideine (46,62,106): H 2HCH 2(CH 5) 2CH0 —.—> + H 20 y-aminobutyraldehyde A *-pyrroline Such c y c l i c compounds are unstable under the best of conditions and techniques f o r t h e i r preparation, assay and characterization have recently been described (5,43,46,72). With the polyamines, the points at which an i n i t i a l enzymatic attack are l i k e l y , are between the carbon-nitrogen bonds. Since there are four of these f o r spermidine and s i x f o r spermine, i t can r e a d i l y be v i s u a l i z e d that the products that might be expected, are several. Tabor et a l (103) found that during the oxidation, of spermine by beef plasma 2.0 jM of NH^ and 2.0 ;uM,of H2O,, were produced f o r every 2.0 of 0 2 consumed when catalase was absent from the reaction mixture. These workers therefore concluded 10 that spermine was deaminated at both ends of the molecule (positions A and A'): A 0 B B 1 C A' I 1 1 i 1 1 H^N-C C H 2 ) 3 - H H - ( CH2) 4 - N H - ( 0H 2) - H H 2 spermine However, the appearance i n t h e i r r e a c t i o n mixtures of putrescine and possibly spermidine and an aminoaldehyde (102,103) indicated that the i n i t i a l conclusions were doubtful since the l a t t e r products required that spermine be s p l i t at C and C . I t i s very l i k e l y that the beef plasma preparation contained several enzymes f o r spermine degradation. More recently, Bachrach and Bar-Or (7) demonstrated that sheep serum contained an enzyme which cleaved spermine at 0 or C to y i e l d spermidine and an u n i d e n t i f i e d aminoaldehyde. The spermidine was then further degraded. Weaver and Herbst (115) have reported that N. perflava extracts degrade spermine and spermidine with the consumption of 0.5 nM 0 2 and the production of 1.0 ^iM of an aminoaldehyde and 1.0 }M of 1,3-diaminopropane. This would require that spermine or spermidine be s p l i t at B or B'. In the case of spermidine such a s p l i t would y i e l d f-aminobutyraldehyde which they assumed to exist as such or to be converted spontaneously to A 1 - p y r r o l i n e . 11 A C B A' H2N-( CH2) 3-NH-( 0H2) ^ KHg More recently Baehrach (5) reported findings similar to Weaver and Herbst. Using a partially'purified cell-free extract from Serratia marcescens, i t was found that spermidine was degraded to equimolar amounts of 1,3-diaminopropane and A'-pyrroline with the consumption of 0.5 equivalents of 0^ when catalase was present. No ammonia was produced. If the report of putrescine (102) as an intermediate i n spermine breakdown i s valid, there may be two general types of degradative enzymes for polyamines—> one type yielding 3-carbon aminoaldehydes and 4-carbon diamines and the other producing 4-earbon aminoaldehy.des and 3-carbon diamines. Amines as substrates for bacteria Gale (29) reported on the a b i l i t y of washed c e l l preparations to oxidize a variety of amines including putrescine, cadaverine, agmatine, histamine and tyramine. Among the organisms tested by this worker, Ps. pyocyanea was found to be the most active while other organisms tested, including E. c o l i . attacked fewer amines. In the case of the former bacterium, putrescine, cadaverine and agmatine were completely oxidized with none of the substrate being assimilated. 12 Satake et a l (87) tested the a b i l i t y of a number of microorganisms to oxidize primary, amines. A l l but one of the organisms tested were found to be capable of ox i d i z i n g putrescine a f t e r adaptive l a g periods. Members of the following genera were tested: S e r r a t i a . Proteus, Escherichia. Plavobacter., Pseudomonas. B a c i l l u s , Achromobacter and Micrococcus. In addition, Achromobacter liquidum. when adapted to putrescine appeared to be simultaneously adapted to fc-aminobutyric a c i d . Razin et a l (76) surveyed some twenty s t r a i n s of microorganisms to t e s t t h e i r a b i l i t y to u t i l i z e d i - and polyamines. Of the stra i n s tested, putrescine and spermine were not metabolized by E. c o l i (2 strains)., K l e b s i e l l a (1 s t r a i n ) , Proteus v u l g a r i s (1 s t r a i n ) , S h i g e l l a (4 species), Salmonella (4 species), Staphylococcus (1 species), Micrococcus (1 species), Streptococcus (2 species) and Mycobacterium p h l e i . Ps. aeruginosa was found to oxidize spermine, spermidine, putrescine, agmatine and cadaverine. S. marcescens oxidized spermidine, putrescine and agmatine while Corynebacterium pseudodiphtherium oxidized only putrescine. Jakoby and co-workers (46,47,89,90) reported that Ps. fluorescens oxidized putrescine and studied the degradation of t h i s compound i n . d e t a i l . Baehrach et a l (9) reported that Myc. smegmatis oxidized spermine, spermidine and putrescine. This confirmed the observations of Roulet and Z e l l e r (84) that t h i s microorganism attacked spermine and 13 putrescine. Other microorganisms reported as o x i d i z i n g spermine and/or spermidine are; Ps. pvoeyanea (92) H. parainfluenzae, H. perflava and P. tularensis (115,116). Studies with washed c e l l preparations have usually employed conventional manometric techniques i n COg-free gas atmospheres. Such studies have indicated that, with c e r t a i n exceptions (29), only portions of the amines are oxidized to O O 2 , B^O and.NH^, the remainder of the molecules presumably, being assimilated. Thus Roulet and Z e l l e r (84) reported that putrescine was only p a r t i a l l y oxidized. Similar r e s u l t s are reported by Razin et a l (76) and Bachrach et a l (9) f o r the oxidation of various d i - and polyamines by a number of bacteria. I t would therefore appear that amines may serve not only as sources of energy f o r many bacteria, but also as s t r u c t u r a l b u i l d i n g blocks. This f a c t has been proven conclusively f o r Ps. iluorescens which can grow on putrescine as a sole carbon, nitrogen and energy source (46). Detailed e l u c i d a t i o n of the pathways f o r the u t i l i z a t i o n of d i - and polyamines i n bacteria are l a r g e l y due to the work of Razin et a l (74,76), Bachrach et a l (9)-, Jakoby and colleagues (46,47,89,90) and Weaver and Herbst (115). The work of the l a s t authors has already been mentioned i n connection with polyamine. metabolism. Studies with Ps.  aeruginosa. Myc. smegmatis and S. marcescens indicated that along with 0 P consumption and the production of HH_ and 00«, 14 certain intermediates were produced which, were either later u t i l i z e d , or accumulated i n the reaction mixture. Thus Ps. aeruginosa degraded spermine by a pathway with spermidine, 1,3-diaminopropane, £-alanine and y-aminobutyric acid as intermediates while S. marcescens produced 1,3-diaminopropane as an intermediate which accumulated during the oxidation of spermidine. Preeze-dried cells of Myc. smegmatis metabolized spermine by a similar pathway to Ps. aeruginosa. The net result of this research led to the following degradative pathway formulated by Razin et a l (76): Spermine -2H +H20 Spermidine /3-aminoproprionaldehyde 1,3-diamino propane fi-alanine The details for the mechanism of the oxidation of Jf-aminobutyric acid and yg-alanine were studied by Baehrach 15 et a l ( 9 ) , Bachrach (6) and Jakoby and colleagues (46,89, 90) using freeze-dried c e l l s and c e l l - f r e e extracts. ,;. - 'V. • ,* In Ps. aeruginosa - and My c. smegmatis, y-aminobutyrie ac i d and $-alanine undergo transamination with keto acids to form succinic and malonic semialdehydes. The semialdehydes are then oxidized to succinic and malonic acids. A s i m i l a r process f o r the u t i l i z a t i o n of putrescine exists i n Ps. fluorescens (46). The diamine i s f i r s t deaminated oxidatively to y i e l d y-aminobutyraldehyde. The l a t t e r which i s thought to e x i s t i n equibrium with i t s spontaneous c y c l i z a t i o n product, A ' - p y r r o l i n e , i s dehydrogenatively oxidized to Jf-aminobutyric a c i d which i n turn undergoes a transamination with ot-ketoglutaric a c i d to form glutamic a c i d and succinic semialdehyde. The l a t t e r compound i s then oxidized v i a an HAD-or NADP-requiring enzyme to succinic a c i d whence i t can enter the t r i c a r b o x y l i c acid cycle and be further degraded and/or assimilated. This series of reactions has been studied i n some d e t a i l and because of the remarkable s p e c i f i c i t y of the enzymes involved i n Ps. fluorescens, the enzymes are suitable for assay methods (44). A rather novel mechanism fo r the metabolism of putrescine i n an E. c o l i mutant has recently been reported (52). In t h i s system putrescine i s not oxidatively deaminated as i s usual during the i n i t i a l degradative steps. Instead, putrescine undergoes a transamination with cfc-ketoglutaric a c i d to y i e l d glutamic acid and Jf-aminobutyraldehyde. 16 From t h i s stage on, the remaining reactions are i d e n t i c a l with those of the other systems discussed. In t h i s system the transaminase appeared to he constitutive while i n most of the other systems examined where putrescine and other amines were oxidized, the systems usually appeared to be induced (9,46,76,87,115). 17 MATERIALS AND METHODS I Organisms Strains of mycobacteria obtained f o r t h i s study were kindly supplied by Dr. H. Vogel, Department of Health, City of New York, N.Y., and A.J. Ross, Western Pish Disease Laboratory, Seattle, Washington. Table 1 l i s t s the cultures obtained which proved suitable f o r the immediate purposes of t h i s project because of t h e i r r e l a t i v e l y rapid rate of growth. Table 1 L i s t of cultures obtained f o r study. Mycobacterium sp. Host Supplier Designation N.C.T.C. s t r a i n #2291 Neon t e t r a , s t r a i n N Chambers Creek s t r a i n Myc. salmoniphilum Halibut Neon t e t r a * Steelhead trout Chinook salmon H. Vogel H. Vogel A.J. Ross A.J. Ross H.A.L. N.T.N. C.C. S.C. * Species of t r o p i c a l f i s h . Some of the c h a r a c t e r i s t i c s of the microorganisms l i s t e d i n Table 1 are described in. papers by Vogel (114) and Ross (83). A l l mycobacterial strains were i n i t i a l l y assayed f o r t h e i r a b i l i t y to metabolize putrescine (1,4-diaminobutane), since according to Z e l l e r et a l (118) t h i s compound i s one of the better substrates f o r demonstrating diamine oxidase 18 a c t i v i t y i n bacteria. However, the studies that followed t h i s general survey were concentrated on strainsN.T.N., C.C. and S.C. II Growth conditions Preliminary work showed that growth i n s t a t i c broth media was granular and i n p e l l i c u l a r form. The addition of surface active agents to such s t a t i c cultures did l i t t l e towards producing uniform suspensions of c e l l s . Uniform suspensions of mycobacterial c e l l s suitable f o r quantitative studies were found to be most r e a d i l y prepared by growing the c e l l s on a wrist-action shaker i n f l u i d media containing T e r g i t o l NPX at a f i n a l concentration of 0.01$ (v/v). Although growth was equally good at 30°C, incubation was rou t i n e l y conducted at room temperature i n cotton-stoppered erlenmeyer f l a s k s . A 5$> inoculum (v/v) of an a c t i v e l y growing culture usually produced a s a t i s f a c t o r y y i e l d of log-phase c e l l s i n three to four days under the conditions already described. III Media In studies designed to demonstrate the a b i l i t y of the microorganisms to grow on amines as the sole carbon source, a minimal s a l t s medium was used (see Appendix, Table l ) . When i t was necessary to show whether the system responsible for the metabolism of putrescine was constitutive or induced, 19 larger volumes of c e l l s were required since such studies involved observations on both i n t a c t c e l l s and t h e i r c e l l -free extracts. In such cases, a r i c h e r synthetic medium was employed which could be f o r t i f i e d with putrescine (see Appendix, Table 2). Where large batches of c e l l s were required f o r preparing c e l l - f r e e extracts containing putrescine-metabolizing enzymes, t r y p t i c soy broth (Difco), supplemented with putrescine at a f i n a l concentration of 5.0 p^/mL, of medium, was employed. This medium resulted i n f a s t e r growth, and better y i e l d s of c e l l s , than either of the previously l i s t e d media. IV Preparation of washed c e l l suspensions Since the mycobacterial c e l l s used i n these experiments were hydrophobic, a l l manipulations involving i n t a c t c e l l s were c a r r i e d out i n suspending media containing 0.01$ (v/v) T e r g i t o l NPX. C e l l s were harvested by centrifugation. The packed c e l l s were washed twice i n large volumes of 0.85$ (w/v) saline and resuspended i n buffer at pH 7.2. Intact c e l l s were studied manometrically at pH 7.2 i n sodium-potassium phosphate or tris-(hydroxymethyl)aminomethane ( t r i s ) buffer. Oxygen uptake studies on i n t a c t c e l l s acting on putrescine i n e i t h e r buffer gave i d e n t i c a l r e s u l t s . Varying the concentration of the suspending buffer from 0.1 M to 0.03 H also f a i l e d to have an e f f e c t on putrescine oxidation. C e l l s 20 were therefore r o u t i n e l y suspended i n 0.1 M t r i s "buffer, pH 7.2. V Standardization of washed c e l l suspensions I t was empirically determined that a c e l l suspension i n 0.1 M t r i s buffer adjusted on a Klett-Summerson photoelectric colorimeter to 250 K l e t t u n i t s ( f i l t e r #4-2) contained the equivalent of 0.8-1.0 mg. of dried c e l l s / m l . The volume of buffer required to adjust 1.0 ml. of a heavy c e l l suspension to 250 K l e t t units was then noted and from t h i s value i t was possible to calculate an appropriate d i l u t i o n so that 1.0 ml. of d i l u t e d c e l l suspension added to a Warburg respirometer vessel would contain the desired number of mg. (dry weight) of c e l l s . C e l l s were dried to constant weight at 100°C using 5.0 ml. samples. Corrections were made f o r the weight of dried buffer s a l t s . VI Preparation of c e l l - f r e e extracts C e l l - f r e e extracts were obtained by grinding frozen c e l l - g l a s s bead mixtures i n a mortar and pestle. Wet, packed c e l l s and glass beads (1.0 g. to 8.0 g . ) were thoroughly mixed and frozen at -20°C. The mixture was then ground at 4°C f o r 5-6 minutes i n 10 g. amounts. Ground preparations were pooled and extracted with 0.05 M phosphate buffer, pH 7.2, containing 0.005 M mercaptoethanol 21 such that 200 mg. of wet c e l l s were extracted with 1.0 ml. of buffer. The supernatant f l u i d was c o l l e c t e d and the glass bead - c e l l mixture was extracted once more with an equal volume of buffer. Both extracts were then combined and centrifuged at 0°C f o r 30 minutes at 10,000 X G. The supernatant f l u i d was then decanted and dialyzed f o r 36 hours, at 0-4°C in. three changes of d i s t i l l e d water t o t a l l i n g 15-20 l i t r e s . The dialyzed extracts were then centrifuged as before, and the supernatants divided into 10 ml. quantities f o r storage at -20°C. Such dialyzed extracts t y p i c a l l y contained 5.0-6.0 mg./ml. of protein when the t r i c h l o r o a c e t i c a c i d (TCA) p r e c i p i t a b l e material was. converted to nitrogen and the l a t t e r converted to protein values by using a f a c t o r of 6.25. No TCA; soluble nitrogen was present. When protein was determined by the turbidimetric method (91), using bovine albumen (Armour) as the standard, the extracts were found to contain 6.0-7.0 mg. prote i n / ml. Glass beads used were Superbrite, type 110, (regular). The beads were covered with 12 H HC1 and allowed to s i t overnight at room temperature. They were then washed free of acid with d i s t i l l e d water, rinsed with 0.1 M phosphate buffer, pH 7.2, and f i n a l l y rinsed with d i s t i l l e d water. 22 VII Manometric measurements o _ Gas exchange was measured at 30 0. 1 Intact c e l l s . Unless otherwise sp e c i f i e d , each Warburg vessel received 3.0-5.0 mg. of c e l l s (dry weight) i n 1.0 ml. of 0.1 M t r i s buffer. Substrate additions were usually i n 0.5 ml. amounts. The t o t a l volume of f l u i d i n each vessel was always 3.2 ml. In 0g uptake studies, 0.2 ml. of 20$ (w/v) K0H was placed i n the centre w e l l . Where COg output was to be determined, 0.2 ml. of 6 N HgSO^ replaced the a l k a l i and was put i n the second sidearm of a double sidearm v e s s e l . GOg production was determined by the d i r e c t "two cup M method followed by an "acid t i p " (113). In experiments where i n h i b i t o r s were used, the c e l l s were preincubated with i n h i b i t o r f o r 30 minutes p r i o r to the addition of substrate. Under these conditions, substrate was added i n 0.1 ml. amounts to avoid any dr a s t i c d i l u t i o n of i n h i b i t o r . Following prolonged manometric experiments, smears of the reaction mixtures were stained by the Ziehl-Heelson acid-f a s t method to ensure that the re s u l t s were not influenced by the appearance of contaminants during the experiment. 23 2 C e l l - f r e e extracts. Warburg vessels usually contained 1.0 ml. of extract and 1.0 ml. of 0.1 M'buffer at the desired pH. The remaining volume was bfought up to 3.0 ml. with water and/or aqueous substrate and/or aqueous i n h i b i t o r . The centre well contained 20$ KOH (w/v). VIII Studies with i n t a c t c e l l s 1 A b i l i t y of washed c e l l s to oxidize putrescine. C e l l s f o r t h i s study were grown i n t r y p t i c soy broth. Washed, standardized c e l l s were assayed i n Warburg respirometer vessels i n order that oxygen consumption could be followed. Pour mycobacterial s t r a i n s were assayed on 0.001 M.and 0.01 M putrescine dihydrochloride ( N u t r i t i o n a l Biochemical Corp.). Since putrescine at each concentration resulted i n i d e n t i c a l rates of oxygen uptake i n i t i a l l y , i t was concluded that the compound did not a f f e c t the c e l l s adversely. The oxygen uptake values i n Table 2 represent oxygen consumption i n the presence of 0.01 M putrescine at 4.5 hours. The corresponding endogenous oxygen uptake values are also l i s t e d . The l a t t e r values represent oxygen consumption with water substituted f o r putrescine. 2 Influence of pre-exposure to putrescine on the oxidation of putrescine• 2 4 The object of t h i s experiment was. to determine whether the i n i t i a l rate of putrescine oxidation could be enhanced by growing c e l l s i n the presence of the compound* C e l l s were grown i n the synthetic medium described i n Table 2 of the Appendix, During the f i n a l 1 6 hours of growth, h a l f of each culture was supplemented with a s t e r i l e s olution of putrescine ( 5 . 0 JJM putrescine/ml. medium). The remaining h a l f of each culture received no additions. The c e l l s were then harvested, washed, standardized, and assayed on 0 . 0 1 M putrescine i n Warburg respirometer vessels. Results f o r the three mycobacterial s t r a i n s tested are presented i n Pigs. 1 - 3• The curves represent oxygen uptake due to added putrescine, endogenous oxygen uptake values having been subtracted from those i n the presence of putrescine. 3 A b i l i t y of washed c e l l s to oxidize a l i p h a t i c amines. Previous r e s u l t s had indicated that washed c e l l s of various mycobacterial strains could oxidize putrescine and that pre-exposure to the compound enhanced the i n i t i a l rate of putrescine oxidation. This experiment was therefore designed to determine whether s i m i l a r r e s u l t s could be obtained f o r the related diamines 1,3-diaminopropane and 1 , 5 -diaminopentane• S t r a i n C.C. c e l l s were grown i n the synthetic medium described i n Table 2 of the Appendix, The medium was supplemented with 5 . 0 nM/ml. of either 1 ,3-diaminopropane or 25 1,5-diaminopentane."'Log-phase c e l l s were harvested, washed, standardized and assayed manometrically on 15 }M of each of the following amines: 1,3-diaminopropane, putrescine and 1,5-diaminopentane. The r e s u l t s recorded i n Table 3 represent the oxygen uptake at s i x hours. In a re l a t e d experiment, s t r a i n C.C. c e l l s were grown i n the same medium supplemented with spermidine (5.0 )M/ml, medium). I t was expected that i f the c e l l s could be adapted to spermidine they might simultaneously be adapted to putrescine and/or 1,3-diaminopropane. The c e l l s were therefore assayed manometrically with spermine, putrescine and 1,3-diaminopropane as substrates. The r e s u l t s are graphically depicted i n F i g . 5. The rea c t i o n mixture supernatants were examined by paper chromatography at the conclusion of the experiments to determine whether the substrates had disappeared or had been modified i n any manner. 4 Amines as sole carbon sources. This experiment was designed to test the a b i l i t y of mycobacterial strains to u t i l i z e a number of amines as sole carbon sources f o r growth. The basal medium used i s described i n Table 1 of the Appendix. The following amines were tested' as t h e i r hydrochloride s a l t s at 0.4$ (w/v): 1,3-diaminopropane, putrescine, cadaverine, p y r r o l i d i n e , piperidine and 26 1-aminobutane• The phosphate s a l t s of the polyamines, spermidine and spermine, were also tested on a smaller scale i n test tubes at a concentration of 0.1$ (w/v). A d i l u t e suspension of washed c e l l s (0.2 ml.) i n 0.067 M phosphate buffer, pH 7.2, was used as inoculum and the cultures were incubated f o r 17 days. Controls, with 0.4$ glucose (w/v) as the sole source of carbon were also included. The r e s u l t s f o r the three mycobacterial strains tested, are presented i n Table 4. 5 Occurrence of amines i n mycobacteria. This experiment was conducted to determine whether mycobacterial c e l l s contain amines i n detectable quantities. Because of the lengthy procedure involved, only one mycobacterial s t r a i n was examined. S t r a i n C.C. c e l l s , grown i n t r y p t i c soy broth, were harvested and washed. The packed c e l l s were combined with glass beads and the mixture thoroughly ground. The mixture was then extracted at room temperature with several volumes of 5$ TCA containing HC1 at a f i n a l concentration of 0.1 U. A protein-free extract, equivalent to 2.0 g. of wet, packed c e l l s was then combined with 10 g. of HaOH p e l l e t s and the mixture steam- d i s t i l l e d f o r 2.5 hours. At t h i s time, no further detectable base appeared to be coming over i n the d i s t i l l a t e . The d i s t i l l a t e (approximately 1 l i t r e ) was a c i d i f i e d with d i l u t e HC1 and the volume reduced by b o i l i n g . 27 P i n a l drying of the d i s t i l l a t e to free i t of excess acid, was accomplished overnight by means of a warm blas t of a i r from a fan. The acid-free residue was dissolved i n a small volume of water and applied, with washings, to a column of Dowex 50 H r e s i n . The column (110 X 12 mm.) had a hold-up capacity of approximately 12 ml. for. 2 H NaOH. The sample was eluted with a l i n e a r concentration gradient of H + ions supplied by 550 ml. of water and 550 ml. of 3 N HC1. Fractions were c o l l e c t e d i n 5.0 ml. amounts on a f r a c t i o n c o l l e c t o r . These were fan-dried overnight i n a wind tunnel and redissolved i n 1.0 ml. of water. Aliquots (0.1 ml.) were then assayed f o r the presence, of amines,with the 2,4-dinitroflourobenzene technique of Dubin (20). Fractions which were p o s i t i v e f o r amines were subjected to paper chromatography i n order to i d e n t i f y the compounds. Id e n t i c a l analyses were performed on a 5.0 g. sample of t r y p t i c soy broth powder and on a synthetic mixture of amines ; containing 1.5 jM of spermine and 1.5 pM. of putrescine. The r e s u l t s are discussed under the appropriate sections of "Experimental Results and Discussion". F i g . 6 shows the eluti o n patterns obtained with the s t r a i n C.C. extract, t r y p t i c soy broth and the synthetic amine mixture respectively. 6 Influence of putrescine on endogenous r e s p i r a t i o n . Indirect techniques were used to determine whether 28 putrescine influenced the rate of endogenous r e s p i r a t i o n . Oxygen consumption toy s t r a i n S.C. was measured i n an experiment where a constant quantity of c e l l s (approximately 2.0 mg.) was allowed to oxidize 1.5, 3.0 and 6.0 jM of putrescine Respectively. S t r a i n N.T.N. was also tested toy t h i s technique with 1.5 and 3.0 ^ iM of. putrescine. In another experiment, three d i f f e r e n t quantities of s t r a i n S.C. c e l l s (approximately 1.0 mg., 2.0 mg. and 3.0 mg.) were each allowed to oxidize 3.0 jM of putrescine. Oxygen consumption was followed on the Wartourg apparatus u n t i l the curves l e v e l l e d o f f at 4.5 hours. C e l l s of tooth s t r a i n s had previously "been grown i n the presence of putrescine to ensure a s a t i s f a c t o r y rate of oxidation of the compound. The r e s u l t s of these experiments, are presented i n Tatoles 5 and 6. 7 Oxygen, ammonia and cartoon dioxide r e l a t i o n s h i p s during putrescine oxidation. C e l l s used i n these experiments, were adapted to putrescine. The studies were designed to determine, quantitatively, the degree of as s i m i l a t i o n occurring during the oxidation of putrescine toy washed c e l l s . Strains U.T.U., C.C. and S.C. were tested. A. Oxygen consumption. Washed c e l l s were allowed to oxidize a given amount of 29 putrescine and the oxygen uptake was followed manometrically. Putrescine oxidation, i n these studies, occurred i n a C0 2-free atmosphere. B. Ammonia production. Ammonia produced during the oxidation of putrescine was quantitatively determined on reaction mixtures obtained from Warburg respirometer vessels a f t e r the oxygen uptake curves had l e v e l l e d o f f . In addition, larger scale reaction mixtures of sim i l a r proportions, to those used i n the Warburg respirometer vessels, were run i n p a r a l l e l on the Warburg respirometer with shaking. The l a t t e r reaction mixtures were run i n cotton-stoppered erlenmeyer f l a s k s which f a c i l i t a t e d sampling at desired i n t e r v a l s , but i t should be noted that i n these flasks reactions occurred under atmospheric conditions., Ammonia determinations were also conducted on reaction mixtures withdrawn from Warburg reaction vessels which contained no a l k a l i and which had been used f o r COg determinations. Such determinations were made a f t e r the oxygen uptake curves had l e v e l l e d o f f . C. Carbon dioxide. Carbon dioxide was determined by the d i r e c t Htwo-cup H method followed by an "acid t i p " . This technique does not take in t o account the influence that the presence, or absence, of C0 o may have on the course of the reactio n . 30 Oxygen consumption curves were used to determine when to stop the reaction. Acid was tipped into the reaction mixture a f t e r the oxygen uptake curve had l e v e l l e d o f f , or at the region of the "break" of the oxygen uptake curve. D. Paper chromatography of reaction mixture supernatants. In order to determine whether products accumulated i n the reaction mixtures during the oxidation of putrescine, the reaction mixture supernatants were subjected to paper chromatography. The reaction mixtures were chromatographed d i r e c t l y , or when necessary, a f t e r the removal of buffer s a l t s by i o n exchange. In order to detect nitrogenous compounds, chromatograms were sprayed with ninhydrin or 2,4-dinitroflourobenzene. Organic acids were sought by spraying chromatograms with an i n d i c a t o r dye. Reaction mixture supematants from Warburg respirometer vessels were examined chromatographieally at the end of the reaction period, while those from erlenmeyer f l a s k s were examined at various i n t e r v a l s during the course of putrescine oxidation. The r e s u l t s of these studies are discussed under "Experimental Results and Discussion". Pigs. 7 and 8 present the oxygen uptake and ammonia curves obtained f o r two s t r a i n s while Pig. 9 shows the course of oxygen uptake f o r a t h i r d s t r a i n . The curves have been corrected f o r 31 endogenous oxygen and ammonia values since e a r l i e r studies indicated endogenous r e s p i r a t i o n to be independent of putrescine oxidation. 8 I n h i b i t o r studies with washed c e l l s . C e l l s used f o r the following experiments were adapted to putrescine. The effects of a number of i n h i b i t o r s on two mycobacterial strains were tested. S t r a i n C.C. c e l l s (approximately 5.0 mg. dry weight/ reaction vessel) were allowed to oxidize putrescine (3.0 JIM) i n the presence of sodium arsenite (0.003 M ) , sodium azide (0.003 M ) , p-phenylenediamine (0.0045 M ) and malonic a c i d (0.005 M), respectively. The effects of these i n h i b i t o r s on the oxidation of putrescine and on the endogenous r e s p i r a t i o n were measured manometrically. The oxygen consumption values for the various treatments are presented i n Table 7. The values represent oxygen consumption a f t e r the curves had l e v e l l e d o f f . Table 8 records the r e s u l t s of s i m i l a r studies with s t r a i n S.C. c e l l s (approximately 3.0 mg./reaction v e s s e l ) . In addition to arsenite, the effects of dihydrostreptomycin (DHS) (0.001 M and 0.006 M ) and of i s o n i c o t i n i c a c i d hydrazide (INH) (0.001 M and 0.006 M ) were measured. Valuable information might have been obtained from a paper chromatographic analysis of the various reaction mi-xtures obtained with the i n h i b i t o r s tested. However, the time required to prepare clean samples suitable for 32 paper chromatography precluded such studies. Instead, t e s t s with a l l other i n h i b i t o r s were abandoned i n favour of a more detailed examination of the a f f e c t s of arsenite. This compound was the only i n h i b i t o r which caused hydrazone-forming substances to accumulate i n the reaction mixtures. In addition, arsenite had the most pronounced a f f e c t s on corrected oxygen uptake values. Pig. 10 represents the e f f e c t of 0 .003 M arsenite on the oxygen uptake of s t r a i n S.C. c e l l s acting on 3 . 0 ;uM of putrescine. P a r a l l e l quantitative determinations on the concentration of carbonyl compounds in. the reaction mixture are presented i n terms of K l e t t u n i t s . Pig. 11 records the ef f e c t of 0.009 H arsenite on the oxygen uptake and ammonia production by s t r a i n S.C. c e l l s ( 3 . 0 mg. dry weight/Warburg vessel) acting on 3 . 0 joM of putrescine. The r e s u l t s of s i m i l a r studies with s t r a i n C.C. ( 2 . 0 mg./Warburg vessel) are.presented i n P i g . 12 . In these experiments, samples f o r analysis were taken from cotton-stoppered erlenmeyer f l a s k s which were run i n p a r a l l e l with the Warburg respirometer vessels as previously described. Samples of the reaction mixtures were used f o r paper chromatography a f t e r the removal of sodium arsenite by ion exchange. Paper chromatography was also employed to i d e n t i f y the hydrazones formed when the carbonyl compounds i n the reaction mixtures reacted with 2,4-dinitrophenylhydrazine 33 (iDPNH). At various times during the course of the reaction, c e l l s obtained from the various reaction mixtures were washed once with cold water and the sedimented c e l l s extracted with c o l d 0,1 N HG1 f o r 30 minutes. Chromatography of such extracts was conducted to obtain some idda as to the nature of the amino acid pool within the c e l l s and to determine whether c e r t a i n amino acids might be derived from putrescine. IX, Studies with c e l l - f r e e extracts Crude, dialyzed, c e l l - f r e e extracts were examined for the presence of enzymes responsible f o r the dismutation of putrescine. No attempts were made to purif y , or to characterize i n d e t a i l , the enzymes encountered. C e l l - f r e e extracts were obtained from s t r a i n C.C. c e l l s adapted to putrescine during growth i n t r y p t i c soy broth. 1 Diamine oxidase (D.O.). The presence of D.O. i n c e l l - f r e e extracts was established by following the oxygen consumption r e s u l t i n g from i t s action on known amounts of putrescine. During the reaction, ammonia and A 1 - p y r r o l i n e were produced. The l a t t e r compound was demonstrated q u a l i t a t i v e l y by paper chromatography. Techniques f o r the quantitative assay of both compounds are dealt with under A n a l y t i c a l Methods. The s e n s i t i v i t y of D.O. to heat and several i n h i b i t o r s 34 was b r i e f l y investigated. The s p e c i f i c i t y of the enzyme was determined f o r a series of a l i p h a t i c mono-, d i - and polyamines. The e f f e c t of pH on the rate of the reaction was also investigated. In heat s t a b i l i t y experiments, the c e l l - f r e e extract (2.0-3.0 ml.), was placed i n tubes containing an equal volume of 0.1 M phosphate buffer, pH 7.2, at 55°C. A f t e r incubation f o r the desired period, the buffered extract was c h i l l e d immediately and assayed. During i n h i b i t o r experiments, enzyme was incubated with i n h i b i t o r at 30°C f o r 20 minutes before the addition of substrate. A t y p i c a l reaction mixture contained, i n a t o t a l of 3.0 ml.: C e l l - f r e e extract, 1.0 ml.; substrate, 6.0-30/JM; buffer, 100 jffl. at the desired pH; water or aqueous i n h i b i t o r . 2 A' -pyrroline dehydrogenase (P.D.). The occurrence of the P.D. enzyme i n crude, dialyzed, c e l l - f r e e extracts could only be demonstrated i f reducing materials were present. This requirement was s a t i s f i e d by adding mercaptoethanol to the reaction mixture. Two techniques were used to follow P.D. a c t i v i t y . The f i r s t method was based on the increase i n o p t i c a l density at 340 mn due to the reduction of added nicotinamide adenine dinucleotide (NAD). Appropriate controls were employed to cancel out the eff e c t on o p t i c a l density of 35 added substrate or cofactor. Such studies were conducted on a Beckman model DU spectrophotometer. A t y p i c a l reaction mixture contained, i n a t o t a l of 3.0 ml.: C e l l - f r e e extract, 1.0 ml.; buffer, pH 9.0, 100 jMi &'-pyrroline, 3.0 ;uM; cofactor, 0.3 mercaptoethanol, 15 ;uM. The second method used to follow P.D. a c t i v i t y , involved coupling dehydrogenase a c t i v i t y with atmospheric oxygen by means of methylene blue ( f i n a l concentration, 4.45 X 10 M). Under such conditions, the coiirse of the reaction could be followed by measuring oxygen consumption manometrically. The a b i l i t y of the enzyme to catalyze the reaction was tested at pH 7.2 and 9.0 with A'-Pyrroline and ^'-piperideine as substrates. The manometric method required that two reaction vessels be used, since the oxidation of mercaptoethanol by methylene blue resulted i n a considerable endogenous oxygen uptake. Por t h i s reason, i t was also necessary to add higher concentrations of mercaptoethanol since t h i s compound was progressively oxidized as the reaction proceeded. Theoretical oxygen consumption values were obtained routinely by correcting f o r endogenous oxygen consumption when the following 3.0 ml. reaction mixtures were employed: C e l l - f r e e extract, 1.0 ml.; buffer, 100 j M at the desired pH; mercaptoethanol, 50'jaM; methylene blue, 1.34 X IO"1-uM; cofactor, 0.3 uM; substrate, 6.0 pM, or water. In manometric experiments, the reaction was i n i t i a t e d 36 by adding putrescine to the complete reaction mixture, i n which case, the oxygen uptake represented the oxygen consumption due to both D.O. and P.D. a c t i v i t y . A l t e r n a t i v e l y , methylene blue was added to the reaction mixture a f t e r the oxygen consumption due to added putrescine was completed. Under the l a t t e r conditions, the oxygen consumption due to the dehydrogenation of enzymatieally synthesized A'-pyrroline could be measured independently. The transformation) of 4*-pyrroline to y-aminobutyric aci d during the P.D. catalyzed reaction, was demonstrated by paper chromatography. 3 Gamma-aminobutyric ac i d - ^ - k e t o g l u t a r i c acid transaminase. C e l l - f r e e extracts did not appear to contain oxidative or dehydrogenative enzymes capable of catalyzing the deamination of y-aminobutyric ac i d to y i e l d succinic semialdehyde and ammonia. Gamma-aminobutyric acid was found to undergo a transamination with oC-ketoglutaric acid to y i e l d succinic semialdehyde and glutamic acid. Succinic semialdehyde produced by the reaction was demonstrated by paper chromatography as i t s 2,4-dinitrophenylhydrazone d e r i v a t i v e . Glutamic a c i d was shown to be the other product, by paper chromatography of the deproteinated, deionized reaction mixture. The rea c t i o n was followed routinely by assaying aliquots 37 of the reaction mixture f o r glutamic ac i d . For t h i s purpose, a s p e c i f i c glutamic decarboxylase preparation was used (see A n a l y t i c a l Methods). The optimum pH was determined by conducting the reaction at various pH values between 7.0 and 9.0 i n phosphate and t r i s buffers. Thirty-minute samples of each' reaction mixture were analysed f o r the amount of glutamic acid produced. The ef f e c t of pH on the amount of glutamic acid produced i n these systems, was also studied a f t e r more extended reaction periods. The s p e c i f i c i t y of the transaminase enzyme was also investigated. Pyruvic and oxalacetic acids were tested with y-aminobutyric a c i d to determine whether they could serve as a l t e r n a t i v e amino-group acceptors. The following amino acids were tested as a l t e r n a t i v e amino-group donors with of-ketoglutaric a c i d : glycine, £-alanine, g-aminovaleric a c i d and f-aminocaproic ac i d . The s p e c i f i c i t y studies were conducted at pH 9.0 i n t r i s buffer f o r 60 minutes at 30°C. A t y p i c a l r eaction mixture f o r the optimum pH experiments contained i n a t o t a l of 3.0 ml.: C e l l - f r e e extract, 1.0 ml.j phosphate or t r i s buffer at the desired pH, 200 >iMj y-aminobutyric acid, 30 jM; oC-ketoglutaric acid, 15 ;oM. A t y p i c a l reaction,mixture f o r the s p e c i f i c i t y experiment, contained i n a t o t a l of 3.0.ml.: C e l l - f r e e 38 extract, 1.0 ml.; t r i s buffer, pH 9.0, 100 nM; amino acid, 15 p&i keto acid, 15 uM. Reaction mixtures from the s p e c i f i c i t y studies were examined q u a l i t a t i v e l y by paper chromatography. In the case of the l a t t e r experiment where «<-ketoglutaric ac i d served as the amino-group acceptor, the amount of glutamic acid formed was determined quantitatively. 4 Succinic semialdehyde dehydrogenase (S.S.D.). Succinic semialdehyde dehydrogenase a c t i v i t y i n dialyzed, c e l l - f r e e extracts, was demonstrated by following the increase i n o p t i c a l density at 340 mu due to the reduction of NAD or NADP (nicotinamide adenine dinucleotide phosphate) i n the presence of succinic semialdehyde. Succinic semialdehyde was prepared chemically (see A n a l y t i c a l Methods) or enzymatically by. the transaminase reaction e a r l i e r described. The dehydrogenative oxidation of succinic semialdehyde yielded succinic a c i d . The product of the reaction was shown to be succinic a c i d by paper chromatography of the ether extract obtained from the a c i d i f i e d , protein-free, reaction mixture, and by the use of a suecin-oxidase enzyme, the preparation of which i s described under A n a l y t i c a l Methods. In an i n i t i a l experiment, paper chromatography indicated that during the transamination reaction between i ©C-ketoglutaric a c i d and K-aminobutyric acid, a new hydrazone-39 forming compound accumulated. The addition of substrate amounts of NAD prevented the accumulation of t h i s compound and allowed the transamination reaction to go to completion (as determined by the amount of glutamic a c i d produced). A second experiment was therefore devised i n which oxalacetic acid was included i n the reaction mixtures to i n h i b i t any breakdown of the succinic a c i d formed. Reaction mixtures were made to contain, i n a t o t a l of 6.0 ml.; G e l l -free extract, 2.0 ml.; t r i s buffer, pH 8.5, 200 ;uM; y-aminobutyrie acid, 6.0 juM; «c-ketoglutaric acid, 12 joM; HAD, 6.0 ;uM. An i d e n t i c a l reaction mixture, with water substituted f o r cofactor, was included as a control. The o reaction was allowed to proceed at 30 C f o r 4 hours under an atmosphere of a i r at which time a 2.0 ml. sample was taken, adjusted to pH 4.8, and analysed quantitatively f o r glutamic acid. The reaction i n the remaining 4.0 ml. of each reaction mixture was stopped by adding 0.1 ml. of 6 N HOI. Each reaction mixture was then heated i n a b o i l i n g water bath f o r 15 minutes i n order to p r e c i p i t a t e the protein and to destroy both the reduced cofactor and oxalacetic a c i d . The mixtures were then centrifuged, the supernatants c o l l e c t e d and adjusted to pH 7.4, and representative aliquots assayed f o r t h e i r succinic acid content with the succin-oxidase preparation. 40 X A n a l y t i c a l methods 1 Ammonia. Ammonia was determined a f t e r micro-diffusion, by Ues s l e r i z a t i o n or by t i t r a t i o n (18). N e s s l e r i z a t i o n was employed mainly i n studies with c e l l - f r e e extracts where under c e r t a i n circumstances, the presence of v o l a t i l e amines might have l e d to erroneus r e s u l t s i f measured by the t i t r a t i o n method. 2 A1 - p y r r o l i n e . A standard curve f o r the quantitative determination of A*- p y r r o l i n e was prepared. The assay was based on the yellow colour produced when -pyrroline and o-aminobengaldehyde were allowed to react (43,46). Known quantities of A'-pyrroline (0.1 to 0.5 jM), prepared from putrescine by D.O. action at pH 7*2, were reacted with a constant amount of o-aminobenzaldehyde at pH 6.5 i n a t o t a l volume of 5.0 ml. After incubation at room temperature f o r 90 minutes, the absorbance of the solutions was read on a Klett-Summerson photoelectric colorimeter ( F i l t e r #42). Under the conditions of the assay, the colour developed bore a l i n e a r r e l a t i o n s h i p to the concentration of A'-pyrroline. An aliquot of the A*-pyrroline solution used to prepare the standard curve was checked f o r 41 i t s A 1-pyrroline content by the manometric method'..outlined e a r l i e r under the A 1-pyrroline dehydrogenase section and the concentration of the compound was found to be within yfo of the t h e o r e t i c a l value. The reagent, o-aminobenzaldehyde, used f o r the colorimetric assay of A'-pyrroline, was prepared by the chemical reduction of commercially a v a i l a b l e o-nitrobenzaldehyde (46). Chemically synthesized -pyrroline and A' -piperideine were used i n the manometric assay method f o r A1 -pyrroline dehydrogenase. These compounds were prepared by reacting ornithine and l y s i n e , respectively, with N-bromosuccinimide ( 4 4 , 4 6 ) . Chemically synthesized A'-pyrroline was also used to characterize the product of putrescine degradation due to D. O . a c t i v i t y . 3 Glutamic a c i d . Quantitative determinations f o r L-glutamic acid were conducted i n KOH-free Warburg reaction vessels. The assay was based on the stoichiometric production of 1.0 ;uM of C0 2 f o r each nM of glutamic a c i d present. Carbon dioxide production was determined manometrically at pH 4.8 i n 0.1 M sodium-acetate buffer. A s p e c i f i c L-glutamic decarboxylase was employed to catalyze the reaction. L-glutamic decarboxylase was prepared as an acetone-dried powder from a prototrophic E. c o l i K 12 s t r a i n , grown according to the directions of ) i 42 Cohen (16). A number of amino acids tested, including aspartic acid, l y s i n e , h i s t i d i n e , arginine and D-glutamic acid, were not attacked under the conditions of the assay. Under the same conditions, there was no oxygen uptake i n the presence of glutamic and y-aminobutyric acids. Organic acids (succinic and cC-ketoglutaric acids) and other amino acids, singly or i n mixtures, served to decrease the rate of the reaction, but at completion, t h e o r e t i c a l carbon dioxide values were obtained. For assay, aliquots of reaction mixtures were adjusted to pH 4.8 with sodium-acetate buffer to stop the reaction. A f t e r temperature e q u i l i b r a t i o n at 30°C, the enzyme (approximately 2.0 mg./reaction vessel) was added from the reaction vessel sidearm. Endogenous controls were always included, although corrections f o r endogenous carbon dioxide production were usually unnecessary. A single preparation of the acetone-dried c e l l s , o stored i n a t i g h t l y stoppered v i a l at -20 C, retained high decarboxylase a c t i v i t y f o r more than a year. 4 Succinic a c i d . Succinic a c i d was determined quantitatively by measuring the oxygen consumption r e s u l t i n g from the enzymatic oxidation of succinic a c i d to fumaric acid. During the reaction, 0.5 ; J M 0 2 were consumed fo r each ;uM of succinic a c i d oxidized. The reaction was catalyzed by a suecin-oxidase enzyme which 43 was prepared from pig heart muscle by the method outlined by Cohen (16). A number of organic acids were tested as substrates; malonic, succinic, g l u t a r i c , oC-ketoglutarie, oxalacetic and pyruvic acids. Succinic acid was the only compound attacked. The oxidation of succinic a c i d was completely i n h i b i t e d by equimolar amounts of oxalacetic acid, while pyruvic a c i d was i n a c t i v e i n t h i s respect. (When, therefore, reaction mixtures containing oxalacetic acid were to be assayed f o r succinic acid, the former was f i r s t converted to pyruvate by heating i n the presence of HC1. The same technique was used to destroy reduced cofactors (1ADH or NADPH) i n such reaction mixtures, since the succin-oxidase preparation contained a system f o r r e o x i d i z i n g these compounds which would otherwise have i n t e r f e r e d with succinic acid determinations. The l a t t e r f a c t was r e a d i l y established when i t was found that the succin-oxidase enzyme preparation could replace methylene blue i n the manometric method f o r the assay of A'-pyrroline dehydrogenase a c t i v i t y . For assay, a representative aliquot of the protein-free reaction mixture was assayed at pH 7.4 i n 0.1 M:phosphate buffer i n Warburg reaction vessel containing 0.2 ml. of 20$ (w/v) KOH i n the centre w e l l . Approximately 4.0 mg. (dry weight) of the preparation were used/assay. Corrections f o r endogenous oxygen consumption were usually unnecessary, even a f t e r extended runs. 44 A batch of the pig heart preparation, stored at - 20°0 i n sealed polyethylene bags, retained high a c t i v i t y with low blank oxygen uptake values f o r over a year. 5 2,4-dinitrophenyl hydrazones. Pyruvate, oC-ketoglutarate and succinic semialdehyde were determined q u a l i t a t i v e l y by paper chromatography as t h e i r 2,4-dinitrophenyl hydrazine (DPNH) der i v a t i v e s . Pyruvic and pt-ketoglutaric acids were determined qua n t i t a t i v e l y as t h e i r DPNH derivatives. Standard curves were prepared f o r t h i s purpose by reacting 0 to 1.0 nM of either a c i d with excess DPNH (54). The hydrazone(s) formed was (were) extracted quantitatively i n t o ethyl acetate (3 X 2.0 ml. a l i q u o t s ) . The pooled ethyl acetate extracts were then quant i t a t i v e l y extracted with 10$ (w/v) NagCO^ (3 X 2.0 ml. a l i q u o t s ) . The pooled Na^CO^ extracts were then washed free of traces of unreacted DPNH with 1.0 ml. of ethyl acetate and the l a t t e r was discarded. The volume of the NagOO^ soluti o n was then brought up to 10 ml. with 4.0 ml. of 2 N NaOH, and the colour formed was read within 10 minutes on a Klett-Summerson photoelectric colorimeter ( F i l t e r #54)• Under the conditions of the assay, there was a l i n e a r r e l a t i o n s h i p between the colour developed and the keto a c i d concentration. A reaction mixture to be examined f o r the presence of hydrazone-forming compounds was f i r s t freed of c e l l s or 45 protein. The reaction mixture was treated with excess DPHH i n 2 HC1 f o r 1.5 hours at room temperature. The hydrazones were then trapped i n 10$ Ua 200^ by the procedure already described. A representative portion of the IfaujCO^ solution was then assayed f o r absorbance as e a r l i e r described and the remaining solution was a c i d i f i e d at 0°0 with concentrated HC1. The hydrazones were then extracted into peroxide-free ethyl ether f o r spotting on paper chromatograms. Accurate quantitative determinations of pyruvate or oC-ketoglutarate using the Klett-Summerson photoelectric colorimeter, were only possible when the keto a c i d occurred alone, or where interference due to colour produced by other hydrazone derivatives, was n e g l i g i b l e . When paper chromatography indicated the presence of more than one hydrazone derivative, the r e s u l t s were expressed i n K l e t t u n i t s , and the r e l a t i v e concentration of each derivative could only be judged grossly by the density of the spots obtained by paper chromatography. Hydrazone derivatives f o r use as standards i n paper chromatography were always f r e s h l y prepared. Standards used were: oC-ketoglutarate, oxalacetate, pyruvate and succinic semialdehyde. Since the l a t t e r compound was not available commercially, i t was synthesized chemically by the crossed Claisen condensation technique described by Jakoby (44)• 46 6 Paper chromatography. A. Solvents. Routine paper chromatography of amines and amino acids was conducted i n n-butanol:acetic acid:water (2:1:1 v/v) (10). A second solvent employed to characterize amines was n-propanoljconcentrated HCl:water (3*1*1 v/v) (22). Amino acids were also separated i n water-saturated phenol prepared by d i s s o l v i n g 500 g. of phenol c r y s t a l s i n 225 ml. of water, allowing the layers to separate and using the lower layer to i r r i g a t e the paper. Amines and amino acids were run as t h e i r hydrochloride s a l t s . Organic acids were chromatographed as t h e i r ammonium s a l t s i n ethanol:NH A0H:water (16:1:3 v/v) (95). 2,4-dinitrophenylhydrazones were separated i n n-butanol saturated with 5 N NHA0H. The lower phase was used to saturate the chromatographic chamber. As opposed to other chromatograms, hydrazones were developed by the ascending technique. Whatman #4 paper was used f o r a l l paper chromatography. B. Detection reagents. Nitrogenous compounds were detected by spraying with 0.5$ (w/v) ninhydrin i n n-butanol, or with 2,4-dinitro-flourobenzene (DNPB). The l a t t e r reagent was prepared by diss o l v i n g 0.65 ml. of DNPB i n 50 ml. of acetone and using 10 ml. of t h i s solution dissolved i n 90 ml. of 2.5$ (w/v) sodium 47 tetraborate (20). The DNPB reagent gave yellow spots with a l i p h a t i c mono- and diamines and brown spots with the polyamines spermidine and spermine. I t suffered the disadvantages of not being as sensitive as ninhydrin and i n not being capable of detecting v o l a t i l e amines. Organic acids were detected with an i n d i c a t o r dye solution: Brom creso l purple 0.04$ (w/v) i n a 1:5 (v/v) mixture of commercial formalin and ethanol. The pH was adjusted to 5.0 with 0.1 N . NaOH (91). C. Preparation of samples f o r paper chromatography. Hydrochloric a c i d extracts of i n t a c t c e l l s were dried to remove excess HC1. The dried s a l t s were redissolved i n water and the aqueous solutions were used f o r spotting chromatograms• While i t was possible to chromatography small samples of c e l l - f r e e reaction mixtures d i r e c t l y , with la r g e r samples., the buffer resulted i n poor separations. Sodium arsenite used to poison i n t a c t c e l l s , and protein p r e c i p i t a n t s l i k e zinc sulphate and TOA, also resulted i n poor separations. In order to obtain reproducible chromatograms i t was therefore necessary to free samples of such i n t e r f e r i n g substances. JOL1 -pyrroline was freed of zinc sulphate by ethyl ether extraction from a l k a l i n e solution. Succinic acid was obtained free from zinc aulphate by extraction into ethyl ether from acid solution. A 1 -pyrroline was then converted 48 to i t s hydrochloride s a l t and succinic a c i d to i t s ammonium s a l t , f o r paper chromatography. C e l l - f r e e reaction mixtures were examined f o r organic acids a f t e r the removal of t r i s buffer. For t h i s purpose, + the sample was applied to a Dowex 50 H column which retained the t r i s buffer while the organic acids appeared/in.the water wash. The l a t t e r was concentrated to a suitable .volume f o r paper chromatography a f t e r n e u t r a l i z a t i o n with d i l u t e HH^OH. C e l l - f r e e reaction mixtures were examined f o r the presence of amino acids a f t e r the elimination of t r i s buffer by ion exchange on Dowex 1 OH columns. In t h i s technique, the column was washed free of t r i s buffer with water. The amino acids which remained on the column were then eluted with HC1. The acid eluate was then dried and the s a l t s were redissolved i n water f o r spotting on the chromatograms. Amino acids were freed of sodium arsenite and t r i s buffer by i o n exchange on both Dowex r e s i n s . The sample was f i r s t applied to a Dowex 1 0H~ column. Washing with water removed sodium i o n and t r i s buffer. The amino acids were then eluted with HC1 and the eluate was dried. The redissolved s a l t s were then applied to a Dowex 50 column where the arsenite i o n was removed by the water wash leaving the amino acids behind on the column. These were then eluted with HC1. The acid eluate was then dried and the s a l t s were dissolved i n water f o r chromatography. A: s i m i l a r technique 49 was used to free amino acid-containing samples from TCA and t r i s buffer, except that the bulk of the TCA was usually f i r s t removed by one or two extractions with ethyl ether. + Dowex 50 H, columns employed f o r delonizing purposes, had a hold-up capacity of 1.5-2.0 ml. f o r 2 N NaOH. Dowex 1 OH" columns had a sim i l a r hold-up capacity f o r 2 N HC1. Ions not exchanged by. these columns, were usually completely washed out of the columns by 3-4 column volumes of water. A, 10 ml. aliquot of 2 N.,HC1 was s u f f i c i e n t to elute a l l the amino acids encountered from either type of column. Further washing with HOI f a i l e d to elute any nitrogenous compounds when the fr a c t i o n s were analyzed f o r t h e i r presence by the DNFB technique of Dubin (20). XI Isotopic studies with i n t a c t c e l l s 1 A A s s i m i l a t i o n studies with uniformly l a b e l l e d C putrescine were not conducted since the compound was not re a d i l y a v a i l a b l e commercially. Instead, a s s i m i l a t i o n was 14 studied using 1,4-C . Labelled putrescine was supplied by the New England Nuclear Corporation. 1 Incubation with substrate. S t r a i n N.T.N, c e l l s , adapted to putrescine, were used f o r these studies. 50 Washed N.T.N, c e l l s (approximately 5.0 mg./Warburg reaction vessel) were allowed to oxidize 3.0^uM of putrescine (1.75 ;uc). The oxygen uptake was followed manometrically and at various times, samples were taken f o r analysis. At each sampling, counts of the r a d i o a c t i v i t y were made on the following samples: ( l ) KOH-trapped C^Og (2) c e l l s and supernatant and (3) supernatant. C e l l s obtained from the samples were also fractionated immediately by a modification of the procedure outlined by Roberts et a l (78). The c e l l f r a c t i o n s were analysed to determine the d i s t r i b u t i o n of the r a d i o a c t i v i t y within the c e l l s . Aliquots of the various samples were plated i n duplicate on s t a i n l e s s s t e e l planchets at i n f i n i t e thinness. Samples were dried under an i n f r a red lamp. Counts were recorded with a Nuclear-Chicago scaler, Model 181A, equipped with a gas-flow counter having a thin-end-window Geiger tube. Corrections were made f o r background and a minimum of 2000 counts were recorded to reduce the r e l i a b l e error to l e s s than 5$. C e l l - f r a c t i o n s were chromatographed and the radioactive areas on the chromatograms were located by running chromatogram s t r i p s through a Nuclear-Chicago Model C 100 B/Actigraph II equipped with a gas-flow counter and a Model 1620 B a n a l y t i c a l Count Rate Meter and Chart Recorder. 51 2 Sampling and c e l l f r a c t i o n a t i o n procedure. At given times,, the reaction was "stopped" by p i p e t t i n g the reaction mixture (5.0 ml.) into i c e - c o l d centrifuge tubes. As a precaution against l o s s by breakage, 1.0 ml. of each reaction mixture was withdrawn and frozen immediately at -20°C. Of the remaining 2.0 ml. of the reaction mixture, 0.2 ml. were taken f o r p l a t i n g on planchets (0.02 ml./ planchet). Counts recorded f o r such samples represented c e l l s and supernatant. The remaining 1.8 ml. of suspension was centrifuged immediately i n the cold, and the supernatant was c o l l e c t e d and counted (0.02 ml./planchet). Traces of supernatant remaining on the walls of the centrifuge tube were removed with a paper swab and the c e l l p e l l e t was resuspended i n 1.8 ml. of i c e - c o l d 0.1 H HG1. The.cells were extracted with HC1 f o r one hour at 0°C after which the sample was centrifuged i n the cold. The supernatant (cold HC1-soluble f r a c t i o n ) was c o l l e c t e d and counted (0.02 ml./planchet). The walls of the centrifuge tube were again dried with a paper swab and the extracted p e l l e t was resuspended i n 1.8 ml. of 75$ ethanol containing HC1 at a f i n a l concentration of o 0.01 H; The sample was incubated f o r 30 minutes at 45 C and then centrifuged. The supernatant (acid alcohol-soluble  f r a c t i o n ) was c o l l e c t e d and counted (0.02 ml./planchet). Residual a c i d ethanol was removed from the centrifuge tube and the p e l l e t was resuspended i n 1.8 ml. of 5$ (w/v) TCA. 52 The suspension was extracted f o r 50 minutes at 90°o and then centrifuged. The supernatant (hot TCA-soluble fraction ) was counted (0.02 ml./planchet). The centrifuge tube was again wiped dry and the c e l l residue was "dissolved" i n 1.8 ml. of 0.5 I KaOH f o r counting (0.02 ml./planchet). determinations were made on the KOH from the centre well of each reaction v e s s e l . F i l t e r paper and KOH were transfered to a 5.0 ml. volumetric f l a s k and the volume was brought up to 5.0 ml. with water rinses from the centre w e l l . The contents of each f l a s k were then thoroughly mixed and transfered to large test tubes. The stoppered tubes were held at -20°C u n t i l 0.05 ml. aliquots were counted. Counts on KOH samples were recorded as soon a f t e r p l a t i n g as possible to avoid any C0 2 exchange with atmospheric COg. The p o s s i b i l i t y of such an exchange was further minimized by keeping the plated samples dry. 53-EXPERIMENTAL RESULTS AND DISCUSSION I Studies with.intact c e l l s ( l ) A b i l i t y of washed c e l l s to oxidize putrescine. The r e s u l t s summarized i n Table 2 indicate that a l l four s t r a i n s of mycobacteria tested were capable of oxid i z i n g putrescine. Washed c e l l s of s t r a i n C.C. appeared to be most active i n t h i s respeet. Table 2 Oxygen uptake by washed c e l l s acting on 0.01 M putrescine at pH 7*2 at 4.5 hours. Mycobacterial s t r a i n Op uptake (nl) with putrescine 0 9 uptake Oul) n«rith water 0 2 uptake Cul) corrected H.A.L. 305 190 115 N.T.N. 325 200 125 C.C. 810 220 590 s.c; 330 210 120 (2) Influence of pre-exposure to putrescine on the oxidation of putrescine. The r e s u l t s of three adaptation studies shown i n Pigs. 1-3, indicate that each of the three mycobacterial st r a i n s tested, oxidize putrescine more r a p i d l y a f t e r p r i o r exposure to the compound. 54 TfME (HOURS) FIG. 1. Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve. 2) s t r a i n N.T.N, c e l l s . 55 * - a 3 4 T 7 M £ (HOURS) FIG. 2. Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n C.C. c e l l s . 56 o i X 3 it-TIMS, (HOURS) FIG. 3. Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n S.C. c e l l s . 57 FIG. 4. Diamine oxidase a c t i v i t y i n c e l l - f r e e extracts obtained from putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n S.C. c e l l s . Reaction mixtures contained i n a volume of 3.0 ml.: C e l l - f r e e extract, 1.0 ml., equivalent to 3.0 mg. protein; putrescine, 3.0 .uM; t r i s buffer, pH 9.0, 100 >uM; H 20. 58 S t r a i n C;C. c e l l s appear to contain a s i g n i f i c a n t amount of a c t i v i t y f o r putrescine even without pre-exposure to putrescine. However, adaptation to putrescine caused a three-fold increase i n the Q Q 2 value ( n l 0 2 consumed/mg. dry weight of c e l l s / h o u r ) . The adaptive nature of the putreseine-metabolizing system i s most pronounced with s t r a i n S.Ci c e l l s ( F i g . 3). In a s i m i l a r , but independent experiment with the l a t t e r s t r a i n , an increase i n the QQ^ value of over eleven-fold was obtained a f t e r prelncubating with putrescine. Because of the remarkable increase i n a c t i v i t y obtained a f t e r adaptation with t h i s s t r a i n , c e l l - f r e e extracts derived from putrescine-adapted and unadapted c e l l s were assayed f o r D.O. a c t i v i t y with putrescine as substrate. The curves i n F i g . 4 indicate that D.O. a c t i v i t y only occurred i n c e l l - f r e e extracts obtained from putrescine-adapted c e l l s . I f one defines an induced system a3 one i n which the l e v e l of a c t i v i t y may be raised by p r i o r exposure to inducing substrate, then i t would appear that the putreseine-metabolizing enzymes constitute an induced system i n the mycobacterial s t r a i n s tested. These findings agree with those of other workers f o r amine-metabolizing systems i n a v a r i e t y of bacteria (9,46,76,87,115). (3) A b i l i t y of washed c e l l s to oxidize a l i p h a t i c amines. In order to determine whether mycobacterial st r a i n s possessed the a b i l i t y to u t i l i z e various amines, diamines 59 clo s e l y related to putrescine were tested. C e l l s of s t r a i n C.C. were grown i n a basal medium (see Appendix, Table 2) supplemented with either 1,3-diaminopropane or 1,5-diamino-pentane and assayed subsequently to determine whether these compounds were oxidized. The r e s u l t s of these experiments are recorded i n Table 3. Table 3 E f f e c t of adaptation to amines on t h e i r subsequent oxidation by s t r a i n CQ. c e l l s . C e l l s grown i n Oxygen uptake (nl) a f t e r 6 hours with 0.005 M basal medium -m  plus: 1,3-diamino- 1,4-diamino- 1,5-diamino- water propane butane pentane Ho amines 175 667 210 244 1,3-diaminopropane 204 569 182 218 1,5-diaminopentane 161 552 171 206 I t was apparent that c e l l s grown i n the presence of 1,3-diaminopropane or 1,5-diaminopentane, were not adapted to either of these compounds even though such c e l l s were capable of oxi d i z i n g putrescine. Oxidation of putrescine by such c e l l s was not as rapid as that obtained with c e l l s grown i n the basal medium alone. In addition, exposure of c e l l s during growth to the 3- and 5-carbon diamines resulted i n a s l i g h t decrease i n the amount of oxygen consumed when 60 compared with that f o r endogenously r e s p i r i n g c e l l s . Any adverse e f f e c t s that these diamines may have caused with growing c e l l s was not evident as the y i e l d s of c e l l s from the diamine-containing media were comparable to those obtained from the basal medium. Tests on the c e l l - f r e e culture media with the MFB technique (20) indicated that substantial amounts of the added diamines were s t i l l present. I t would therefore appear that 1,3-diaminopropane and 1,5-diaminopentane were present i n the media as r e l a t i v e l y i n e r t ingredients. Under s i m i l a r conditions with putrescine as inducing substrate, the putrescine disappeared completely. Pig. 5 shows the r e s u l t of adaptation during growth to spermidine. I t was thought that i f c e l l s could be adapted to spermidine, a simultaneous adaptation to 1,3-diaminopropane and/or putrescine might be accomplished. The curves i n Pig. 5 indicate, however, that such c e l l s f a i l to oxidize both spermidine and 1,3-diaminopropane. Spermidine i n the growth medium did not appear to i n h i b i t growth. An examination of the growth medium a f t e r the c e l l s were harvested f o r study, showed the presence of much r e s i d u a l spermidine. Spermidine "adapted" c e l l s were capable of oxi d i z i n g putrescine a f t e r a l a g period. Equimolar amounts of l,3Tdiaminopropane did not a f f e c t the rate of putrescine oxidation. In the presence of 7.5 ;uM of putrescine, the oxygen consumption at the end of the experiment was 425 >il (equivalent to 46$ of the maximum t h e o r e t i c a l oxygen 61 TIME ( HOURS) PIG. 5. E f f e c t of pre-exposure to spermidine on the subsequent oxidation by s t r a i n C.C. c e l l s of 7.5 juM of putrescine (curve 1), 3.25 uM of each of putrescine and 1,3-diaminopropane (curve 2), 7.5 uM of 1,3-diaminopropane (curve 3), and 7.5 ,nM of spermidine (curve 4). 62 consumption*). With 3.25 ;uM of putrescine i n the presence of an equal amount of 1,3-diaminopropane, the oxygen consumption at the end of the experiment amounted to 225 jnl (or 48.7$ of the maximum t h e o r e t i c a l value). This s l i g h t increase i n the percentage of the maximum possible oxygen uptake was not necessarily e n t i r e l y due to 1,3-diaminopropane oxidation, but probably also r e f l e c t e d the fa c t that with twice as much putrescine to be oxidized, the time required to reach a s i m i l a r l e v e l of oxidation would be longer. Paper chromatography of the reaction mixture supernatants obtained from the Warburg respirometer vessels at the conclusion of the experiment showed that while putrescine had disappeared, 1,3-diaminopropane and spermidine were s t i l l present. These experiments indicate that s t r a i n C.C. c e l l s are r e l a t i v e l y l i m i t e d i n the range of amines that can be u t i l i z e d as substrates. Whether i n a b i l i t y of such c e l l s to adapt to 1,3-diaminopropane, 1,5-diaminopentane and spermidine r e f l e c t s a complete lack of the genetic c a p a b i l i t y to synthesize the system(s) which metabolize the compounds, or whether the c e l l s only lack the a b i l i t y to synthesize a permease, i s not known. As w i l l be shown l a t e r , c e l l - f r e e extracts obtained from putrescine-adapted c e l l s , oxidize 1,5-diaminopentane at a s i g n i f i c a n t rate. Since "unadapted * Based on a value of 5.5 jM Ovj/nM putrescine. 63 s t r a i n C.C, c e l l s possess some a b i l i t y to oxidize putrescine, a c e r t a i n amount of a c t i v i t y could have been expected when such c e l l s were incubated with 1,5-diaminopentane. The f a i l u r e to demonstrate such a c t i v i t y with the l a t t e r compound could indicate that the c e l l s are not permeable to 1,5-diaminopentane, or that intermediates derived from i t are strongly i n h i b i t o r y to oxidative processes occurring within the c e l l s . ( 4 ) Amines, as sole carbon sources. Various amines were tested i n d i v i d u a l l y f o r t h e i r a b i l i t y to support the growth of three mycobacterial s t r a i n s when present as the sole carbon source. The r e s u l t s of t h i s study are recorded i n Table 4» Table 4 Amines as sole carbon sources f o r growth*. Mycobacterial s t r a i n Carbon source N.T.N. C.C. S.C. 1.3- diaminopropane _ -1.4- diaminobutane (putrescine) + + + 1.5- diaminopentane (cadaverine) - - -1-aminobutane - -Py r r o l i d i n e - -Pipe r i d i n e - - -Spermidine - -Spermine - -Glucose + + + * + ss Growth within 17 days; - = No growth within 17 days« 64 Good growth was obtained f o r each s t r a i n tested with glucose and with putrescine within 5-8 days. The experiment was concluded a f t e r 17 days, at which time none of the other amines had produced any detectable growth. Among the mycobacterial strains tested, the a b i l i t y to u t i l i z e amines appeared to be r e s t r i c t e d to putrescine. The f a c t that putrescine was the only amine attacked supports the findings of Z e l l e r et a l (118) who reported t h i s compound to be one of the better b a c t e r i a l diamine substrates. Razin et a l (76) also found that putrescine was the most frequently u t i l i z e d amine among the na t u r a l l y occurring amines tested with various bacteria. Among the mycobacteria, the a b i l i t y to oxidize amines varies considerably. Putrescine and histamine were reported not to be attacked by Myc. tuberculosis. M. rubrum. M.  smegmatis and M. p h l e i (26). The l a t t e r organism was also found not to oxidize spermidine (76). On the other hand, strai n s of M. smegmatis studied by Roulet and Z e l l e r (60) and Bachrach et a l (9) were found to oxidize histamine, putrescine, cadaverine, agmatine, spermidine and spermine. Avian tubercle b a c i l l i (4,73) and M. tuberculosis var.  hominis (73) oxidized putrescine while M. l a c t i c o l a attacked both putrescine and histamine (26). (5) Occurrence of amines i n mycobacteria. Pig. 6 shows the eluti o n patterns obtained f o r (40 w> 100 50 o •a V 100 o © So V*i © Or i too AUP EXT&flcT OF STRRIM C.C CELLS J TRYP7IC S O Y BROTH P o W D E R (VlFCO) PVTR£SC/N£ AfJb SpBRtAfNE M I X T U R E So F R A C T I O N S f S - O F / a . 6. SePflKAT/ON O F A M I N E S B Y f O K E X C H A N G E . S A M P L E S WEfU ELUT5D F R O M COLUMN OF DOWEX 50 RESIN W'TH /I LINEARLY /A/CflEAS/A/Q CONC £NTR (\ Tio N OF H Ct ^0 - /-S /S/). 66 nitrogenous compounds contained i n ste a m - d i s t i l l a t e s obtained from s t r a i n C.C. e e l l extracts, t r y p t i c soy broth powder and a synthetic mixture of putrescine and spermine. The f i r s t peaks (f r a c t i o n s 20 to 30) represent ammonia. Both n e s s l e r i z a t i o n and the DHFB technique gave corresponding peaks. In the case of t r y p t i c soy broth, ammonia (as the hydrochloride s a l t ) accounted f o r over 99*9$ of the weight of the dried s a l t s obtained from the s t e a m - d i s t i l l a t e . Fractions 35-50 contained r e l a t i v e l y large amounts of p y r r o l i d i n e and trace quantities of piperidine and 1-aminobutane. P y r r o l i d i n e gave a yellow colour i n i t i a l l y with ninhydrin on paper chromatograms. Later the colour turned brown. The R'f value i n the solvent n-butanol:aeetic a c i d : water (2:1:1 v/v), was 0.69. These properties were shared by commercially obtained p y r r o l i d i n e which was used as a standard. P i p e r i d i n e and 1-aminobutane had Rf values of 0.74 and 0.72 respectively, i n the same solvent. The commercially a v a i l a b l e compounds migrated with i d e n t i c a l Rf values. The colour reactions with ninhydrin were also c h a r a c t e r i s t i c . Piperidine gave l i l a c - p u r p l e spots while 1-aminobutane yielded mauve-red spots. The coloured spots due to these compounds tended to fade gradually while colour with p y r r o l i d i n e was more permanent. P y r r o l i d i n e , p i p e r i d i n e and 1-aminobutane were not r e a d i l y detectable on paper chromatograms when sprayed with alkaline-DNFB solution, i n d i c a t i n g that these compounds were r e l a t i v e l y 67 v o l a t i l e under a l k a l i n e conditions. These compounds also formed derivatives with DNPB that were soluble i n aqueous acid-dioxane. While i t was reasonable to expect that p y r r o l i d i n e and 1-aminobutahe might occur i n c e l l - e x t r a c t s and t r y p t i c soy broth powder, possibly as decomposition products of compounds containing r e l a t e d structures, i t was d i f f i c u l t to conceive of a mechanism by which a 5-carbon structure l i k e piperidine could be derived from compounds l i k e putrescine and spermine which constituted the synthetic amine mixture. I t was therefore concluded that the presence of such compounds was due to contamination (possibly from the washed corks used i n the steam d i s t i l l a t i o n apparatus), Tryptic soy broth powder contained an amine, the DHP-derivative of which was only very s l i g h t l y soluble i n aqueous acid-dioxane. Such derivatives are t y p i c a l of those of d i - and polyamines. The amine was c o l l e c t e d i n fr a c t i o n s 81-89. Commercial putrescine was eluted i n f r a c t i o n s 72-82 but s l i g h t differences i n the concentration gradient of the elut i n g f l u i d may have accounted f o r t h i s . The amine had an Rf of 0.23 i n common with putrescine i n the n-butanols a c e t i c acidswater solvent. In the solvent n-propanol: 12 H:H01:water ( 3 s l * l v/v), the amine migrated l i k e putrescine (Rf 0.34). Unfortunately, not enough sample was obtained f o r enzymatic assay with D.O. prepared from s t r a i n C.C. putrescine-adapted c e l l s . 68 Acid extracts of s t r a i n C.C. c e l l s did not contain any d i - or polyamines. Herbst et a l (37) reported s i m i l a r findings with Myc. p h l e i and Myc. smegmatis. I f , therefore, d i - and polyamines occur i n mycobacterial c e l l s , they must be present i n extremely small quantities. (6) Influence of putrescine on endogenous r e s p i r a t i o n . In order to determine whether putrescine affected the rate of endogenous r e s p i r a t i o n i n mycobacterial c e l l s , two var i a t i o n s of the i n d i r e c t manometric technique were employed. In the f i r s t method, a single concentration of s t r a i n S.C. or N.T.N, c e l l s was allowed to oxidize various concentrations of putrescine. I f putrescine had a s i g n i f i c a n t e f f e c t on the rate of endogenous r e s p i r a t i o n , then the eff e c t should be r e f l e c t e d i n the degree of substrate oxidation when the l a t t e r value was based on the oxygen uptake corrected f o r endogenous oxygen consumption. The r e s u l t s of such studies, are recorded i n Table 5* The data indicate that i f endogenous oxygen uptake i s neglected, with s t r a i n S.C. c e l l s , there i s a 33.5% v a r i a t i o n i n the degree of putrescine oxidation depending on the concentration of the substrate. With N.T.N, c e l l s , a v a r i a t i o n of 37% was obtained. The l a t t e r s t r a i n also consumed more oxygen than was t h e o r e t i c a l l y possible with 1.5 jM of putrescine. When, however, the degree of substrate oxidation- was based on the oxygen uptake corrected 69 Table 5 E f f e c t of putrescine on endogenous r e s p i r a t i o n of s t r a i n S.C, and N.T.N, c e l l s : one concentration of c e l l s acting on various concentrations of putrescine. Myco- Putrescine Total Maximum <$> corrected \1.#V.^  -bac- GuM/cup) uptake theor- theor- uptake theor-t e r i a l 0*1 C>>) e t i c a l e t i c a l (jil 0 2) e t i c a l s t r a i n uptake* uptake - corrected O i l 0 2) uptake S.C, 1.5 172 184.8 93.5 87 47.1 3.0 262 369.6 71.0 177 47.9 6.0 443 739.2 60.0 358 48.5 N.T.N. 1.5 219 184.8 118.7 88 47.6 3.0 302 369.6 81.7 171 46.4 * Based on 5.5 ;uM 02/uM putrescine. f o r endogenous oxygen consumption, f a i r l y constant values f o r the degree of substrate oxidation were obtained. The data strongly suggest that putrescine, at the concentrations employed, had l i t t l e or no e f f e c t on the rate of endogenous r e s p i r a t i o n . In the second method, s t r a i n S.C. c e l l s were used at three concentrations with substrate (putrescine) at a single concentration. I n t h i s technique, the l e v e l of endogenous r e s p i r a t i o n should vary with the c e l l concentration. I f the endogenous r e s p i r a t i o n was not affected by putrescine, the t o t a l oxygen consumed with a constant amount of substrate 70 should he the same when the l a t t e r values were corrected f o r t h e i r respective endogenous oxygen uptake values. The r e s u l t s are summarized i n Table 6. Table 6 E f f e c t of putrescine on endogenous r e s p i r a t i o n of s t r a i n S.C. c e l l s : three concentrations of c e l l s acting on one concentration of putrescine. C e l l Endogenous Putrescine f> maximum* corrected $ maximum concen- 0 2 uptake 3 jaM th e o r e t i c a l uptake t h e o r e t i c a l t r a t i o n Cwl) 0 2^uj|ake uptake gal) uptake I X 70 231 62.5 161 43.6 2 X 127 290 78.5 163 44.2 3 X 186 353 95.5 167 45.3 * Based on 5.5 «»M 02/uM putrescine or 396.6 jul 02/3.0 nM putrescine. The r e s u l t s i n Table 6 indicate that a v a r i a t i o n of 33$ i n the degree of substrate oxidation occurs i f the endogenous r e s p i r a t i o n i s not taken into account. I f , however, the endogenous r e s p i r a t i o n i s assumed to continue unabated i n the presence of putrescine, and the oxygen uptake due to putrescine oxidation i s calculated by sub-t r a c t i n g the endogenous oxygen consumption from the t o t a l oxygen uptake, the degree of substrate oxidation appears to be r e l a t i v e l y constant f o r a l l c e l l concentrations. Stated 71 i n another way, the t o t a l oxygen uptake i n the presence of a single concentration of substrate, increases proportionately with an increase i n endogenous r e s p i r a t i o n . The data i n Tables 5 and 6 are consistent with the f a c t that endogenous r e s p i r a t i o n continues normally i n the presence of putrescine. In reporting oxygen uptake, i t therefore appears j u s t i f i a b l e to report oxygen uptake values corrected f o r endogenous r e s p i r a t i o n . Similar techniques have been used to determine the status of endogenous r e s p i r a t i o n i n the presence of substrate (71) although Blumenthal (12) has pointed out that the premise on which the manometric techniques are based, requires further t e s t i n g . (7) Oxygen, ammonia and carbon dioxide relationships during putrescine oxidation. Data showing i n p a r a l l e l the consumption of oxygen and the production of ammonia by N.T.N, c e l l s during the oxidation of 3.0 juM putrescine are i l l u s t r a t e d i n F i g . 7. Oxygen consumption was rapid i n i t i a l l y . The break i n the oxygen uptake curve occurred at 160 n l 0 2 (equivalent to 2.38 jaM 02/nM putrescine or to 43.3$ of the maximum th e o r e t i c a l l e v e l of oxidation). At the end of the experiment, the oxygen consumption had proceeded to 44.2$ of the maximum degree of oxidation. At t h i s point, the curve f o r ammonia which had already l e v e l l e d o f f , showed the reaction mixture supernatant to contain 1.52 }M NEz/ml. Since the 72 PIG. 7. Oxygen consumption and ammonia production during putrescine oxidation by s t r a i n N.T.N, c e l l s . Curve 1: Oo uptake with 3.0 ;uM putrescine (1.0 juM/ml.) Curve 2: NH^ production with putrescine (1.0 uM/ml.). 73 maximum possible concentration of ammonia was 2.0 JUM NH^/ ml., than at t h i s stage of the reaction, 76$ of the ammonia had been released from the putrescine molecule. Ammonia production was not influenced by the presence or absence of carbon dioxide i n the reaction mixtures. During the oxidation of putrescine by N.T.N, c e l l s i n the presence of atmospheric carbon dioxide, 1 . 5 2 ^ NH^/ml. of reaction mixture ware produced. The corresponding value f o r ammonia production under COg-free conditions was 1.50 pK NH^/ml. At the end of the 5-hour experiment, ammonia i n the endogenous f l a s k s amounted to 0.30 pM. NH3/ml. of reaction mixture. However, since zero time ammonia determinations were not made, endogenous ammonia production was probably somewhat l e s s than indicated above. In an independent study to determine carbon dioxide production, by N.T.N, c e l l s , the c e l l s were allowed to oxidize 3.0 ;uM of putrescine. At the break i n the oxygen uptake curve (160 jol 0^ or 43.3$ of the maximum l e v e l of oxidation), the carbon dioxide production amounted to 1.06 juM C0p/)aM putrescine. At a point further along the oxygen uptake curve corresponding to 46.2$ oxidation, the C0 2 output was 1.14 jM COg/uM putrescine. Thus, as expected, there i s a sharp decrease i n the rate of carbon dioxide production a f t e r the break i n the oxygen uptake curve. During the r a p i d oxygen uptake portion of the curve, the respiratory quotient (R.Q.) was 0.445. However, i n r e a l i t y the R.Q. may be somewhat 74 higher (at l e a s t 0.60) since radioactive studies conducted 14-subsequently with 1,4-C, putrescine showed that under COg-free conditions, approximately 72$ of the terminal carbons of putrescine were evolved as carbon dioxide at the. break region of the oxygen uptake curve. During studies on the oxidation of putrescine by Myo. smegmatis, Roulet and Z e l l e r (60) determined carbon dioxide by the i n d i r e c t manometric method. Since these workers did not know to what extent the presence and absence of carbon dioxide i n t h e i r reaction systems might have influenced the course of the oxidation, these workers were concerned about the v a l i d i t y of the carbon dioxide values they obtained. They observed, however, that values f o r ammonia obtained from the reaction mixtures, with and without carbon dioxide, were i d e n t i c a l and therefore i n f e r r e d that differences i n the carbon dioxide pressure i n t h e i r reaction systems had not influenced carbon dioxide production. In the l i g h t of the present r e s u l t s , however, such an assumption may have been i n v a l i d . Thus under CO^-free conditions, more carbon dioxide i s ac t u a l l y evolved than i s indicated by the i n d i r e c t manometric method, even though ammonia production i n the reaction flasks, with,, and without carbon dioxide, may be i d e n t i c a l . F i g . 8 shows the oxygen uptake curve obtained with s t r a i n S.C. c e l l s acting on 6.0}M of putrescine together with ammonia production determined i n p a r a l l e l . The oxygen uptake curve broke at 305;ul Og (2.27 nM Og/uM putrescine 75 TIME (HOURS) FIG. 8. Oxygen c o n s u l tion and ammonia production during putrescine oxidation by s t r a i n S.C. c e l l s . Curve 1: Op uptake with 6.0 uM putrescine (2.0 nM/ml.). Curve 2: NH^ production with putrescine (2.0 uM/ml.). 76 or at 41.3$ of the maximum l e v e l of oxidation). At the conclusion of the experiment, the net oxygen consumption amounted to 2.48 }M Q^/jM putrescine (or 45.1$ of the maximum degree of oxidation). The break i n the ammonia output curve coincided with that of the oxygen uptake curve and remained e s s e n t i a l l y at the same l e v e l thereafter. At the end of the experiment, a t o t a l of 2.84 ^iM NH^/ml. were produced.out of a possible 4.0 p& HH^/ml. of reaction mixture. This meant that 1.42 p& NHy'uM putrescine (or 71$ of* the ammonia) had been released from the putrescine molecule. At the end of the 5-hour experiment, ammonia i n the control f l a s k s amounted to 0.37 joM/ml. of reaction mixture. Carbon dioxide production with s t r a i n S.C. c e l l s was determined i n an independent experiment. At a point a f t e r the break i n the oxygen uptake curve corresponding to 47.2$ of the maximum l e v e l of oxidation, carbon dioxide production amounted to 1.14 juM. COg/^ uM putrescine. With t h i s organism, the presence or absence of carbon dioxide had no ef f e c t on the amount of ammonia produced since 69$ of the t h e o r e t i c a l maximum amount of ammonia was produced i n each of the Warburg reaction vessels used to determine carbon dioxide production by the i n d i r e c t manometric method. In other independent studies with t h i s mycobacterial s t r a i n , the break i n the oxygen consumption curve occurred 77 at 2.4 }M 02/uM putrescine, and the percentage of the maximum ammonia production ranged from 69 to 76$. Pig. 9 shows the oxygen uptake curve obtained f o r s t r a i n C.C. c e l l s acting on 6.0 jaM of putrescine. By extrapolation, the curve appeared to break at 305 ;ul 0^ (equivalent to 2.27 p& 02/wM putrescine or to 41.3$'of the th e o r e t i c a l maximum oxidation l e v e l ) . This s t r a i n seldom gave sharp breaks i n the oxygen uptake curves and consequently determinations of the break-points:were more d i f f i c u l t . Oxygen consumption continued a f t e r the break u n t i l at the end of the experiment 342 jil 0^ were consumed. This corresponded to 2.54 uM 02/uM putrescine or to 46.3$ of the maximum l e v e l of oxidation. At t h i s point, a t o t a l of 1.26 juM of C0 2 were produced per jaM of putrescine. P a r a l l e l ammonia determinations were not conducted during t h i s experiment but at the end of the reaction, of a possible 4.0 }M NH^/ml. of reaction mixture, 2.75;uM NHj/ml., or 69$ of the maximum possible ammonia were ac t u a l l y l i b e r a t e d . With t h i s s t r a i n , the ammonia curve broke i n the region of the oxygen uptake curve break, a f t e r which the l e v e l of ammonia remained r e l a t i v e l y constant. An example of an ammonia curve f o r s t r a i n C.C. i s shown i n the next section (Pig. 12) where the effect of arsenite on ammonia production was studied. At the end of the experiment, ammonia i n the endogenous flasks amounted to 0.30 /uM/ml. of reaction mixture. 78 HOO t TIMB (HOVRS) FIG. 9. Oxygen consumption during the oxidation of 6.0 pM. of putrescine by s t r a i n C.C. c e l l s . 79 An examination of the reac t i o n mixture supematants by paper chromatography f a i l e d to reveal the presence of any organic acids or nitrogenous compounds. Such examinations were made at various times during the course of the reaction with each s t r a i n . To summarize, a l l three mycobacterial strains oxidized putrescine a f t e r adaptation to the compound. The break i n the oxygen.uptake curves occurred at points ranging from 2.27 to 2.40 jffl., Og/uM putrescine. Thereafter oxygen consumption proceeded more slowly u n t i l at the conclusion of the experiments the l e v e l of the maximum t h e o r e t i c a l oxidation had reaohed 43.3 to 47.2$. During the experiments, only two products appeared i n the supernatantms HH^ and COg. Ammonia production reached a maximum l e v e l and remained at t h i s l e v e l corresponding to 69-76$ of the t h e o r e t i c a l maximum f o r ammonia output. Presumably, the rest of the ammonia was assimilated by the c e l l s since no other nitrogenous compounds were detectable i n the supernatants. A l l putrescine-carbon not assimilated by the ce l l s , appeared to be accounted f o r as carbon dioxide. C e l l s at the conclusion of the experiments appeared to have assimilated somewhat over 2.0 ;uM of carbon out of a possible 4.0 nM of carbon. The f i n d i n g that part of the putrescine molecule i s assimilated, i s i n agreement with growth studies with putrescine as a sole source of carbon. Since the c e l l s also 80 assimilated 24%, or more, of the nitrogen, i t i s possible that putrescine could serve as both a carbon and nitrogen source. This i s reported to be the case with a s t r a i n of Ps. fluorescens (46). The incomplete oxidation of putrescine by Myc. smegmatis (60) i s i n accord with the r e s u l t s obtained i n t h i s study. During the oxidation of putrescine by Myc. smegmatis. the compound was 65% oxidized. Carbon dioxide and ammonia production accounted f o r 39% and 72%, respectively, of the putrescine molecule (60). Putrescine and r e l a t e d polyamines were also found to be incompletely oxidized by other bacteria tested (76). However, while the incomplete oxidation of amines may be the general r u l e , Ps. pyocyanea was shown to oxidize putrescine to completion (29). (8) I n h i b i t o r studies with i n t a c t c e l l s . Table 7 summarizes the r e s u l t s obtained from studies with s t r a i n C.C. c e l l s acting on putrescine i n the presence of various i n h i b i t o r s . The data represent oxygen consumption a f t e r the oxygen uptake curves had l e v e l l e d o f f . Malonic a c i d and p-phenylenediamine did not exert any s i g n i f i c a n t e f f ects on putrescine oxidation or on endogenous r e s p i r a t i o n . This was possibly due to the f a c t that the c e l l s were not r e a d i l y permeable,to the compounds. These agents have been reported to i n h i b i t succinic dehydrogenase a c t i v i t y (88) and were used i n an attempt to demonstrate that succinic a c i d was produced as an intermediate during putrescine degradation. 81 Table 7 The e f f e c t of various i n h i b i t o r s on the oxygen consumption by s t r a i n S.C. c e l l s acting on 3.0 of putrescine at 4 hours.. Treatment* C e l l s plus: Og uptake Net 0 o uptake 2 Oa) PUT HgO 490 302 188 PUT + MAI (0.005 M) HgO + MAL (0.005 M) 501 316 185 PUT + PPD (0.0045 M) H 20 + PPD (0.0045 M) 493 300 193 PUT + Azide (0.003 M) H 20 + Azide (0.003 M) 560 400 160 PUT + Arsenite (0.003 M) HgO + Arsenite (0.003 M) 315 192 123 * PUT = putrescine; MAL = malonic acid; PPD =B p-phenylenediamine. Sodium.azide stimulated endogenous oxygen consumption. A s i m i l a r e f f e c t on the oxygen uptake i n the presence of putrescine was also observed. The net ef f e c t of azide, was to prevent the slow increase i n oxygen consumption that normally occurred a f t e r the break i n the oxygen uptake curve. Thus, 82 a f t e r t h i s point, the oxygen uptake remained at a l e v e l corresponding to 160 ;ul Og. Of the i n h i b i t o r s tested, sodium arsenite exerted the most pronounced ef f e c t s on the net oxygen consumption. Both endogenous r e s p i r a t i o n , and oxygen consumption due to putrescine, were i n h i b i t e d . Reaction mixture supernatants from each of the i n h i b i t o r studies with the C.C. s t r a i n were examined f o r the presence of carbonyl compounds. I t was found that substances reacting-with 2,4-dinitrophenylhydrazine (DPNH) only accumulated when arsenite was present. Endogenously r e s p i r i n g c e l l s poisoned with arsenite, excreted oC-ketoglutaric a c i d (0.13 .nM/ml. of reaction mixture) i n addition to traces of pyruvate. Under s i m i l a r conditions, but with putrescine as substrate, only traces of «*-ketoglutarate accumulated. Under the l a t t e r conditions, pyruvic, a c i d was the dominant keto acid released by the c e l l s (0.18 ;uM/ml.)• Such r e s u l t s suggested that s t r a i n C.C. c e l l s contained a r e l a t i v e l y a r s e n i t e - i n s e n s i t i v e system which u t i l i z e d pc-ketoglutaric acid when putrescine, or an intermediate of putrescine degradation, was present. C e l l - f r e e extracts contained a transaminase enzyme f o r which efc-ketoglutaric a c i d and df-aminobutyric acid were.substrates. Since the l a t t e r compound was also shown to be an intermediate i n the d i s s i m i l a t i o n of putrescine, i t i s probable that the conversion of eC-ketoglutaric acid to glutamic acid by t h i s system explained the observed f a c t s . 83 The increase i n the concentration of excreted pyruvate observed when putrescine was present as substrate, indicated that at l e a s t a portion of the putrescine molecule was metabolized v i a pyruvic a c i d . Table 8 records the r e s u l t s of i n h i b i t o r studies with s t r a i n S.C. c e l l s acting on putrescine. The data represent the oxygen uptake a f t e r 4 hours at which time the break i n the oxygen uptake curves had already occurred. Dihydrostreptomycin and i s o n i c o t i n i c a c i d hydrazide exerted no s i g n i f i c a n t e f f e c t s on the net oxygen consumption due to putrescine oxidation. The former may have i n h i b i t e d the net oxygen uptake s l i g h t l y while the l a t t e r appeared to. produce a small increase i n the net oxygen consumption. Neither of the reaction mixtures treated with these a n t i -mycobacterial agents contained any compounds reacting with DPNH. The e f f e c t of arsenite at 0.003 M on the net oxygen uptake by. S.C. c e l l s r e s p i r i n g endogenously, and i n the presence of putrescine, was not very marked. However, an examination of the reaction mixtures obtained at the conclusion of the experiment indicated that endogenously r e s p i r i n g c e l l s , poisoned with 0.003 M arsenite, had excreted 0.16 jM of pyruvate/ml. The corresponding value f o r c e l l s with putrescine as substrate, was 0.33 .uM of pyruvate/ ml. At t h i s concentration of i n h i b i t o r , no leakage of o<-ketoglutarate occurred. Arsenite at 0.009 M exerted a 84 Table 8 The e f f e c t of various i n h i b i t o r s on the oxygen consumption by s t r a i n S.C. c e l l s acting on 3.0 nM.of putrescine. Treatment* C e l l s plusi 3« uptake Net 0 o uptake 2 oui) 2 (£D PUT H 20 337 166 171 PUT + DHS H 20 + DBS (0.001 M) (0.001 M) 324 167 157 PUT + DHS (0.006 M) H 20 + DHS (0.006 M) 322 158 164 PUT + IHH H 20 + IHH (0.001 M) (0.001 M) 355 171 184 PUT + IHH (0.006 M) HgO + IHH (0.006 M) 354 161 193 PUT + Arsenite (0.003 M) H 20 + Arsenite (0.003 M) 264 104 160 PUT + Arsenite (0.009 M) H o0 + Arsenite (0.009 M) 228 85 143 * DHS = dihydrostreptomycin; IHH a i s o n i c o t i n i c acid hydrazide. 85 more pronounced ef f e c t on the net amount of the oxygen consumed hut there was a concurrent reduction i n the quantity of DPNH-reacting materials excreted into the reaction mixtures. Table 9 expresses the concentrations of the coloured DPNH-reacting substances found i n the reaction mixture supematants i n terms of K l e t t units ( F i l t e r #54). Table 9 The e f f e c t of arsenite on s t r a i n S.C. c e l l s : accumulation of UPKH-reacting materials. Treatment C e l l s plus: Colour due to DPHH derivatives i n 1.0 ml. of reaction mixture at 4 hours (K l e t t units) PUT* H 20 14 13 PUT + Arsenite HgO + Arsenite (0.003 M) (0.003 M) 195 85 PUT + Arsenite HgO + Arsenite (0.009 M) (0.009 M) 68 56 Blank 15 * PUT - putrescine (0.001 M). Endogenously r e s p i r i n g c e l l s treated with arsenite at 0.009 M produced both pyruvate and ot-ketoglutarate i n approximately equal amounts as judged by the density of the 86 spots obtained on paper chromatograms. The same was true f o r c e l l s incubated with putrescine and arsenite at 0.009 M. I t therefore appeared that by increasing the concentration of i n h i b i t o r from 0.003 M to 0.009 M, not only was there a decrease i n the amount of the excreted keto acids, but also there must have been an accumulation of some other oxi(Sizable material, the l o s s of which from the c e l l might account f o r the reduction i n the net oxygen uptake. The fact that putrescine did not prevent the accumulation of ©c-ketoglutarate, suggested that under the conditions of higher l e v e l s of arsenite, the system that u t i l i z e d oC-ketoglutarate was now being i n h i b i t e d . Concurrent studies demonstrated that arsenite decreased ammonia output (Pigs. 11 and 12), I t therefore appeared l i k e l y that the suspected leakage material might be nitrogenous. As w i l l be shown l a t e r , several amino acids were found to be excreted by c e l l s poisoned with arsenite. P i g . 10 shows the oxygen consumption by arsenite- 1 poisoned s t r a i n S.C. c e l l s acting on putrescine i n comparison with that f o r unpoisoned c e l l s . The coincident accumulation of carbonyl compounds i n the reaction mixture supernatant i s also i l l u s t r a t e d . Endogenously r e s p i r i n g c e l l s (1.0 mg. dry weight/Warburg v e s s e l ) , poisoned with 0.003 M arsenite, excreted traces of ot-ketoglutaric a c i d along with measurable amounts of pyruvic acid (0.08 jiM/ml. of reaction mixture). When putrescine was present as substrate, s i m i l a r l y treated c e l l s excreted only pyruvic a c i d (0.33 ;aM/ml. of rea c t i o n 87 in -200 - 100 © o N or a 3 T / M £ (HOURS) F I G . 10. E f f e c t o f a r s e n i t e on t h e m e t a b o l i s m o f p u t r e s c i n e by s t r a i n S . C . c e l l s . C u r v e 1: 0 2 u p t a k e w i t h 3 . 0 juM p u t r e s c i n e ( 1 . 0 u M / m l . ) . C u r v e 2: Same a s c u r v e 1, but a r s e n i t e ( 3 . 0 u M / m l . ) p r e s e n t , C u r v e 3*. L e a k a g e o f p y r u v a t e w i t h p u t r e s c i n e (1 .0 u M / m l . ) . C u r v e 4: Same a s c u r v e 3» b u t a r s e n i t e ( 3 . 0 u M / m l . ) p r e s e n t , 88 mixture). Curves 3 and 4 i n F i g . 10 represent hydrazone colour with added substrate measured i n K l e t t units ( F i l t e r #54) corrected f o r hydrazone colour due to endogenously produced pyruvate. At the end of the experiment, the net amount of pyruvate excreted amounted to 0.25 juM/ml. of reaction mixture. Unpoisoned c e l l s did not excrete carbonyl compounds. F i g . 11 shows the eff e c t of 0.009 M arsenite on the oxygen uptake and ammonia production by s t r a i n S.C. c e l l s acting on 3.0 of putrescine. Both oxygen consumption and ammonia output were decreased. Similar e f f e c t s on the oxygen consumption and ammonia production are recorded i n F i g . 12 f o r the oxidation of putrescine by s t r a i n C.C. c e l l s i n the presence of arsenite. With s t r a i n S.C. c e l l s , arsenite reduced the ammonia output from 71.1$ of a possible maximum f o r unpoisoned c e l l s , to 53.1$ with poisoned c e l l s . The corresponding values f o r poisoned and unpoisoned s t r a i n i CkC.:; c e l l s were 68.7$ and 50.3$ respectively. Oxygen uptake and ammonia output were plotted as the t o t a l oxygen uptake or ammonia output observed with putrescine and arsenite, corrected f o r the corresponding endogenous values obtained f o r c e l l s incubated with arsenite. The data recorded i n Figs. 11 and 12 were obtained by using high enough l e v e l s of arsenite to cause the accumulation of only ot-ketoglutarate i n reaction mixtures containing endogenously r e s p i r i n g c e l l s . With putrescine as substrate 89 200 TIM£ (HOURS). FIG. 11. E f f e c t of arsenite on oxygen consumption and ammonia output by s t r a i n S.C. c e l l s acting on putrescine. Curve 1: 0 2 uptake with 3.0 nM putrescine (1.0 juM/ml.). Curve 2: Same as curve 1, but arsenite (9.0;uM/ml.) present. Curve 3: NH5 output with putrescine (1.0 nM/ml.). Curve 4: Same as curve 3, but arsenite (9.0 uM/ml.)present. 90 loo -1 ISO ;£ / oo K o -•—+-So -rO x 3 r / M £ ( HOURS) FIG. 12. E f f e c t of arsenite on oxygen consumption and ammonia output by s t r a i n C.C, c e l l s acting on putrescine. Curve 1: 0 2 uptake with 3.0 ;uK putrescine (1.0 JuM/ml.). Curve 2: Same as curve 1, but arsenite (3.0/jM/ml.) present. Curve 3: NH, production with putrescine (1.0 ^ iM/ml.) present. Curve 4: Same as curve 3, but arsenite (3.0 ;jM/ml.) present. 9 1 s i m i l a r l y treated c e l l s excreted pyruvate i n addition to cG-ketoglutarate. Since the percentage of the ammonia released had been reduced to 50-53% of the possible maximum, the data supported the view that only one out of a possible two micromoles of ammonia had been released from the putrescine molecule. Thus, i f one postulated that ^-aminobutyric acid was the intermediate r e t a i n i n g the r e s i d u a l nitrogen, t h i s compound, or amino-compounds derived from i t , should be present i n the reaction mixture as the excreted product(s). A paper chromatographic examination of the reaction mixtures obtained at the conclusion of the experiments with s t r a i n C.C. and S.C. c e l l s showed that i n the presence of putrescine and arsenite, the c e l l s had excreted trace amounts of aspartic acid, glutamic acid and valine, together with l a r g e r amounts of alanine and £-aminobutyric acid. In the case of s t r a i n C.C. c e l l s , y-aminobutyric acid was the amino acid leaked i n highest concentration. With s t r a i n S.C. c e l l s , the concentration of alanine approached that of y-aminobutyric acid as judged by the density of the ninhydrin-p o s i t i v e areas on paper chromatograms. Supernatants from endogenous reaction mixtures containing arsenite contained no jf-aminobutyric acid, but traces of aspartic acid, glutamic acid, alanine and valine were detected. C e l l s not incubated with arsenite did not excrete any amino acids i n t o the reaction mixture sxipernatants. In order to determine the nature of the amino acid pool 92 within the c e l l s , and to determine whether c e r t a i n amino acids were derived from putrescine, washed samples of c e l l s , obtained at various periods during the course of the reaction, were extracted with HC1 and the acid extracts were chromatographed. The separated compounds were developed with ninhydrin. C e l l s examined i n t h i s manner included endogenously r e s p i r i n g c e l l s , with and without arsenite, and c e l l s o x i d i z i n g putrescine, with and without arsenite. Acid extracts corresponding to 2.0 mg. dry weight of s t r a i n C.C. c e l l s were examined by paper chromatography and the amino compounds observed are l i s t e d i n Table 10. Table 10 ffinhydrin-positive areas on chromatograms* obtained from HC1 extracts.of v a r i o u s l y treated s t r a i n C.C. c e l l s . Ninhydrino-positive spot Treatments c e l l s plus: Rf Probable i d e n t i t y HgO PUT HgO+Arsenite PUT+Arsenite 0.416 Glutamic a c i d 0.582, y-aminobutyric a c i d 0.692 Valine + + + + + + + + + + * Solvent: n-butanol:acetic acid:water (2:1:1 v/v). The data i n Table 10 indicated that y-aminobutyric a c i d was an intermediate i n putrescine degradation since the former 93 compound only occurred when putrescine was present as substrate. Unfortunately, too l i t t l e material was spotted on chromatograms with the r e s u l t that only those amino compounds present i n r e l a t i v e l y large quantities were detected. However, the chromatograms demonstrated that while the concentrations of glutamic a c i d and vali n e remained e s s e n t i a l l y constant, ^-aminobutyric acid disappeared completely i n unpoisoned c e l l s . The disappearance coincided with the region of the break of the oxygen uptake curve. In c e l l s poisoned with arsenite, traces of y-aminobutyric acid were s t i l l evident at the conclusion of the experiment. The absence of alanine on these chromatograms was somewhat surprising i n view of the f a c t that r e l a t i v e l y large amounts of the amino a c i d were excreted i n t o the external medium by poisoned c e l l s metabolizing putrescine. I t i s possible, however, that i n h i b i t e d c e l l s excreted most, or a l l of the alanine formed during putrescine d i s s i m i l a t i o n , and that i n uninhibited c e l l s , alanine was u t i l i z e d as r a p i d l y as i t was synthesized. Table 11 records the r e s u l t s of paper chromatographic studies on a c i d extracts obtained from various treatments of s t r a i n S*0. c e l l s . In t h i s experiment, an amount of a c i d extract corresponding to about 6.0 mg. dry weight of c e l l s was applied to each spot on the chromatogram so that a more complete picture of the acid-soluble pool might be obtained. The data i n Table 11 again suggested that y-aminobutyric acid was an intermediate of putrescine d i s s i m i l a t i o n since 94 Table 11 Ninhydrin-positive areas on chromatograms* obtained from HC1 extracts of variously treated s t r a i n S.C. c e l l s . Ninhydrin-positive spot Treatments c e l l s plus Rf Probable i d e n t i t y H 20 PUT H P0+Arsenite PUT+Arsenite 0.178 Unknown +- +- +- + 0.252 Putrescine + + 0.318 Aspartic acid + + + + 0.414 Glutamic a c i d + + + + 0.515 Alanine + + + + 0.582 y-aminobutyric acid - + - + 0.680 Valine + + + + 0.771 Leucine +- +- +- +-* Solvents n-butanol:acetic acid:water (2:1:1 v/v). the former only occurred when putrescine was present. Gamma-aminobutyric acid disappeared more slowly i n acid extracts obtained from arsenite-poisoned c e l l s . The l a t t e r observation was equally true f o r the disappearance of putrescine. Glutamic ac i d occurred i n high concentrations throughout the experiment. Aspartic acid, v a l i n e and leucine were present i n lower, but constant amounts. The most notable e f f e c t observed with arsenite was a large accumulation of alanine i n acid-extracts from poisoned c e l l s . This b u i l d -up of alanine was p a r t i c u l a r l y marked i n the case of poisoned c e l l s where putrescine was present as substrate. A sim i l a r 95 accumulation of an u n i d e n t i f i e d ninhydrin-positive compound (Rf 0.178) occurred with poisoned c e l l s which was more noticeable when putrescine was present. Unfortunately i n s u f f i c i e n t sample was available to allow any further characterization of the compound and the experiment was not repeated. However, the unknown was not detected as an excreted product - perhaps because the product was not excreted. Or, since reaction mixtures were examined by paper chromatography a f t e r anion and cation exchange, i t was possibly l o s t with the water wash of the anion exchange column - i n which case the compound might have been an amine. Arsenite i n h i b i t i o n of putrescine oxidation by H.T.H. c e l l s , produced r e s u l t s that d i f f e r e d from those obtained with c e l l s of st r a i n s S.C. and C.C. With N.I.H. c e l l s , arsenite at 0.001 M and 0.003 M caused the oxygen uptake to proceed very slowly so that by the end of a 4-hour experiment, the oxygen uptake had not yet approached the break-point i n the oxygen uptake curve. No carbonyl compounds or amino acids were detected i n the reaction mixture supernatants. This was probably because the system most sensitive to arsenite i n these c e l l s did not involve the u t i l i z a t i o n of these compounds. I t therefore seemed l i k e l y that the arsenite-s e n s i t i v e system occurred early i n the reaction sequence, possibly at the A * - p y r r o l i n e dehydrogenase l e v e l . A block at t h i s point would not be unexpected since arsenite has 96 been reported to i n h i b i t aldehyde dehydrogenases (45). Furthermore, i f ^ ' - p y r r o l i n e had accumulated i n arsenite containing reaction mixtures, i t would have been l o s t during the anion and cation exchange procedure employed to eliminate Na ions and AsO^ ions from the reaction mixtures and therefore would not have been detected on paper chromatograms. Hydrochloric acid extracts of non-poisoned H.T.N, c e l l s metabolizing putrescine contained s i x ninhydrin-positive areas corresponding to: putrescine (which disappeared eventually), aspartic acid, glutamic acid, alanine, ^-aminobutyric a c i d (which l a t e r vanished) and v a l i n e . Similar extracts from endogenously r e s p i r i n g c e l l s were q u a l i t a t i v e l y i d e n t i c a l except that putrescine and ^ -aminobutyric a c i d were absent. The oxidation of putrescine by N.T.N, c e l l s which had been frozen and thawed once, proceeded normally when judged by oxygen consumption. However, reaction mixtures were found to contain excreted alanine. Freezing and thawing therefore appeared to impair to some extent the a b i l i t y of the c e l l s to metabolize alanine. The fact that the excretion of alanine by such c e l l s did not a f f e c t the oxygen consumption indicated that either alanine was not metabolized oxidatively or that the amount of the compound exdreted was too small to be r e f l e c t e d i n the oxygen uptake. An examination of the reaction mixtures obtained from s i m i l a r c e l l s r e s p i r i n g endogenou.sly, f a i l e d to reveal the presence of any amino acids. During the oxidation of putrescine by fre s h l y prepared c e l l s , no excretion 97 products other than ammonia and carbon dioxide were detected. N.T.N. c e l l s therefore appear to metabolize putrescine i n a s i m i l a r manner to that of the other two mycobacterial s t r a i n s . During the degradation of putrescine, y-aminobutyric a c i d was a common intermediate produced by each s t r a i n . Another amino acid which appeared to be derived from putrescine was alanine. The build-up of an u n i d e n t i f i e d ninnydrin-p o s i t i v e compound (Ef 0.178) on i n h i b i t i o n with arsenite was only observed with the S.C. s t r a i n . Alanine formation probably occurred as follows: Putrescine $-aminobutyric acid + « -ketoglutaric a c i d (from an ^ endogenous source) of-ketoglutaric a c i d succinic semialdehyde + glutamic a c i d i succinic a c i d — » 00^ + pyruvic acic + alanine C e l l - f r e e extracts obtained from s t r a i n C»C. c e l l s adapted to putrescine, were capable of catalyzing the transamination, of oxalacetic and pyruvic acids with glutamic acid to y i e l d aspartic and alanine respectively. Under the conditions of the assay (pH 9.0) the reactions were not as rapid as the transamination between o<-ketoglutarie acid and y-aminobutyric a c i d . I t i s therefore not d i f f i c u l t to v i s u a l i z e that putrescine may become equilibrated r a p i d l y with a number of amino acids. 98 I I Studies with c e l l - f r e e extracts (1) Diamine oxidase (D.O.). When crude, dialyzed, c e l l - f r e e extracts were incubated with putrescine, oxygen was consumed and ammonia was produced. The stoichiometry of the reaction was determined by allowing the extract to oxidize 15 }M of putrescine. At completion of the reaction, 167.2 pi Og were consumed. The expected 0^ uptake (assuming catalase to be present i n the extract) was 168.0 ;ul. Measurements fo r ammonia indicated that 14.94 )M were produced instead of a t h e o r e t i c a l 15.0 ;aM. The other product of the reaction was shown by paper chromatography to be A'-Pyrroline. In the solvent, n-butanol:acetic acid:water, chemically synthesized d'-pyrroline had an Rf value of 0.30. The compound could be eluted from the chromatogram and reacted with o-aminobenzaldehyde to produce the c h a r a c t e r i s t i c yellow complex. On chromatograms, the colour produced with ninhydrin was i n i t i a l l y yellow. Later, the colour turned tan-brown. These properties were also shared by the compound produced from putrescine by D.O. action. Extraction of A 1 - p y r r o l i n e into ethyl ether from a l k a l i n e solution appeared to cause some destruction of the compound. Two additional ninhydrin-positive areas occurred on paper chromatograms: a tan-brown spot, Rf 0.15, and a grey-brown spot, Rf 0.60. The degradation of -pyrroline was accompanied 99 by a c e r t a i n amount of streaking on chromatograms and the decomposition could not be prevented by conducting the extraction at 0°C. The i n s t a b i l i t y of A'-pyrroline has been well documented (44,72), When, therefore the stoichiometric production of A'-pyrroline from putrescine was studied, the reaction was conducted at pH 7»2 to avoid the decomposition of the former compound. The oxygen uptake due to D.O. action on 6.0 pM. of putrescine was followed i n order to determine when the reaction, had proceeded to completion. At t h i s time, the reaction was stopped w±th zinc sulphate, the preci p i t a t e d protein removed by centrifugation, and a representative aliquot of the supernatant assayed f o r A'-pyrroline with o-aminobenzaldehyde. Instead of a t h e o r e t i c a l 6.0 ;oM of A * -pyrroline, 5.7 of A'-pyrroline were a c t u a l l y found to be present. The p o s s i b i l i t y that -pyrroline existed i n equilibrium with y-aminobutyraldehyde (44), with the former compound as the quan t i t a t i v e l y dominant species, was b r i e f l y investigated with inconclusive r e s u l t s . Protein-free reaction mixtures containing A ,""Py r r°H n e» were allowed to react with DPNH at room temperature f o r up to 6 hours i n order to trap ^-aminobutyraldehyde as i t s hydrazone derivative. A l l colour was extractable i n t o ethyl acetate from acid solution. Since the hydrazone\should have possessed a p o s i t i v e l y charged amino group under acid conditions, i t should have had a tendency 100 to remain i n the aqueous phase unless the large hydrophobic portion of the molecule was able to overcome the i o n i c e f f e c t , That some hydrazone formation may have occurred, was suggested by the f a c t that one or two extra extractions with ethyl acetate were required to remove a l l colour from the aqueous phase. In the/ absence of /l*-pyrroline, s i m i l a r l y treated controls required fewer extractions with ethyl acetate f o r removal of DPNH. The r e s u l t s support the conclusion that during the oxidation of 1.0/xM of putrescine by the D.O. of crude, c e l l - f r e e extracts, 0.5 )M Og are consumed and 1.0 ;aM of and 1.0 jM of ^ - p y r r o l i n e are produced: D.O. Putrescine * Jf-aminobutyraldehyde + NEL catalase ^ - H 20 t +H20 A' - p y r r o l i n e Gamma-aminobutyraldehyde has been included i n the reaction i l l u s t r a t e d even though i t s existence was not conclusively demonstrated. By analogy with r e l a t e d reactions i t i s assumed to be the immediate product of the oxidative deamination (43,44,46,115). That catalase was present i n crude c e l l - f r e e extracts was indicated i n an e a r l i e r experiment which was i n i t i a l l y intended to investigate the heat s t a b i l i t y of the D.O. enzyme. The r e s u l t s i n Table 12 not only demonstrate that the enzyme 101 i s r e l a t i v e l y heat-stable, but also that heat treatment f o r o 15 minutes at 55 0 caused a doubling i n the oxygen consumption without a f f e c t i n g the expected ammonia production. The l a t t e r observation strongly suggested that while the heat treatment did not a f f e c t the D.O. enzyme, catalase a c t i v i t y i n the c e l l - f r e e extract was completely destroyed. Table 12 The ef f e c t of heat on 33.0. a c t i v i t y * i n c e l l - f r e e extracts. Treatment c e l l - f r e e extract of 0„ uptake Oil) at 30 min. Expected 0« uptake with < catalase: present absent at 30 min. Expected NH OA Unheated 3 3 33 66 2.92 2.95 Heated 15 mins., 55°,C 64 33 66 2.90 2.95 Heated 30 mins.., 55°C 61 33 66 3.01 2.95 * A c t i v i t y measured at pH 7.2 i n phosphate buffer with putrescine (0.01 M) as substrate. To further characterize the putreseine-metabolizing enzyme as t y p i c a l of a D.O. enzyme, the ef f e c t s of several i n h i b i t o r s were b r i e f l y studied. The r e s u l t s are recorded i n Table 13. The data i n Table 13 in d i c a t e that the oxidation of putrescine was i n h i b i t e d by carbonyl reagents l i k e 102 Table 15 The effects of various i n h i b i t o r s on D.O. a c t i v i t y * i n c e l l - f r e e extracts. I n h i b i t o r I n h i b i t o r Concentration 0 2 uptake (pi) at 15 minutes Hydroxylamine 0.01 M 0 0.001 M 0 Semicarbazide 0.01 H 0 0.001 H 9 Ephedrine 0.01 M 20 0.001 M 21 Water - 20 * A c t i v i t y measured at pH 7.2 i n phosphate buffer with putrescine (0.01 M) as substrate. hydroxylamine and semicarbazide. Such r e s u l t s are i n accord with the reports of D.O. s u s c e p t i b i l i t y to carbonyl reagents (117). The fact that ephedrine sulphate f a i l e d to i n h i b i t D.O. a c t i v i t y was not surprising since t h i s compound i s reported to be a monoamine oxidase i n h i b i t o r (117). The curve r e l a t i n g D.O. a c t i v i t y to pH i s shown i n Pig. 13. D.O. a c t i v i t y was studied over the pH range 4.5-9.0. Ci t r a t e buffer was employed from pH 4.5 to pH 6.0. Phosphate and t r i s buffers were used f o r the pH ranges 6.0 to 8.0 and 7.5 to 9.0 respe c t i v e l y . 103 PIG. 13. The optimum pH for diamine oxidase a c t i v i t y . Reactions conducted i n c i t r a t e (pH 4.5 - 6.0) and phosphate (pH 6.0 - 8.0) buffers (curve 1) and i n t r i s buffer (pH 7.5 - 9.0) (curve 2). 104 The pH optimum fo r D.O. a c t i v i t y was found to l i e i n the pH region 8.0 to 9.0. T r i s buffer exerted a marked i n h i b i t o r y e f f e c t at pH 7.5 which was correlated with a s i g n i f i c a n t amount of p r e c i p i t a t i o n of c e l l - f r e e extract material. This phenomenon occurred consistently i n d i f f e r e n t batches of t r i s buffer at pH 7.5 hut was not observed with phosphate buffer at the same pH. At pH 8.0, D.O. a c t i v i t y was greater i n phosphate buffer than i n t r i s buffer but at t h i s pH no protein p r e c i p i t a t i o n was evident with the l a t t e r buffer. I t may be that some enzyme denaturation occurred with t r i s buffer at pH 8.0 which went undetected, or i t i s possible that phosphate buffer stimulated D.O. a c t i v i t y . Stimulation of D.O. a c t i v i t y by phosphate ions has been reported by other workers (11). At pH 8.5-9.0 i n t r i s buffer, the oxygen uptake curves proceeded upwards i n a l i n e a r fashion and l e v e l l e d o f f abruptly when substrate was exhausted. At pH 8.0 i n phosphate buffer, the l e v e l l i n g o f f of the oxygen uptake curve due to depletion of substrate, occurred more gradually. These observations were made during short and extended experiments although the effects were not as marked during the shorter reaction periods. I t was therefore f e l t that enzyme i n a c t i v a t i o n could not e n t i r e l y explain t h i s phenomenon. I t appeared l i k e l y that at higher pH values, some of the product (A*-pyrroline) was removed from the reaction by spontaneous decomposition. In the presence of oxygen, and under a l k a l i n e 105 conditions, 4*-pyrroline i s reported to be very unstable (44,72). During assays f o r D.O. a c t i v i t y , the net r e s u l t of A'-pyrroline decomposition would be to cause the reaction to proceed to completion more r a p i d l y . An alte r n a t i v e explanation could be that the equilibrium constant of the reaction favoured completion of the reaction at higher pH values. Table 14 records the r e s u l t s of a study of the substrate s p e c i f i c i t y of the D.O. enzyme contained i n c e l l - f r e e extracts obtained from putrescine-adapted s t r a i n 0.0. c e l l s . The reaction was conducted at pH 8.5 with each substrate at 0.005 M (except spermine 0.001 M). Table 14 A l i p h a t i c amines as substrates f o r D.O. i n c e l l - f r e e extracts obtained from putrescine-adapted c e l l s . Substrate* 0 2 uptake (pi) 0 2 uptake (pi) at 15 minutes at 120 minutes 1-amino ethane 2 4 1-aminopropane 1 2 1-aminobutane 2 4 1-aminop entane 3 5 l-amino|texane 1 3 1,2-diaminoethane 1 5 1,3-diaminopropane 2 5 1,4-diaminobutane (putrescine) 20 134 1,5-diaminopentane (cadaverine) 7 52 1,6-diaminohexane 1 1 Spermidine 1 0 Spermine 1 0 Water 0 3 * Mono- and diamines as t h e i r hydrochloride s a l t s ; spermidine and spermine as t h e i r phosphate s a l t s . 106 The data i n Table 14 indicate that the D.O. enzyme i s f a i r l y s p e c i f i c . Ho mono- or polyamines were attacked. Of the diamines, only putrescine and cadaverine were oxidized with the l a t t e r compound being attacked at an approximately one-third of the rate f o r that of putrescine. D.O. a c t i v i t y was not appreciably reduced by d i a l y s i s . Prolonged d i a l y s i s (48 hours at 4°C) versus 100 volumes of d i s t i l l e d water resulted i n a decrease of only 5-8% of the o r i g i n a l a c t i v i t y . Such losses i n a c t i v i t y were more l i k e l y a t tributable to the p r e c i p i t a t i o n of some active material rather than to a l o s s of cofactor. I t was concluded that, as i s t y p i c a l of other D.O. preparations (117), the prosthetic group of the D.O. enzyme i s t i g h t l y bound to the enzyme. D.O. a c t i v i t y was not sedimented by centrifugation f o r 1 hour at 10,000 I S , When the unwashed p e l l e t (debris) was resuspended i n the o r i g i n a l volume and an aliquot assayed f o r D.O. a c t i v i t y with putrescine, i t was found to be one-nineteenth as active as a comparable portion of the' supernatant f r a c t i o n . Much of the a c t i v i t y i n the sedimented debris may have been due to unbroken c e l l s since smears of t h i s material showed numerous i n t a c t acid-fast b a c i l l i to be present. C e l l - f r e e extracts did not contain IADH oxidizing a b i l i t y which indicated that i n t a c t mitochondria were eliminated by centrifugation. I t i s possible, however, that the D.O. enzyme was attached to p a r t i c u l a t e material since the g r a v i t a t i o n a l forces used would not have sedimented 107 ribosomes and small mitochondrial fragments. (2) A * - p y r r o l i n e dehydrogenase (P.D.) Pig. 14 shows the P.D, catalyzed reduction of NAD and NADP (curves 1 and 2, respectively) with chemically synthesized A *-pyrroline as substrate. The reactions were conducted at pH 9.0 i n the presence of 0.005 M mercaptoethanol and assayed spectrophotometrically at 340 mjti. The enzyme employed f o r the assay was obtained from putrescine-adapted s t r a i n C.C. c e l l s . When the i n i t i a l reaction rates were compared, the enzyme was found to be about six times more active with NAD than with NADP. The eff e c t on the reaction rate of. omitting mercaptoethanol from, the system i s shown i n curves 3 and 4 f o r NAD and NADP, respectively. The marked decreases i n P.D. a c t i v i t y observed i n the absence of mercaptoethanol, have been reported f o r the same enzyme obtained.from Ps. fluorescens (44). The r e s u l t s of i d e n t i c a l studies with c e l l - f r e e extracts obtained from putrescine-adapted s t r a i n S.C. c e l l s , are presented i n _ P i g . 15. In the presence of 0.005 M mercaptoethanol, the P.D. enzyme was approximately 3.3 times as active with NAD as with NADP. A*-pyrroline employed as substrate f o r t h i s assay, was synthesized from putrescine with the D.O. enzyme contained i n s t r a i n S.C. c e l l - f r e e extracts. A *-pyrroline dehydrogenase a c t i v i t y was also measured 108 TIME (MIHUT£S) PIG. 14. Strain C.C. A'-pvrroline dehydrogenase a c t i v i t y (pH 9.0). Curve 1: Mercaptoethanol + NAD. Curve 2: Mercaptoethanol + NADP. Curve 3: NAD. Curve 4: NADP. O X *r 6 % IO PIG. 15. Strain S.C. A'-pyrroline dehydrogenase a c t i v i t y (pH 9.0). Curve 1: Mercaptoethanol + NAD. Curve 2: Mercaptoethanol + NADP. 109 manometrically using methylene blue (MB) to couple the reaction to molecular oxygen* The substrate, £ 1 - p y r r o l i n e , was either synthesized chemically, or i t was prepared enzymatically from putrescine using crude, dialyzed c e l l -free extracts obtained from putrescine-adapted s t r a i n C.C. c e l l s : A B 1 Putrescine » 1 ^ * - p y r r o l i n e » l v-amino-D.O. P.D. butyric NAD a c i d catalase catalase M.B. R-SH. In the reactions shown, the oxidation of 1.0 ;uM of putrescine to 1.0 juM of y-aminobutyric acid would require the consumption of 22.4^1 0 2 (1.0 pM 0 g) since each step (A,B) consumes 11.2 pi 0^. A l t e r n a t i v e l y , the reactions A and B could be separated so that the oxygen uptake due to each step could be observed i n d i v i d u a l l y . Pig. 16 i l l u s t r a t e s the r e s u l t s obtained at pH 7.2 from manometric studies on P.D. a c t i v i t y with putrescine (6.0 ;uM) as the precursor of /Q'-pyrroline. The c e l l - f r e e extract used, was obtained from s t r a i n C.C. c e l l s . Curve 1 shows the oxygen uptake with the "complete system": C e l l - f r e e extract, mercaptoethanol, NAD, MB, and putrescine. The oxygen consumption l e v e l l e d o f f at 136 yd 0n (theory: 134.4 pi Og). Curve 2 i l l u s t r a t e s the oxygen uptake obtained with an i d e n t i c a l reaction mixture except that NAD was omitted. I t 110 T I M E (MINUTES) FIG. 16. Manometric assay of s t r a i n C.C. A'-pyrroline dehydrogenase a c t i v i t y (pH 7.2). See text f o r d e t a i l s . I l l can be seen that the oxygen consumption proceeded to completion (133^1 © 2 ) although i n the upper portion of the curve, i t did so more elowly. This f i n d i n g was surprising since the ommision of HAD should have reduced the f i n a l oxygen uptake by 50% (67.2 jal Og). Furthermore, previous spectrophotometric studies with the dialyzed enzyme preparation had f a i l e d to reveal an increase i n absorbance at 340 mn when A 1 - p y r r o l i n e was added as substrate. This was taken to indicate that the extract was free of HAD or HADP. In the l i g h t of the manometric r e s u l t s , however, i t appeared that amounts of cofaetor small enough to go undetected by spectrophotometry remained i n the c e l l - f r e e preparation. Under conditions where the cofaetor was continually reoxidized by MB, i t s presence was demonstrated. Curve 3, Pig. 16 demonstrates the almost absolute requirement of the P.D. enzyme f o r reducing substances. The reaction system contained: C e l l - f r e e extract, HAD, MB and substrate (putrescine). Mercaptoethanol was not present. The oxygen uptake curve proceeded to 69 jul 0^ instead of 134.4.#1 0g i n d i c a t i n g that -pyrroline was not attacked. Curve 4 shows the r e s u l t of allowing the oxidation of putrescine to go to completion i n the presence of mercaptoethanol and HAD (68;ul 0 2 instead of 67.2 JJ! 0 2 ) . At t h i s stage (indicated by the arrow), the addition of MB from the second sidearm of a double sidearm reaction vessel, allowed the accumulated A 1 -pyrroline to be oxidized quantitatively. 112 Pig. 17 records the re s u l t s of si m i l a r manometric studies on the P.D. enzyme at pH 9.0. Curve 1 represents the oxygen uptake du£ to 6.0 ;uM of putrescine i n the presence of the "complete system". The oxygen consumption l e v e l l e d o f f at 141 ;ol Og (4«9$ higher than the expected value). Curve 2 demonstrates the eff e c t of omitting mercaptoethanol from an otherwise "complete system". Once again, the oxygen uptake at completion, was somewhat higher than expected although the requirement of the P.D. enzyme f o r mercaptoethanol was c l e a r l y demonstrated. Although no attempt was made i n t h i s experiment to separate D.O. a c t i v i t y from that of the P.D. enzyme, an idea of the reaction rate due to the l a t t e r enzyme i s obtained from curve 3. Curve 3 was calculated by subtracting the oxygen uptake values f o r curve 2 from those of curve 1. The calculated reaction rate f o r the P.D. enzyme i s v a l i d since the rate of oxygen uptake i n the "complete system" was double that f o r the mereaptoethanol-deficient system. Ho a c t i v i t y was demonstrated when chemically synthesized A'-piperideine was tested as substrate at pH 9.0. However, i t i s not known whether t h i s was due to substrate s p e c i f i c i t y on the part of the P.D. enzyme. The p o s s i b i l i t y exists that traces of bromine, formed during the chemical synthesis of A'-piperideine, may have remained i n the substrate solution. I f such was the ease, the P.D. enzyme may have been inactivated by bromine. 113 TIME. (MINUTES) FIG. 17. Manometric assay of s t r a i n C.C. A»-pyrroline dehydrogenase a c t i v i t y (pH 9-0). See text for d e t a i l s . 114 The r e s u l t s indicated that the P.D. enzyme was active at pH 7.2 and 9.0. HAD was the preferred cofactor. The enzyme exhibited an almost absolute requirement f o r free t h i o l groups. Paper chromatography of "complete reaction mixtures" obtained at the end of the experiments, showed the presence of a single ninhydrin-positive area which corresponded to JC-aminobutyric acid (Rf 0.582 i n butanol:acetic acid: water, 2:1:1 v/v; Rf 0.750 i n water-saturated phenol). This compound contained a c i d i c and basic functi.onal groups since i t was retained by anion and cation exchange r e s i n s . C e l l - f r e e extracts therefore contain two enzymes responsible f o r the synthesis of ^-aminobutyrie acid from putrescine. (3) Gamma-aminobutyrie ac i d - ©O-ketoglutaric acid transaminase. Intact c e l l s were shown to release up to 1.5 jM that the remaining 0.5 ' )M NH^ might possibly have been derived frofir y-aminobutyric a c i d . Consequently, c e l l - f r e e extracts derived from putrescine-adapted s t r a i n C.C. c e l l s were examined fo r t h e i r a b i l i t y to metabolize t h i s compound. C e l l - f r e e extracts did not consume oxygen at pH 7.0, 8.0 or 9.0 with If-aminobutyrie a c i d as substrate. Under si m i l a r conditions i n the presence of HAD or HADP, there was 115 no increase i n absorbance at 340 mn. An examination of the reaction mixtures obtained from such experiments, indicated that ammonia was not produced. I t was therefore concluded that i n t a c t c e l l s produced ammonia from y-aminobutyric a c i d i n d i r e c t l y , since c e l l - f r e e extracts were not capable of catalyzing the oxidative, or dehydrogenative deamination of the compound. Similar r e s u l t s have been reported by Bachrach (6) f o r c e l l - f r e e preparations obtained from Ps. aeruginosa. Gamma-aminobutyric ac i d was metabolized by c e l l - f r e e extracts when oc-ketoglutarate was present. An i n i t i a l experiment i n which 3.0 juM of Jf-aminobutyric a c i d and 6.0 jM of o<-ketoglutaric acid were incubated at pH 8.5 f o r 4 hours at 30°C showed that transamination had occurred. Paper chromatography of the deproteinated, deionized reaction mixture demonstrated glutamic acid to be a product (Rf 0.414 i n the n-butanol:acetic acid:water, 2:1:1 v/v solvent; Rf 0.231 i n water-saturated phenol solvent). A measurement of the glutamic a c i d produced during the rea c t i o n showed that the reaction had proceeded 76.3% to completion. Paper chromatography of the hydrazones formed with DPNH indicated the appearance of a second hydrazone (Rf 0.439 i n n-butanol saturated with 5 N NH.OH). This compound migrated l i k e the 4 hydrazone prepared from chemically synthesized succinic semialdehyde. The optimum pH f o r transaminase a c t i v i t y was determined f o r the pH range 7.0-9.0 employing phosphate buffer (pH 7.0) . 116 and t r i s buffer (pH 8 . 0 - 9 . 0 ) . Transaminase a c t i v i t y was determined by measuring quantitatively, the amount of glutamic acid produced. The r e s u l t s i l l u s t r a t e d i n H g . 18 indicate that the optimum pH l i e s i n the pH range 8.5-9.0. However, on prolonging the reaction time, the reaction proceeded further to completion at pH 9.0 than at lower pH values. This f a c t i s shown i n Pigs. 18 and 1 9 . This e f f e c t was not further investigated but i t may have been due to enzyme i n a e t i v a t i o n at lower pH values, or to destruction of one of the reaction products at higher pH (succinic semialdehyde?). A l t e r n a t i v e l y , the equilibrium point may have favoured completion of the r e a c t i o n at a l k a l i n e pH. The r e s u l t s of substrate s p e c i f i c i t y studies with the transaminase enzyme are summarized i n Table 15. Table 15 The a b i l i t y of s t r a i n C.G. c e l l - f r e e extracts to catalyze the transamination between various fcl-amino acids andc<-ketoglutaric ac i d . Amino acid Glutamic a c i d (jM) synthesized at 1 hour Glycine 0 <8 -alanine 0 Jr-aminobutyric acid 4.95 S-aminovaleric a c i d 2.80 £-aminocaproic acid 0.27 117 PIG. 19. E f f e c t of pH and reaction time on >f-aminobutyric acid - «c-ketoglutaric acid transaminase a c t i v i t y . 118 The r e s u l t s i n Table 15 were further confirmed q u a l i t a t i v e l y by paper chromatography. Although y-aminobutyric acid appeared to be the most e f f i c i e n t amino group donor, other (tf-amino acids were capable of undergoing transamination i n t h i s system. The r e s u l t s disagree with those f o r a s i m i l a r transaminase enzyme obtained from Ps. fluorescens i n which the enzyme was s p e c i f i c f o r y-aminobutyrie acid (44). When ^-aminobutyrie acid was incubated with oxalacetate or pyruvate, no aspartate or alanine was detected by chromatography. The enzyme therefore appeared to be s p e c i f i c i n the requirement f o r oC-ketoglutarate. Similar r e s u l t s were obtained f o r the same transaminase i n Ps. fluorescens (18) but c e l l - f r e e preparations from Ps.  aeruginosa were capable of transaminations between Jr-aminobutyrie a c i d and pyruvate, oxalacetate and ©c-ketovalerate i n addition to ©c*-ketoglutarate (6). (4) Succinic semialdehyde dehydrogenase (S.S.D.). Pig. 20 shows the S.S.D.-catalyzed reduction of HAD and HADP (curves 1 and 2, respectively) by a c e l l - f r e e preparation obtained from putrescine-adapted s t r a i n G.G. c e l l s . Succinic semialdehyde used i n t h i s assay, was prepared by incubating equimolar amounts of Jr^-aminobutyric acid and oC-ketoglutaric a c i d with the c e l l - f r e e extract f o r 30 minutes p r i o r to the addition of cofactor. The reaction was conducted at pH 9.0. The curves demonstrate that HAD i s the more 119 a <f *S 8 io TIME ( M I N U T E S ) PIG. 20. Strain C.C. succinic semialdehyde dehydrogenase a c t i v i t y (pH 9.0). Curve 1: NAD Curve 2: NADP. T I M E (MINUT£$) PIG. 21. Strain S.C. succinic semialdehyde dehydrogenase a c t i v i t y (pH 9.0). Curve 1: NAD. Curve 2: NADP. 120 e f f i c i e n t cofaetor i n the S.S.D. system. The r e s u l t s of simi l a r experiments with the S.S.D. enzyme obtained from putrescine-adapted s t r a i n S.C. c e l l s are i l l u s t r a t e d i n Pig. 21. With t h i s s t r a i n , HAD and HADP appeared to be equally as e f f i c i e n t as cofactors i n the dehydrogenation. Chemically synthesized succinic semialdehyde was employed as substrate f o r the reaction shown i n Pig. 21. Although Jakoby (18) reported that S.S.D. from Ps. fluorescens required mercaptoethanol f o r optimal a c t i v i t y , the eff e c t of t h i s compound on the a c t i v i t y of mycobacterial S.S.D. was not tested since good a c t i v i t y was obtained i n i t s absence, even a f t e r prolonged storage of the enzyme at -20°0» In Ps. fluorescens (147) and Ps. aeruginosa (106), HADP appeared to be the more e f f i c i e n t cofaetor f o r the S.S.D. enzyme. In the l a t t e r organism, there appeared to be two S.S.D. enzymes, one requiring HAD, and the other requiring HADP (68). In an i n i t i a l experiment, the product of the S.S.D.-catalyzed reaction was shown to be succinic a c i d . For t h i s experiment, the substrate, succinic semialdehyde, was prepared enzymatidally using c e l l - f r e e extract obtained from putrescine-adapted s t r a i n C.C. c e l l s . The reaction mixture contained: ^-aminobutyric acid (12 ;uM), oC-ketoglutarie acid (6.0 jM)i HAD (6.0 uM) and malonic acid (12 jM), Malonie acid was employed to i n h i b i t the further possible breakdown of any succinic acid formed. The reaction was conducted at 121 pH 8.5 f o r 2.5 hours at 30°C, at which time, a representative aliquot was examined for i t s succinic semialdehyde content. Succinic semialdehyde was determined by measuring the amount of glutamic acid present since these compounds would have been produced i n equimolar amounts during the transamination. The remaining reaction mixture was freed of protein with zinc sulphate (95) and a portion was examined f o r DPNH-reacting materials. The rest of the reaction mixture was a c i d i f i e d , extracted with ethyl ether", and the ether extract was ehromatographed to demonstrate the presence of succinic acid. Similar techniques were employed to examine a control reaction mixture i n which water was substituted f o r NAD. The,results of the glutamic acid assay indicated that transamination had proceeded to completion i n the NAD-containing system. The corresponding value f o r the control reaction mixture was 66.5$. Paper chromatography of the hydrazones formed with DPNH showed that the NAD-containing system was devoid of both succinic semialdehyde and c<-ketoglutarate while both of these compounds remained i n the cofactor-deficient system. These r e s u l t s were consistent with the fa c t that succinic semialdehyde was quantitatively removed from the peaction system when substrate amounts of cofactor were present, and coincident with the dismutation of succinic semialdehyde, the transaminase reaction was induced to proeeed to completion. Paper chromatography of the ether extracts demonstrated that succinic a c i d was the product of the 122 dehydrogenative oxidation (Hf 0.254 i n ethanol :NHA0H:H20, 16:1s3 v/v). In the preceding experiment, no quantitative estimation was made on the succinic acid produced "because of the tedious procedure required to separate malonate from succinate before the assay of the l a t t e r could be accomplished by enzymatic means. In a subsequent experiment, however, i t was shown that during the S.S.D.-catalyzed reaction, 1.0 )M of succinate was produced from 1.0 )M of succinic semialdehyde. In t h i s experiment oxalacetate was used i n place of malonate, since the former was r e a d i l y destroyed and since the products of i t s destruction were shown not to i n t e r f e r e with the enzymatic assay of succinate. The actual r e s u l t obtained showed that i n the presence of 1.04 ^iM of succinic semialdehyde (measured as glutamic acid), 0.93 ;uM of succinic acid were produced during the S.S.D. reaction. I l l Isotopic studies with i n t a c t c e l l s Table 16 records the d i s t r i b u t i o n of r a d i o a c t i v i t y that occurred i n the reaction system when putrescine-14 adapted s t r a i n N.T.N, c e l l s were allowed to oxidize 1,4-C; putrescine under C0 9-free conditions. 123 Table 16 14 D i s t r i b u t i o n of C; occurring during the oxidation 14 , v of 1,4-C; putrescine (3.0 jM) by putrescine-adapted s t r a i n N.T.N, c e l l s . Time % of t o t a l counts added to the reaction, system i n : (minutes) C e l l s + Supernatant C e l l s C0 p C e l l s + Supernatant (difference) Supernatant + eo 2 25 72.0 31.0 41.0 19.5 91.5 50 41.8 1.3 40.5 62.1 103.9 65 33.0 1.2 31.8 72.2 105.2 120 29.5 1.2 28.3 78.6 108.2 Table 17 shows the incorporation of the r a d i o a c t i v i t y that occurred i n the various c e l l f r a c t i o n s during the course of the reaction. By summation of the counts contained i n the various c e l l f r a c t i o n s that comprised the i n t a c t c e l l , a second estimate of the c e l l r a d i o a c t i v i t y was made possible. When these counts f o r c e l l s were added to the counts contained i n supernatant and carbon dioxide samples, the percentage of the counts obtained approximated more clos e l y the counts act u a l l y added to the reaction system than did the corresponding 124 values shown i n Table 16. Table 17 14 Incorporation of C i n t o various N.T.N, c e l l f r a c t i o n s 14 / N during the oxidation of 1,4-C putrescine (3.0 ;uM). i> of t o t a l counts i n c e l l f r a c t i o n : Time (minutes) 25 50 65 120 Cold HCl Aeid alcohol Hot TCA Residue 24.5 14.7 4.9 4.8 8.1 10.6 6.5 11.2 5.3 7.9 6.8 11.8 2.9 6.5 5.4 10.0 % of t o t a l counts i n : Combined c e l l f r a c t i o n s Supematants co2 48.9 31.0 19.5 36,4 1.3 62.1 31.8 1.2 72.2 24.8 1.2 78.6 % of t o t a l counts obtained 99.4 99.8 105.2 104.6 The data presented i n Table 16 indicated that by 50 minutes, no substrate remained i n the reaction mixture supernatant and that no subsequent release of non-volatile 14 C; -material occurred. This f i n d i n g confirmed the r e s u l t s obtained from e a r l i e r studies with non-radioactive putrescine. 125 During the period of active oxygen consumption, the c e l l s contained up to 41-48.9$ (Tables 16 and 17.) of the radio-a c t i v i t y supplied. As w i l l be shown l a t e r (Table 18), as much as 50$ of t h i s r a d i o a c t i v i t y occurred i n the cold  HOl-soluble f r a c t i o n as undegraded putrescine and as amino acids. As the reaction proceeded, the l e v e l of the counts i n the c e l l s decreased u n t i l at the end of two hours, the c e l l s contained between 21.4-28.3$ of the r a d i o a c t i v i t y supplied. The average of these values (24.8$), i s the value obtained by adding the counts contained i n the various \ 14 c e l l f r a c t i o n s f o r t h i s sampling time, (Table 16). A l l G. l o s t by the c e l l s appeared i n the a l k a l i i n the centre well as carbon dioxide. H g . 22 i l l u s t r a t e s the d i s t r i b u t i o n of G14" that occurred during the reaction among c e l l , supernatant and carbon dioxide samples. In addition, oxygen consumption that occurred i s shown i n p a r a l l e l . 14 The amount of terminal G assimilated by the c e l l s agreed very well f o r the amount of putrescine-nitrogen incorporated by c e l l s of t h i s s t r a i n . As indicated by previous studies 24-25$ of the amino-nitrogen of putrescine was assimilated at t h i s stage of the reaction. Such r e s u l t s suggested that t h i s portion of the putrescine molecule might have been assimilated i n t o nitrogenous materials. Unfortunately, the fate of carbons 2 and 3 of the putrescine molecule i s uncertain since they were unlabelled and since the i n d i r e c t technique f o r carbon dioxide production gave lower values 126 PIG. 22. D i s t r i b u t i o n of r a d i o a c t i v i t y during the oxidation of 1,4-C putrescine by putrescine-adapted s t r a i n N.T.N, c e l l s . Reaction mixture supernatant (curve 1); combined c e l l fractions (curve 2); c e l l s , calculated as the difference.between c e l l s + supernatant and supernatant (curve 3): C 0 2 ( ° u r v e 4); 0 2 uptake occurring during the reaction (curve 5)» 127 than a c t u a l l y were true f o r carbon dioxide production under COg-free conditions. However, only a small portion of these carbons may have been released as carbon dioxide since the oxygen uptake at the conclusion of the experiment corresponded to 44.4% of the l e v e l f o r maximum oxidation. Table 18 expresses the r a d i o a c t i v i t y of the c e l l f r a c t i o n s as a percentage of the counts occurring within the i n t a c t c e l l s . Table 18 14 D i s t r i b u t i o n of incorporated C i n various c e l l f r a c t i o n s obtained from s t r a i n l.T.H. c e l l s incubated with 1,4-C 1 4 putrescine (3.0 ;iM). C e l l Counts per f r a c t i o n as a % of eounts i n f r a c t i o n : whole c e l l s at time (minutes): 25 50 65 120 16.7 11.7 24.8 26.2 21.4 21.8 37.1 40.3 % Total counts 100.0 100.0 100.0 100.0 i n c e l l s Cold HCl 50.1 22.2 Acid alcohol 30 .1 29.2 Hot TCA 1 0 . 0 17 .8 Residue 9 . 8 3 0 . 8 I t can be seen that i n i t i a l l y , 50.1% of the counts contained i n the c e l l s occurred i n the cold HOT-soluble 14 f r a c t i o n . At the end of two hours, the l e v e l of C i n t h i s 128 f r a c t i o n was reduced to 11.7$. Coincident with the lo s s i n r a d i o a c t i v i t y i n the cold HCl-soluble f r a c t i o n , there was a gain i n that of the hot TCA-soluble and residue f f a c t i o n s . In E. c o l i , the former f r a c t i o n was found to consist l a r g e l y of nucleic acid, while the l a t t e r contained mainly protein (78). Presumably the lo s s i n r a d i o a c t i v i t y i n the cold  HCl-soluble f r a c t i o n was p a r t l y due to the formation of macromolecules (nucleic acid and protein). The acid alcohol- soluble f r a c t i o n retained a f a i r l y constant proportion of the c e l l r a d i o a c t i v i t y throughout the reaction. According to Roberts et a l (78), the l a t t e r f r a c t i o n , i n E. c o l i consisted l a r g e l y of protein and l i p i d materials. E f f o r t s were made to determine the composition of the fraction s obtained from N.T.N, c e l l s . Paper chromatography of the cold HCl-soluble f r a c t i o n ( i n n-butanol:acetic acid:water 2:1:1 v/v), demonstrated the presence i n i t i a l l y of six ninhydrin-positive compounds which migrated l i k e putrescine, aspartic acid, glutamic acid, alanine, jf-aminobutyrie acid and va l i n e . At 2 5 minutes, f i v e peaks of r a d i o a c t i v i t y occurred on the chromatograms. These peaks corresponded to the f i r s t f i v e compounds l i s t e d . Radioactivity i n the sample obtained at 120 minutes revealed that a l l the counts of the f r a c t i o n were present i n glutamic acid. No materials absorbing i n the u l t r a - v i o l e t range were detected. Paper chromatography of the unhydrolysed, two-hour, 129 acid-ethanol soluble f r a c t i o n , was conducted i n sec-butanol: formic acid:water (7:1:2 v/v) (78). Most of the r a d i o a c t i v i t y i n t h i s f r a c t i o n remained at the o r i g i n of the chromatogram while a l e s s e r amount of C 1 4 material migrated with the solvent front. The a c t i v i t y occurring at the o r i g i n probably represented protein since the material was feebly ninhydrin-p o s i t i v e . The material migrating with the solvent front d i d not react with ninhydrin, According to Roberts et a l (78), the materials at the o r i g i n and at the solvent front represent protein and l i p i d , respectively. Three additional ninhydrin-positive areas occurred on the chromatogram at Rf values corresponding to: 0.155, 0.380, and 0.580. Low l e v e l s of r a d i o a c t i v i t y were associated with the compound at Rf 0.380. I f these compounds represented amino acids that were not completely removed by the f i r s t f r a c t i o n a t i o n step, then the compound at Rf 0.380 was probably glutamic aci d . No U.V.-absorbing materials were evident on the chromatogram. The hot TOA-soluble f r a c t i o n obtained at two hours, was freed of the bulk of the TCA by extracting with ethyl ether. The sample was chromatographed a f t e r p a r t i a l hydrolysis i n 1.0 N HCl at 100°C f o r 1 hour. Chromatography was conducted i n sec-butanol:formic aeid:water (7:1:2 v/v). When the dried chromatogram was viewed under u l t r a - v i o l e t l i g h t , three absorbing areas were detected (Rf: 0.188, 0.267 and 0.420). Radioactivity was associated with the absorbing 14 areas. In addition, C occurred at the o r i g i n and at 130 ninhydrin-positive areas with Rf values of 0.151 and 0.455. The f r a c t i o n therefore appeared to contain protein i n addition to nucleic acid materials. Satisfactory f r a c t i o n a t i o n of the residue was not achieved. The residue could not be completely dissolved i n warm 0.5 NNaOH. Extraction of the residue from basic or acid solution with ethyl ether, resulted i n the formation of a s o l i d layer of material at the interface of the l i q u i d s . Although a small percentage (3-5$) of the counts i n the f r a c t i o n was extractable into the organic phase, the bulk of the counts was associated with the non-ether-soluble residue. When the acid-aqueous and organic phases were centrifuged i n the cold, the l i q u i d s separated cleanly with a layer of compacted residue remaining at the int e r f a c e . o Hydrolysis of t h i s material was conducted overnight at 100 C i n 5 N-.'HC1. Paper chromatography of the hydrolysate ( i n n-butanol:acetic acid:water 2:1:1 v/v) showed the presence of ninhydrin-positive areas from Rf 0.20 to 0.80. This f r a c t i o n therefore contained large amounts of protein but since the material appeared to possess d e f i n i t e l i p o p h i l i c c h a r a c t e r i s t i c s , the protein was probably associated with hydrophobic materials-presumably the waxy substances responsible f o r the hydrophobic nature of i n t a c t mycobacterial c e l l s . When putrescine-adapted c e l l s were allowed to oxidize 1,4-C14" putrescine, the r a d i o a c t i v i t y disappeared from the medium and appeared i n the c e l l s and i n carbon dioxide. An 131 analysis of various c e l l f ractions showed that a l l f r a c t i o n s were r a p i d l y l a b e l l e d . I n i t i a l l y , the bulk of the r a d i o a c t i v i t y appeared to be associated with simple nitrogenous compounds (amino aeias), but as the reaction was allowed to 14 progress, larger proportions of the 0 appeared i n fr a c t i o n s containing nucleic aeid, l i p i d and protein. These r e s u l t s were consistent with those of e a r l i e r studies which showed that putrescine could support growth when present as the sole source of carbon. The l i b e r a t i o n of G.. Og and the l a b e l l i n g of glutamic acid indicated that at lea s t a portion of the putrescine molecule was oxidized v i a the t r i c a r -boxylic acid cycle. Judging from oxygen uptake occurring during the reaction, over 50% of the added putrescine was assimilated. However, since the substrate was not uniformly l a b e l l e d , the fate of carbons 2 and 3 remains i n some doubt. 132 GENERAL CONCLUSIONS AND DISCUSSION Mycobacteria i s o l a t e d from poikilothermie animals appear to be remarkably r e s t r i c t e d i n t h e i r a b i l i t y to u t i l i z e amines as carbon sources f o r growth. Of a series of a l i p h a t i c and c y c l i c amines tested, only putrescine was found to support growth. In mycobacterial c e l l s , the system responsible f o r the oxidation of putrescine, was shown to be adaptive. This f a c t , together with the f i n d i n g that such c e l l s do not appear to contain any d i - and polyamines, probably indicates that such compounds are not normally present i n mycobacterial c e l l s as metabolic intermediates. The f a i l u r e to demonstrate the presence of amines i n mycobacterial c e l l s i n t h i s and other studies (37) may mean that mycobacteria do not store amines. I f t h i s i s so, the role (or roles) that these compounds play i n other microorganisms are not required i n mycobacteria. A l t e r n a t i v e l y , i n mycobacteria, some other substance (or substances) may function i n place of the amines. During the oxidation of putrescine, the endogenous r e s p i r a t i o n appeared to function normally. Calculations f o r the degree of putrescine - oxidation were therefore made possible. When putrescine was oxidized, oxygen consumption usually proceeded to approximately 45$ of the maximum l e v e l of oxidation. During t h i s process, 69-76$ of the amino-nitrogen of the putrescine molecule was released as ammonia, the remaining 24-31$ being assimilated. Carbon dioxide production, estimated by the "direct method", resulted i n carbon dioxide values which were unreliable since i n the 133 absence of carbon dioxide there appeared to be increased carbon dioxide production. When 1,4-C 1 4 putrescine was incubated with c e l l s under COg-free conditions, approximately 75% (or 1.5 .uM COg/uM putrescine) of the terminal carbons, were evolved as carbon dioxide. The p o s s i b i l i t y exists that the release of carbon dioxide may have even been higher since the fate of the two remaining unlabelled carbons of the putrescine molecule i s uncertain. Studies with c e l l - f r e e extracts obtained from putrescine-adapted c e l l s indicated the presence of four enzymes which were involved i n converting putrescine into succinic a c i d . Enzymes performing a si m i l a r series of reactions i n Ps. fluorescens have recently been described i n d e t a i l by Jakoby (44). Putrescine was f i r s t oxidatively deaminated to y i e l d y -aminobutyraldehyde. This compound was detected i n i t s A ' - p y r r o l i n e form. The l a t t e r compound was then oxidized to -aminobutyric a c i d by a A ' - p y r r o l i n e dehydrogenase enzyme which required NAD or NADP. Gamma-aminobutyric acid was found to undergo a transamination with oC-ketoglutaric acid to y i e l d succinic semialdehyde and glutamic a c i d . I t i s assumed that i n t a c t c e l l s supply the oC-ketoglutarate from endogenous sources since c e l l - f r e e extracts appeared to contain no alte r n a t i v e system f o r the metabolism of y-aminobutyric a c i d . In addition, i n h i b i t o r studies with arsenite showed that endogenously. r e s p i r i n g c e l l s producede<-ketoglutarate. Succinic semialdehyde produced as a r e s u l t of the transamination, was oxidized to succinic acid by a 134 dehydrogenase enzyme requiring NAD or NADP, Up to t h i s stage, no reactions responsible f o r the production of carbon dioxide had occurred. Decarboxylation reactions that occurred l a t e r , resulted from the further u t i l i z a t i o n of products derived from putrescine, Amino-nitrogen derived from putrescine was assimilated i n d i r e c t l y by way of glutamic acid which yielded other amino acids (aspartic acid and alanine) by other transaminase reactions. Transaminase enzymes were demonstrated i n the c e l l - f r e e extracts which could accomplish the transaminations required to produce these amino acids. That y-aminobutyric a c i d was produced by i n t a c t c e l l s during the degradation of putrescine was demonstrated by paper chromatography of HC1 extracts obtained from c e l l s 14 metabolizing putrescine. When 1,4-C putrescine was employed as substrate, the ^ -aminobutyric acid produced was radioactive. C e l l s i n h i b i t e d with arsenite were shown to produce pyruvic a c i d . When putrescine was included i n such systems, the amount of pyruvic a c i d formed was increased. This f i n d i n g suggested that part of the putrescine molecule was metabolized by way of pyruvate. C e l l s incubated with putrescine showed the presence of aspartic acid, glutamic 14 acid and alanine i n HC1 extracts. When 1,4-C putrescine was employed as substrate these amino acids were found to be radioactive. Since aspartic and glutamic acids are derived from oxalacetate and 06-ketoglutarate respectively, and since 135 the l a t t e r compounds are intermediates i n the t r i c a r b o x y l i c acid cycle, the f i n d i n g that the l i s t e d amino acids were radioactive, indicated that some of the putrescine carbon-skeleton was metabolized v i a the t r i c a r b o x y l i c acid cycle. Carbon dioxide production was to be expected when i n t e r -mediates of putrescine degradation were metabolized v i a t h i s cycle. When mycobacterial c e l l s were allowed to oxidize 1,4-C 1 4 putrescine under COg-free conditions the c e l l s assimilated approximately 25% of the r a d i o a c t i v i t y . A l l 14-C, not assimilated by the c e l l s appeared as G. Og. The 25% of assimilated r a d i o a c t i v i t y was di s t r i b u t e d among frac t i o n s containing l i p i d , nucleic a c i d and protein. These findings are consistent with the a b i l i t y of mycobacterial c e l l s to u t i l i z e putrescine as the sole source of carbon f o r growth. Since amines are reported to occur i n nature usually i n r e l a t i v e l y low concentrations, i t might be of advantage f o r bacteria to u t i l i z e only those amines which (within t h e i r genetic capacity) require the minimum expenditure of stored energy and reserve materials. Putrescine meets these requirements, since the synthesis of only four enzymes i s required to produce succinic acid - an intermediate metabolite which the c e l l machinery can normally handle. On the other hand, i f diamines l i k e 1,3-diaminopropane and cadaverine were metabolized by an analogous series of reactions, 136 they would y i e l d malonic and g l u t a r i c acids, respectively. But since these intermediate products do not normally occur within c e l l s , t h e i r e f f e c t i v e u t i l i z a t i o n would require addit i o n a l enzyme, synthesis. Otherwise, a s i t u a t i o n would a r i s e i n which an i n i t i a l waste of energy l e d to the accumulation of substances, some of which might be detrimental to the c e l l . Malonic acid, f o r instance, i s known to impair the e f f i c i e n t operation of the t r i c a r b o x y l i c a c i d cycle. The i n a b i l i t y of the tested mycobacterial strains to u t i l i z e c e r t a i n amines appears at f i r s t sight to be a handicap to s u r v i v a l . But act u a l l y when the c e l l environment includes c e r t a i n amines, i n addition to acceptable substrates,, a complete i n a b i l i t y (rather than p a r t i a l a b i l i t y ) to u t i l i z e the former may be more advantageous to the c e l l . 137 APPENDIX  Table 1 Composition of the medium employed to t e s t the a b i l i t y of mycobacterial c e l l s to u t i l i z e amines as sole sources of carbon. Amine (as hydrochloride s a l t or phosphate s a l t ) 1.0 g. or 4.0 F e r r i c ammonium c i t r a t e 50.0 mg. Magnesium sulphate 10.0 mg. Calcium chloride 0.5 mg. Zinc sulphate 0.1 mg. Copper sulphate 0.1 mg. Disodium phosphate 2.5 &> Monopotassium phosphate 1.0 g. Ammonium n i t r a t e 1.0 g. Aqueous T e r g i t o l (1$ v/v) 10.0 ml. Water 1000.0 ml. The medium was s t e r i l i z e d at 121 C fo r 15 minutes. 133 Table 2 Composition of the medium used f o r growing mycobacterial c e l l s i n order to test f o r the inductive nature of the D.O. enzyme. Asparagine (Difco) 5.0 g« Glucose* 2.0 &• F e r r i c ammonium c i t r a t e 50.0 mg. Magnesium sulphate 10.0 mg. Calcium chloride 0.5 mg. Zinc sulphate 0.1 mg. Copper sulphate 0.1 mg. Disodium phosphate 2.5 g» Monopotassium phosphate 1.0 g-Aqueous T e r g i t o l (1% v/v) 10.0 ml. Water 1000.0 ml. * Glucose was prepared as a 50% w/v solution and s t e r i l i z e d separately at 121°C f o r 15 minutes. The medium was autoclaved at 121°C f o r 20 minutes i n 100 ml. amounts. To each 100 ml. of medium was added 0.4 ml. of s t e r i l e glucose solution. 139 BIBLIOGRAPHY 1. ALARCON, R.A., POLEY, G.E., and E.J. MOLEST. 1961. Ef f e c t s of spermine on mammalian c e l l s . Arch. Biochem. Biophys. 94:540-541. 2. AMES, B.N., and D.T. DUBIN. I960. 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A common s i t e of action f o r polyamines and streptomycin. Biochim. Biophys. Acta. 62:202-204. 59. MAGER, J . , TRAUB, A., and N. GROSSOWICZ. 1954. C u l t i v a t i o n of Pasteurella t u l a r e n s i s i n chemically defined media: E f f e c t of buffers and spermine. v Nature. 174:747-748. 144 60. MAHLER, H.R., MEHROTRA, B.D., and C.W. SHARP. 1961 Ef f e c t s of diamines on thermal t r a n s i t i o n of MA. Biochem. Biophys. Research. Commun. 4:79-82. 61. MANDEL, M. 1962. The i n t e r a c t i o n of spermine and native deoxyribonucleic acid. J . Molecular B i o l . 5:435-441. 62. MANN, P.J.G., and W.R. SMITHIES. 1955. Plant enzyme reactions leading to the formation of heter o c y c l i c compounds. 1. The formation of unsaturated p y r r o l i d i n e and piperidine compounds. Biochem. J . 61:89-100. 63. MARQUARDT, H. 1949. Mutationsauslosumg durch Putrescin-Hydrochlorid und Kaltextract aus uberalterten Qehotherasamen. Experientia. 5:401-403. 64. 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Enzymic exchange of protein amide groups. Arch. Biochem. Biophys. 77:227-229. 71. NORRIS, P.C., CAMPBELL, J.J.R., and P.W. NEY. 1949. The intermediate metabolism of Pseudomonas aeruginosa. I. The status of the endogenous r e s p i r a t i o n . Can. J . Research, C-Botanical. 27:157-164. 145 72. OKUYAMA, T., and Y. KOBAYASHI. 1961. Determination of diamine oxidase a c t i v i t y by l i q u i d s c i n t i l l a t i o n counting. Arch. Biochem. Biophys. 95:242-250. 73. OWEN, O.A., J r . , KARLSON, A.G., and E.A.ZELLER. 1951. Enzymology of tubercle b a c i l l i and other mycobacteria. V. Influence of streptomycin and other basic substances on the diamine oxidase of various bacteria. J . Bacterioib. 62:53-62. 74. RAZIN, S., BACHRACH, U., and I. GERY. 1958. Formation o f f i - a l a n i n e from spermine and spermidine by Pseudomonas aeruginosa. Nature. 181:700-701. 75. RAZIN, S., COHEN, A., and R. ROZANSKY. 1958. Antimycotic e f f e c t of spermine. Proc. Soc. Exp t l . B i o l . N.Y. 99:459-462. 76. RAZIN, S., GERY, I., and U. BACHRACH. 1959. The degradation of natural polyamines and diamines by bacteria. Biochem. J . 71:441-558. 77. RAZIN, S., and R. ROZANSKY. 1959. Mechanism of the a n t i b a c t e r i a l action of spermine. Arch. Biochem. Biophys. 81:36-54. 78. ROBERTS, R.B., ABELSON, P.H., COWIE, D.B., BOLTON, E.T., and R.J. BRITTEN. 1957. Studies of the biosynthesis i n Escherichia c o l i . Carnegie Inst. Wash. Publ. no. 607. 79. ROSENTHAL, S.M., and D.T. DUBIN. 1962. Metabolism of polyamines by Staphylococcus. J . B a c t e r i o l . 84:859-863. 80. ROSENTHAL, S.M., and D.T. DUBIN. I960. Acetylation of polyamines by Staphylococcus. Ped. Proc. 19:2 81. ROSENTHAL, S.M., FISHER, E.R., and E.F. STOHLMAN. 1952. Nephrotoxic action of spermine. Proc. Soc. Exptl. B i o l . Med. 80:432-434. 82. ROSENTHAL, S.M., and C.W. TABOR. 1956. The pharmacology of spermine and spermidine. D i s t r i b u t i o n and excretion. J . Pharmacol, and E x p t l . 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The oxidation of polyamines by Neisseria perflava. J . B i o l . Chem. 231:647-655. 116. WEAVER, R.H., and E.J. HERBST. 1958. Metabolism of diamines and polyamines i n microorganisms. J . B i o l . Chem. 231:637-646. 117. ZELLER, E.A. 1951. Oxidation of amines, p.536-558. In J.B. Sumner and K. Myrback (ed^, The enzymes,vol I I part 1| Acad. Press Inc., New York. 118. ZELLER, E.&., OWEN, CA. Sr., and A.G. KARLSON. 1951. Diamine oxidase of Mycobacterium smegmatis:Teffeet.of streptomycin and dihydrostreptomycin. J . B i o l . Chem. 188:623-630. Sf 54 tooh as i 2 3 TfMG (HOURS) FIG. 1. Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n N.T.N. c e l l s . 55 ' - * 3 4 T 7 M £ ^HOURS) FIG. 2. Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n O.C. c e l l s . 56 loo h 200 h tOO h I X 3 T»M£ (HOURS) FIG. 3« Oxidation of putrescine by putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n S.C. o e l l s . 57 TIMS (MINUTES) [PIG. 4. Diamine oxidase a c t i v i t y i n c e l l - f r e e extracts obtained from putrescine-adapted (curve 1) and unadapted (curve 2) s t r a i n S.C. c e l l s . Reaction mixtures contained i n a volume of 3.0 ml.: C e l l - f r e e extract, 1.0 ml., equivalent to 3.0 mg. protein; putrescine, 3.0 jaM; t r i s buffer, pH 9.0, 100 ;uM; HgO. 61 * * £ 6 TIME (HOURS) FIG. 5 . E f f e c t of pre-exposure to spermidine on the subsequent oxidation by s t r a i n CO. c e l l s of 7 .5 juM of putrescine (curve 1 ) , 3.25 uM of each of putrescine and 1,3-diaminopropane (curve 2 ) , 7.5 )M of 1,3-diaminopropane (curve 3 ) , and 7 .5 jJM of spermidine (curve 4 ) . 65 (SJINfl VflOlOO 3NIHti-dNG 7.2 FIG. 7. Oxygen consumption and ammonia production during putrescine oxidation by s t r a i n H.T.N, c e l l s . Curve It Op uptake with 3.0 jM putrescine (1.0 juM/ml.). Curve 2: HH^ production with putrescine (l.O uM/ml.). 75 TIME (HOURS) FIG. 8. Oxygen consumption and ammonia production during putrescine oxidation by s t r a i n S.C. c e l l s . Curve I J OO uptake with 6.0 uM putrescine ( 2 . 0 juM/ml.). Curve 2: NH3 production with putrescine ( 2 . 0 ;oM/ml.). TIKIS (HOURS) FIG. 9. Oxygen consumption during the oxidation of 6.0 nM of putrescine by s t r a i n C.C. c e l l s . T/M£ (HOURS) FIG. 10. E f f e c t of arsenite on the metabolism of putrescine by s t r a i n S.C. c e l l s . Curve Is 0 2 uptake with 3.0 nM putrescine (1.0 uM/ml.). Curve 2: Same as curve 1, but arsenite (3.0 nM/ml.) present. Curve 3s Leakage of pyruvate with putrescine (1.0 uM/ml.). Curve 4s Same as curve 3, but arsenite (3.0 pM/ml.) present. 89 FIG. 11. E f f e c t of arsenite on oxygen consumption and ammonia output by s t r a i n S.C. c e l l s acting on putrescine. Curve 1: 0 2 uptake with 3.0 nM putrescine (1.0 juM/ml.). Curve 2: Same as curve 1, but arsenite (9.0 juM/rnl.) present. Curve 3: NH^ output with putrescine (1.0 jaM/ml.). Curve 4: Same as curve 3, but arsenite (9.0 nM/ml.)present. 77 ME ( HOURS) FIG. 12. E f f e c t of arsenite on oxygen consumption and ammonia output by s t r a i n C.C. c e l l s acting on putrescine. Curve 1: 0 2 uptake with 3.0 pK putrescine (1.0 JuH/ml.). Curve 2: Same as curve 1, but arsenite (3.0juM/ml.) present. Curve 3. NH* production with putrescine (1.0 pk/mL.) present. Curve 4: Same as curve 3, but arsenite (3.0 jjM/ml.) present. 103 HO r 5> PIG. 13. The optimum pH f o r diamine oxidase a c t i v i t y . Reactions conducted i n c i t r a t e (pH 4.5 - 6.0) and phosphate (pH 6.0 - 8.0) buffers (curve l ) and i n t r i s buffer (pH 7.5 - 9.0) (curve 2). 108 TIME. (MINUTES) FIG. 14. Strai n C.C. A'-pyrroline dehydrogenase a c t i v i t y (PH9.0). Curve 1: Mercaptoethanol + NAD. Curve 2: Mercaptoethanol + NADP. Curve 3: NAD. Curve 4: NADP. o x q 6 % to TIME (MINUTES) FIG. 15. Strai n S.C.4'-pyrroline dehydrogenase a c t i v i t y (pH 9.0). Curve 1: Mercaptoethanol + NAD. Curve 2: Mercaptoethanol + NADP. 110 TfM£ (MINUTES) PIG. 16. Manometric assay of s t r a i n C.C. A*-pyrroline dehydrogenase a c t i v i t y (pH 7.2). See text f o r d e t a i l s . 113 TIME (MINUTES') PIG. 17. Manometric assay of s t r a i n C.C. A'-pyrroline dehydrogenase a c t i v i t y (pH 9.0). See text f o r d e t a i l s . 117 FIG. 19 . E f f e c t of pH and reaction time on Y-aminobutyrie a c i d - «c-ketoglutarie acid transaminase a c t i v i t y . 119 i o a * 6 % io TIME (MlJMOTfis) FIG. 20. St r a i n C.C. succinic semialdehyde dehydrogenase a c t i v i t y (pH 9.0). Curve 1: NAD Curve 2: NADP. o X if (, 8 to T I M E (MINUTES) FIG. 21. Strai n S.C. succinic semialdehyde dehydrogenase a c t i v i t y (pH 9.0). Curve 1: NAD. Curve 2: NADP. 126 T/M£ (MINUTES) 3?IG. 22. D i s t r i b u t i o n of r a d i o a c t i v i t y during the oxidation of 1,4-0 putrescine by putrescine-adapted s t r a i n N.T.N, c e l l s . Reaction mixture supernatant (curve 1); combined c e l l f r a c t i o n s (curve 2); c e l l s , calculated as the difference., between c e l l s + supernatant and supernatant (curve 3); C^Og (curve 4); 0 2 uptake occurring during the reaction (curve 5). 

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