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UBC Theses and Dissertations

Disphosphopyridine nucleotide-nitrate reductase in Beta vulgaris L. Yang, Kuan Jen 1964

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DIPHOSPHOPYRIDINE NUCLEOTIDE-NITRATE REDUCTASE i n B e t a v u l g a r i s L. by KUAN-JEN YANG B.Sc. Taiwan Normal U n i v e r s i t y , C h i n a , 1956 M.Sc. N a t i o n a l Taiwan U n i v e r s i t y , C h i n a , 1959 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f BIOLOGY AND BOTANY We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d . THE UNIVERSITY OF BRITISH COLUMBIA November, 1964 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 o f t h e r e q u i r e m e n t s f o r an advanced d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by t h e Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f B i o l o g y and B otany The U n i v e r s i t y o f B r i t i s h C o l u m b i a , Vancouver 8, Canada. Date: November 2, 1964. i ABSTRACT A s o l u b l e D P N H - n i t r a t e r e d u c t a s e (NRase) o f t h e s u g a r b e e t has been p u r i f i e d and c h a r a c t e r i z e d . The o c c u r r e n c e o f N0~ as an end p r o d u c t and t h e i n s e n s i t i v i t y o f t h e enzyme t o oxygen i n d i c a t e t h a t t h e s u g a r b e e t NRase i s o f t h e n i t r a t e a s s i m i l a t i o n t y p e . The enzyme was n o t a s s o c i a t e d w i t h any c e l l p a r t i c l e and a l l NRase a c t i v i t y p r e s e n t i n t h e homogenate o f s u g a r b e e t l e a v e s was r e c o v e r e d i n t h e 20,000 x g s u p e r n a t a n t . A s i x t y f o l d p u r i f i c a t i o n was a c c o m p l i s h e d by ammonium s u l f a t e p r e c i p i t a t i o n f o l l o w e d by a d s o r p t i o n on c a l c i u m phosphate g e l . A t room t e m p e r a t u r e s and h i g h e r t h e enzyme was h e a t l a b i l e , b u t was r e l a t i v e l y s t a b l e a t -15°C. D i a l y s i s a t 4° C. d i d n o t r e s u l t i n an a p p r e c i a b l e l o s s o f a c t i v i t y . The optimum pH was 7.0. The NRase was s e n s i t i v e t o heavy m e t a l i n h i b i t o r s b u t i t was n o t p o s s i b l e t o show t h a t Mo was t h e s p e c i f i c p r o s t h e t i c m e t a l . I t was d e m o n s t r a t e d , however, t h a t c h e m i c a l l y r e d u c e d Mo c o u l d s e r v e as an e l e c t r o n donor. Thus Mo may be a c o f a c t o r f o r t h e enzyme. The r e v e r s a l o f p - c h l o r o m e r c u r i b e n z o a t e i n h i b i t i o n by t h e s u l f h y d r y l r e a g e n t s g l u t a t h i o n e and c y s t e i n e , c o u p l e d w i t h s t r o n g i n h i b i t i o n by i o d o a c e t a t e and c u p r i c s u l f a t e i n d i c a t e d t h e s u l f -h y d r y l n a t u r e o f t h e enzyme. The p a r t i a l l y p u r i f i e d NRase was s t i m u l a t e d t o a c o n -s i d e r a b l y g r e a t e r degree by FAD t h a n by FMN. Rf v a l u e s and c o -chromatography i n d i f f e r e n t s o l v e n t s showed t h a t a s u b s t a n c e l i b e r a t e d from t h e enzyme p r e p a r a t i o n by a c i d and h e a t was n o t i i r i b o f l a v i n o r FMN b u t v e r y p r o b a b l y was FAD. I t i s s u g g e s t e d t h a t , i n common w i t h o t h e r a s s i m i l a t o r y NRases o f h i g h e r p l a n t s , t h e f l a v i n n u c l e o t i d e p r o s t h e t i c group o f sugar b e e t NRase i s FAD. The p r e s e n c e o f two NRases i n t h e su g a r b e e t was i n d i -c a t e d by t h e f a c t t h a t t h e c r u d e "enzyme" was s t i m u l a t e d t o t h e same e x t e n t by t h e a d d i t i o n o f DPNH o r TPNH, t h a t t h e r a t i o o f a c t i v i t i e s r e s u l t i n g from t h e a d d i t i o n o f t h e two p y r i d i n e n u c l e o t i d e s changed w i t h t h e degree o f p u r i t y o f t h e enzyme, and t h a t t h e enzyme f i n a l l y o b t a i n e d by c a l c i u m p h o s p h a t e g e l a d s o r p -t i o n and e l u t i o n was D P N H - s p e c i f i c . That p u r i f i c a t i o n was n o t co m p l e t e was shown by t h e p r e s e n c e o f DPNH-quinone r e d u c t a s e and DPNH-cytochrome c r e d u c t a s e a c t i v i t y i n t h e NRase p r e p a r a t i o n . A low NRase a c t i v i t y and a h i g h n i t r a t e c o n t e n t were measured i n su g a r b e e t l e a v e s d u r i n g growth i n d a r k n e s s . The r e v e r s e o c c u r r e d i n l i g h t . I t i s s u g g e s t e d t h a t t h e d i t t r n a l v a r i a t i o n i n NRase a c t i v i t y may be t h e r e s u l t o f t h e f a l l o f l e a f t i s s u e pH d u r i n g d a r k n e s s and i t s r i s e t o a p p r o x i m a t e t h e enzyme's optimum pH i n l i g h t . The p o s s i b l e p a r t i c i p a t i o n o f t h e NRase i n a f l a v i n n u c l e o t i d e - c a t a l y z e d e n z y m a t i c p h o t o r e d u c t i o n o f n i t r a t e was i n d i c a t e d by t h e c o u p l i n g o f p h o t o r e d u c t i o n o f FAD w i t h t h e r e d u c t i o n o f n i t r a t e by NRase. i i i ACKNOWLEDGEMENTS I t i s my p l e a s u r e t o acknowledge t h e h e l p I have r e c e i v e d f r o m P r o f . D. J . Wort. D u r i n g t h e p e r i o d when t h i s r e s e a r c h was c o n d u c t e d he has been a c o n s t a n t s o u r c e o f encouragement and a d v i c e . I n a d d i t i o n I w i s h t o thank him f o r r e v i e w i n g t h e m a n u s c r i p t c r i t i c a l l y . My thanks a r e due t o P r o f . J . J . R. Cam p b e l l f o r much h e l p f u l d i s c u s s i o n and a d v i c e and f o r p e r m i t t i n g t h e use o f h i s l a b o r a t o r y f a c i l t i e s t h r o u g h o u t t h e c o u r s e o f t h i s s t u d y . I w o u l d a l s o l i k e t o thank P r o f . W. J . P o l g l a s e f o r h i s c o n t i n u i n g i n t e r e s t i n t h e prob l e m and f o r h i s u n s t i n t e d h e l p and c r i t i c i s m d u r i n g 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 . Thanks a r e a l s o due t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada and t h e B r i t i s h C o l u m b i a Sugar R e f i n i n g Co. whose f i n a n c i a l s u p p o r t made t h i s s t u d y p o s s i b l e . i v TABLE OF CONTENTS A b s t r a c t i Acknowledgement •• i i i T a b l e o f C o n t e n t s i v L i s t o f F i g u r e s v i i i L i s t o f T a b l e s i x HISTORICAL I . DISCOVERY OF NITRATE REDUCTASE 1 I I . CLASSIFICATION OF NITRATE REDUCTASE 5 I I I . ASSIMILATORY NITRATE REDUCTASE 15 1. Methods o f E x t r a c t i o n 15 2. P u r i f i c a t i o n o f Enzyme 21 3. Measurement o f A c t i v i t y 24 4. S t a b i l i t y o f Enzyme 30 5. E l e c t r o n Donors 31 6. FAD as P r o s t h e t i c Group. 33 7. S u l f h y d r y l P r o p e r t y 35 8. Molybdenum C o n s t i t u e n t 35 9. Phosphate S t i m u l a t i o n 38 10. I n h i b i t o r s 40 11. Mechanism o f A c t i o n 41 12. L o c a l i z a t i o n o f Enzyme 43 13. A d a p t i v e F o r m a t i o n o f Enzyme 44 14. E f f e c t o f P l a n t Age on Enzyme A c t i v i t y 46 15. N i t r a t e R e d u c t a s e A c t i v i t y i n Roots 47 16. L i g h t and N i t r a t e R e d u c t a s e A c t i v i t y 49 V TABEE OF CONTENTS ( c o n t ' d ) EXPERIMENTAL PROCEDURE MATERIAL AND METHODS 53 1. S o u r c e o f Enzyme 53 2. E x t r a c t i o n o f Enzyme 53 3. C o f a c t o r s and Ot h e r S u b s t a n c e s 54 4. P r e p a r a t i o n o f C a l c i u m G e l 55 5. P r e p a r a t i o n o f A l u m i n a C r G e l 55 6. P r e p a r a t i o n o f E l e c t r o n Donors • 56 7. D e t e r m i n a t i o n o f P r o t e i n 56 8. D e t e r m i n a t i o n o f C h l o r o p h y l l C o n t e n t 57 9. D e t e r m i n a t i o n o f N i t r a t e C o n t e n t 57 10. S t a n d a r d N i t r a t e R e d u c t a s e A s s a y 58 11. D e t e r m i n a t i o n o f DPNH-Menadione R e d u c t a s e A c t i v i t y . . . . . . 59 12. D e t e r m i n a t i o n o f DPNH-Quinone R e d u c t a s e A c t i v i t y ....... 59 13. D e t e r m i n a t i o n o f DPNH-Cytochrome c R e d u c t a s e A c t i v i t y . . . 60 14. D e t e r m i n a t i o n o f DPNH-Diaphorase A c t i v i t y . 60 15. I s o l a t i o n and I d e n t i f i c a t i o n o f Enzyme Bound F l a v i n . . . . . 61 16. F r a c t i o n a t i o n o f C e l l u l a r Components. 61 17. P h o t o c h e m i c a l R e d u c t i o n o f F l a v i n N u c l e o t i d e s . 62 EXPERIMENTAL RESULTS I . PURIFICATION OF ENZYME 64 1. F i r s t Ammonium S u l f a t e F r a c t i o n a t i o n 64 2. Second iSmmonium S u l f a t e F r a c t i o n a t i o n 65 3. C a l c i u m P hosphate G e l A d s o r p t i o n 66 4. T h i r d Ammonium S u l f a t e F r a c t i o n a t i o n 67 v i TABLE OF CONTENTS (cont'd) II. CHARACTERIZATION OF NITRATE REDUCTASE 71 1. Stability of Enzyme .........*..*... 71 2. Time Course of Enzymatic Reaction 74 3. Enzyme Activities with Enzyme Concentration «.... 74 4. pH Optimum . * 74 5. Substrate A f f i n i t y 74 6. Specificity for Electron Donors 79 7. Effect of Temperature on Activity 79 8. FAD as Prosthetic Group 84 9. Sulfhydryl Nature 86 10. Dialysis of Enzyme 88 11. Phosphate Stimulation 90 12. Inhibition 93 13. Separation of DPNH-Nitrate Reductase from TPNH-Nitrate.. 99 14. Other Enzyme Activities in Enzyme Preparation 99 15. Products and Stoichiometry 100 III. EFFECT OF AGE OF PLANT ON ENZYME ACTIVITY 106 IV. DISTRIBUTION OF ENZYME IN SUGAR BEET PLANT 107 V. SUB-CELLULAR LOCALIZATION OF ENZYME 108 VI. FLAVIN NUCLEOTIDE-CATALYZED ENZYMATIC PHOTOREDUCTION OF NITRATE 110 1. Photoreduction of Flavin Nucleotides 110 2. Characterization of Flavin Nucleotide-Catalyzed Enzymatic Photoreduction of Nitrate 110 v i i TABLE OF CONTENTS (cont'd) VII. DIURNAL CHANGES IN NITRATE CONTENT AND NITRATE REDUCTASE ACTIVITY IN SUGAR BEET LEAVES 114 VIII. OTHER METHODS OF ENZYME PURIFICATION 117 1. Extraction of Nitrate Reductase from Acetone Powder..., 117 2. Removal of Nucleoprotein from the Enzyme Preparation... 118 3. Adsorption of Nitrate Reductase on Alumina C r Gel ..... 119 DISCUSSION 121 BIBLIOGRAPHY 129 v i i i L I S T OF FIGURES FIGURE PAGE 1. E f f e c t o f p r o t e c t a n t i n t h e e x t r a c t i o n medium on t h e l e v e l o f s u g a r b e e t n i t r a t e r e d u c t a s e a c t i v i t y ..... 72 2. E f f e c t o f t i m e and s t o r a g e on t h e a c t i v i t y o f n i t r a t e r e d u c t a s e 73 3. P r o p o r t i o n a l i t y o f enzyme a c t i v i t y w i t h t i m e 75 4. P r o p o r t i o n a l i t y o f enzyme a c t i v i t y w i t h enzyme c o n c e n t r a t i o n 76 5. E f f e c t o f pH on n i t r a t e r e d u c t a s e a c t i v i t y 77 6. E f f e c t o f n i t r a t e c o n c e n t r a t i o n on n i t r a t e r e d u c t a s e a c t i v i t y 78 7. E f f e c t o f DPNH and TPNH on t h e a c t i v i t y o f n i t r a t e r e d u c t a s e 80 8. V a r i a t i o n o f a c t i v i t y w i t h t e m p e r a t u r e 83 9. E f f e c t o f FAD and FMN on t h e a c t i v i t y o f n i t r a t e r e d u c t a s e 85 10. Time c o u r s e o f n i t r a t e r e d u c t a s e r e a c t i o n 105 11.. P h o t o r e d u c t i o n o f FAD and FMN w i t h EDTA I l l 12. P h o t o r e d u c t i o n o f FAD and FMN w i t h m e t h i o n i n e 112 i x L I S T OF TABLES TABLE PAGE I . Comparison o f A s s i m i l a t o r y N i t r a t e R e d u c t a s e and R e s p i r a t o r y N i t r a t e R e d u c t a s e 14 I I . Summary o f Methods o f E x t r a c t i n g A s s i m i l a t o r y N i t r a t e R e d u c t a s e 19 I I I . Summary o f Measurement o f A s s i m i l a t o r y N i t r a t e A c t i v i t y by C o l o r i m e t r i c Method 26 IV. Summary o f P r o p e r t i e s o f A s s i m i l a t o r y N i t r a t e R e d u c t a s e 34 V. A d s o r p t i o n o f N i t r a t e R e d u c t a s e by C a l c i u m P h o s p h a t e G e l 66 V I . Summary o f P u r i f i c a t i o n o f Sugar Beet N i t r a t e R e d u c t a s e 70 V I I . E f f e c t o f V a r i o u s Hydrogen Donors on N i t r a t e R e d u c t a s e 81 V I I I . E f f e c t o f Temperature on N i t r a t e R e d u c t a s e A c t i v i t y 82 I X . Paper Chromatography o f N i t r a t e R e d u c t a s e F l a v i n 86 X. I n h i b i t i o n o f N i t r a t e R e d u c t a s e by p - C h l o r o -m e r c u r i b e n z o a t e and i t s R e v e r s a l by S u l f h y d r y l Compounds 87 X I . I n f l u e n c e o f D i a l y s i s A g a i n s t V a r i o u s Reagents on t h e A c t i v i t y o f N i t r a t e R e d u c t a s e 89 X I I . A c t i v a t i o n o f N i t r a t e R e d u c t a s e by Phosphate 92 X I I I . The S u b s t i t u t i o n o f O t h e r A n i o n s f o r Pho s p h a t e 92 XIV. E f f e c t o f Heavy M e t a l s on t h e A c t i v i t y o f N i t r a t e R e d u c t a s e 95 XV. E f f e c t o f V a r i o u s M e t a l - c h e l a t i n g Agents on t h e A c t i v i t y o f N i t r a t e R e d u c t a s e 96 X X V I . E f f e c t o f N o n - s p e c i f i c I n h i b i t o r s on t h e A c t i v i t y o f N i t r a t e R e d u c t a s e 97 X V I I . E f f e c t o f P h e n o l i c Compounds on t h e A c t i v i t y o f N i t r a t e R e d u c t a s e 98 X V I I I . S e p a r a t i o n o f D P N H - N i t r a t e R e d u c t a s e from TPNH-N i t r a t e R e d u c t a s e 101 XIX. O t h e r E n z y m a t i c A c t i v i t i e s P r e s e n t i n P a r t i a l l y P u r i f i e d N i t r a t e R e d u c t a s e 102-XX. S t o i c h i o m e t r y o f N i t r a t e R e d u c t a s e R e a c t i o n 104 X X I . E f f e c t o f Age o f P l a n t on N i t r a t e R e d u c t a s e A c t i v i t y 106 X X I I . D i s t r i b u t i o n o f N i t r a t e R e d u c t a s e A c t i v i t y i n B e t a v u l g a r i s 107 X X I I I . S u b - c e l l u l a r L o c a l i z a t i o n o f Sugar Beet N i t r a t e R e d u c t a s e 109 XXIV. C h a r a c t e r i z a t i o n o f F l a v i n N u c l e o t i d e - C a t a l y z e d Enzymic P h o t o r e d u c t i o n o f N i t r a t e 113 XXV. D i u r n a l Change i n N i t r a t e C o n t e n t and N i t r a t e R e d u c t a s e A c t i v i t y i n Sugar Beet Leaves .......... 116 XXVI. E x t r a c t i o n o f N i t r a t e R e d u c t a s e from A c e t o n e Powder 118 X X V I I . Removal o f N u c l e o p r o t e i n from Enzyme P r e p a r a t i o n 119 X X V I I I . A d s o r p t i o n o f N i t r a t e R e d u c t a s e i n A l u m i n a C r G e l 120 XXIX. Comparison o f t h e P r o p e r t i e s o f N i t r a t e R e d u c t a s e from B e t a v u l g a r i s and from Other H i g h e r P l a n t s . . . 126 1. I . DISCOVERY OF NITRATE REDUCTASE By t h e p r o c e s s e s o f a m m o n i f i c a t i o n and n i t r i f i c a t i o n t h e n i t r o g e n o f s o i l o r g a n i c m a t t e r i s c o n v e r t e d t o ammonia and f i n a l l y t o n i t r a t e . N i t r a t e i s t h e m a i n s o u r c e o f n i t r o g e n u t i l i z e d by h i g h e r p l a n t s . The n i t r o g e n o f n i t r a t e , w i t h an o x i d a t i o n number o f +5, i s i n a h i g h l y o x i d i z e d s t a t e , b u t t h e n i t r o g e n o f t h e amino group and ammonia w i t h an o x i d a t i o n number o f - 3 , i s i n a h i g h l y r e d u c e d s t a t e . The r e d u c t i o n o f n i t r o g e n i s thus t h e f i r s t s t e p i n t h e s y n t h e s i s o f amino a c i d s and o t h e r o r g a n i c n i t r o g e n o u s compounds when n i t r a t e i s t h e s o u r c e o f n i t r o g e n . A l t h o u g h o t h e r n i t r o g e n s o u r c e s such as ammonia, n i t r i t e and amino a c i d s c a n be u t i l i z e d by h i g h e r p l a n t s , n i t r a t e u s u a l l y s u p p o r t s b e t t e r growth t h a n t h e s e s o u r c e s . One of t h e e a r l i e s t i n v e s t i g a t i o n s , r e p o r t e d i n 1903 by P o z j i i E s c o t , d e m o n s t r a t e d t h a t t h e aqueous e x t r a c t s o f t h e stem o f t h e b u r d o c k had t h e power t o r e d u c e n i t r a t e s t o n i t r i t e s and ammonia. K a s t l e and E l v o v e (1904) a l s o f ound t h a t t h e r e d u c t i o n t o o k p l a c e most r a p i d l y a t 40° t o 50°C and t h a t a l d e h y d e was one o f t h e most e f f e c t i v e o f v a r i o u s " a c c e l e r a t o r s " o f t h e a c t i o n . I r v i n g and H a n k i n s o n (1908) c l a i m e d t o have f o u n d a n i t r a t e r e d u c i n g enzyme i n E l o d e a , Potamogeton, and numerous members o f Gramineae. They c o n s i d e r e d t h a t t h e n i t r a t e r e d u c t i o n r e q u i r e d o n l y t h e enzyme and a s u i t a b l e c a r b o h y d r a t e . E c k e r s o n (1924) f o r t h e f i r s t t i m e s u g g e s t e d t h a t h i g h e r 2. p l a n t s c o n t a i n an enzyme " r e d u c a s e " r e s p o n s i b l e f o r t h e r e d u c t i o n o f n i t r a t e . The " r e d u c a s e " a c t i v i t y was c a l c u l a t e d from t h e r a t e o f p r o d u c t i o n o f n i t r i t e f rom n i t r a t e . A t t h e same t i m e Anderson (1924) d e t e c t e d n i t r i t e i n t h e s h o o t s o f 25 o f t h e 105 s p e c i e s exam-i n e d . The n i t r a t e - r e d u c i n g s u b s t a n c e , p r e s e n t i n t h e f o l i a r p a r t o f s u c h p l a n t s as s u n f l o w e r , a r t i c h o k e , pea, r a d i s h , tomato, c l o v e r and wheat, was found t o be t h e r m o l a b i l e and o x i d i z a b l e . Wu and Loo r e p o r t e d i n 1950 t h a t an enzyme " r e d u c t a s e " had been d e t e c t e d i n t h e r o o t s and l e a v e s o f 11 h i g h e r p l a n t s , b u t n o t i n t h e stem. T h i s enzyme had an optimum pH o f 5.2 and an optimum t e m p e r a t u r e o f 52°C. The r e d u c t i o n o f n i t r a t e t o n i t r i t e i n v i t r o was a f i r s t o r d e r r e a c t i o n . R e d u c t a s e from soybean s p r o u t s a p p e a r e d t o use l a c t a t e as an e l e c t r o n donor. D e l w i c h e (1952) f o u n d t h a t c e l l - f r e e e x t r a c t s o f r o o t s and l e a v e s o f v a r i o u s p l a n t s c o u l d c o n v e r t N ^ - l a b e l e d n i t r a t e and n i t r i t e t o ammonia. Optimum a c t i v i t y was o b t a i n e d w i t h a d i a l y z e d p r e p a r a t i o n . C i t r a t e o r o t h e r K r e b ' s i n t e r m e d i a t e s were a b l e t o s t i m u l a t e t h e r e a c t i o n . The s y s t e m was i n h i b i t e d by Cu , AsO^ , and i o d o a c e t a m i d e . Nason and Evans (1953) and Evans and Nason (1953) s u c c e s s -f u l l y p u r i f i e d n i t r a t e r e d u c t a s e (NRase) from N e u r o s p o r a and soybean l e a v e s , r e s p e c t i v e l y . I t p r o v e d t o be a m e t a l l o f l a v o p r o t e i n c o n t a i n i n g FAD, molybdenum and a c t i v e s u l f h y d r y l g r o u p s . D P N H 2 o r T P N H 2 s e r v e d as h y d r o g e n donor. NRase was later shown to occur in many plants: Higher Plants Rice Tang and Wu (1957) Cauliflower Candela, Fisher and Hewitt (1957) Wheat leaf Anacker and Stoy (1958) Wheat embryo Rijven (1958); Spender (1959) Tomato roots Vardyanathan and Street (1959) Corn seedling Hageman and Flesher (1960) Tobacco Fukuzumi and Tamaki (1962) Vegetable Cresswell, Hageman and Hewitt (1962) marrow Tomato leaf Sanderson and Cocking (1964) Green Algae Ankistrodesmus Anabaena Chlorella Fungi Neurospora Hansenula Bacteria Achromobacter flschSfci Czyan (1963) Hattori (1962) Syrett and Morris (1963) Shafter, Baker and Thompson (1961) Nicholas and Scawin (1956) Kinsky and McElroy (1958) Silver (1956) Sadana and McElroy (1957) A z o t o b a c t e r v i n l a n d i i T a n i g u i c h i and Ohmachi (1960) B e c k i n g (1962) E s c h e r i c h i a c o l i N i c h o l a s and Nason (1955b) T a n i g u c h i , S a t o and Egami (1956) F a r k a s - H i m s l e y and Artman (1957) Medina and H e r e d i a (1958) T a n i g u c h i and I t a g a k i (1959) R h i z o b i u m j a p o n i c a Evans ( 1 9 5 4 ) ; C h e n i a e and Evans (1955, 1956, 1957, 1959, 1960); Lowe and Evans (1964) R h o d o s p i r i l i u m r u b r u n K a t o (1963) Pseudomonas a e r u g i n o s a Fewson and N i c h o l a s (1961) Ps eudomonas d e n i t r i f i c a n s V ernon (1956) I I . CLASSIEICATION OF NITRATE REDUCTASE From t h e l i t e r a t u r e i t i s a p p a r e n t t h a t a v a r i e t y o f t y p e s o f n i t r a t e r e d u c t i o n o c c u r s i n t h e o r g a n i s m s w h i c h have been s t u d i e d . The d i s s i m i l a r i t y o f e l e c t r o n t r a n s p o r t sequences and of t h e end p r o d u c t o f r e d u c t i o n i n d i c a t e t h a t t h e NRase w h i c h c a t a l y z e s n i t r a t e r e d u c t i o n o c c u r s i n s e v e r a l d i f f e r e n t f orms. S e v e r a l c l a s s i f i c a t i o n s have been p r o p o s e d f o r t h e n i t r a t e r e d u c t i o n t y p e s , V e r h o e v e n (1956) s u g g e s t e d t h r e e t y p e s o f n i t r a t e r e d u c t i o n f o r m i c r o o r g a n i s m : a s s i m i l a t i o n , i n c i d e n t a l d i s s i m i l a t i o n and t r u e d i s s i m i l a t i o n . S a t o (1956) c l a s s i f i e d n i t r a t e r e d u c i n g m i c r o -o rganisms on t h e b a s i s o f t h e b e h a v i o u r o f t h e i r c ytochromes tow a r d n i t r a t e . Egami e t a l . ( 1 9 5 7 ) d e s c r i b e d t h r e e t y p e s o f n i t r a t e r e d u c t i o n i n g e r m i n a t i n g s e e d l i n g s o f V i g i n i a s e s q u i p e d a l i s , e.g. n i t r a t e a s s i m i l a t i o n , n i t r a t e r e s p i r a t i o n and n i t r a t e f e r m e n t a t i o n . The c l a s s i f i c a t i o n o f n i t r a t e r e d u c t i o n by Nason (1962) i s w i d e l y used a t t h e p r e s e n t . He d i v i d e d n i t r a t e r e d u c t i o n i n t o two c l a s s e s : 1) n i t r a t e a s s i m i l a t i o n , o r a s s i m i l a t o r y n i t r a t e r e d u c t i o n , i n w h i c h n i t r a t e i s r e d u c e d t o ammonia o r t h e amino l e v e l w i t h t h e p r o d u c t s b e i n g u s e d f o r t h e b i o s y n t h e s i s o f n i t r o g e n - c o n t a i n i n g c e l l c o n s t i t u e n t s , f o r example, p r o t e i n s and n u c l e i c a c i d s ; 2) n i t r a t e r e s p i r a t i o n , o r d i s s i m i l a t o r y n i t r a t e r e d u c t i o n , i n w h i c h n i t r a t e i s u s e d as t h e t e r m i n a l e l e c t r o n a c c e p t o r i n p l a c e o f o x y g e n , u s u a l l y u nder a n a e r o b i c c o n d i t i o n s . N a s o n 1 s two t y p e s o f NRase a r e d i s -c u s s e d b r i e f l y . 6. 1, A s s i m i l a t o r y NRase A s s i m i l a t o r y NRase c a t a l y z e s t h e f i r s t s t e p i n t h e sequence o f r e d u c t i o n by w h i c h n i t r a t e i s c o n v e r t e d t o t h e ammonia o r amino a c i d l e v e l f o r t h e u l t i m a t e s y n t h e s i s o f p r o t e i n , n u c l e i c a c i d s and o t h e r n i t r o g e n - c o n t a i n i n g c e l l c o n s t i t u e n t s . The enzyme o b t a i n e d by Evans and Nason (1953) and Nason and Evans (1953) f r o m N e u r o s p o r a and soybean l e a v e s i s t h e t y p i c a l a s s i m i l a t o r y enzyme. W i t h p u r i f i e d NRase from N e u r o s p o r a , N i c h o l a s and Nason (1954) have shown t h a t , d u r i n g t h e e n z y m a t i c t r a n s f e r o f e l e c t r o n s f rom TPNH t o n i t r a t e , b o t h FAD ( o r FMN) and molybdenum f u n c t i o n as e l e c t r o n c a r r i e r s . They p r o p o s e d t h e f o l l o w i n g r e d u c t i o n sequence: TPNH > FAD ( o r FMN) > Mo » NC~ An a s s i m i l a t o r y t y p e o f p y r i d i n e n u c l e o t i d e - N R a s e s i m i l a r t o t h a t d e s c r i b e d f o r N e u r o s p o r a and soybean l e a v e s , has been r e p o r t e d t o o c c u r i n t i s s u e s o f h i g h e r p l a n t s e.g. wheat l e a v e s ( A n a c k e r and S t o y , 1 9 5 8 ) , wheat embryo ( S p e n c e r , 1959) and tomato l e a v e s ( S a n d e r s o n and C o c k i n g , 1 9 6 4 ) , e t c . N i c h o l a s and Nason (1955b) r e p o r t e d t h e p r e s e n c e o f an a s s i m i l a t o r y NRase i n E. c o l i . The enzyme was D P N H - l i n k e d and r e q u i r e d FAD and p r o b a b l y molybdenum as c o f a c t o r s . S i l v e r (1957) p u r i f i e d and c h a r a c t e r i z e d a p y r i d i n e n u c l e o t i d e - N R a s e i n t h e e x t r a c t s o f t h e y e a s t , H a n s e n u l a anomala, 7. grown on a n i t r a t e - c o n t a i n i n g medium. The enzyme was s i m i l a r t o t h a t f o u n d i n N e u r o s p o r a and soybean l e a v e s . The p o s s i b i l i t y t h a t cytochromes p l a y a d i r e c t p a r t i n n i t r a t e r e d u c t i o n by H a n s e n u l a has been e l i m i n a t e d by d i r e c t s p e c t r o p h o t o m e t r i c o b s e r v a t i o n s o f l i v i n g c e l l s . T a n i g u c h i and Ohmachi (1960) d e s c r i b e d an i n d u c i b l e p y r i d i n e n u c l e o t i d e NRase o f s u l f h y d r y l C O - i n s e n s i t i v e m e t a l l o e n z y m e n a t u r e i n t h e l a r g e p a r t i c l e s from n i t r a t e - g r o w n A z o t o b a c t e r c e l l s . The a c t i v i t y was s t i m u l a t e d 1.5 t o 2 . 0 - f o l d by t h e a d d i t i o n o f FAD o r FMN. E x c e p t f o r i t s p a r t i c u l a t e n a t u r e , t h e enzyme s t r o n g l y r e s e m b l e d t h a t o f N e u r o s p o r a and h i g h e r p l a n t s and i t was a p p a r e n t l y o f t h e a s s i m i l a t o r y t y p e . The f o l l o w i n g e l e c t r o n - t r a n s f e r sequence was p r o p o s e d : Cytochrome > Cytochrome o x i d a s e > 0£ s y s t e m t DPNH FAD N i t r a t e r e d u c t a s e N0~ ( o r F M N ) ( C O - s e n s i t i v e h e a v y ^ ^ m e t a l ) Reduced n i l e b l u e 2. R e s p i r a t o r y NRase A c c o r d i n g t o Nason's c l a s s i f i c a t i o n , r e s p i r a t o r y NRase c a t a l y z e s t h e f i r s t s t e p i n n i t r a t e r e s p i r a t i o n by w h i c h n i t r a t e i s u s e d as t h e t e r m i n a l e l e c t r o n a c c e p t o r i n p l a c e o f 8. oxygen by m i c r o o r g a n i s m s under a n a e r o b i c o r p a r t i a l l y a n a e r o b i c c o n d i t i o n s . The end p r o d u c t s o f n i t r a t e r e s p i r a t i o n may i n c l u d e n i t r i t e , n i t r i c o x i d e , n i t r o u s o x i d e , m o l e c u l a r o r o t h e r o x i d a t i o n s t a t e s o f n i t r o g e n . Most o f t h e r e d u c t i o n p r o d u c t s a r e n o t f u r t h e r u t i l i z e d and f o r t h e most p a r t a r e e x c r e t e d i n t o t h e growth medium. The d i s t i n g u i s h i n g c h a r a c t e r i s t i c s o f r e s p i r a t o r y NRase i s t h e p a r t i c i p a t i o n o f cytochrome i n i t s e l e c t r o n t r a n s p o r t sequence. T a n i g u c h i e t a l . (1956) f o r m u l a t e d t h e f o l l o w i n g scheme t o v i s u a l i z e e l e c t r o n t r a n s p o r t pathways i n the p a r t i c u l a t e p r e p a r a -t i o n o f E. c o l i . DPNH • FAD • i f o r m a t e Cytochrome b.. • — O x i d a s e N i t r a t e r e d u c t a s e r r e d u c e d by m e t h y l e n e b l u e ->N0„ The p a r t i c i p a t i o n o f cytochrome b^ i n t h e n i t r a t e r e d u c i n g mechanism i n E. c o l i was b a s e d on t h e s p e c t r o s c o p i c o b s e r v a t i o n t h a t t h e a d d i t i o n o f n i t r a t e t o a c e l l - f r e e e x t r a c t o f t h i s o r g a n i s m ( i n t h e a bsence o f oxygen) c a u s e d t h e c h a r a c t e r i s t i c bands o f t h e 9. r e d u c e d cytochrome t o f a d e , and t h a t t h i s a n a e r o b i c o x i d a t i o n o f cytochrome b^ was p r e v e n t e d by c y a n i d e . They were a b l e t o o b t a i n more d i r e c t e v i d e n c e f o r t h i s w i t h t h e a i d o f a s p e c i f i c i n h i b i t o r o f cytochrome b and b^, 2 - h e p t y l - 4 - h y d r o x y q u i n o l i n e - N -o x i d e (HOQNO). When f o r m a t e was u s e d as t h e hydrogen donor, t h e r e d u c t i o n o f n i t r a t e was s t r o n g i n h i b i t e d by t h i s HOQNO. I n t h i s t y p e o f e l e c t r o n t r a n s f e r s y s t e m , NRase a c t s as a t e r m i n a l enzyme, m e d i a t i n g t h e d i r e c t t r a n s f e r o f e l e c t r o n s t o n i t r a t e . The f i n d i n g o f an u n i d e n t i f i e d s o l u b l e f a c t o r n e c e s s a r y t o t h e DPNH- o r formate-NRase s y s t e m l e d I i d a and T a n i g u c h i (1959) t o m o d i f y t h e sequence p r o p o s e d by T a n i g u c h i e t a l . ( 1 9 5 6 ) . T h i s u n i d e n t i f i e d f a c t o r c o u l d n o t be r e p l a c e d by FAD, menadione o r f e r r o u s i o n . The marked i n h i b i t i o n o f t h e t e r m i n a l NRase by s e v e r a l m e t a l - c h e l a t i n g a g e n t s s u g g e s t e d t h e p r e s e n c e o f a m e t a l component i n t h e t e r m i n a l s t e p . DPNH • DPNH -> FAD Cytochrome b n Formate {->• F o r m i c —>(FAD) dehydrogenase O x i d a s e S o l u b l e f a c t o r i - ( P a r t i c u l a t e system) N i t r a t e r e d u c t a s e (unknown m e t a l ) 4* .NO Reduced m e t h y l e n e b l u e 10. I t a g a k i and T a n i g u c h i (1959) e x t r a c t e d a s o l u b l e DPNH-NRase from E. c o l i grown i n s y n t h e t i c medium under a e r o b i c c o n d i t i o n s . I n c o n t r a s t t o t h e p a r t i c u l a t e p r e p a r a t i o n t h i s s o l u b l e e x t r a c t was u n a b l e t o use f o r m a t e as an e l e c t r o n donor. I t was s e n s i t i v e t o HOQNO and s t i m u l a t e d by t h e a d d i t i o n o f FAD t o t h e d i a l y z e d enzyme. The f e r r o u s i o n c o u l d a c t as an e l e c t r o n donor f o r n i t r a t e v i a c ytochrome b^. An unknown heavy m e t a l p a r t i c i p a t i o n i n t h e t e r m i n a l s t e p was p r o p o s e d because o f t h e i n h i b t i o n by c y a n i d e and a z i d e . FAD 2+ NRase DPNH-» o r --manadione, Fe — ^ C y t o c h r o m e b 1 >-N0 FMN 1 (unknown heavy m e t a l ) 3 r e d u c e d dyes Medina and H e r e d i a (1960) e x t r a c t e d a p a r t i c u l a t e E. c o l i NRase w h i c h r e q u i r e d DPNH o r TPNH as e l e c t r o n donor and v i t a m i n as e l e c t r o n c a r r i e r . They s u g g e s t e d two mechanisms by w h i c h n i t r a t e c a n be r e d u c e d t o n i t r i t e . V i t a m i n a n a l o g u e DPNH dehydrogenase \ Menadione r e d u c t a s e DPNH—< ) > NRas e — > NO" 3 FAD—^ cytochrome b ^ — O x i d a s e 11. Under a e r o b i c as w e l l as a n a e r o b i c c o n d i t i o n s , v i t a m i n K3 o r i t s a n a l o g u e c a n a c t as t h e e l e c t r o n c a r r i e r w i t h o u t t h e p a r t i c i p a t i o n o f f l a v i n s . Under a n a e r o b i c c o n d i t i o n s n i t r a t e r e d u c t i o n t a k e s p l a c e t h r o u g h an e l e c t r o n - t r a n s p o r t c h a i n i n v o l -v i n g FAD and DPNH-oxidase c h a i n . Evans (1954) and C h e n i a e and Evans (1956, 1957, 1959, 1960) r e p o r t e d t h a t a p a r t i c u l a t e DPNH-NRase had been e x t r a c t e d f rom R h i z o b i u m japonium o f soybean n o d u l e s . T h e i r r e s u l t s i n d i -c a t e d t h a t t h i s enzyme c a t a l y z e s t h e r e d u c t i o n o f n i t r a t e t o n i t r i t e w i t h e i t h e r DPNH o r s u c c i n a t e as e l e c t r o n donor. When DPNH i s u s e d as a s o u r c e o f e l e c t r o n s f o r t h e s y s t e m , t h e e v i d e n c e i n d i c a t e s i n v o l v e m e n t o f a v i t a m i n K, o r a r e l a t e d q u i n o n e , a c y t o -chrome, an A n t i m y c i n A - s e n s i t i v e s i t e , two c y a n i d e - s e n s i t i v e s i t e s , and NRase. When s u c c i n a t e i s us e d as t h e e l e c t r o n donor f o r t h e s y s t e m i t i s s u g g e s t e d t h a t s u c c i n i c d ehydrogenase, a c y t o c h r o m e , an A n t i m y c i n A - s e n s i t i v e s i t e , two c y a n i d e s i t e s , and NRase a r e i n v o l v e d i n t h e e l e c t r o n t r a n s p o r t t o n i t r a t e . Enzyme a c t i v i t y i s p r o b a b l y c o r r e l a t e d w i t h t h e c a p a c i t y t o f i x a t m o s p h e r i c n i t r o g e n . The p a r t i c u l a t e enzyme e x h i b i t e d p r o p e r t i e s s i m i l a r t o t h o s e o f t h e n i t r a t e r e s p i r a t i o n s y s t e m from E. c o l i d e s c r i b e d by T a n i g u c h i and I t a g a s k i ( 1 9 6 0 ) . Lowe and Evans (1964) d e v e l o p e d a method f o r t h e p r e p a r a -t i o n o f a s o l u b l e NRase e x t r a c t o f R h i z o b i u m japonium^ c e i l s . T h i s s o l u b l e enzyme was p u r i f i e d about e l e v e n f o l d by ammonium s u l f a t e 12. f r a c t i o n a t i o n and chromatography on a c a l c i u m p h o s p h a t e column. DPNH and s u c c i n a t e were c o m p l e t e l y i n e f f e c t i v e donors f o r n i t r a t e r e d u c t i o n w i t h t h e s o l u b l e p r e p a r a t i o n . Reduced b e n z y l v i o l o g e n was employed as t h e e l e c t r o n d o nor. J u d g i n g from t h e s i m i l a r i t y o f t h e i r s u b s t r a t e a f f i n i t y and o f pH optimum, t h e s o l u b l e NRase and t h e NRase p o r t i o n o f t h e p a r t i c u l a t e complex from R. japonicum c e l l s a r e t h e same enzyme. Sadana and M c E l r o y (1957) e x t r a c t e d a n i t r a t e r e d u c i n g s y s t e m f r o m t h e s a l t - w a t e r luminous b a c t e r i u m , Achromobacter f i s h e r i , and s u g g e s t e d t h e f o l l o w i n g e l e c t r o n t r a n s p o r t system: 3+ DPNH FAD * Fe =• B a c t e r i a l 0 (TPNH) (FMN) cytochrome The n i t r a t e - r e d u c i n g s y s t e m from t h i s b a c t e r i u m was s o l u b i l i z e d by o s m o t i c r u p t u r e and s e p a r a t e d i n t o two s o l u b l e f r a c t i o n s , a DPNH-cytochrome c r e d u c t a s e m o i e t y w i t h a r e q u i r e -ment f o r FAD o r FMN, and a t e r m i n a l NRase. The enzyme c o u l d c a t a l y z e t h e r e d u c t i o n o f n i t r a t e by u s i n g r e d u c e d cytochrome o r b e n z y l v i o l o g e n . T h i s enzyme i s q u i t e s i m i l a r t o t h e s o l u b l e r e s p i r a t o r y NRase from E. c o l i . Reduced b e n z y l Fewson and N i c h o l a s (1961) p u r i f i e d and c h a r a c t e r i z e d 13. a p a r t i c u l a t e r e s p i r a t o r y NRase fr o m t h e d e n i t r i f y i n g b a c t e r i u m , Pseudomonas a e r u g i n o s a . Cytochrome c and molybdenum were r e q u i r e d as f u n c t i o n a l components. The f o l l o w i n g sequence o f e l e c t r o n t r a n s p o r t was s u g g e s t e d : DPNH > F A D — > Cytochrome c — > Mo I Cytochrome o x i d a s e -~9- NO, -> 0, A summary c o m p a r i s o n o f t h e p r o p e r t i e s o f a s s i m i l a t o r y and r e s p i r a t o r y NRase i s g i v e n i n T a b l e I . 14, T a b l e 1 Comparison o f A s s i m i l a t o r y NRase w i t h R e s p i r a t o r y NRase P r o p e r t i e s A s s i m i l a t o r y N i t r a t e R e d u c t a s e R e s p i r a t o r y N i t r a t e R e d u c t a s e O r i g i n a l Form S o l u b l e o r p a r t i c u l a t e S o l u b l e o r p a r t i c u l a t e S e n s i t i v i t y t o oxygen Enzyme-bound Components F l a v i n Cytochrome O t h e r Component None FAD None Molybdenum P r e s e n t Unknown T y p i c a l E l e c t r o n T r ans f e r S equence TPNH-* FAD -» Mo -^NO* I n h i b i t e d FAD P r e s e n t (Cytochrome b^ i n E. c o l i ) P r e s e n t (1 atom/molecule i n E. c o l i ) I r o n (40 a toms/molecule i n E. c o l i ) DPNH 4/ 2+ FAD, ( M e n a d i o n e ) ( F e ) cytochrome b. I Mo N0„ Km f o r n i t r a t e P h y s i o l o g i c a l f u n c t i o n 1.4 x 10 M (Neurospora) N i t r a t e a s s i m i l a t i o n 5,1 x 1 0 _ 4 M ( E . c o l i ) N i t r a t e r e s p i r a t i o n 15. I I I . ASSIMILATORY NITRATE REDUCTASE As a b a c k g r o u n d f o r t h e s t u d y o f t h e a s s i m i l a t o r y NRase o f t h e sugar b e e t , a l i t e r a t u r e r e v i e w o f t h e ex-t r a c t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f NRase o f a v a r i e t y o f o t h e r p l a n t s i s p r e s e n t e d i n t h e f o l l o w i n g s e c t i o n . 1. Methods o f E x t r a c t i o n Nason and Evans (1953) i s o l a t e d NRase from N e u r o s p o r a by t h e f o l l o w i n g p r o c e d u r e . A f t e r f r e e z i n g a t -15° C, N e u r o s p o r a m y c e l i a were ground i n a Ten B r o e c k homogenizer i n t h r e e t i m e s t h e i r w e i g h t o f 0.1 M K^HPO^ b u f f e r , pH 7.0, and c e n t r i f u g e d a t 20,000 x g f o r 10 m i n u t e s a t 4° C. About 857. of t h e t o t a l a c t i v i t y was o b t a i n e d i n t h e s u p e r n a t a n t . The enzyme f r o m soybean l e a v e s (Evans and Nason, 1953) was e x t r a c t e d by g r i n d i n g t h e l e a v e s i n a W a r i n g B l e n d o r i n t h e p r e s e n c e o f phosphate b u f f e r , pH 9.0, and a l u m i n a powder. The m i x t u r e was f u r t h e r ground f o r t h r e e m i n u t e s i n a Ten B r o e c k homogenizer a t 0 - 4° C, t h e n c e n t r i f u g e d a t 20,000 x g f o r 10 m i n u t e s a t 4° C. About 107. o f t h e enzyme was l o s t by t h i s p r o c e d u r e . N i c h o l a s and Nason (1955a) o m i t t e d t h e a l u m i n a powder and added c y s t e i n e and EDTA, each a t a f i n a l c o n c e n t r a t i o n o f 10"^ M. T h i s i m p r o v e d t h e s t a b i l i t y and y i e l d o f t h e enzyme from soybean l e a v e s by p r o t e c t i n g i t a g a i n s t i n a c t i v a t i o n by 16. heavy m e t a l s o r o x i d a t i o n o f s u l f h y d r y l g r o u p s , N i c h o l a s and Nason (1955b) i s o l a t e d a s o l u b l e a s s i m i -l a t o r y NRase from E. c o l i . The f r o z e n c e l l s were homogenized i n a c o l d m o r t a r u s i n g an e q u a l w e i g h t o f a l u m i n a powder ( A l c o a A-301). A f t e r g r i n d i n g f o r 10 m i n u t e s , t h r e e t i m e s t h e i r w e i g h t o f c o l d 0.1 M l^HPO^, pH 7.0, c o n t a i n i n g sodium v e r s e n a t e ( 1 0 " ^ M f i n a l c o n c e n t r a t i o n ) was s l o w l y added and g r i n d i n g c o n t i n u e d f o r a n o t h e r 5 m i n u t e s . A f t e r c e n t r i f u g a t i o n f o r 15 m i n u t e s a t 4° C., t h e s u p e r n a t a n t s o l u t i o n c o n t a i n e d 757. o f t h e enzyme a c t i v i t y o f t h e homo-ge n a t e . S i l v e r (1956) e x t r a c t e d from t h e y e a s t H a n s e n u l a  anomala a s o l u b l e enzyme w h i c h was c o n c e r n e d w i t h t h e a s s i m i -l a t i o n o f n i t r a t e . The c e l l s were suspended i n s u f f i c i e n t 0.2 M pho s p h a t e b u f f e r , pH 7.5, t o make a t h i c k cream. A 50 -ml a l i q u o t c o f t h e cream and 5 g o f 200-mesh powdered p y r e x were p l a c e d i n a 9-KC R a y t h r e o n s o n i c o s c i l l a t o r and t h e c e l l s d i s r u p t e d f o r 40 m i n u t e s i n t h e c o l d . Unbroken c e l l s , l a r g e d e b r i s , and g l a s s were removed by low speed c e n t r i f u g a t i o n and t h e s u p e r n a t a n t was s u b j e c t e d t o 107,000 x g i n a p r e p a r a t i v e S p i n c o c e n t r i f u g e f o r 50 m i n u t e s . The s u p e r n a t a n t c o n t a i n e d 95 t o 1007. o f t h e e n z y m a t i c a l l y a c t i v e m a t e r i a l o f t h e w h o l e homogenate. 17. Candela et a l . (1957) extracted NRase from cauliflower by grinding fresh tissue for 15 minutes with neutral acid-washed s i l i c a sand and three times its weight of extractant at 0°C in a chilled mortar. The extracting reagent was 0.1 M -4 phosphate buffer, pH 7.0, containing 10 M cysteine and ethylene diamine tetracetate (EDTA). The homogenate was fil t e r e d through muslin and the f i l t r a t e was centrifuged at 20,000 x g at 0° C. Spencer (1959) separated wheat embryos from the endo-sperm after 48 hours germination on f i l t e r paper moistened with 10 mM potassium nitrate, and ground them in a cold mortar at 0 - 2° C with ten times their weight of 0.1 M K^PO^/KHgPO^ pH 7.5, containing 10 M glutathione. The grindate was then centrifuged at 5000 x g for 20 minutes. Hageman and Flesher (1960) extracted NRase from maize seedlings. The seedlings were cut into small pieces and ground in a Omnimixer at maximum speed for 2 minutes. The grinding medium was 0.1 M Tris-(hydroxymethyl)-amino-methane -4 (Tris), 0.01 M cysteine and 3 x 10 M EDTA at a pH of 7.3 to 7.8. Four ml of cold (2° C) grinding medium was added for each gram of tissue. The homogenate was pressed through cheesecloth and centrifuged for 15 minutes at 20,000 x g. The supernatant was decanted through glass wool and assayed. 18. Sanderson and C o c k i n g (1964) ground tomato l e a v e s i n a c o l d m o r t a r c o n t a i n i n g a c i d - w a s h e d s a n d . The g r i n d i n g medium was 0.1 M T r i s - H C l b u f f e r c o n t a i n i n g 1 0 M c y s t e i n e a t pH 7.5. The m a c e r a t e was p r e s s e d t h r o u g h c h e e s e c l o t h and t h e f i l t r a t e was c e n t r i f u g e d a t 1750 x g f o r 20 m i n u t e s . T a b l e II summarizes t h e methods o f e x t r a c t i o n em-p l o y e d by numerous i n v e s t i g a t o r s . 19. T a b l e I I Summary o f Methods o f E x t r a c t i n g A s s i m i l a t o r y NRase S o u r c e o f E x t r a c t i o n M l medium/ Method o f R e f e r e n c e Enzyme Medium g t i s s u e E x t r a c t i o n Soybean 0.1 M Add 2 w t s , a l u m i n a , Evans and l e a v e s K2HP0 4 3a W a r i n g B l e n d o r 2 Nason ( 1 s t t r i - (pH 9.0) m i n , a t 0-4° C, (1953) f o l i a t e ) Ten B r o e c k homogen-a t i o n , c e n t r i f u g e d 20,000 x g. 10 m i n . a t 4°C. 907. a c t i -v i t y o b t a i n e d . N e u r o s p o r a 0.1 M Ten B r o e c k homoge- Nason and c r a s s a K 2 H P 0 4 3:1 n a t i o n a t 0-4°C, Evans (pH 7.5) c e n t r i f u g e d a t (1953) 20,000 x g. 857. a c t i v i t y o b t a i n e d . Soybean 0.1 M As above, f o r N i c h o l a s l e a v e s K 2 H P 0 4 (pH 9.0) 3:1 soybean b u t o m i t and Nason 1 0 " 2 M EDTA + a l u m i n a . (1955a) 10" M c y s t e i n e Y e a s t 0.2 M phosphate Raytheon s o n i c S i l v e r H a n s e n u l a b u f f e r (pH 7.5) O s c i l l a t o r f o r 40 (1956) anomala m i n . , c e n t r i f u g e d a t 107,000 x g. 95-1007. a c t i v i t y r e c o v e r e d . C a u l i - 0.1 M p h o s p h a t e Ground i n m o r t a r C a n d e l a f l o w e r b u f f e r (pH 7.0) 3:1 a t o°C w i t h s a n d . e t , a l . 1 0 " 4 M EDTA + C e n t r i f u g e d a t (1957) 1 0 ~ 4 c y s t e i n e 20,000 x g. N. c r a s s a 0.1 M K^HPO^ Homogenized'in K i n s k y and (pH 7.5) S e r v a l l Omnimixer M c E l r o y 0-4°C, c e n t r i f u g e d (1958) a t 10,000 x g. 907. a c t i v i t y o b t a i n e d . 20. T a b l e I I ( c o n t ' d ) S o u r c e o f E x t r a c t i o n M l medium/ Method o f R e f e r e n c e Enzyme Medium g t i s s u e E x t r a c t i o n Wheat 0,1 M phosphate Ground i n c h i l l e d S pencer embryo b u f f e r (pH 7.5) 10:1 m o r t a r , c e n t r i - (1959) 1 0 " 3 M fuged a t 5000 x g. g l u t a t h i o n e Wheat 0.025 M K 2 H P 0 4 3:1 90 s e c . i n B l e n d o r , Anacker l e a v e s 5 x 10"* M EDTA 3 min. i n P o t t e r - and S t o y E l v e h j e m M a c e r a t o r (1958) a t 4°C M a i z e 0.1 M T r i s b u f f e r Ground i n Omnimixer, Hageman l e a v e s (pH 7.3 - 7.8) 4:1 c e n t r i f u g e d a t and 3 x 10"" 4 M EDTA 30,000 x g. F l e s h e r and 10"2 M c y s t e i n e (1960) Tomato 0.1 M T r i s Ground i n c h i l l e d S a nderson l e a v e s (pH 7.5) 4:1 m o r t a r , c e n t r i f u g e d and 10-3 M EDTA a t 1750 x g f o r C o c k i n g 20 m i n . 80-1007. (1964) a c t i v i t y r e c o v e r e d . 21. 2. P u r i f i c a t i o n o f Enzyme NRase enzyme p u r i f i c a t i o n has been a c c o m p l i s h e d by ammonium s u l f a t e p r e c i p i t a t i o n and c a l c i u m p h o s p h a t e g e l a d s o r p t i o n . Column chromatography and e l e c t r o p h o r e s i s have a l s o been used. Nason and Evans (1953) p u r i f i e d N e u r o s p o r a NRase by ammonium s u l f a t e f r a c t i o n a t i o n and c a l c i u m p h o s p h a t e g e l a d s o r p t i o n . A l l s t e p s o f p u r i f i c a t i o n p r o c e d u r e were c a r r i e d o u t a t 0-4° C. C e n t r i f u g a t i o n was p e r f o r m e d a t a p p r o x i m a t e l y 3000 x g. The v a r i o u s p e r c e n t a g e s o f s a t u r a t i o n were o b t a i n e d by t h e a d d i t i o n o f a s a t u r a t e d ammonium s u l f a t e s o l u t i o n t o t h e e x t r a c t . The f i r s t f r a c t i o n , o b t a i n e d w i t h 0-437. s a t u r a t i o n , c o n t a i n e d 507. o f t h e i n i t i a l a c t i v i t y w i t h a t w o f o l d i n c r e a s e i n p u r i t y . The se c o n d ammonium s u l f a t e f r a c t i o n a t i o n u s e d 24-467. s a t u r a t i o n . The a c t i v i t y o f second f r a c t i o n (24-467. s a t u r a t i o n ) r e p r e s e n t e d 207o o f t h e u n i t s i n the c r u d e s t a r t i n g m a t e r i a l and a s e v e n f o l d p u r i f i c a t i o n . The s e c o n d f r a c t i o n was t r e a t e d w i t h c a l c i u m p h o s p h a t e g e l , aged 9 months. The g e l - p r o t e i n r a t i o was about 2:5. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and washed t w i c e w i t h c o l d 0.1 M p y r o p h o s p h a t e b u f f e r , pH 7.0. The p y r o p h o s -p h a t e e l u a t e showed about 107. o f t h e t o t a l a c t i v i t y o f t h e o r i g i n a l c r u d e e x t r a c t , and an o v e r a l l p u r i f i c a t i o n o f 40 t o 50 t i m e s . The t h i r d ammonium s u l f a t e f r a c t i o n a t i o n i n v o l v e d 22. 0-60% s a t u r a t i o n . T h i s f i n a l f r a c t i o n c o n t a i n e d 10% o f t h e u n i t s i n t h e c r u d e s t a r t i n g m a t e r i a l and t h e p u r i f i c a t i o n was 60- t o 7 0 - f o l d . P u r i f i c a t i o n o f NRase from soybean l e a v e s (Evans and Nason, 1953) employed c a l c i u m p h o s p h a t e g e l a d s o r p t i o n f o l l o w e d by ammonium s u l f a t e p r e c i p i t a t i o n . N i n e t y - f i v e m l o f c a l c i u m p h o s p h a t e g e l (11 mg p e r ml) was added t o 190 ml o f c r u d e e x t r a c t . The enzyme was t h e n e l u t e d from t h e g e l by 0.1 M p y r o p h o s p h a t e b u f f e r a t pH 7.0. T h i s f r a c t i o n a c o n t a i n e d 4 0 % o f t h e t o t a l a c t i v i t y . The p u r i f i c a t i o n was 2 4 - f o l d . A f t e r two ammonium s u l f a t e p r e c i p i t a t i o n s , t h e enzyme p r e p a r a t i o n c o n t a i n e d 77. o f t h e t o t a l a c t i v i t y and was p u r i f i e d 6 5 - f o l d . C h a r c o a l (Darco G 60) has been u s e d by N i c h o l a s and S c a w i n (1956) t o t r e a t N e u r o s p o r a NRase p r e p a r a t i o n s w h i c h had a l r e a d y been f r a c t i o n a t e d by ammonium s u l f a t e p r e c i p i t a t i o n and c a l c i u m p hosphate g e l a d s o r p t i o n . When t h e enzyme p r e -p a r a t i o n was t r e a t e d w i t h "Darco G 60" (30 mg/ml enzyme) and c e n t r i f u g e d a t 3000 x g f o r 15 m i n u t e s , t h e e n z y m a t i c a c t i v i t y was p r e s e n t i n s u p e r n a t a n t . T h i s f r a c t i o n c o n t a i n e d 0.87. o f t h e i n i t i a l a c t i v i t y and r e p r e s e n t e d an o v e r a l l p u r i f i c a t i o n o f 8 4 - f o l d . 23. A n a c k e r and S t o y (1958) u s e d a column o f c a l c i u m p h osphate t o f r a c t i o n a t e wheat l e a f NRase. The a d s o r p t i o n column was 35 mm i n d i a m e t e r and c o n t a i n e d 5 g o f c a l c i u m p h o s p h a t e i n 30 m l . The enzyme was e l u t e d from t h e column by i n c r e a s i n g c o n c e n t r a t i o n s o f I^HPO^ b u f f e r , pH 7.0. The most a c t i v e f r a c t i o n was o b t a i n e d by e l u t i o n w i t h t h e phosphate b u f f e r a t 0.05 M. Rechromatography and g r a d i e n t e l u t i o n w i t h sodium p y r o p h o s p h a t e 0.001-0.1 M, pH 7.0, r e s u l t e d i n a 1 0 0 - f o l d i n c r e a s e i n a c t i v i t y . The enzyme was t h e n p r e c i p i t a t e d by ammonium s u l f a t e 0-357. s a t u r a t i o n , w i t h a f u r t h e r t w o - f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y . The p r e p a r a t i o n was n o t homogeneous i n t h e u l t r a c e n t r i f u g e , b u t t h e a c t i v i t y was a s s o c i a t e d w i t h p r o t e i n h a v i n g a sedimen-t a t i o n o f 19S and a m o l e c u l a r w e i g h t o f 500,000-600,000. K i n s k y and M c E l r o y (1958) d e m o n s t r a t e d t h a t n u c l e i c a c i d i n enzyme p r e p a r a t i o n s from N e u r o s p o r a c o u l d be s u c c e s s -f u l l y removed w i t h p r o t a m i n e s u l f a t e . I n t h e subsequent ammonium s u l f a t e p r e c i p i t a t i o n , t h e y o b t a i n e d a h i g h l y p u r i f i e d enzyme, w i t h s p e c i f i c a c t i v i t y about 10,000. They a l s o r e p o r t e d t h a t when t h i s p u r i f i e d p r e p a r a t i o n o f NRase was examined i n t h e P e r k i n - E l m e r e l e c t r o p h o r e s i s a p p a r a t u s , a minimum o f t h r e e d i s t i n c t peaks were d i s c e r n i b l e . 24 Spencer (1959) p u r i f i e d wheat embryo NRase w i t h t h r e e ammonium s u l f a t e p r e c i p i t a t i o n s (0-55, 28-40 and 29-35% s a t u r a t i o n ) . The f i n a l f r a c t i o n showed a s e v e n f o l d i n c r e a s e i n s p e c i f i c a c t i v i t y w i t h a r e c o v e r y o f 587. o f t h e a c t i v i t y o f t h e c r u d e c e l l - f r e e e x t r a c t . S a n d e r s o n and C o c k i n g (1964) p a s s e d t h e c r u d e enzyme p r e p a r a t i o n from tomato l e a v e s t h r o u g h Sephadex column -3 c o n t a i n i n g 10 M c y s t e i n e . From 80 t o 1007. o f t h e enzyme was n o r m a l l y r e c o v e r e d i n t h e 6 ml o f e l u a t e f o l l o w i n g t h e f i r s t 12 m l . The e l u a t e r e p r e s e n t e d t w o f o l d p u r i f i c a t i o n . 3. Measurements o f A c t i v i t y T hree methods have been used t o a s s a y NRase a c t i v i t y : c o l o r i m e t r i c , m a n o m e t r i c , and s p e c t r o p h o t o m e t r y . C o l o r i m e t r i c method: T h i s method i s ba s e d on t h e d i a z o t i z a t i o n o f an a r o m a t i c amino compound by n i t r i t e i n a c i d s o l u t i o n and c o u p l i n g w i t h a s u i t a b l e r e a g e n t t o g i v e an i n t e n s i v e r e d - p u r p l e c o l o r . T h i s method f o r q u a n t i t a t i v e d e t e r m i n a t i o n o f n i t r i t e was o r i g i n a l l y d e s c r i b e d by S n e l l and S n e l l ( 1 9 4 9 ) . The p r o c e d u r e i s as f o l l o w s : a l l o w 5 m i n u t e s o f i n c u b a t i o n o f r e a c t i o n m i x t u r e ; add 0.9 ml o f rL^O and 0.5 m l o f 17. s u l f a n i l a m i d e i n N H C l t o s t o p t h e r e a c t i o n ; f o l l o w by 0.5 m l o f 0.027. N - ( l - n a p h t h y l ) - e t h y l e n e -d i a m i n e h y d r o c h l o r i d e t o d e v e l o p t h e c o l o r . A f t e r 10 m i n u t e s , 25. r e a d t h e d e n s i t y o f t h e c o l o r i n a c o l o r i m e t e r w i t h a g r e e n (540 mu) f i l t e r . U s i n g t h i s method, Evans and Nason (1953) f o u n d t h a t t h e r e was a s m a l l n o n - e n z y m a t i c d i s a p p e a r a n c e o f n i t r i t e i f TPNH and n i t r i t e were a l l o w e d t o r e m a i n i n c o n t a c t w i t h t h e a c i d s u l f a n i l a m i d e r e a g e n t f o r a few m i n u t e s b e f o r e a d d i n g t h e s o l u t i o n o f N - ( l - n a p h t h y l ) - e t h y l e n e - d i a m i n e h y d r o c h l o r i d e . Medina and N i c o l a s (1957) r e p o r t e d t h a t DPNH and TPNH r e a c t e d w i t h t h e d i a z o t i s e d p r o d u c t o f n i t r i t e and s u l p h a n i l a m i d e , i n a c i d s o l u t i o n . I t i s supposed t h a t DPNH i n a c i d s o l u t i o n r e d u c e s d i a z o t i s e d s u l f a n i l a m i d e t o a p h e n y l h y d r a z i d e d e r i v a t i v e w h i c h i s n o t a b l e t o c o u p l e w i t h t h e n a p h t h y l r e a g e n t . T h i s s o u r c e o f i n t e r f e r e n c e can be overcome by r e m o v a l o f r e s i d u a l DPNH and TPNH a t t h e end o f t h e r e a c t i o n . B a rium a c e t a t e and e t h a n o l were u s e d t o p r e c i p i t a t e t h e p y r i d i n e n u c l e o t i d e s . Evans and Nason (1953) f o u n d t h a t t h e NRase p r e s e n t i n th e r e a c t i o n m i x t u r e a l s o c a u s e d t h e d i s a p p e a r a n c e o f n i t r i t e . 10"^ M h y d r o x y l a m i n e h y d r o c h l o r i d e e f f e c t i v e l y i n h i b i t e d t h e s l i g h t n i t r i t e r e d u c t a s e a c t i o n i n soybean e x t r a c t s b u t a l s o c a u s e d a 12% i n h i b i t i o n o f NRase. S i l v e r and M c E l r o y (1954) i n c l u d e d c y a n i d e a t about 3 x 10 ^ M t o i n h i b i t t h e enzyme. N i c h o l a s e t aJL. (1954) a l s o u s e d c y a n i d e o f a low c o n c e n t r a t i o n (4 x 1 0 " 5 M). A v a r i e t y o f methods e m p l o y i n g c o l o r i m e t r y a r e summarized i n T a b l e I I I . 26. T a b l e I I I Summary o f Measurements o f A s s i m i l a t o r y NRase A c t i v i t y by C o l o r i m e t r i c Method P l a n t M a t e r i a l R e a c t i o n M i x t u r e Time Temp. U n i t R e f e r e n c e (min.) (0°) Soybean 10 uM KNO3, 55 uM l e a v e s p h o s p h a t e b u f f e r , pH 7.0, 1.3 muM FAD, 80 muM TPNH, 0.2 m l enzyme e x t r a c t i n 0.5 m l . 10 28 1 muM NO2/5 m i n . Evans and Nason (1953) N. c r a s s a 10 uM KNO3, 52 uM p y r o p h o s p h a t e b u f f e r , pH 7.0, 0,75 muM FAD, 80 muM TPNH, 0.05 m l enzyme e x t r a c t i n 0.5 m l . N. c r a s s a 1 Q ^ 4 g ^ p y r o p h o s p h a t e b u f f e r , pH 7.0, 50 uM FMN, 80 uM TPNH, 0.05 ml enzyme e x t r a c t . 23-28 1 muM NO^/S min. 10 Room 1 muM temp. NO^/IO min. Nason and Evans (1953) N i c h o l a s e t a l . (1954) N. c r a s s a 10 uM KN0 2, 23 uM T r i s b u f f e r , pH 7.5, 0.02 m l b o i l e d T r i s - e x t r a c t o f p i g h e a r t , 0.02 ml a c e t o n e powder. 100 muM TPNH i n 0.5 m l . N i c h o l a s and S c a w i n (1956) H a n s e n u l a 10 uM KNO3, 0.05 ml anomala b o i l e d e x t r a c t o f 10 p i g l i v e r , 0.01 - 0.05 m l enzyme e x t r a c t . 0.2 M p h o s p h a t e b u f f e r , pH 7.5, i n 0.5 m l . Room temp 1 muM NO"/10 min. S i l v e r (1956) 27 T a b l e I I I ( c o n t ' d ) P l a n t R e a c t i o n Time Temp. U n i t R e f e r e n c e M a t e r i a l M i x t u r e (min.) (0°) N. c r a s s a 10 uM NaN0 3, 20-29 10 Room 0.19 muM K i n s k y and uM phosphate b u f f e r , temp NO"/30 m i n . M c E l r o y pH 7-7.5, 50 muM FAD (1958) 100 uM TPNH, 0.1-1.0 ml enzyme e x t r a c t i n 0.5 m l . Wheat 10 uM KN0 3, 100-250 uM ph o s p h a t e b u f f e r , 30 30 1 muM Spencer pH 7.5, 0.5 muM FAD, NO"/30 m i n . (1959) 200 muM DPNH, 0.05-0.2 m l enzyme e x t r a c t w a t e r t o 0.5 m l . M a i z e 20 uM KNO3, 100 uM p h o s p h a t e b u f f e r , 15 27 1 muM Hageman ph 7.3, 0.68 uM DPNH, NO^/hr./g and 0.2 ml enzyme e x t r a c t f r e s h w t . F l e s h e r w a t e r t o 2.0 m l . (1960) A z o t o - 30 uM KNO3, 1.5 uM 10 30 1 uM T a n i g u c h i b a c t e r DPNH, 0.2-0.6 m l 30 NO^/hr. and vin> - enzyme e x t r a c t , Ohmachi l a n d i i 0.05M p h o s p h a t e b u f f e r (1960) t o 3 m l i n a Thunberg t u b e , use CuSO^ t o reduc e i n t e r f e r e n c e o f DPNH Tomato 10 uM KN0 3, 50 uM l e a f p hosphate b u f f e r , 20 27 1 muM Sanderson pH 7.3, 270 muM NO2/2O m i n . and DPNH, 0.05-0.2 m l C o c k i n g enzyme e x t r a c t , (1964) w a t e r t o 0.8 ml 28. Manometric method: R o b i n s o n (1954) d e s c r i b e d a method o f manometric d e t e r m i n a t i o n o f NRase a c t i v i t y . I t i s b a s e d on t h e r e a c t i o n o f n i t r i t e w i t h s u l f a n i c a c i d i n s u l f u r i c a c i d s o l u t i o n w i t h subsequent l i b e r a t i o n o f m o l e c u l a r n i t r o g e n i n a c c o r d a n c e w i t h t h e f o l l o w i n g r e a c t i o n : N H 2S0 3H + HN0 2 > E2S°U + H 2 ° + N 2 N i t r i t e was d e t e r m i n e d m a n o m e t r i c a l l y u s i n g Warburg equipment. The v e s s e l s c o n t a i n e d t h e NaNO^ i n 2.0 ml 0.05 M phos p h a t e b u f f e r , pH 7.6, w i t h 0.2 ml o f t h e s u l f a n i c a c i d s o l u t i o n i n t h e s i d e arm. A f t e r e q u i l i b r a t i o n a t 30° C t h e two s o l u t i o n s were m i x e d and n i t r o g e n e v o l u t i o n was d e t e r m i n e d . The same p r o c e d u r e was us e d f o r t h e d e t e r m i n a t i o n o f NRase a c t i v i t y i n t h e h a l o p h i l i c b a c t e r i u m , V i b r i o c o s t i c o l u s . Double-arm Warburg r e a c t i o n v e s s e l s c o n t a i n e d t h e c e l l s u s p en-s i o n o r c e l l - f r e e e x t r a c t i n p h o s p h a t e b u f f e r , pH 7.6, w i t h a p p r o p r i a t e c o n c e n t r a t i o n s o f N a C l , Sodium s u c c i n a t e (10 uM) as h y d r o g e n donor and NaNO^ (5.0 uM) were p i p e t t e d i n t o one s i d e arm and t h e s u l f a n i c a c i d s o l u t i o n i n t o t h e o t h e r . A f t e r e q u i l i b r a t i o n , t h e s u b s t r a t e and enzyme were combined, t h e r e a c t i o n m i x t u r e was i n c u b a t e d f o r 30 m i n . i n n i t r o g e n , and t h e s u l f a n i c a c i d s o l u t i o n was f i n a l l y t i p p e d i n t o t he r e a c t i o n m i x t u r e . The manometric method o f H e w i t t and H a l l a s (1959) 29. depended on n i t r i t e r e a c t i o n w i t h a z i d e i n a c i d s o l u t i o n as f o l l o w s : B0~ + N~ + 2H+ > N 2 0 + N 2 + H 20 I n t h i s method, one uM n i t r i t e y i e l d e d 44.8 u l o f g a s . U s i n g t h i s method, H e w i t t and H a l l a s o b t a i n e d 82-86 u l o f gas from 2 uM n i t r i t e i n a f l u i d volume o f 4.5 m l a t 27°C i n an atmosphere o f n i t r o g e n . S p e c t r o p h o t o m e t r i c method: Nason and Evans (1953) measured t h e changes i n o p t i c a l d e n s i t y a t 340 mu a s s o c i a t e d w i t h o x i d a t i o n o f TPNH o r DPNH t o f o l l o w t h e r e a c t i o n i n t h e NRase s y s t e m o f N e u r o s p o r a . The a s s a y s y s t e m c o n t a i n e d 0.83 uM TPNH, 0.3 uM FMN, 100 uM KNO3, and 424 u n i t s o f enzyme i n 3.6 ml o f 0.03 M p y r o p h o s p h a t e b u f f e r . A change o f p p t i c a l d e n s i t y o f 0.06 p e r m i n u t e , a t 340 mu, was p r o d u c e d o v e r a p e r i o d o f 15 m i n u t e s . Evans and Nason (1953) o b s e r v e d a v e r y r a p i d o x i d a t i o n o f TPNH i n p r e s e n c e o f t h e NRase s y s t e m and a f a i r l y r a p i d endogenous r a t e when a l l c o n s t i t u e n t s , e x c e p t KNO-j were p r e s e n t . S i n c e n i t r i t e was formed i n t h e c o m p l e t e s y s t e m o n l y , t h e o x i d a t i o n o f TPNH was c a u s e d by some f a c t o r o t h e r t h a n n i t r a t e . Owing t o t h e p r e s e n c e o f TPNH o r DPNH o x i d a s e s y s t e m i n t h e p a r t i a l l y p u r i f i e d enzyme, t h i s method o f a s s a y i s n o t s u f f i c i e n t l y s e n s i t i v e . 4. S t a b i l i t y o f Enzyme Most NRase p r e p a r a t i o n s a r e r e l a t i v e l y t h e r m o l a b i l e and a r e e a s i l y d e n a t u r e d . The enzyme from N e u r o s p o r a , o b t a i n e d by Nason and Evans (1953) l o s t about 507. o f i t s a c t i v i t y i n 5 m i n u t e s a t 40°C, and a l l o f i t s a c t i v i t y a t 50°C i n 5 m i n u t e s . D i a l y s i s o f t h e enzyme a g a i n s t phosphate b u f f e r , r u n n i n g t a p w a t e r , o r t r i p l y - d i s t i l l e d w a t e r r e s u l t e d i n a c o m p l e t e l o s s o f a c t i v i t y w i t h i n one h o u r , t h e s h o r t e s t t i m e i n t e r v a l t r i e d . When t h e enzyme was exposed t o washed c e l l o p h a n e d i a l y z i n g membrane (5,0 ml o f enzyme, 0.5 gm o f s h r e d d e d membrane) f o r 2 m i n u t e s , 957. o f t h e o r i g i n a l a c t i v i t y was l o s t . A t t e m p t s t o r e s t o r e t h e a c t i v i t y by a d d i t i o n o f b o i l e d c r u d e e x t r a c t , ashed c r u d e e x t r a c t , g l u t a t h i o n e , o r 2 — 2+ i n o r g a n i c i o n s s u c h as MoO^ , Fe e t c . were u n s u c c e s s f u l . The p a r t i a l l y p u r i f i e d enzyme from soybean l e a v e s o b t a i n e d by Evans and Nason (1953) was v e r y h e a t - s e n s i t i v e . T w e n t y - e i g h t , 46, 71 and 1007. i n a c t i v a t i o n r e s u l t e d from 5-minute exposures a t 20°, 30°, 40° and 50°C r e s p e c t i v e l y . The enzyme a c t i v i t y was r e d u c e d t o 50 t o 707. o f i t s o r i g i n a l a c t i v i t y d u r i n g a s t o r a g e p e r i o d o f one week a t -15° C. Spencer . (1959) r e p o r t e d t h a t wheat embryo NRase was i n a c t i v a t e d 38, 96 and 1007. by h e a t i n g t o 30°, 45°, and 60° C, r e s p e c t i v e l y . A p p r o x i m a t e l y 507. o f t h e e n z y m a t i c a c t i v i t y was l o s t a f t e r s t o r a g e f o r one week a t -15° C. 31. I t has been shown t h a t i n o r d e r t o o b t a i n maximal s p e c i f i c a c t i v i t y and good y i e l d s , t h e a d d i t i o n o f p r o t e c t i v e a g e n t s s u c h as c y s t e i n e , g l u t a t h i o n e and EDTA t o t h e e x t r a c t i o n medium i s n e c e s s a r y . N i c h o l a s and Nason (1955a) r e p o r t e d t h a t t h e a d d i t i o n -4 o f c y s t e i n e and EDTA, each a t f i n a l c o n c e n t r a t i o n o f 10 M, improved t h e s t a b i l i t y and y i e l d o f t h e enzyme by p r o t e c t i n g i t a g a i n s t i n a c t i v a t i o n by heavy m e t a l s o r o x i d a t i o n o f s u l f h y d r y l g r o u p s . E x t r a c t i o n from c a u l i f l o w e r l e a f by H e w i t t et a l . (1955) was c a r r i e d o u t w i t h 10"^ M c y s t e i n e and EDTA. Spencer (1959)] d e m o n s t r a t e d t h a t when t h e e x t r a c t i n g medium i n c l u d e d 10"^ M g l u t a t h i o n e a s l i g h t i n c r e a s e i n a c t i v i t y r e -s u l t e d . Thus i t i s advantageous t o m a i n t a i n low c o n c e n t r a t i o n s o f t h e s e p r o t e c t i v e a g e n t s d u r i n g s t a g e s o f f r a c t i o n a t i o n . 5. E l e c t r o n Donors I n a fungus such as N e u r o s p o r a t h e NRase i s e s s e n t i a l l y T P N H - s p e c i f i c (Nason and Evans 1 9 5 3 ) , b u t i n many h i g h e r p l a n t s , e.g. s o y b e a n , b a r l e y c a u l i f l o w e r and v e g e t a b l e marrow, DPNH and TPNH f u n c t i o n e q u a l l y w e l l (Evans and Nason, 1953; C a n d e l l a e t a l . 1957; C r e s s w e l l e t a l . ( 1 9 6 2 ) . The wheat embryo sy s t e m (Spencer 1959); and m a i z e l e a f s y s t e m (Hageman and F l e s h e r , 1960) a r e D P N H - s p e c i f i c b u t t h e wheat l e a f s y s t e m f u n c t i o n s w i t h DPNH (Anacker and S t o y , 1958) o r TPNH ( S p e n c e r , 1959). The a c t i v i t y 32. i n c o r n r o o t s was o b t a i n e d w i t h TPNH o r DPNH i n t h e work o f Evans a n d Nason ( 1 9 5 3 ) . Nason and Evans (1953) i n d i c a t e d t h a t s u c c i n a t e , c y s t e i n e , g l u t a t h i o n e o r a s c o r b i c a c i d c o u l d n o t r e p l a c e TPNH as e l e c t r o n donors f o r NRase f r o m N e u r o s p o r a . V a i d y a n a t h a n and S t r e e t ( 1 9 5 9 ) , however, o b t a i n e d s l o w r e d u c t i o n o f n i t r a t e by a c r u d e p r e p a r a t i o n from e x c i s e d tomato r o o t s when a s c o r b a t e and f e r r o u s i r o n were p r e s e n t . Hageman e t a l . (1962) have shown t h a t NRase o b t a i n e d from v e g e t a b l e marrow l e a v e s can f u n c t i o n w i t h r e d u c e d b e n z y l v i o l o g e n i n p l a c e o f p y r i d i n e n u c l e o t i d e as t h e p r i m a r y e l e c t r o n donor. Nason and Evans (1953) p r e s e n t e d e v i d e n c e t h a t energy f o r n i t r a t e r e d u c t i o n can be d e r i v e d from t h o s e s u b s t r a t e s , f o r t h e most p a r t i n t e r m e d i a t e s o f c a r b o h y d r a t e m e t a b o l i s m , w h i c h a r e o x i d i z e d by TPNH enzyme s y s t e m s . Spencer (1959) u s e d c r y s t a l l i n e a l c o h o l dehydroganase a t pH 7.5 i n t h e p r e s e n c e o f e t h a n o l and DPNH f o r a s s a y s on c r u d e wheat embryo e x t r a c t . Hageman e_t a l . (1962) l i n k e d g l u c o s e - 6 - p h o s p h a t e and i t s dehydrogenase and c a t a l y t i c amounts of TPN t o NRase o b t a i n e d from v e g e t a b l e marrow. Evans and Nason (1953) were a b l e t o l i n k soybean NRase w i t h p h o t o r e d u c t i o n o f TPNH, t h r o u g h t h e m e d i a t i o n o f c h l o r o -p l a s t g r a n a . S t o y (1956) has shown t h a t p h o t o c h e m i c a l r e d u c t i o n o f r i b o f l a v i n can be l i n k e d d i r e c t l y t o NRase a c t i o n w i t h o u t t h e a d d i t i o n o f a p y r i d i n e n u c l e o t i d e . 33. 6. PAD as P r o s t h e t i c Group The n e c e s s i t y o f f l a v i n as a p r o s t h e t i c group o f NRase was d e m o n s t r a t e d by Evans and Nason ( 1 9 5 3 ) . T h e i r r e p o r t showed t h a t t h e a c t i v i t y o f t h e t w i c e - p r e c i p i t a t e d soybean NRase was m a r k e d l y s t i m u l a t e d by FAD, b u t v e r y s l i g h t l y by FMN. T e s t s w i t h D-amino a c i d o x i d a s e and f l u o r o m e t r i c a n a l y s e s i n d i c a t e d t h a t enzyme s o l u t i o n s c o n t a i n e d FAD and t h a t 89 t o 9 8 7 o o f t h e t o t a l f l a v i n p r e s e n t i n t h e enzyme e x t r a c t was FAD. They c o n -c l u d e d t h a t t h e enzyme e x t r a c t c o n t a i n e d a maximum o f 3.4 x lO"""* uM FMN p e r m l . T h i s o r g r e a t e r q u a n t i t i e s o f FMN were n o t s u f -f i c i e n t t o pro d u c e an a p p r e c i a b l e s t i m u l a t i o n o f t h e a c t i v i t y o f t w i c e - p r e c i p i t a t e d enzyme i n t h e s t a n d a r d a s s a y p r o c e d u r e . A s i m i l a r c o n c l u s i o n a p p l i e d t o N e u r o s p o r a NRase o b t a i n e d by Nason and Evans ( 1 9 5 4 ) . Spencer (1859) showed t h a t t h e a c t i v i t y o f NRase o f c r u d e c e l l - f r e e e x t r a c t s o f embryos from g e r m i n a t i n g wheat was d o u b l e d by t h e a d d i t i o n o f FAD. A f t e r i s o l a t i o n o f t h e enzyme by ammonium s u l f a t e f r a c t i o n a t i o n , a d d i t i o n o f FAD ca u s e d a t h r e e -f o l d s t i m u l a t i o n a t a f i n a l c o n c e n t r a t i o n o f 10"^ M, FMN was i n e f f e c t i v e as t h e c o f a c t o r . I n t h e work o f Hageman and F l e s h e r ( 1 9 6 0 ) , no r e s p o n s e t o added f l a v i n s was o b t a i n e d w i t h t h e c r u d e p r e p a r a t i o n o f NRase fr o m c o r n s e e d l i n g s . I t i s p r o b a b l e t h a t t h e f l a v i n and p r o t e i n were n o t e a s i l y d i s s o c i a t e d as was a l s o t h e c a s e w i t h t h e enzyme f r o m E. c o l i ( N i c h o l a s and Nason, 1955b). T a b l e IV summarizes t h e n u c l e o t i d e s p e c i f i c i t y o f a s s i m i -l a t o r y NRases from s e v e r a l s o u r c e s . 34 Table IV Summary of Properties of Assimilatory NRase Plant material Original form Pyridine nucleotide specificity Flavin nucleotide specificity References Soybean leaves Soluble Non-specific FAD Evans and Nason (1953) Neuro-spora Soluble TPNH-specific FAD Nason and Evans (1953) E. c o l i Soluble DPNH-specific FAD Nicholas and Nason (1954) Hansenula Soluble Non-specific FAD Silver (1956) Wheat leaves Soluble Non-specific FAD Anacker and Stoy (1958) Wheat embryo Soluble DPNH-specific FAD Spencer (1959) Azoto-bacter Particulate Non-specific FAD Taniguchi and Ohmachi (1960) Tomato leaves Soluble DPNH-specific - Sanderson and Cocking (1964) 35. 7. S u l f h y d r y l P r o p e r t y The s u l f h y d r y l n a t u r e o f NRase was shown by p-c h l o r o m e r c u r i b e n z o a t e i n h i b i t i o n and i t s r e v e r s a l by s u l f h y d r y l r e a g e n t s s u c h as g l u t a t h i o n e and c y s t e i n e (Evans and Nason, 1953). S t u d i e s o f t h e p o s s i b l e r o l e o f s u l f h y d r y l groups by N i c h o l a s and Nason (1954) i n d i c a t e d t h a t t h e s e groups a r e n e c e s s a r y f o r t h e enzymic t r a n s f e r o f e l e c t r o n s from TPNH t o f l a v i n . T h i s was i n d i c a t e d by t h e c o m p l e t e i n h i b i t i o n o f enzyme a c t i v i t y by t h e mercury r e a g e n t and i t s r e v e r s a l by g l u t a t h i o n e . Subsequent e l e c t r o n t r a n s p o r t t o n i t r a t e , u s i n g EMNH2 as t h e e l e c t r o n d o n o r , i s i n h i b i t e d o n l y 257. by p - c h l o r o m e r c u r i b e n z o a t e . These e x p e r i m e n t s have shown t h e -SH groups o f t h e p r o t e i n a r e n e c e s s a r y l a r g e l y f o r t h e e n z y m a t i c r e d u c t i o n o f FMN by TPNH, p r o b a b l y by t h e b i n d i n g o f TPNH and u n r e d u c e d f l a v i n t o t h e enzyme. That t h e s u l f h y d r y l groups a r e p r o b a b l y n o t s e r v i n g as e l e c t r o n c a r r i e r s , was i n d i c a t e d by t h e f a c t t h a t t h e y do n o t s e r v e as e l e c t r o n donors i n t h i s e n z y m a t i c system. NRase has n o t been p u r i f i e d t o t h e homogenous p r o t e i n l e v e l , hence t h e number o f s u l f h y d r y l groups w h i c h a r e i n v o l v e d i n enzyme f u n c t i o n i s n o t known. 8. Molybdenum C o n s t i t u e n t N i c h o l a s and Nason (1954) p r e s e n t e d t h e f i r s t e v i d e n c e t h a t molybdenum i s t h e m e t a l c o n s i t u e n t o f NRase i n N e u r o s p o r a . 36. 1) When N e u r o s p o r a NRase was f r a c t i o n a t e d w i t h ammonium s u l f a t e , t h e h i g h e s t a c t i v i t y o c c u r r e d i n t h e 24 t o 407. ammonium s u l f a t e f r a c t i o n w h i c h a l s o had t h e g r e a t e s t molybdenum c o n t e n t . I n c r e a s e d enzyme a c t i v i t y i n v a r i o u s p r o t e i n f r a c t i o n s was accompanied by i n c r e a s e d molybdenum c o n t e n t . 2) D i a l y s i s o f NRase a g a i n s t c y a n i d e r e s u l t e d i n a d e c r e a s e o f molybdenum c o n t e n t and a d e a c t i v a t i o n o f t h e enzyme. The a d d i t i o n o f molybdenum t r i o x i d e o r sodium m o l y b d a t e r e a c t i v a t e d t h e enzyme t o 857. o f t h e o r i g i n a l v a l u e . P r e i n c u b a t i o n w i t h o t h e r t r a c e m e t a l s , i n c l u d i n g i r o n , z i n c , manganese, n i c k l e , c o b a l t , m e r c u r y , t u n g s t e n , u r a n i u m , vanadium, and b o r o n , r e s p e c t i v e l y , d i d n o t r e s t o r e t h e a c t i v i t y o f n i t r a t e r e d u c t a s e . I n t h e i r s t u d y o f t h e mechanism o f t h e N e u r o s p o r a NRase r e a c t i o n , N i c h o l a s and Nason (1954) f o u n d t h a t t h e molybdenum-f r e e enzyme had l o s t i t s a b i l i t y t o c a t a l y z e t h e r e d u c t i o n o f n i t r a t e t o n i t r i t e by e i t h e r TPNH o r r e d u c e d f l a v i n . I t s t i l l p o s s e s s e d t h e a b i l i t y t o c a t a l y z e t h e r e d u c t i o n o f f l a v i n by TPNH. They a l s o i n d i c a t e d t h a t d i t h i o n i t e - t r e a t e d m o l y b d a t e c a n r e d u c e n i t r a t e t o n i t r i t e a n a e r o b i c a l l y i n t h e p r e s e n c e o f NRase w i t h o u t t h e p a r t i c i p a t i o n o f TPNH o r r e d u c e d f l a v i n . Evans and H a l l (1955) p r e s e n t e d d i r e c t e v i d e n c e o f a s s o c i a t i o n o f molybdenum w i t h n i t r a t e r e d u c t i o n from soybean l e a v e s . They g e r m i n a t e d soybean seeds i n a f l a t o f san d . 37. F o u r days a f t e r t h e seeds were p l a n t e d , t h e f l a t s were t r e a t e d 99 w i t h a s o l u t i o n c o n t a i n i n g 3.5 mc o f Mo p e r gram. When t h e s e e d l i n g s were 10 days o l d , l e a v e s were h a r v e s t e d . The e x t r a c t s were f r a c t i o n a t e d w i t h ammonium s u l f a t e p r e c i p i t a t i o n and c a l c i u m p hosphate a d s o r p t i o n . The a c t i v i t y o f NRase from each f r a c t i o n was a s s a y e d , u s i n g DPNH as t h e hydr o g e n donor. An a l i q u o t from each f r a c t i o n was ashed i n a m u f f l e f u r n a c e and c o u n t e d f o r r a d i o a c t i v i t y w i t h a G e i g e r - M u l l e r c o u n t e r . The r e s u l t s i n d i c a t e d t h a t a c l o s e c o r r e l a t i o n between enzyme a c t i v i t y and r a d i o a c t i v i t y o f t h e f r a c t i o n o f h i g h e s t p u r i t y e x i s t e d . I n o r d e r t o o b t a i n f u r t h e r e v i d e n c e c o n c e r n i n g t h e m e t a l r e q u i r e m e n t o f NRase, Evans and H a l l (1955) d i a l y z e d p a r t i a l l y p u r i f i e d soybean NRase a g a i n s t phosphate b u f f e r c o n -t a i n i n g g l u t a t h i o n e and KCN, f o l l o w e d by d i a l y s i s f o r 8 h o u r s a g a i n s t a s i m i l a r b u f f e r s o l u t i o n c o n t a i n i n g g l u t a t h i o n e b u t no KCN. They found t h a t NRase a c t i v i t y was s t i m u l a t e d by t h e a d d i t i o n o f molybdenum. The most r e d u c e d v a l e n c e s t a t e o f Mo i n t h e d i t h i o n i t e r e d u c e d s o l u t i o n o f sodium m o l y b d a t e was shown by paper chromatography t o be +5, ( N i c h o l a s and S t e v e n s , 1 9 5 5 ) . M0 +^ p e n t a c h l o r i d e , f r e e d from t h e o t h e r v a l e n c e s t a t e s o f Mo by column c h r o m a t o g r a p h y , a c t s as an e f f e c t i v e e l e c t r o n donor f o r t h e enzymic r e d u c t i o n o f n i t r a t e . N i c h o l a s and Stevens (1955) 4+ f o u n d t h a t Mo i s s t a b l e i n s t r o n g e t h a n o l o n l y , and t h a t i t 38. d i s m u t e s r e a d i l y i n t h e p r e s e n c e of a t r a c e o f w a t e r t o g i v e 5+ 3+ 4+ Mo and Mo . I t i s most u n l i k e l y t h a t Mo i s i n v o l v e d i n 3+ any p h y s i o l o g i c a l p r o c e s s . Mo r e d u c e d n i t r a t e n o n - e n z y m a t i c a l l y . The r a t e o f c h e m i c a l r e d u c t i o n was a p p r o x i m a t e l y t w i c e t h a t o f 3-l~ Sm\" NRase a c t i o n . Mo i s o x i d i z e d i n s t a n t a n e o u s l y by Mo . 3+ R e d u c t i o n o f n i t r a t e by Mo i n N e u r o s p o r a i s u n l i k e l y s i n c e 3+ Mo has n o t been d e t e c t e d i n t h i s o r g a n i s m . I t w o u l d thus appear t h a t one o f t h e f u n c t i o n s o f Mo i n t h e enzyme i s t o c o u p l e t h e f l a v i n t o n i t r a t e by a one e l e c t r o n t r a n s p o r t c h a r g e whereby Mo"** i s o x i d i z e d Mo^ +. C a n d e l a e t a l . (1955) r e p o r t e d t h a t c a u l i f l o w e r grown w i t h o u t molybdenum p o s s e s s e d much l o w e r t o t a l and s p e c i f i c NRase a c t i v i t y t h a n t h e n o r m a l p l a n t s . I n f i l t r a t i o n o f molybdenum i n t o l e a v e s o f d e f i c i e n t p l a n t s r e s u l t e d i n i n c r e a s e d a c t i v i t y o f t h e enzyme i n 18 t o 24 h o u r s . 2+ 3+ N i c h o l a s and Nason (1954) r e p o r t e d t h a t Fe o r Fe t r e a t e d w i t h Na2S20^ f a i l e d t o s e r v e as an e l e c t r o n donor f o r t h e e n z y m a t i c r e d u c t i o n o f n i t r a t e i n t h e p r e s e n c e o r absence o f m o l y b d a t e . T h i s f a c t r u l e s out t h e r o l e o f i r o n as an e l e c t r o n c a r r i e r i n t h e N e u r o s p o r a NRase system. 9. Phosphate S t i m u l a t i o n P h o sphate s t i m u l a t i o n o f N e u r o s p o r a NRase has been o b s e r v e d by N i c h o l a s and Scawin (1956) and K i n s k y and M c E l r o y ( 1 9 5 8 ) . A s i m i l a r a c t i o n by p h o s p h a t e on t h e wheat embryo NRase was r e p o r t e d by Spencer ( 1 9 5 9 ) . 39. N i c h o l a s and Scawin (1956) f r a c t i o n a t e d N e u r o s p o r a NRase by ammonium s u l f a t e p r e c i p i t a t i o n , c a l c i u m phosphate g e l a b s o r p t i o n , and c a r b o n a b s o r p t i o n . Maximal enzyme a c t i v i t y was o b t a i n e d by t h e a d d i t i o n o f 2.5 uM phosphate t o 0.1 ml o f t h e enzyme. The phosphate c o u l d be r e p l a c e d by s e l e n a t e , t e l l u r a t e o r a r s e n a t e , p a r t i a l l y by s u l f a t e o r s i l i c a t e , b u t n o t by p y r o p h o s p h a t e , a d e n o s i n e t r i p h o s p h a t e , b o r a t e , v a n a d a t e m o l y b d a t e and t u n g s t a t e . The i n v e s t i g a t o r s s u g g e s t e d t h a t one o f t h e r o l e s o f p h o s p h a t e i n NRase i s t o b i n d t h e Mo t o t h e apoenzyme because t h e a n i o n s w h i c h c a n a c t as d i d p h o s p h a t e o have s i m i l a r a t o m i c r a d i i t o phosphate (2.76 A ) , b e i n g w i t h i n o t h e range 2.4 t o 2.8 A. They a l s o form complexes w i t h m o l y b d a t e s . K i n s k y and M c E l r o y (1958) o b s e r v e d t h a t a r s e n a t e and t u n g s t a t e , b u t n o t s i l i c a t e , c o u l d s u b s t i t u t e f o r p h o s p h a t e i n t h e c a s e o f N e u r o s p o r a NRase. They a l s o p r e s e n t e d e v i d e n c e t h a t p hosphate had no e f f e c t on t h e T P N H - t o - f l a v i n n u c l e o t i d e r e a c t i o n , whereas t h e s e c o n d s t e p , from f l a v i n n u c l e o t i d e , i s d e f i n i t e l y s t i m u l a t e d by p h o s p h a t e . They s u g g e s t e d t h a t molybdenum combines w i t h p h o s p h a t e , a r s e n a t e and t u n g s t a t e , t o form phosphomolybdo-, arsenomolybdo- and t u n g s t o m o l y b d o -complexes w h i c h presumably have o x i d a t i o n - r e d u c t i o n p o t e n t i a l s d i f f e r e n t from t h a t o f t h e molybdenum i o n . T h i s a l t e r n a t i o n o f p o t e n t i a l due t o complexes may p e r m i t i n t e r a c t i o n w i t h e n z y m a t i c a l l y o r c h e m i c a l l y r e d u c e d f l a v i n n u c l e o t i d e . 4 0 . Spencer ( 1 9 5 9 ) a l s o r e p o r t e d t h a t i n o r g a n i c p hosphate was e s s e n t i a l f o r maximum e n z y m a t i c a c t i v i t y . The p r e s e n c e o f - 2 1 0 M p o t a s s i u m phosphate c a u s e d a 3 0 7 . i n c r e a s e i n t h e a c t i v i t y o f wheat embryo NRase. 1 0 . I n h i b i t o r s NRase i s v e r y s e n s i t i v e t o m e t a l - c h e l a t i n g a g e n t s . Evans and Nason ( 1 9 5 3 ) d e m o n s t r a t e d t h a t KCN a t a f i n a l coneen-- 3 - 4 t r a t i o n o f 1 0 and 1 0 M i n h i b i t e d t h e a c t i v i t y o f soybean NRase 8 3 and 2 7 7 . r e s p e c t i v e l y ; and NaN^ a t t h e s e c o n c e n t r a t i o n s - 4 i n h i b i t e d 8 9 and 3 9 7 . r e s p e c t i v e l y . A c o n c e n t r a t i o n o f 2 x 1 0 M p o t a s s i u m e t h y l x a n t h a t e i n h i b i t e d t h e a c t i v i t y by 4 9 7 o , and - 4 1 0 M CuSO^ i n h i b i t e d i t by 3 2 7 . . There was no i n h i b i t i o n by - 3 1 0 M NaF, Ot h e r m e t a l - c h e l a t i n g agents s u c h as 8 - h y d r o x y q u i n o -l i n e , d i e t h y l d i t h i o c a r b a m a t e , EDTA, a , a - d i p y r i d y l and p h e n a n t h r o l i n e a l s o i n h i b i t e d a t d i f f e r e n t r a t e s . A co m p l e t e l a c k o f i n h i b i t i o n by 1 0 0 7 . CO s u g g e s t s t h e absence o f cytochrome i n t h e enzyme s y s t e m . S u l f h y d r y l r e a g e n t s , s u c h as p - c h l o r o m e r c u r i b e n z o a t e and heavy m e t a l s , s t r o n g l y i n h i b i t e d NRase. T h i s i n h i b i t i o n c o u l d be r e v e r s e d by t h e a d d i t i o n o f g l u t a t h i o n e . N i c h o l a s and Scawin ( 1 9 5 6 ) showed t h a t t h e N e u r o s p o r a NRase was n o t i n h i b i t e d by u n c o u p l i n g r e a g e n t s , e.g. d i n i -- 5 - 4 - 4 t r o p h e n o l a t 1 0 and 1 0 M, Aureomycin a t 5 x 1 0 M, o r - 2 sodium a r s e n a t e a t 1 0 M. Thus, t h e r e was no e v i d e n c e f o r a 41. p h o s p h o r y l a t i o n mechanism d u r i n g n i t r a t e r e d u c t a s e a c t i o n . V a nadate i o n has been shown to be a p o w e r f u l i n h i b i t o r o f t h e enzyme from wheat embryo ( S p e n c e r , 1959). A f i n a l c o n c e n t r a t i o n o f a p p r o x i m a t e l y 3 x 10 ^ M sodium o r t h o v a n a d a t e c a u s e d 507. i n h i b i t i o n . T h i s i n h i b i t i o n was n o t r e v e r s e d by a 2 5 - f o l d e x c e s s o f sodium m o l y b d a t e . 11. Mechanism o f A c t i o n I n t h e c l a s s i c paper "Mechanism o f a c t i o n o f n i t r a t e r e d u c t a s e from N e u r o s p o r a " , N i c h o l a s and Nason (1954) s u g g e s t e d t h e f o l l o w i n g sequence o f e l e c t r o n t r a n s f e r f o r t h e enzyme: TPNH > FAD ( o r FMN) > Mo >• N0~ N / 2, 3, 6 - t r i c h l o r o i n d o p h e n o l The enzyme c a t a l y z e s t h e t r a n s f e r o f e l e c t r o n s from TPNH t o n i t r a t e v i a e l e c t r o n c a r r i e r s FAD ( o r FMN) and molybdenum. When i n t r o d u c e d i n t o t h i s s y s t e m , 2 , 3 , 6 - t r i c h l o r o -i n d e o p h e n o l may a c t as an a r t i f i c i a l c a r r i e r between f l a v i n and molybdenum. Molybdenum i s e s s e n t i a l f o r t h e f i n a l s t e p i n e l e c t r o n d o n a t i o n t o n i t r a t e . I f i t i s s e p a r a t e d from t h e enzyme by d i a l y s i s a g a i n s t c y a n i d e , n e i t h e r TPNH n o r FADH2 can r e d u c e n i t r a t e t o n i t r i t e . Reduced m o l y b d a t e p r e p a r e d w i t h e i t h e r t h e r e d u c i n g agent N a 2 S 2 0 4and H^ o r p a l l a d i u m and H 2 can r e d u c e n i t r a t e t o n i t r i t e a n a e r o b i c a l l y i n t h e p r e s e n c e o f NRase. I r o n , c o p p e r , v a n a d a t e o r chromate when t r e a t e d w i t h 42. Na2S20^ and f a i l e d t o s u b s t i t u t e f o r d i t h i o n i t e - t r e a t e d m o l y b d a t e . Fewson and N i c h o l a s (1961) , by measurement o f e l e c t r o n s p i n r e s o n a n c e s i g n a l s , c o n f i r m e d t h e f o r m a t i o n o f Mo^ + d u r i n g NRase a c t i o n i n P. a e r u g i n o s a . V a l e n c e changes w h i c h o c c u r r e d 5+ , „ 6+ were presumed t o i n v o l v e Mo and Mo . A c c o r d i n g t o K i n s k y and M c E l r o y ' s (1958) s u g g e s t i o n , p h osphate and o t h e r a n i o n s combine w i t h molybdenum t o fo r m a complex. FAD o r FMN may behave as one o f t h e s p e c i f i c r e d u c i n g a g e n t s c a p a b l e o f r e d u c i n g o n l y molybdenum complexes w i t h p h o s p h a t e , a r s e n a t e , t u n g s t a t e and s i l i c a t e . I n s u p p o r t o f t h e i r s u g g e s t i o n f o r t h e e l e c t r o n t r a n s -f e r sequence o f NRase, N i c h o l a s and Nason (1954) were a b l e t o show t h a t DPNH can be r e p l a c e d by FADH 2. FADH 2 p r e p a r e d e n z y m a t i c a l l y by t h e DPNH o x i d i z i n g s y s t e m f r o m C l o s t r i d i u m  k l u w e r i , c o u l d s e r v e , under a n a e r o b i c c o n d i t i o n s , as e l e c t r o n donor f o r t h e r e d u c t i o n o f n i t r a t e by N e u r o s p o r a NRase. C h e m i c a l l y r e d u c e d f l a v i n s a l s o were a c t i v e as e l e c t r o n d o n o r s . The f o l l o w i n g scheme i s a summation o f v a r i o u s c o n -t r i b u t i o n s d i s c u s s e d above: c y a n i d e DPNH-. ^ F A D ^ . PT 2 M o 5 + i n h i b i t i o n (TPNH) \ / (FMN) \J +2H+ + NO. i \ , FADH 2 / ^ 2 M o 6 + J V NO" + H O (TPN +) ^ (FMNH 2) -SH dependent Phosphate s t i m u l a t i o n o 43. 12. L o c a l i z a t i o n o f Enzyme W i t h t h e e x c e p t i o n o f some b a c t e r i a l enzymes r e d u c i n g n i t r a t e , a l l t h e a s s i m i l a t o r y NRase s t u d i e d thus f a r seem t o be s o l u b l e . Nason and Evans (1953) i n d i c a t e d t h a t 857. o r more o f t h e NRase o f t h e homogenate from N e u r o s p o r a was p r e s e n t i n t h e c e l l - f r e e e x t r a c t . S p e n c e r (1959) ground embryos from g e r m i n a t i n g wheat i n 0.4 M s u c r o s e c o n t a i n i n g 0.05 M p o t a s s i u m p h o s p h a t e , pH -3 7.4 and 10 M g l u t a t h i o n e . The g r i n d a t e was c e n t r i f u g e d a t low speed ( x 100 g) t o remove unbroken c e l l s . The s u p e r n a t a n t s o l u t i o n was f u r t h e r c e n t r i f u g e d a t 105,000 x g i n a S p i n c o u l t r a c e n t r i f u g e f o r 30 m i n u t e s . He f o u n d t h a t a l l t h e NRase p r e s e n t i n t h e s u p e r n a t a n t s o l u t i o n from t h e low speed c e n t r i -f u g a t i o n was r e c o v e r e d i n t h e s u p e r n a t a n t s o l u t i o n f o l l o w i n g h i g h speed c e n t r i f u g a t i o n . The enzyme was thus l o c a t e d i n s o l u b l e , c y t o p l a s m i c f r a c t i o n o f t h e c e l l , r a t h e r t h a n i n t h e p a r t i c u l a t e f r a c t i o n . I n s t u d i e s o f d i s t r i b u t i o n o f NRase i n c e l l - f r e e e x t r a c t s o f A z o t o b a c t e r v i n e l a n d i i , T a n i g u c h i and Ohmachi (1960) f o u n d t h a t 907. o f NRase a c t i v i t y r e s i d e d i n l a r g e p a r t i c l e s (2000 - 14,000 x g) r e g a r d l e s s o f t h e e l e c t r o n d o n o r s , and 107. i n s m a l l p a r t i c l e s (14,000 - 105,000 x g) and s o l u b l e s u p e r n a t a n t . I t i s a p p a r e n t l y a p a r t i c u l a t e a s s i m i l a t o r y NRase. 44. The s o l u b l e o r p a r t i c u l a t e n a t u r e o f NRases from v a r i o u s s o u r c e s i s summarized i n T a b l e IV (page 3 4 ) . 13. A d a p t i v e F o r m a t i o n o f Enzyme Evans and Nason (1952) f i r s t r e p o r t e d t h a t N e u r o s p o r a NRase app e a r e d t o be a d a p t i v e s i n c e l i t t l e o r no a c t i v i t y c o u l d be d e t e c t e d i n e x t r a c t s o f m y c e l i a grown on an ammonium n i t r o g e n medium i n t h e absence o f exogenous n i t r a t e . I n 1953, t h e i r s t u d i e s o f e f f e c t s o f d i f f e r e n t s o u r c e s o f n i t r o g e n on t h e a c t i v i t y o f N e u r o s p o r a NRase i n d i c a t e d t h a t t h e enzyme a c t i v i t y o c c u r r e d o n l y i n m y c e l i a grown i n t h e p r e s e n c e o f n i t r a t e o r n i t r i t e and t h a t no a c t i v i t y e x i s t e d i n m y c e l i a grown w i t h ammonia o r a l a n i n e as a s o l e n i t r o g e n s o u r c e . They s u g g e s t e d t h a t some o f t h e n i t r i t e was o x i d i z e d t o n i t r a t e w h i c h was r e s p o n s i b l e f o r t h e i n d u c t i o n o f t h e enzyme. Tang and Wu (1957) o b t a i n e d more d i r e c t e v i d e n c e o f t h e a d a p t i v e p r o p e r t y o f t h e enzyme i n r i c e s e e d l i n g s . They d e m o n s t r a t e d t h a t no NRase a c t i v i t y was p r e s e n t i n 4- t o 6-day o l d s e e d l i n g s when grown i n q u a r t z sand w i t h e i t h e r 0.033 o r 0.066 M p h o s p h a t e b u f f e r . T h i s a c t i v i t y c o u l d be demon-s t r a t e d i n b o t h e t i o l a t e d and g r e e n s e e d l i n g s when n i t r a t e was added t o c u l t u r e s o l u t i o n , o r when t h e g r a i n s were t r e a t e d w i t h n i t r a t e s o l u t i o n b e f o r e g e r m i n a t i o n . Under o p t i m a l c o n -d i t i o n s s u c h as pH 5.0, 30° C, and 6 mM n i t r a t e , t h e NRase f o r m a t i o n c o u l d be i n d u c e d i n 1 day, i n 5 h o u r s o r even i n 3 45. h o u r s , i n e t i o l a t e d i n t a c t s e e d l i n g s , i n t a c t g r e e n s e e d l i n g s , and e x c i s e d r o o t s o f t h o s e s e e d l i n g s , r e s p e c t i v e l y . When t h e s e e d l i n g s were t r a n s f e r r e d back t o a n i t r a t e - f r e e medium,de-a d a p t a t i o n o c c u r r e d i n about 2 days. Ammonium s u l f a t e i s i n e f f e c t i v e i n i n d u c i n g t h e f o r m a t i o n o f NRase and i s i n h i b i t o r y i n t h e p r e s e n c e o f n i t r a t e . Low oxygen tens-ion o f 1 t o 57. does not s i g n i f i c a n t l y impede NRase f o r m a t i o n as compared w i t h 10% oxygen t e n s i o n . H e w i t t and A f r i d i (1959) have found i n numerous t e s t s t h a t f o r m a t i o n o f NRase a c t i v i t y can be r a p i d l y i n d u c e d i n s m a l l f ragments o f l e a f e x c i s e d from c a u l i f l o w e r , w h i t e m u s t a r d and s u n f l o w e r . T h e i r r e s u l t s showed t h a t when n i t r a t e o r m o l y b d a t e , o r b o t h , were i n t r o d u c e d by vacuum f i l t r a t i o n i n t o e x c i s e d fragments o f l e a v e s o f c a u l i f l o w e r , r a p i d p r o d u c t i o n o f enzyme a c t i v i t y o c c u r r e d . The i n t r o d u c t i o n o f molybdenum i n t o l e a v e s o f p l a n t s d e f i c i e n t i n Mo and grown w i t h n i t r a t e had a s i m i l a r e f f e c t . T i s s u e s o f p l a n t s grown w i t h ammonium s u l f a t e b u t w i t h o u t molybdenum, r e s p o n d e d l i t t l e o r n o t a t a l l t o e i t h e r f a c t o r a l o n e , b u t d e v e l o p e d a c t i v i t y r a p i d l y when b o t h were p r e s e n t . T h e i r r e s u l t s a l s o i n d i c a t e d t h a t some o f t h e i n h i b i t o r s o f p r o t e i n s y n t h e s i s , s u c h as l , 2 - d i c h l o r o - 4 - ( p - n i t r o b e n z e n e -s u l f o n y l a m i d e ) - 5 - n i t r o b e n z e n e (DCDNS), a c t i d i o n e , and 46. p o l y m y x i n - b - s u l f a t e , m a r k e d l y i n h i b i t e d t h e s y n t h e s i s o f NRase. They s u g g e s t e d t h a t p r o t e i n s y n t h e s i s was n e c e s s a r y f o r development o f enzyme a c t i v i t y and t h e p r o c e s s seemed a n a l o g o u s t o enzyme i n d u c t i o n i n o t h e r s y s t e m s . The p r e s e n c e o f ammonia i n t h e n u t r i e n t medium may cause s u p p r e s s i o n o f enzyme a c t i v i t y i n some o r g a n i s m , e.g. i n t h e fungus S c o p u l a r i o p s i s , even when n i t r a t e i s a l s o g i v e n ( M a r t o n , 1956). K i n s k y (1961) s t u d i e d i n d u c t i o n and r e p r e s s i o n o f n i t r a t e r e d u c t i o n by N e u r o s p o r a . He f o u n d t h a t t h e n i t r a t e -i n d u c e d enzyme f o r m a t i o n was c o m p l e t e l y i n h i b i t e d by c i t r a t e and a c e t a t e , and t h a t t h e r a t e o f enzyme f o r m a t i o n was dependent on t h e pH o f t h e i n d u c t i o n medium. K i n s k y u s e d " f e e d - b a c k r e p r e s s i o n " t o e x p l a i n ammonia r e p r e s s i o n o f NRase f o r m a t i o n . 14. E f f e c t o f P l a n t age on Enzyme A c t i v i t y I t i s w e l l known t h a t t h e m e t a b o l i c a c t i v i t y o f t i s s u e s v a r i e s w i t h t h e i r age, as w e l l as w i t h o t h e r p h y s i o l o g i c a l f a c t o r s . Evans and Nason (1953) r e p o r t e d t h a t NRase appeared t o be more a c t i v e i n t h e m e r i s t e m a t i c and young t r i f o l i a t e d l e a v e s o f 1 7 - d a y - o l d soybean p l a n t s and s u g g e s t e d t h a t t h e enzyme i s c o n c e n t r a t e d i n young, m e t a b o l i c a l l y a c t i v e t i s s u e . C a n d e l a e t a l . (1957) showed t h a t t h e a c t i v i t y o f c a u l i f l o w e r NRase was g r e a t e s t i n mature f u l l y expanded 47. l e a v e s , a d j a c e n t t o t h e o l d e s t two o r t h r e e l e a v e s r e m a i n i n g on t h e p l a n t a t t h e t i m e . The n e t t o t a l a c t i v i t y i n l e a v e s o f c a u l i f l o w e r was c o n s i d e r a b l y l e s s f o r p l a n t s aged 20 weeks t h a n t h o s e aged 12 weeks sampled a t t h e same t i m e . The s p e c i f i c a c t i v i t y , however, was s i m i l a r i n b o t h age groups due t o t h e l o w e r p r o t e i n e x t r a c t e d from t h e o l d e r p l a n t s . S pencer (1959) f o u n d a r e l a t i v e h i g h a c t i v i t y o f NRase i n wheat embryo. I t was o b s e r v e d t h a t , on t h e b a s i s o f f r e s h w e i g h t , 2300 muM n i t r a t e was p r o d u c e d p e r h o u r . A v a l u e o f about 200 muM was o b t a i n e d by Sanderson and C o c k i n g (1964) f o r 3 3 - o l d wheat l e a v e s . 15. N i t r a t e R e d u c t a s e A c t i v i t y i n Roots Based on h i s f i n d i n g t h a t n i t r a t e was p r e s e n t o n l y i n t h e f i n e s t p a r t s o f t h e r o o t s o f t h e a p p l e t r e e , Thomas (1927) s u g g e s t e d t h a t most o f n i t r a t e r e d u c t i o n t a k e s p l a c e i n r o o t t i s s u e s o f t h i s p l a n t . The r e d u c t i o n o f n i t r a t e i n e x c i s e d r o o t s and l e a v e s o f wheat l e d B u r s t r o m (1946) t o c o n c l u d e t h a t i n t h e i n t a c t p l a n t n i t r a t e r e d u c t i o n o c c u r s i n b o t h t h e s e o r g a n s . Bonner (1952) i n d i c a t e d t h a t i n asparagus and n a r c i s s u s n i t r a t e r e d u c t i o n i s o r d i n a r i l y c o n f i n e d t o t h e r o o t , and t h a t upward t r a n s l o c a t i o n o f n i t r a t e was o b s e r v e d o n l y when i t was s u p p l i e d t o t h e r o o t i n l a r g e amounts. I n s t i l l o t h e r s p e c i e s s u c h as tomato, t o b a c c o , c u r c u r b i t s and c e r e a l s t h e r o o t s a r e 48. n o t p a r t i c u l a r l y a c t i v e i n t h e r e d u c t i o n o f n i t r a t e b u t on t h e c o n t r a r y , n i t r a t e i s t r a n s l o c a t e d t h r o u g h t h e t i s s u e o f t h e e n t i r e p l a n t w i t h r e d u c t i o n t a k i n g p l a c e i n t h e l e a v e s as w e l l as i n t h e r o o t s . Evans and Nason (1953) r e p o r t e d t h e p r e s e n c e o f NRase a c t i v i t y i n e x t r a c t s o f r o o t s o f seven s p e c i e s o f p l a n t s namely: p o t a t o , b a r l e y , muskmelon, wheat, tomato, c o r n and soybean. A r e l a t i v e l y h i g h r a t e o f a c t i v i t y was f o u n d i n t h e e x t r a c t o f tomato roots:.: When TPNH was u s e d as hydrogen donor i n t h e a s s a y s y s t e m , t h e a c t i v i t y o f NRase i n t h e r o o t s o f t h e s e p l a n t s was as h i g h as i n t h e i r l e a v e s . The p r e s e n c e i n r o o t s o f enzyme systems c a p a b l e o f r e d u c e d p y r i d i n e n u c l e o t i d e p r o d u c t i o n w o u l d make t h e r e d u c t i o n o f n i t r a t e p o s s i b l e . V a i d y a n a t h a n and S t r e e t (1959) have shown t h a t e x t r a c t s from e x c i s e d n i t r a t e - g r o w n r o o t s d i d n o t r e d u c e n i t r a t e t o n i t r i t e i n s p i t e o f a d d i t i o n o f Mo, DPNH and FMN. However, t h e f u r t h e r a d d i t i o n o f a l d e h y d e - f r e e e t h a n o l , l a c t a t e , s u c c i n a t e o r g l u t a m a t e l e d t o t h e f o r m a t i o n o f n i t r i t e . S a n d e r s o n and C o c k i n g (1964) r e p o r t e d t h a t t h e a c t i v i t y o f NRase i n t h e r o o t e x t r a c t s o f such h i g h e r p l a n t s as tomato, b a r l e y , c o r n , k i d n e y bean, p o t a t o , t o b a c c o and wheat, was one-q u a r t e r o f t h e a c t i v i t y i n t h e i r l e a f e x t r a c t s . I n o r d e r t o de m o n s t r a t e w i t h c e r t a i n t y t h a t t h i s a c t i v i t y was due t o enzymic a c t i v i t y endogenous t o t h e r o o t s , e x c i s e d r o o t s were grown i n s t e r i l e n u t r i e n t s o l u t i o n c u l t u r e . The r e s u l t s showed 49. c l e a r l y t h a t tomato r o o t s do c o n t a i n NRase and t h a t t h e l e v e l o f a c t i v i t y i s dependent on t h e l e v e l o f n i t r a t e s u p p l i e d d u r i n g c u l t u r e . 16. L i g h t and N i t r a t e R e d u c t a s e A c t i v i t y A l t h o u g h t h e e f f e c t o f l i g h t on t h e n i t r a t e r e d u c t i o n i n g r e e n p l a n t s h a d been o b s e r v e d by p l a n t p h y s i o l o g i s t s f o r o v e r a c e n t u r y , t h e mechanism o f t h e r e a c t i o n i s s t i l l unknown. That p h o t o c h e m i c a l p r o c e s s e s may f u r n i s h t h e h y d r o g e n d o n o r s , i . e . r e d u c e d p y r i d i n e n u c l e o t i d e s , f o r n i t r a t e r e d u c t i o n was shown by Evans and Nason ( 1 9 5 3 ) . I n t h e i r h i s t o r i c a l e x p e r i -ment, t h e y showed t h a t t h e c o m b i n a t i o n o f NRase and g r a n a from soybean l e a v e s c o u l d r e d u c e n i t r a t e t o n i t r i t e i n l i g h t i f c a t a l y t i c amounts o f TPN were added. The s y s t e m was i n a c t i v e i n t h e d a r k u n l e s s TPN was r e p l a c e d by TPNH i n s t o i c h i o m e t r i c amounts, I n t h i s p h o t o c h e m i c a l r e d u c t i o n , n i t r a t e a c c e p t s t h e hyd r o g e n o f the r e d u c e d c a r r i e r formed d u r i n g t h e p h o t o l y s i s o f w a t e r . The f o l l o w i n g r e a c t i o n s were u s e d t o e x p l a i n t h i s mechanism. H 20 + TPN W t and g r a n a > ^ ^ + ^ + R + TPNH + H + + N 0 - N i t r a t e r e d u c t a s e ^ N Q - + T p N + + ^ J a g e n d o r f (1956) showed t h a t t h e h i g h l y p u r i f i e d c h l o r o -p l a s t s a r e c a p a b l e o f r e d u c i n g d y e s , t h e u s u a l H i l l o x i d a n t s , s u c h as f e r r i c y a n i d e and TPN, and t h a t t h e r e d u c t i o n o f TPN was c o u p l e d t o s p i n a c h NRase u s i n g t h e s y s t e m o f Evans and Nason (1953), 50. However, c h l o r o p l a s t s do n o t r e d u c e DPN under c o n d i t i o n s where g r a n a a r e p e r f e c t l y c a p a b l e o f c a r r y i n g o u t t h i s p h o t o r e d u c t i o n o f TPN. R e c e n t l y , R a m i r e x e t a l . (1964) r e p o r t e d t h e f i n d i n g s o f a new t y p e o f n o n - c y c l i c p h o t o s y n t h e t i c e l e c t r o n f l o w , i n w h i c h f l a v i n - n u c l e o t i d e s m e d i a t e t h e d i r e c t t r a n s f e r o f e l e c t r o n s from i l l u m i n a t e d g r a n a t o n i t r a t e w i t h t h e a i d o f NRase. I n t h e i r e x p e r i m e n t , t h e p h o t o e v o l u t i o n o f oxygen had been s u p p r e s s e d by h e a t i n g t h e g r a n a a t 55° C f o r 5 m i n u t e s and t h e e l e c t r o n s were s u p p l i e d by t h e s y s t e m o f a s c o r b a t e -d i c h l o r o p h e n o l - i n d o p h e n o l ( D P I P ) . The r e a c t i o n d i d n o t p r o c e e d i n t h e d a r k and r e q u i r e d FAD i n a d d i t i o n t o NRase. Manadione and f e r r e d o x i n were n o t c a p a b l e o f r e p l a c i n g t h e f l a v i n n u c l e o t i d e , b u t b e n z y l v i o l o g e n was an e f f e c t i v e e l e c t r o n c a r r i e r i n t h e s y s t e m . They s u g g e s t e d t h e f o l l o w i n g sequence o f e l e c t r o n t r a n s f e r i n p h o t o s y n t h e t i c r e d u c t i o n o f n i t r a t e . Reduced DPIP N i t r a t e r e d u c t a s e Water > C h l o r . I . » C h l o r . I I > FAD o r FMN > N i t r a t e ( l i g h t ) ( l i g h t ) A c o n c e p t b a s e d upon c o m p e t i t i o n between n i t r a t e and c a r b o n d i o x i d e f o r r e d u c i n g power has been p r o p o s e d by Van N e i l e t a l . ( 1 9 5 3 ) . They o b s e r v e d t h a t i n C h l o r e l l a a t low l i g h t i n t e n s i t y t h e u p t a k e o f c a r b o n d i o x i d e , b u t n o t t h e p r o d u c t i o n 51. o f oxygen was d e c r e a s e d i n t h e p r e s e n c e o f n i t r a t e . S t o y ' s e x p e r i m e n t s gave a n o t h e r c l u e f o r t h e u n d e r -s t a n d i n g o f t h e p h o t o r e d u c t i o n o f n i t r a t e . He f i r s t o b s e r v e d (1955) n i t r a t e r e d u c t i o n by d e t a c h e d wheat l e a v e s i n t h e b l u e and v i o l e t p a r t s o f t h e s p e c t r u m and s u g g e s t e d i n v o l v e m e n t o f a y e l l o w pigment. He a l s o o b s e r v e d t h a t p h o t o s y n t h e s i s and n i t r a t e a s s i m i l a t i o n p a r a l l e l e d each o t h e r i n r e d and green l i g h t . Based on t h i s , he s u g g e s t e d t h a t r i b o f l a v i n s e r v e d as a l i g h t - a b s o r b i n g c a t a l y s t and hydrogen c a r r i e r i n b i o l o g i c a l p h o t o r e d u c t i o n as d e s c r i b e d by B r a n n e r and B r a n n e r ( 1 9 5 4 ) . S t o y ' s e x p e r i m e n t d e m o n s t r a t e d t h a t p h o t o r e d u c e d r i b o f l a v i n was a c t u a l l y a more e f f i c i e n t r e d u c i n g agent t h a n DPNH. EDTA was r e q u i r e d f o r t h e r i b o f l a v i n - c o u p l e d s y s t e m and c o u l d n o t be r e p l a c e d by c y s t e i n . The f o l l o w i n g r e a c t i o n scheme was p r o p o s e d . hv DPN > DPNH > FAD —> Mo > N0~ g r a n a hv R i b o f l a v i n >• Reduced f l a v i n (EDTA r i b o f l a v i n ) No d i r e c t l i g h t a c t i v a t i o n o f t h e f l a v i n - c o n t a i n i n g n i t r a t e r e d u c t a s e was o b s e r v e d . C a n d e l a jet _ a l . (1957) f o u n d t h a t when c a u l i f l o w e r p l a n t s were t r a n s f e r r e d t o d a r k n e s s , NRase a c t i v i t y i n t h e e x t r a c t d e c r e a s e d t o n i l a f t e r 5 t o 6 days. I t was found 52. t h a t when n o r m a l l i g h t was r e s t o r e d t h e r e were r a p i d i n c r e a s e s i n t h e t o t a l and s p e c i f i c a c t i v i t i e s . Hageman and F l e s h e r (1960) d e m o n s t r a t e d t h a t when c o r n s e e d l i n g s were p l a c e d i n t h e d a r k , t h e NRase dropped t o l e s s t h a n 107. o f t h e o r i g i n a l l e v e l o f a c t i v i t y a f t e r 48 h o u r s . NRase was n o t d e t e c t a b l e i n t h e e x t r a c t s from c o r n s e e d l i n g s a f t e r 90 hours o f d a r k n e s s . No i n c r e a s e i n NRase was d e t e c t a b l e w i t h i n 2 h o u r s a f t e r t h e p l a n t s were r e t u r n e d t o f u l l s u n l i g h t a f t e r 65 hours o f d a r k n e s s . The e x t r a c t i o n , p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f sugar b e e t NRase, d e s c r i b e d i n t h i s t h e s i s , was u n d e r t a k e n as p a r t o f t h e o v e r a l l s t u d y o f s u g a r b e e t m e t a b o l i s m c o n d u c t e d a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a o v e r a p e r i o d o f y e a r s and s u p p o r t e d by r e s e a r c h g r a n t s from t h e N a t i o n a l R e s e a r c h C o u n c i l o f Canada and t h e B r i t i s h C o l u m b i a Sugar R e f i n i n g Co., V a n c o u v e r , B. C. A knowledge o f t h e p r o p e r t i e s o f t h e enzyme was p a r t i c u -l a r l y d e s i r a b l e as an e n t r y i n t o t h e b r o a d e r f i e l d o f n i t r o g e n m e t a b o l i s m o f t h e b e e t as r e l a t e d t o l a t e s e a s o n growth w h i c h r e s u l t s i n a d e p l e t i o n o f s u c r o s e s t o r e d i n t h e r o o t . MATERIALS AND METHODS 53. 1. S o u r c e o f Enzyme The c r u d e enzyme was o b t a i n e d from t h e b l a d e s o f l e a v e s o f 40- t o 6 0 - d a y - o l d s u g a r b e e t ( B e t a v u l g a r i s L.) p l a n t s . The seeds were s u p p l i e d by t h e B r i t i s h C o l u m b i a Sugar R e f i n i n g Co., V a n c o u v e r , B r i t i s h C o l u m b i a , Canada. The p l a n t s , grown i n wooden f l a t s f i l l e d w i t h v e r m i c u l i t e , were s u p p l i e d once e v e r y day w i t h n u t r i e n t s o l u t i o n c o n t a i n i n g 0.005 M C a ( N 0 3 ) 2 , 0.0005 M K H 2 P 0 4 , 0 . 0 0 2 M MgSO^, 0.05 M KNO3, 0.5 ppm Fe as i r o n c h e l a t e , 0.04 ppm o f Cu as CuSO^, 0.25 ppm o f Mo as Na MoO^, 2 . 0 ppm o f B as E^BO^, 0 . 2 ppm o f Zn as ZnSO^ and 0.5 ppm o f Mn as M n C l 2 . The pH was a d j u s t e d t o 6.5. The p l a n t s were grown i n a c o n t r o l l e d e n vironment room w i t h t h e f o l l o w i n g c o n d i t i o n s : p h o t o p e r i o d , 16 h r s . ; t e m p e r a t u r e , day 21-26° C, n i g h t 1 8 - 2 2 ° C; r e l a t i v e h u m i d i t y , day 62-70%, n i g h t 65-80%; l i g h t i n t e n s i t y 1800 f o o t c a n d l e s a t t h e t o p o f th e p l a n t s . S l i m l i n e c o o l w h i t e f l u o r e s c e n t tubes were s u p p l e -mented by 60 w a t t i n c a n d e s c e n t l i g h t s . 2, E x t r a c t i o n o f Enzyme Crude c e l l - f r e e e x t r a c t s were p r e p a r e d by g r i n d i n g one w e i g h t o f f r e s h l e a f b l a d e s w i t h f o u r w e i g h t s o f c o l d 0 . 1 M F^HPO^ b u f f e r (pH 7.8 a p p r o x i m a t e l y ) c o n t a i n i n g 10 M o f r e d u c e d g l u t a t h i o n e , i n a W a r i n g B l e n d o r ( a t f u l l speed) f o r 1 t o 2 m i n u t e s , a t 0 - 4° C. The homogenate was s t r a i n e d t h r o u g h f o u r l a y e r s o f c h e e s e c l o t h and t h e n c e n t r i f u g e d i n a S e r v a l l c e n t r i f u g e a t 20,000 x g f o r 20 m i n u t e s a t 0 t o 4° C The r e s u l t i n g g r e e n , c e l l - f r e e s u p e r n a t a n t s o l u t i o n ( c r u d e enzyme) w h i c h was shown t o c o n t a i n 100% o f t h e NRase a c t i v i t y p r e s e n t i n t h e whole homogenate, was u s e d f o r p u r i f i c a t i o n . 3. C o f a c t o r s and Ot h e r S u b s t a n c e s DPNH ( a p p r o x i m a t e l y 97.5% p u r e ) , TPNH ( 9 6 % p u r e ) , FAD ( 8 0 % pure) and FMN (100% pure) were o b t a i n e d from t h e Sigma C h e m i c a l Company o f S t , L o u i s , M i s s o u r i . The c o n e e n t i r a -t i o n s o f DPNH and TPNH were d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y 6 — 1 6 w i t h 6.22 x 10 s q . cm. x mole and 6.24 x 10 s q . cm. x mole ^ as t h e e x t i n c t i o n c o e f f i c i e n t a t 340 mu r e s p e c t i v e l y ( H o r e c k e r and R o m b e r g , 1948). The c o n c e n t r a t i o n o f f l a v i n s o l u t i o n s was d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y by t h e use o f t h e e x t i n c t i o n c o e f f i c i e n t o f 1.13 x 10^ s q . cm. x mole ^ a t 455 mu (Warburg and C h r i s t i a n , 1938). B e n z y l v i o l o g e n was o b t a i n e d from Mann R e s e a r c h L a b o r a t o r i e s o f New Y o r k , N.Y. M e t h y l e n e b l u e and t h e sodium s a l t o f 2 , 6 - d i c h l o r o p h e n o l i d o p h e n o l was o b t a i n e d from Kodak Company o f R o c h e s t e r , N. Y. P h e n a z i n e m e t h o s u l f a t e and a l l o t h e r s u b s t a n c e s were o b t a i n e d from t h e N u t r i t i o n a l B i o c h e m i c a l C o r p o r a t i o n o f C l e v e l a n d , O h i o . 55. 4. P r e p a r a t i o n o f C a l c i u m Phosphate G e l The t r i c a l c i u m p hosphate g e l was p r e p a r e d by t h e method o f K e i l i n and H a r t e e ( 1 9 3 8 ) . One hundred f i f y ml o f C a C ^ s o l u t i o n (88.5 g C a C l ^ p e r l i t e r ) was d i l u t e d t o about 1600 ml w i t h d e m i n e r a l i z e d w a t e r and shaken w i t h 150 ml t r i s o d i u m phosphate s o l u t i o n (152 g Na^PO^. 1 2 ^ 0 p e r l i t e r ) . The pH o f t h e m i x t u r e was b r o u g h t t o 7.5 w i t h a c e t i c a c i d and a l l o w e d t o s t a n d f o r 10 m i n u t e s . The s u p e r n a t a n t was d e c a n t e d and t h e g e l c e n t r i f u g e d . The g e l was t h e n washed t h r e e t i m e s w i t h l a r g e volumes o f d i s t i l l e d w a t e r t o remove CI i o n s . A f t e r f i n a l c e n t r i f u g a t i o n i t was r e s u s p e n d e d i n 500 ml d i s t i l l e d w a t e r and t h e d r y w e i g h t p e r ml o f t h e s u s p e n s i o n d e t e r m i n e d . D i s t i l l e d w a t e r was added t o b r i n g t h e co n c e n -t r a t i o n t o 20 mg p e r ml o f s u s p e n s i o n . A f t e r t h r e e months s t o r a g e a t 4°C t h e g e l was c e n t r i f u g e d down and r e s u s p e n d e d i n t h e same volume o f phosphate b u f f e r , pH 7.5. 5. P r e p a r a t i o n o f A l u m i n a C^ G e l A l u m i n a C^ g e l was p r e p a r e d by a m o d i f i c a t i o n o f t h e method o f C o l o w i c k ( 1 9 5 5 ) . A h o t s o l u t i o n o f 340 grams o f aluminum s u l f a t e i n 500 ml o f w a t e r was p o u r e d a l l a t once i n t o 3.25 l i t e r s o f ammonium s u l f a t e - a m m o n i a w a t e r a t 60° C. The l a t t e r r e a g e n t c o n t a i n e d 100 g o f ammonium s u l f a t e and 215 ml o f 207. ammonia. D u r i n g t h e p r e c i p i t a t i o n and f o r an a d d i t i o n a l 15 m i n u t e s , t h e m i x t u r e was s t i r r e d v i g o r o u s l y and the t e m p e r a t u r e was k e p t a t 50° C. A f t e r t h e f i r s t v o l u m i n o u s 56. p r e c i p i t a t e g r a d u a l l y became f l o c c u l e n t , t h e m i x t u r e was d i l u t e d t o 10 l i t e r s w i t h d e m i n e r a l i z e d w a t e r and t h e s u p e r n a t a n t f l u i d d e c a n t e d as soon as t h e p r e c i p i t a t e had s e t t l e d o u t . The w a s h i n g w i t h d e m i n e r a l i z e d w a t e r and d e c a n t a t i o n was r e p e a t e d s i x t i m e s . To s o l u b i l i z e any r e m a i n i n g b a s i c aluminum s u l f a t e 40 ml o f 20% ammonia was added d u r i n g t h e f o u r t h w a s h i n g . A f t e r r e p e a t e d w a s h i n g s , t h e g e l was suspended i n 200 ml d i s t i l l e d w a t e r and k e p t a t 4° C f o r t h r e e months b e f o r e u s i n g . 6. P r e p a r a t i o n o f E l e c t r o n Donors Reduced f l a v i n s were o b t a i n e d by t h e method o f N i c h o l a s and Nason (1954b). Two mg o f sodium h y d r o s u l f i t e was added t o 0.1 t o 0.5 uM FAD o r FMN c o n t a i n e d i n 3 m l o f 0.1 M VVV°r, p h o s p h a t e b u f f e r , pH 7.5, i n a Thunberg t u b e . Hydrogen gas was th e n b u b b l e d t h r o u g h t h e s o l u t i o n f o r 2 m i n u t e s a t 4° C, f o l l o w e d by e v a c u a t i o n . The dyes were r e d u c e d t o t h e i r l e u c o form by t h e same p r o c e d u r e . 7. D e t e r m i n a t i o n o f P r o t e i n The p r o t e i n c o n t e n t o f enzyme p r e p a r a t i o n was measured a c c o r d i n g t o t h e method o f Lowery e t a l . ( 1 9 5 1 ) . To 0.4 ml o f t h e sample was added 2 ml o f a l k a l i n e c opper s o l u t i o n . T h i s s o l u t i o n was p r e p a r e d by t h e m i x t u r e o f 50 ml o f 2% Na^CO^ i n 0.1 N NaOH and 1 m l o f 0.57. CuS0^.5H 20 i n 17. sodium p o t a s s i u m t a r t r a t e . The c o n t e n t s were m i x e d . A f t e r 10 m i n u t e s 0.2 ml o f 1 N F o l i n - C i o c a l t e a u p h e n o l r e a g e n t was added and t h e 57. c o n t e n t s mixed. O p t i c a l d e n s i t y r e a d i n g s a t 500 mu were t a k e n w i t h a Beckman Model B S p e c t r o p h o t o m e t e r . P r o t e i n c o n t e n t , as mg p e r m l , was o b t a i n e d by c o m p a r i s o n w i t h a s t a n d a r d c u r v e p r e p a r e d by t h e use o f c r y s t a l l i z e d b o v i n e a l b u m i n . 8. D e t e r m i n a t i o n o f C h l o r o p h y l l C o n t e n t C h l o r o p h y l l c o n t e n t was measured by t h e method de-s c r i b e d by Arnon ( 1 9 4 9 ) . 0.1 ml o f c r u d e enzyme e x t r a c t was d i l u t e d t o 20 m l w i t h 807. a c e t o n e (20 m l o f H 20 made t o 100 m l w i t h a c e t o n e ) , and f i l t e r e d t h r o u g h Whatman No. 1 f i l t e r p a p e r . The o p t i c a l d e n s i t y was r e a d a g a i n s t 807. a c e t o n e a t 625 mu (1-cm l i g h t p a t h ) i n a Beckman Model B S p e c t r o -p h o t o m e t e r . C h l o r o p h y l l s o l u t i o n s i n a c e t o n e were p r o t e c t e d from ambient l i g h t , O.D. x 5.8 gave mg o f c h l o r o p h y l l p e r ml o f c r u d e enzyme e x t r a c t . 9. D e t e r m i n a t i o n o f N i t r a t e C o n t e n t N i t r a t e c o n t e n t was e s t i m a t e d by t h e method o f W o o l l e y e t a l . ( 1 9 6 0 ) . F o u r ml o f e x t r a c t was d i l u t e d t o 20 m l . To 1 m l o f t h i s d i l u t e d s o l u t i o n was added 9 ml o f 207. a c e t i c a c i d s o l u t i o n c o n t a i n i n g 0.2 ppm Cu as CuSO^. By t h e use o f a m e a s u r i n g scoop 0.8 g o f an i n t i m a t e m i x t u r e o f 75 g o f c i t r i c a c i d , 10 g manganous s u l f a t e d i h y d r a t e , 4 g. o f s u l f a n i l i c a c i d , 2 g o f z i n c powder, and 2 g o f 1-naphtylamine was added. The 58. sample was t h e n shaken f o r about 15 seconds and was a g a i n shaken t h r e e m i n u t e s l a t e r . A f t e r t h r e e m i n u t e s more t h e sample was shaken f o r t h e t h i r d t i m e and was c e n t r i f u g e d f o r t h r e e m i n u t e s a t 1000 x g. The s u p e r n a t a n t s o l u t i o n was p o u r e d t h r o u g h a s m a l l l o o s e p l u g o f b o r o s i l i c a t e g l a s s w o o l , the l i g h t a b s o r b a n c e was measured a t 520 mu, and t h e amount o f n i t r a t e was c a l c u -l a t e d from a s t a n d a r d c u r v e , 10. S t a n d a r d N i t r a t e R e d u c t a s e A s s a y The NRase was measured by a m o d i f i c a t i o n o f t h e method d e s c r i b e d by Evans and Nason ( 1 9 5 3 ) . A t z e r o t i m e 0.05 - 0.2 ml o f enzyme added t o a r e a c t i o n m i x t u r e c o n t a i n i n g 0.1 m l o f 0.1 M KN0 3, 0.05 m l 2 x 1 0 " 5 M FAD, 0.05 m l 2 x 1 0 " 3 M DPNH, and 0.1 M phosphate b u f f e r , pH 7.0, t o g i v e a volume o f 0.5 m l . A f t e r 15 m i n u t e s i n c u b a t i o n a t 30° C, 1 ml o f ^ 0 and 1 ml o f 17. W/V s u l f a n i l a m i d e r e a g e n t were added t o s t o p t h e r e a c t i o n . One m l o f 0.22 "L W/V N - ( l - n a p h t h y l ) - e t h y l e n e d i a m i n e h y d r o c h l o r i d e r e a g e n t was added and t h e c o n t e n t s m i x e d by i n -v e r t i n g t h e t u b e s . The c o l o r was a l l o w e d t o d e v e l o p f o r 15 m i n -u t e s . A Beckman Model B s p e c t r o p h o t o m e t e r was u s e d t o d e t e r m i n e t h e O.D. o f each sample and i t s b l a n k ( c o m p l e t e e x c e p t f o r DPNH) a t 540 mu. The enzyme e x h i b i t e d no d i f f e r e n c e i n a c t i v i t y when t h e a s s a y was c o n d u c t e d under a n a e r o b i c o r a e r o b i c c o n d i t i o n s . T h e r e was no c h e m i c a l r e d u c t i o n o f n i t r a t e t o n i t r i t e by DPNH. 59. One u n i t o f NRase i s d e f i n e d as t h a t amount o f enzyme w h i c h -3 r e s u l t s i n t h e f o r m a t i o n o f 10 uM o f n i t r i t e under t h e above c o n d i t i o n s o f t h e a s s a y . S p e c i f i c a c t i v i t y i s e x p r e s s e d as u n i t s p e r mg o f p r o t e i n . 11. D e t e r m i n a t i o n o f DPNH-Menadione R e d u c t a s e A c t i v i t y DPNH-menadione r e d u c t a s e was d e t e r m i n e d by a m o d i f i -c a t i o n o f t h e p r o c e d u r e o f W o s i l a i t and Nason ( 1 9 5 4 ) . The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f enzyme t o a m i x t u r e c o n t a i n i n g 0.3 uM menadione, 0.35 uM DPNH and 200 uM pho s p h a t e b u f f e r , pH 8.3, t o g i v e a f i n a l volume o f 3.0 m l . A f t e r t h e a d d i t i o n o f t h e enzyme, t h e d e c r e a s e i n o p t i c a l d e n s i t y a t 340 mu was measured a t 30 s e c o n d i n t e r v a l s f o r t h e f i r s t t h r e e m i n u t e s . One u n i t o f menadione r e d u c t a s e i s d e f i n e d as t h a t amount o f enzyme w h i c h r e s u l t s i n a change i n o p t i c a l d e n s i t y o f 0.001 p e r m i n u t e . The no n - e n z y m a t i c r a t e was a l s o d e t e r m i n e d s p e c t r o -p h o t o m e t r i c a l l y a t 340 mu as above, e x c e p t t h a t t h e enzyme was o m i t t e d from t h e r e a c t i o n m i x t u r e . The s p e c i f i c a c t i v i t y i s e x p r e s s e d as u n i t s p e r mg o f p r o t e i n . 12. D e t e r m i n a t i o n o f DPNH-Quinone R e d u c t a s e A c t i v i t y DPNH-quinone r e d u c t a s e a c t i v i t y was d e t e r m i n e d by a m o d i f i c a t i o n o f t h e p r o c e d u r e o f W o s i l a i t and Nason ( 1 9 5 4 ) . The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f enzyme t o a m i x t u r e c o n t a i n i n g 0.45 uM o f p-quinone s o l u t i o n , 0.35 uM DPNH and 200 60. uM phosphate b u f f e r , pH 7.0, t o g i v e a f i n a l volume o f 3.0 m l . A f t e r t h e a d d i t i o n o f the enzyme, t h e d e c r e a s e i n o p t i c a l d e n s i t y a t 340 mu was measured a t 30-second i n t e r v a l s f o r t h e f i r s t t h r e e m i n u t e s . One u n i t o f qu i n o n e r e d u c t a s e i s d e f i n e d as t h a t amount o f enzyme w h i c h r e s u l t s i n a change i n o p t i c a l d e n s i t y o f 0.001 p e r m i n u t e . The non-enzymatic r a t e was a l s o d e t e r m i n e d s p e c t r o p h o t o m e t r i c a l l y a t 340 mu as above, e x c e p t t h a t t h e enzyme was o m i t t e d from t h e r e a c t i o n m i x t u r e . The s p e c i f i c a c t i v i t y i s e x p r e s s e d as u n i t s p e r mg o f p r o t e i n . 13. D e t e r m i n a t i o n o f DPNH-Cytochrome c R e d u c t a s e A c t i v i t y A c t i v i t y o f DPNH-cytochrome c r e d u c t a s e was d e t e r m i n e d by a m o d i f i c a t i o n o f t h e p r o c e d u r e o f M a h l e r e t a l . ( 1 9 5 4 ) . The r e a c t i o n m i x t u r e c o n t a i n e d 0.2 uM DPNH, 0.03 uM FMN, 0.1 uM o f cytochrome c and 150 mM o f T r i s b u f f e r , pH 7.5, i n a f i n a l volume o f 3 m l . The r e a c t i o n was s t a r t e d by a d d i t i o n o f enzyme and t h e o p t i c a l d e n s i t y a t 550 mu was f o l l o w e d w i t h a Beckman Model B s p e c t r o p h o t o m e t e r . The u n i t o f enzyme a c t i v i t y i s d e f i n e d as an o p t i c a l d e n s i t y change o f 1.00 p e r m i n u t e under t h e above c o n d i t i o n s . 14. D e t e r m i n a t i o n o f DPNH-Diaphorase A c t i v i t y DPNH-diaphorase a c t i v i t y was d e t e r m i n e d by a m o d i f i -c a t i o n o f t h e p r o c e d u r e o f A v r o n and J a g e n d o r f ( 1 9 5 6 ) . The r e a c t i o n m i x t u r e c o n t a i n e d 0.2 uM DPNH, 15 uM o f T r i s b u f f e r , pH 8.0, and 0.3 uM 2, 6 - d i c h l o r o p h e n o l i n d o p h e n o l and 0.05 m l 61. o f enzyme i n a f i n a l volume o f 3 m l . The r e a c t i o n was s t a r t e d by t h e a d d i t i o n o f t h e enzyme. The d e c r e a s e i n o p t i c a l d e n s i t y a t 620 mu was measured a t 3 0 r s e c o n d i n t e r v a l s f o r t h r e e m i n u t e s . One u n i t i s d e f i n e d as t h a t amount o f enzyme w h i c h r e s u l t e d i n a n e t change i n o p t i c a l d e n s i t y o f 1.00 p e r m i n u t e a t 620 mu under t h e above c o n d i t i o n s . 15. I s o l a t i o n and I d e n t i f i c a t i o n o f Enzyme-bound F l a v i n The f l a v i n o f NRase was r e l e a s e d by t h e method des-c r i b e d by Rao e t a l . ( 1 9 6 2 ) . F i v e m l o f F r a c t i o n V enzyme was h e a t e d i n a b o i l i n g w a t e r b a t h f o r 5 m i n u t e s , f o l l o w e d by c o o l i n g t o 5° C and a c i d i f i c a t i o n w i t h p e r c h l o r i c a c i d t o a f i n a l c o n c e n t r a t i o n o f about 10%. A f t e r c e n t r i f u g a t i o n a t 5000 x g f o r 10 m i n u t e s , t h e p r e c i p i t a t e was d i s c a r d e d and t h e y e l l o w s u p e r n a t a n t f l u i d was a d j u s t e d t o pH 7.8 w i t h 6 N KOH. The p o t a s s i u m p e r c h l o r a t e was removed by c e n t r i f u g a t i o n a t 5000 x g f o r 10 m i n u t e s . The s u p e r n a t a n t was condensed i n t h e d a r k w i t h a vacuum pump. D e s c e n d i n g paper chromatography o f f l a v i n s was p e r f o r m e d on 20 x 40 cm s h e e t s o f Whatman No. 1 p a p e r , w i t h • n - b u t a n o l - a c e t i c a c i d - w a t e r ( 4 : 1 : 5 , volume f o r volume) o r 5% Na^HPO^ as t h e s o l v e n t s y s tem. The f l a v i n s were i d e n t i f i e d under an u l t r a v i o l e t lamp. 16. F r a c t i o n a t i o n o f C e l l u l a r Components F r a c t i o n a t i o n o f c e l l u l a r components was c a r r i e d o u t by t h e m o d i f i c a t i o n o f t h e method o f P i e r p o i n t ( 1 9 6 3 ) . F i v e g of s u g a r b e e t l e a f b l a d e s w i t h m i d r i b s removed was c u t i n t o 62. s m a l l p i e c e s and ground f o r 1 m i n u t e i n a homogenizer w i t h 20 ml o f e x t r a c t i o n medium. The l a t t e r c o n s i s t e d o f 0.4 M s u c r o s e , 0.2 M T r i s , 0.01 M phosphate b u f f e r , pH 7.5, 0.01 M EDTA and 0.02 M sodium n i t r a t e , pH 7.5. The homogenate was s t r a i n e d t h r o u g h two l a y e r s o f c h e e s e c l o t h and t h e f i l t r a t e was c e n t r i f u g e d a t 200 x g f o r two m i n u t e s t o remove i n t a c t c e l l s , c e l l w a l l and n u c l e i . The d e c a n t e d s u p e r n a t a n t s o l u t i o n was f u r t h e r c e n t r i f u g e d f o r 7 m i n u t e s a t 1000 x g t o g i v e a sediment c o n t a i n i n g c h l o r o p h y l l o u s m a t e r i a l s . T h i s i s r e f e r r e d t o as t h e " C h l o r o p l a s t f r a c t i o n " . The s u p e r n a t a n t was f u r t h e r c e n t r i f u g e d f o r 30 m i n u t e s a t 10,000 x g t o g i v e a g r e e n " m i t o c h r o n d r i a l " f r a c t i o n and a r e l a t i v e l y c l e a r s u p e r n a t a n t . The m i t o c h o n d r i a - f r e e s u p e r n a t a n t was c e n t r i -f u g e d i n a S p i n c o Model L p r e p a r a t i v e c e n t r i f u g e f o r 30 m i n u t e s a t 105,000 x g, t o o b t a i n m i c r o s o m a l and s u p e r n a t a n t f r a c t i o n s . The t e m p e r a t u r e was k e p t a t 0-4° C. Each f r a c t i o n , v i z . , n u c l e i , c h l o r o p l a s t , m i t o c h o n d r i a and microsome, was washed w i t h 5 m l o f t h e e x t r a c t i o n medium and t h e was h i n g s were added t o t h e s u p e r n a t a n t f r a c t i o n . The washed p a r t i c u l a t e m a t e r i a l s were suspended i n 10 m l o f 0.01 M p o t a s s i u m phosphate b u f f e r a t pH 7.5. 17. P h o t o c h e m i c a l R e d u c t i o n o f F l a v i n N u c l e o t i d e s The p h o t o c h e m i c a l r e a c t i o n s d e s c r i b e d i n t h i s e x p e r i -ment were p e r f o r m e d under a n a e r o b i c c o n d i t i o n s . I n o r d e r 63. t h a t p r e c i s e s p e c t r o s c o p i c measurements c o u l d be made under a n a e r o b i c c o n d i t i o n s , Thunberg tubes were m o d i f i e d i n such a f a s h i o n t h a t a p y r e x a b s o r p t i o n tube was j o i n e d t o t h e Thunberg Tube. T h i s a l l o w e d d i r e c t measurement o f o p t i c a l d e n s i t y t o be made w i t h a Bausch & Lamb s p e c t r o p h o t o m e t e r . A n a e r o b i c c o n d i t i o n s were o b t a i n e d by means o f e v a c u a t i o n w i t h vacuum pump. F o r i n t e n s e i l l u m i n a t i o n , a 375 w a t t S y l v a n i a f l o o d l i g h t a t d i s t a n c e s o f 10 t o 15 cm was u s e d . To f i l t e r o u t i n f r a r e d waves, t h e l i g h t beam was p a s s e d t h r o u g h a b l u e l i g h t f i l t e r ( K l e t t - S u m m e r s o n L i g h t F i l t e r No. 4 2 ) . The Thunberg tubes were immersed i n a 30° C w a t e r b a t h d u r i n g i l l u m i n a t i o n . 64. I . PURIFICATION OF ENZYME 1. F i r s t Ammonium S u l f a t e F r a c t i o n a t i o n The c r u d e enzyme was d i v i d e d i n t o f o u r p o r t i o n s by f r a c t i o n a l p r e c i p i t a t i o n w i t h ammonium s u l f a t e . The f r a c t i o n s were 0-30, 30-50, 50-70 and 70-90% o f s a t u r a t i o n , and s u p e r -n a t a n t . The a c t i v i t y was f o u n d i n t h e 0-30 and 30-50 f r a c t i o n s : S a t u r a t i o n o f A c t i v i t y o f T o t a l Ammonium S u l f a t e Enzyme Volume (7.) ( U n i t s / m l ) (ml) 177 0-30 30-50 50-70 70-90 S u p e r n a t a n t 122 0 0 0 20 3Q. 20 20 80 T o t a l A c t i v i t y ( U n i t s ) 3540 . 3660 0 0 0 I n a n o t h e r e x p e r i m e n t u s i n g 0-20, 20-45 and 45-907. s a t u r a t i o n s w i t h ammonium s u l f a t e t h e major a c t i v i t y was c o n c e n t r a t e d i n t h e 0-20 and 20-45 f r a c t i o n s : S a t u r a t i o n o f Ammonium S u l f a t e 0-20 20-45 45-90 S u p e r n a t a n t A c t i v i t y o f Enzyme ( U n i t s / m l )  51.3 145.0 0 0 T o t a l T o t a l Volume (ml) A c t i v i t y ( u n i t s ) 20 40 20 80 1026 5800 0 0 65. 2. Second Ammonium S u l f a t e F r a c t i o n a t i o n The f i r s t ammonium s u l f a t e f r a c t i o n ( 0 - 4 5 % s a t u r a t i o n ) was d i v i d e d i n t o f o u r p o r t i o n s by f r a c t i o n a t i o n w i t h ammonium s u l f a t e . The f r a c t i o n s were 0-20, 20-30, and 30-40% s a t u r a t i o n , and s u p e r n a t a n t . The a c t i v i t y was fo u n d t o be i n t h e 20-30 and 30-40 f r a c t i o n s : S a t u r a t i o n o f Ammonium S u l f a t e (%) A c t i v i t y o f Enzyme ( U n i t s / m l ) T o t a l Volume (Ml) T o t a l A c t i v i t y ( U n i t s ) 0-20 12.3 20 246 20-30 189.0 20 3600 30-40 75.0 20 1500 S u p e r n a t a n t 5.4 40 216 I n a n o t h e r e x p e r i m e n t u s i n g 0-22, 22-35, and 35-50% s a t u r a t i o n w i t h ammonium s u l f a t e t h e ma j o r a c t i v i t y was fo u n d i n t h e 22-35 f r a c t i o n : S a t u r a t i o n o f Ammonium S u l f a t e (%) A c t i v i t y o f Enzyme ( U n i t s / m l ) T o t a l Volume (ml) T o t a l A c t i v i t y ( u n i t s ) 0-22 15.1 20 302 22-35 210.0 20 4200 35-50 31.3 20 626 S u p e r n a t a n t 0.0 40 0 66. 3. C a l c i u m Phosphate G e l A d s o r p t i o n The f r a c t i o n w h i c h was b r o u g h t down between 22-357. s a t u r a t i o n was a d s o r b e d by c a l c i u m phosphate g e l . To each 2.0 ml o f t h e enzyme s o l u t i o n , v a r i o u s amounts o f c a l c i u m phosphate s u s p e n s i o n (10 mg d r y w e i g h t p e r ml) and phosphate b u f f e r (pH 7.0) were added. The t o t a l volume was 4.0 m l . The m i x t u r e s were k e p t i n an i c e b a t h and s t i r r e d w i t h s t i r r i n g r o d f o r 5 m i n u t e s , a f t e r w h i c h t h e y were c e n t r i f u g e d and enzyme a c t i v i t y and p r o t e i n were d e t e r m i n e d on t h e s u p e r -n a t a n t . The r e s u l t s a r e g i v e n i n T a b l e V. I t was found t h a t 1.0 ml o f c a l c i u m p h o s p h a t e g e l had a d s o r b e d most o f t h e enzyme p r e s e n t i n t h e m i x t u r e s . T a b l e V A d s o r p t i o n o f N i t r a t e R e d u c t a s e by C a l c i u m Phosphate G e l Enzyme P r e p a r a t i o n C a l c i u m Phosphate Phosphate B u f f e r T o t a l Volume Enzyme A c t i v i t y (ml) (ml) (ml) (ml) ( u n i t s / m l ) 2.0 0.00 2.00 4.00 751 2.0 0.10 1.90 4.00 710 2.0 0.20 1.80 4.00 650 2.0 0.50 1.50 4.00 201 2.0 1.00 1.00 4.00 78 2.0 2.00 0.00 4.00 33 67. . 4. T h i r d Ammonium S u l f a t e F r a c t i o n a t i o n The enzyme e l u t e d from c a l c i u m p hosphate g e l by 0.1 M py r o p h o s p h a t e b u f f e r , pH 7.0, was d i v i d e d i n t o f o u r p o r t i o n s by f r a c t i o n a l p r e c i p i t a t i o n w i t h ammonium s u l f a t e . The f r a c t i o n s were 0-25, 25-50, 50-90 and s u p e r n a t a n t . The a c t i v i t y was found t o be i n t h e 0-25 and i n t h e 25-50 f r a c t i o n s . S a t u r a t i o n o f Ammonium S u l f a t e 1 (7.) A c t i v i t y o f Enzyme ( u n i t s / m l ) T o t a l Volume (ml) T o t a l A c t i v i t y ( u n i t s ) 0-25 184 5 920 25-50 275 5 1350 50-90 0 5 0 S u p e r n a t a n t 0 20 0 As a r e s u l t o f t h e above e x p e r i m e n t , t h e method o f p u r i f i c a t i o n a d o p t e d was as f o l l o w s . U n l e s s o t h e r w i s e s p e c i f i e d , a l l s t e p s o f t h e f r a c t i o n a t i o n were c a r r i e d o u t a t 0-4° C. The p r e c i p i t a t e s were c e n t r i f u g e d a t a p p r o x i m a t e l y 5000 x g a t 0° C f o r 10 m i n u t e s . A p p r o x i m a t e l y 80 g o f t h e l e a v e s o f 5 0 - d a y - o l d s u g a r b e e t p l a n t s was ground i n a W a r i n g B l e n d e r w i t h 320 m l o f 0.1 M -3 p o t a s s i u m phosphate b u f f e r , a t pH 7.8, c o n t a i n i n g 10 M r e d u c e d g l u t a t h i o n e . The homogenate was c e n t r i f u g e d f o r 20 m i n u t e s a t 20,000 x g. The c l e a r , b r o w n i s h y e l l o w s u p e r -n a t a n t s o l u t i o n c o m p r i s e d t h e c r u d e , c e l l - f r e e e x t r a c t ( F r a c t i o n I ) . 68. To t h i s F r a c t i o n I was added 100.80 g s o l i d ammonium s u l f a t e , t h e s u s p e n s i o n was a l l o w e d t o s t a n d f o r 10 m i n u t e s , t h e n was c e n t r i f u g e d f o r 10 m i n u t e s . T h i s 0-457. ammonium s u l f a t e p r e c i p i t a t e i s r e f e r r e d t o as F r a c t i o n I I . The p r e -c i p i t a t e was d i s s o l v e d i n 160 m l o f c o l d 0.1 M p o t a s s i u m -3 p h o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 10 M r e d u c e d g l u t a -t h i o n e . To t h e s o l u t i o n o f F r a c t i o n I I was added 24.60 g s o l i d ammonium s u l f a t e , and a f t e r s t a n d i n g 10 m i n u t e s t h e p r e c i p i t a t e was c e n t r i f u g e d down and d i s c a r d e d . The s u p e r n a t a n t s o l u t i o n was t r e a t e d w i t h a f u r t h e r 14.6 g ammonium s u l f a t e and a g a i n c e n t r i f u g e d . T h i s p r e c i p i t a t e , w h i c h i s t h e 22-357. ammonium s u l f a t e p r e c i p i t a t e ( F r a c t i o n I I I ) , was d i s s o l v e d i n 40 ml o f -3 0.1 M p o t a s s i u m p h o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 10 M r e d u c e d g l u t a t h i o n e , and s t o r e d a t -17° C. A f t e r t h a w i n g , t h e F r a c t i o n I I I s o l u t i o n was d i l u t e d t o 80 ml w i t h p o t a s s i u m phosphate b u f f e r and t r e a t e d w i t h 40 m l o f c a l c i u m p h o s p h a t e g e l (10 mg d r y w e i g h t p e r m l ) . A f t e r 15 m i n u t e s , t h e p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n , washed once w i t h a 5 m l p o r t i o n o f c o l d 0.1 M pho s p h a t e b u f f e r , pH 7.5, and e l u t e d t w i c e w i t h 20 ml o f c o l d 0.1 M p y r o p h o s p h a t e b u f f e r , pH 7.0. The p y r o p h o s p h a t e e l u a t e ( F r a c t i o n IV) showed about 17.37o o f t h e t o t a l a c t i v i t y o f t h e o r i g i n a l c r u d e e x t r a c t and an o v e r a l l p u r i f i c a t i o n o f 20 t i m e s . To F r a c t i o n IV (40 ml) 14 g o f s o l i d ammonium s u l f a t e was added t o g i v e 507o s a t u r a t i o n . A f t e r s t a n d i n g f o r 15 m i n u t e s a t 0 ° C, t h e p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n and d i s s o l v e d i n 20 ml o f c o l d 0.1 M -3 phosphate b u f f e r , pH 7.0, c o n t a i n i n g 10 M r e d u c e d g l u t a t h i o n e . The enzyme a c t i v i t y on t h i s f i n a l f r a c t i o n ( F r a c t i o n V) was 157o o f t h e u n i t i n t h e c r u d e s t a r t i n g m a t e r i a l s . The o v e r a l l p u r i f i c a t i o n was 6 0 - f o l d . Table VI Summary of Purification of Sugar Beet NRase Fraction Total volume Total protein Total units Specific activity Percent recovery (ml) (mg) I. Crude enzyme 320 1856 39,000 21 100 II. 0-45% Ammonium sulfate ppt, 160 • 518 26,400 51 67.7 III. 22-35% Ammonium sulfate ppt. 80 112 14,000 125 35.9 IV. Calcium phosphate gel eluate 40 15 6,750 450 17.5 V. 0-50% ammonium 20 5 6,250 1250 16.0 sulfate ppt. I I . CHARACTERIZATION OF NITRATE REDUCTASE 1, S t a b i l i t y o f Enzyme The e f f e c t o f p r o t e c t a n t s d u r i n g t h e e x t r a c t i o n o f t h e enzyme from s u g a r b e e t l e a v e s i s shown i n F i g . 1. I t -3 i n d i a t e s t h a t i n t h e p r e s e n c e o f 10 M c y s t e i n e i n t h e e x t r a -c t i o n medium, t h e a c t i v i t y o f NRase i s t h r e e t i m e s g r e a t e r t h a n i n t h e media w h i c h c o n t a i n e d no p r o t e c t a n t and t h a t g l u t a t h i o n e i s as e f f e c t i v e as c y s t e i n e . The d a t a r e l a t i v e t o l o s s o f t h e enzyme a c t i v i t y d u r i n g s t o r a g e a r e p r e s e n t e d i n F i g . 2. The p e r c e n t a g e r e s i d u a l a c t i v i t y o f t h e enzyme when s t o r e d a t t h e v a r i o u s tern-p e r a t u r e s has been p l o t t e d a g a i n s t t i m e . I n g e n e r a l , t h e enzyme was found t o be e x t r e m e l y u n s t a b l e , p a r t i c u l a r l y a t t h e h i g h e r t e m p e r a t u r e s . The h i g h e r t h e s t o r a g e t e m p e r a t u r e , t h e f a s t e r t h e r a t e o f i n a c t i v a t i o n . The e x t e n t o f i n a c t i v a t i o n was a l s o d i r e c t l y r e l a t e d t o t h e l e n g t h o f s t o r a g e t i m e . When t h e enzyme was s t o r e d i n a f r o z e n s t a t e (-15° C ) , a l o s s o f o n l y 10 t o 207. o f i t s a c t i v i t y was o b s e r v e d i n t h r e e weeks, r e v e a l i n g t h a t i t was q u i t e s t a b l e i n t h e f r o z e n s t a t e . S t o r a g e a t iP C r e s u l t e d i n a l o s s o f 30 and 607. o f i t s a c t i v i t y i n 6 and 9 h o u r s , r e s p e c t i v e l y . A t 25° C, t h e enzyme l o s t i t s a c t i v i t y a l m o s t c o m p l e t e l y i n 6 h o u r s . 72. CO t 3 NONE EDTA CYSTEINE GLUTATHIONE - H C L I0" 3M 10"3M IO"3M F i g . l . E f f e c t o f p r o t e c t a n t s i n t h e e x t r a c t i o n media on the l e v e l o f n i t r a t e r e d u c t a s e a c t i v i t y i n s u g a r b e e t e x t r a c t s . 73 • 3 6 9 12 15 H O U R S F i g . 2, S t a b i l i t y o f n i t r a t e r e d u c t a s e ( F r a c t i o n V) i n phosphate b u f f e r , pH 7.0, c o n t a i n i n g 10"^ M g l u t a t h i o n e s t o r e d a t v a r i o u s t e m p e r a t u r e s f o r d i f f e r e n t l e n g t h s o f t i m e . 74. 2. Time Course o f E n z y m a t i c R e a c t i o n A t y p i c a l t i m e - c o u r s e o f t h e n i t r i t e f o r m a t i o n from n i t r a t e i s shown i n F i g . 3. I n t h e p r e s e n c e o f t h e c o m p l e t e s y s t e m t h e r a t e o f n i t r a t e r e d u c t i o n i s e s s e n t i a l l y c o n s t a n t w i t h i n 25 m i n u t e s . 3. Enzyme A c t i v i t y and Enzyme C o n c e n t r a t i o n F i g . 4 i l l u s t r a t e s t h e r e l a t i o n s h i p o f t h e concen-t r a t i o n o f enzyme t o t h e amount o f n i t r i t e formed. I t can be seen t h a t a c t i v i t y i s l i n e a r f o r q u a n t i t i e s o f enzyme up t o 80 u n i t s . 4. pH Optimum The e f f e c t o f plH on t h e a c t i v i t y o f t h e enzyme i s shown i n F i g . 5. The enzyme c a t a l y z e d t h e r e d u c t i o n o f n i t r a t e o v e r a w i d e pH r a n g e , 6.0 t o 10.0, w i t h a maximum a c t i v i t y a t pH 7.0. The c u r v e i n d i c a t e s t h a t t h e enzyme has a ••• v e r y s h a r p optimum peak. Below Ph 5.0 no enzyme a c t i v i t y c o u l d be d e t e c t e d . 5. S u b s t r a t e A f f i n i t y The e f f e c t o f n i t r a t e c o n c e n t r a t i o n upon t h e r e a c t i o n v e l o c i t y i s shown i n F i g . 6. The maximum r a t e was o b t a i n e d a t 10 uM p e r m l , and t h e amount n e c e s s a r y f o r t h e o n e - h a l f maximal r a t e was a p p r o x i m a t e l y 0.5 uM p e r m l . The M i c h a e l i s 75 5 10 15 20 25 TINE SN MINUTES F i g . 3. P r o p o r t i o n a l i t y o f enzyme a c t i v i t y w i t h t i m e . ( The s t a n d a r d a s s a y p r o c e d u r e was used w i t h 35 u n i t s o f F r a c t i o n V enzyme. I 76. UNITS OF E N Z Y N E ADDED F i g . 4. P r o p o r t i o n a l i t y o f enzyme a c t i v i t y w i t h enzyme c o n c e n t r a t i o n . The s t a n d a r d a s s a y p r o c e d u r e was used w i t h v a r i a t i o n i n volume o f enzyme ( F r a c t i o n V ) . 77. 5 6 7 3 9 10 pH F i g . 5. E f f e c t o f pH on n i t r a t e r e d u c t a s e a c t i v i t y . The s t a n d a r d a s s a y p r o c e d u r e was u s e d , e x c e p t f o r v a r y i n g pH v a l u e s . 42 u n i t s o f enzyme ( F r a c t i o n V) was u s e d . N I T R A T E CONCENTRATION u M / M L F i g . 6. E f f e c t o f n i t r a t e c o n c e n t r a t i o n o n n i t r a t e r e d u c t a s e a c t i v i t y . 79. c o n s t a n t , o b t a i n e d by t h e method o f L i n e w e a v e r and Burk ( 1 9 3 4 ) , was Km = 4.5 x 1 0 " 4 M p e r l i t e r . 6. S p e c i f i c i t y f o r E l e c t r o n Donor F i g . 7 shows t h a t t h e h i g h l y p u r i f i e d n i t r a t e r e d u c t a s e from sugar b e e t l e a v e s appeared t o be s p e c i f i c f o r DPNH as a s o u r c e o f e l e c t r o n TPNH was c o m p l e t e l y i n a c t i v e as an e l e c t r o n donor. The Km v a l u e f o r DPNH as e s t i m a t e d from t h e c u r v e i n F i g . 7 i s 5.0 x 10 M. As shown i n T a b l e V I I , r e d u c e d FAD, FMN, and m o l y b d a t e were a l l e f f e c t i v e e l e c t r o n donors f o r n i t r a t e r e d u c t a s e . Among t h e a r t i f i c i a l d o n o r s , p y o c y a n i d e and p h e n a z i n e metho-s u l f a t e , b o t h o n e - e l e c t r o n donors w i t h v e r y low r e d o x p o t e n t i a l , g i v e a low r a t e o f enzymic a c t i v i t y . R e d u c i n g a g e n t s as g l u t a t h i o n e and a s c o r b a t e were i n a c t i v e as e l e c t r o n d o n o r s . 7. E f f e c t o f Temperature on A c t i v i t y I n o r d e r t o e v a l u a t e t h e a c t i v i t y o f t h e enzyme a t d i f f e r e n t t e m p e r a t u r e s , t h e r e a c t i o n m i x t u r e s were i n c u b a t e d f o r 15 m i n u t e s a t v a r i o u s t e m p e r a t u r e s . The r e s u l t s a r e shown i n T a b l e V I I I . I t i s shown t h a t a t 0° C t h e r e i s no d e t e c t a b l e e n z y m a t i c a c t i v i t y . From 10° t o 30° C, e n z y m a t i c a c t i v i t y i s p r o p o r t i o n a l t o t h e t e m p e r a t u r e i n c r e a s e . A t 50° C, 33% o f t h e a c t i v i t y r e m a i n e d , a t 60° C o n l y 47.. 80. I I I ! I I 0 1 2 3 4 5 COENZYME CQNCENTRATIONCMXSO"4} F i g . 7. E f f e c t o f r e d u c e d p y r i d i n e n u c l e o t i d e c o n c e n t r a t i o n on n i t r a t e r e d u c t a s e a c t i v i t y . The s t a n d a r d a s s a y p r o c e d u r e was used w i t h 42 u n i t s o f F r a c t i o n V enzyme. 81. Table VII The Effect of Various Hydrogen Donors on NRase Activity Fraction V enzyme was used. The reaction mixtures were incubated anaerobically with the appropriate hydrogen donor in Thunberg tubes. The hydrogen donor was dissolved in 0.1 M phosphate buffer, pH 7.0. Hydrogen donor Hydrogen donor Relative f i n a l activity concentration (M) (%) DPNH 2 x lO" 4 100 FAD* 2 X 85 FMN* 2 X io-* 80 Sodium molybdate* 2 X 10-4 80 Benzyl viologen* 2 X lO" 4 10 Methylene blue 2 X lO" 4 15 Pyocyanime* 1 X io-3 5 Phenazine methosulfate* 1 X 10"3 5 Mammalian cytochrome c* 2 X 10-4 10 Glutathione 1 X 10"3 0 Sodium ascorbate 1 X 10-3 0 Reduced by sodium hydrosulfite 82. T a b l e V I I I E f f e c t o f Temperature on A c t i v i t y o f NRase Temperature N i t r i t e formed R e l a t i v e a c t i v i t y (°C) (m^M) (%) 0 0.00 0 10 8.40 37 20 16.00 74 30 22.80 100 40 28.00 122 50 7.60 33 60 1.00 4 84. F i g u r e 8 i s an A r r h e n i u s p l o t o f t h e v a r i a t i o n o f t h e a c t i v i t y o f t h e enzyme ( F r a c t i o n V) w i t h t e m p e r a t u r e s . D e s p i t e t h e f a c t t h a t NRase i s u n s t a b l e a t h i g h e r t e m p e r a t u r e s a l i n e a r r e l a t i o n s h i p was o b t a i n e d between 20° t o 40° C b e c a u s e o f t h e s h o r t d u r a t i o n o f a s s a y (15 m i n u t e s ) . 8. FAD as P r o s t h e t i c Group The enzyme a c t i v i t y o f F r a c t i o n I was n o t a p p r e c i a b l y a f f e c t e d by t h e a d d i t i o n o f FAD o r FMN a t a f i n a l c o n c e n t r a t i o n -5 o f 10 M. A v e r y marked r e s p o n s e t o FAD, however, was d e m o n s t r a t e d by F r a c t i o n V. The a d d i t i o n o f FAD a t a f i n a l •"6 c o n c e n t r a t i o n o f 10 M c a u s e d a t h r e e f o l d s t i m u l a t i o n . One h a l f maximum r e a c t i o n o f t h e enzyme was o b t a i n e d w i t h FAD a t a p p r o x i m a t e l y 1 x 10 ^ M and w i t h FMN a t about 5 x 10"^ M. T h i s g r e a t e r e f f e c t i v e n e s s o f FAD i n a c t i v a t i n g t h e s u g a r b e e t n i t r a t e r e d u c t a s e s u g g e s t e d t h a t t h e p r o s t h e t i c group o f t h e enzyme i s FAD. The r e l e a s e and chromatography o f t h e f l a v i n from F r a c t i o n V by t h e method o f Rao e t a l . (1962) r e s u l t e d i n a s p o t w h i c h showed a g r e e n i s h f l u o r e s c e n c e . The R f v a l u e s o f t h i s m a t e r i a l and t h o s e o f r i b o f l a v i n , FMN, and FAD r u n i n two s o l v e n t systems ( T a b l e IX) s u g g e s t t h a t t h e s u b s t a n c e r e l e a s e d from F r a c t i o n V NRase i s n o t r i b o f l a v i n o r FMN and t h a t i t may be FAD. 85. 0! I I l I I 2 4 6 8 10 FLAVIN CONCENTRATION ( M X 1 0 " 6 ) F i g . 9. E f f e c t o f f l a v i n n u c l e o t i d e c o n c e n t r a t i o n on n i t r a t e r eductase a c t i v i t y . P r e i n c u b a t e d f o r 5 minutes a t 4° C w i t h a l l components of the r e a c t i o n mixture except DPNH p r i o r to the standard assay. 86. N i t r a t e r e d u c t a s e i s n o t t h e o n l y f l a v i n - c o n t a i n i n g p r o -t e i n p r e s e n t i n t h e enzyme p r e p a r a t i o n , hence p r e c i s e i d e n t i f i c a t i o n o f t h e p r o s t h e t i c group must a w a i t f u r t h e r p u r i f i -c a t i o n o f t h e enzyme. As shown i n T a b l e XV, t h e enzyme a c t i v i t y was s t r o n g l y i n h i b i t e d by a t e b r i n and r i b o f l a v i n , known f l a v o p r o t e i n i n h i b i t o r s (Haas, 1944; W r i g h t and S a b i n e , 1944; H e l l e m a n , L i n d s a y and B o v a r m i c k , 1 9 4 8 ) , a t a f i n a l c o n c e n t r a t i o n o f 10 4 M. The i n h i b i t i o n by a t e b r i n c o u l d be r e v e r s e d by t h e a d d i t i o n of FAD. T a b l e I X Paper Chromatography o f N i t r a t e R e d u c t a s e F l a v i n S o l v e n t System R i b o f l a v i n R f x 100 FMN FAD Compound from F r a c t i o n V s u g a r b e e t NRase n - B u t a n o l a c e t i c a c i d w a t e r (4:1:5) 50 32 19 20 Na 2HP0 4- (5%) 33 55 38 40 9. S u l f h y d r y l N a t u r e P a r t i a l l y p u r i f i e d NRase o f t h e s u g a r b e e t was f o u n d t o be s h a r p l y i n h i b i t e d by p - c h l o r o m e r c u r i b e n z o a t e . T h i s i s 87. s i m i l a r t o t h e r e s u l t s o b t a i n e d by Evans and Nason (1953) f o r NRase o f soybean l e a v e s . T a b l e X summarizes t h e r e s u l t s o f t h e e x p e r i m e n t d e m o n s t r a t i n g t h e i n h i b i t i o n by p-c h l o r o m e r c u r i b e n z o a t e and i t s r e v e r s a l by c y s t e i n e and g l u t a -t h i o n e . P r e i n c u b a t i o n o f t h e enzyme w i t h c o n c e n t r a t i o n s o f -3 -5 10 M and 10 M p - c h l o r o m e r c u r i b e n z o a t e r e s u l t e d i n 100 and 78% i n h i b i t i o n r e s p e c t i v e l y . T a b l e X I n h i b i t i o n o f NRase by p - c h l o r o m e r c u r i b e n z o a t e and i t s R e v e r s a l by S u l f h y d r y l Compounds R e a c t i o n m i x t u r e c o n t a i n e d p - c h l o r o m e r c u r i b e n z o a t e i n d i c a t e d , n i t r a t e r e d u c t a s e ( F r a c t i o n IV) and a l l c o n s t i t u e n t s w i t h t h e e x c e p t i o n o f t h e s u l f h y d r y l compound and e l e c t r o n donor. The m i x t u r e s were p r e i n c u b a t e d f o r 10 m i n u t e s i n i c e b a t h . C y s t e i n e h y d r o c h l o r i d e o r g l u t a t h i o n e was added as i n d i c a t e d , f o l l o w e d by an e q u i v a l e n t amount o f Na^CO^. When no s u l f h y d r y l r e a g e n t s were u s e d , w a t e r was added t o m a i n t a i n e q u a l volume. The m i x t u r e s were a g a i n i n c u b a t e d f o r 5 m i n u t e s a t 4° C and t h e n a s s a y e d f o r n i t r i t e r e d u c t a s e a c t i v i t y i n t h e u s u a l manner. R e a c t i o n p - c h l o r o m e r - C y s t e i n e - H C l G l u t a t h i o n e I n h i b i t i o n c u r i b e n z o a t e No. F i n a l Conc.(M) F i n a l Conc.(M) F i n a l Conc.(M) (%) 1 10"3 - _ 100 2 i o - 5 _ _ 78 3 i o - 3 i o " 3 48 4 i o " 5 i o " 3 18 5 i o " 3 - I O " 3 36 6 i o " 5 _ i o " 3 11 88. 10. D i a l y s i s o f Enzyme N i c h o l a s and Nason (1953) have i n d i c a t e d t h a t t h e d i a l y s i s o f N e u r o s p o r a NRase a g a i n s t c y a n i d e r e s u l t e d i n an i n a c t i v a t i o n o f t h e enzyme and t h a t t h e d i a l y z e d enzyme was r e a c t i v a t e d by molybdenum s a l t s . T h i s was t a k e n t o i n d i c a t e t h a t t h e m e t a l c o n s t i t u e n t s o f t h i s NRase was molybdenum. I n o r d e r t o s t u d y t h e m e t a l c o n s t i t u e n t o f s u g a r b e e t n i t r a t e r e d u c t a s e , t h e f o l l o w i n g e x p e r i m e n t s were c a r r i e d o u t : (a) D i a l y s i s o f N i t r a t e R e d u c t a s e As recommended by N i c h o l a s and Nason (1953) , some o f t h e c e l l o p h a n e d i a l y z i n g tubes t o be employed i n t h e d i a l y s i s o f t h e enzyme were f i r s t u s e d t o d i a l y z e a 2°U s o l u t i o n o f egg a l b u m i n f o r 12 h o u r s a g a i n s t a p h o s p h a t e b u f f e r , pH 7.0, and t h e n were b a t h e d f o r 12 h o u r s i n t h e v a r i o u s d i a l y z i n g s o l u t i o n s l i s t e d i n T a b l e X I . T h r e e m l o f t h e p a r t i a l l y p u r i f i e d enzyme ( F r a c t i o n I I I ) was d i a l y z e d a g a i n s t 500 ml o f t h e d i a l y z i n g s o l u t i o n a t 4^ C. T a b l e X I summarizes t h e r e s u l t s o f t h e e x p e r i m e n t s . D i a l y s i s r e s u l t e d i n a h i g h e r a c t i v i t y o f t h e enzyme. T h i s c o u l d have been t h e consequence o f t h e r e m o v a l o f ammonium s u l f a t e o r o f an endogenous i n h i b i t o r from t h e p r e p a r a t i o n . P r e -t r e a t m e n t o f t h e d i a l y z i n g tubes d i d n o t r e s u l t i n more e f f e c t i v e -3 d i a l y s i s . The p r e s e n c e o f 10 M g l u t a t h i o n e i n t h e d i a l y z i n g s o l u t i o n r e s u l t e d i n a much g r e a t e r a c t i v i t y o f t h e enzyme a t t h e end o f 6 and 12 h o u r s d i a l y s i s . 89 T a b l e X I I n f l u e n c e o f D i a l y s i s A g a i n s t V a r i o u s Reagents on t h e A c t i v i t y o f NRase D i a l y s i s A c t i v i t y o f enzyme* P e r i o d o f d i a l y s i s ( h r s ) 0 3 6 12 E x p e r i m e n t A No d i a l y s i s 25 18 15 9 U n t r e a t e d t u b e . D i a l y s i s a g a i n s t 0,1 M p h o s p h a t e 25 38 21 5 T r e a t e d t u b e . * * D i a l y z e d a g a i n s t 0.1 M p h o s p h a t e c o n t a i n i n g 10"3 M g l u t a t h i o n e 25 40 32 18 E x p e r i m e n t B No d i a l y s i s 23 15 12 10 T r e a t e d t u b e . * * D i a l y z e d a g a i n s t 0.1 M p h o s p h a t e c o n t a i n i n g I O - 3 M g l u t a t h i o n e and I O - 3 M c y a n i d e 10 6 0 0 * U n i t s o f enzyme a c t i v i t y p e r 0.1 m l o f F r a c t i o n I I I enzyme ** Tube b a t h e d f o r 12 h o u r s i n s o l u t i o n l i s t e d and 3 m l o f enzyme p r e p a r a t i o n s u b s e q u e n t l y d i a l y z e d a g a i n s t 500 m l o f t h e same s o l u t i o n a t pH 7.0, a t 4° C. 90. (b) Attempt t o r e a c t i v a t e t h e C y a n i d e - d i a l y z e d Enzyme The p a r t i a l l y p u r i f i e d enzyme ( F r a c t i o n I I I ) was d i a l y z e d f o r 6 hours a t 4° C a g a i n s t 0.1 M phosphate b u f f e r , -3 -3 pH 7.0, c o n t a i n i n g 10 M g l u t a t h i o n e and 10 M p o t a s s i u m c y a n i d e . I t was t h e n r e d i a l y z e d f o r 3 hours a g a i n s t 0.1 M -3 p h osphate b u f f e r , pH 7.0, c o n t a i n i n g 10 M g l u t a t h i o n e t o remove KCN. D i s t i l l e d w a t e r , w h i c h was u s e d t o p r e p a r e t h i s b u f f e r s o l u t i o n , had been t r e a t e d t o remove m e t a l s by t h e method of N i c h o l a s ( 1 9 5 2 ) . A t t h e end o f d i a l y s i s , v a r i o u s m e t a l s were added t o t h e enzyme s o l u t i o n a t a f i n a l c o n c e n t r a t i o n o f 1 mM and i t was p r e i n c u b a t e d a t 4° C f o r 10 m i n u t e s b e f o r e t e s t i n g i t s a c t i v i t y . The f o l l o w i n g compounds were added t o t h e r e a c t i o n m i x t u r e : F e S 0 4 , AgN0 3, N a ^ r ^ , C o C l 2 > N i S 0 4 > MnS0 4, F e C l 3 > Na 2W0 4 Z n S 0 4 , N a 2 M o 0 4 # M p 0 3 # I t was f o u n d t h a t t h e a d d i t i o n o f t h e s e compounds f a i l e d t o r e s t o r e enzyme a c t i v i t y . 11. P h osphate S t i m u l a t i o n I n o r d e r t o i d e n t i f y t h e r e q u i r e m e n t o f phosphate i n t h e r e d u c t i o n o f n i t r a t e , t h e NRase was e x t r a c t e d w i t h 0.1 M T r i s b u f f e r , pH 7.8, and s u b j e c t e d t o 0-45 and 22-357. ammonium s u l f a t e f r a c t i o n a t i o n s . The f i n a l p r e p a r a t i o n was d i s s o l v e d i n 0.1 M T r i s b u f f e r , pH 7.0, and d i a l y z e d a g a i n s t 0.01 M T r i s b u f f e r , pH 7.0, f o r one h o u r a t 4° C. The e f f e c t o f a d d i n g g r a d e d amounts o f phosphate t o 0.1 ml o f t h e p a r t i a l l y p u r i f i e d enzyme i s shown i n T a b l e X I I . I n t h e e x p e r i m e n t , maximal a c t i v i t y was o b t a i n e d a t l e v e l o f a p p r o x i m a t e l y 1.0 uM ph o s p h a t e . I t i s n o t c e r t a i n whether t h e r e q u i r e m e n t f o r ph o s p h a t e i s a b s o l u t e , a p r e p a r a t i o n d i a l y z e d t o r e d u c e p h o s p h a t e may c o n t a i n enough t o a c c o u n t f o r t h e r e s i d u e a c t i v i t y . The r e s u l t s i n T a b l e X I I I show t h a t t h e phosphate c a n be r e p l a c e d p a r t i a l l y by a r s e n a t e and s e l e n a t e . I n s t e a d o f s t i m u l a t i n g , v a n a d a t e and s i l i c a t e i n h i b i t e d t h e enzyme a c t i v i t y . 92. T a b l e X I I A c t i v a t i o n o f NRase by Phosphate Phosphate f i n a l c o n c e n t r a t i o n N i t r i t e formed (uM) (muM) 0.00 40.8 0.05 52.4 0.10 58.0 0.20 60.8 0.50 64.0 1.00 T a b l e X I I I 6§.2 The S u b s t i t u t i o n o f Other A n i o n s f o r P h osphate i n t h e A c t i v a t i o n o f NRase A d d i t i o n F i n a l c o n c e n t r a t i o n N i t r i t e formed S u b s t i t u t i o n (uM) (uM) None 40.0 P o t a s s i u m phosphate 1.0 62.4 -Sodium s u l f a t e 10.0 36.8 0 Sodium t u n g s t a t e 10.0 32.0 0 Sodium a r s e n a t e 10.0 52.0 53 ATP 10.0 34.0 0 Sodium b o r a t e 10.0 21.0 0 Sodium s e l e n a t e 10.0 42.4 10 Sodium o r t h o v a n a d a t e 10.0 10.0 0 Sodium s i l i c a t e 10.0 16.0 0 Sodium m o l y b d a t e 10.0 24.0 0 93. 12. I n h i b i t i o n (a) I n h i b i t i o n by Heavy Metals Sugar beet NRase a c t i v i t y was i n h i b i t e d i n the presence of heavy metals (Table XIV). The addi t i o n of cupric -4 s u l f a t e at 10 M r e s u l t e d i n an i n h i b i t i o n of 1007. i n the -4 rate of reactio n . Sodium vanadate at 10 M also caused a complete i n h i b i t i o n . Other metal compounds such as sodium chromate, cobalt c h l o r i d e , zinc s u l f a t e and sodium asenate also strongly i n h i b i t e d the a c t i v i t y of t h i s enzyme. (b) I n h i b i t i o n by Metal-chelating Agents E f f e c t s of various metal-chelating agents are sum-marized i n Table XV. Sugar beet NRase i s very s e n s i t i v e to KCN and i s i n h i b i t e d approximately 607. at 10 ^ M f i n a l concentration. A number of other chelating agents at higher concentration i n h i b i t e d the enzyme, i n d i c a t i n g the presence of metal as an active constituent i n the system. No i n h i b i t i o n was observed on addition of EDTA or sodium f l u o r i d e and only s l i g h t i n h i b i t i o n was obtained with thiourea. (c) I n h i b i t i o n by Amytal and Atebrin Amytal, a well-known i n h i b i t o r of mitochondria -4 electron transport, at 10 M f i n a l concentration caused approxi-mately 507. i n h i b i t i o n . Atebrin i s a competitive i n h i b i t o r with respect to f l a v i n i n the assay system of n i t r a t e reductase. Its i n h i b i t i o n could be reversed by the addition of FAD, as shown i n Table XVI. (d) I n h i b i t i o n by Phenolic Compounds Several quinones have previously been shown to serve as electron c a r r i e r s for flavoprotein enzymes. Indeed, menadione was found to mediate anaerobic reduction of n i t r a t e ky Ii* c o l i r e s p i r a t o r y NRase. However, the analogy between respi r a t o r y NRase and assimilatory NRase f a i l e d i n t h i s instance, since menadione i s s t r i k i n g l y e f f e c t i v e as an i n h i b i t o r of sugar beet NRase, an assimilatory enzyme. The enzyme was 507. i n h i b i t e d by a menadione concentration of -4 10 M. In view of the unexpected i n h i b i t i o n of sugar beet NRase by manadione, i t appeared of i n t e r e s t to assay the effects of various phenolic compounds on NRase of t h i s plant. Of p a r t i c u l a r i n t e r e s t were the findings with benzoquinone and pyrocatechol. As shown i n Table XVII, the presence of 10 M p-benzoquinone or pyrocatechol resulted i n 507. i n h i b i t i o n of n i t r a t e reduction. Other phenolic compounds such as hydroxyquinone, 4-carboxycatechol, phloroglucinol and pyro-g a l l o l a l l i n h i b i t e d the enzyme a c t i v i t y at d i f f e r e n t rates. 95. T a b l e XIV The E f f e c t o f Heavy M e t a l s on t h e A c t i v i t y o f NRase R e a c t i o n m i x t u r e s c o n t a i n e d t h e compounds i n d i c a t e d , F r a c t i o n V enzyme, and a l l c o n s t i t u e n t s o f t h e s t a n d a r d a s s a y m i x t u r e w i t h t h e e x c e p t i o n o f DPNH. These were p r e i n c u b a t e d f o r 10 m i n u t e s a t 4°C; t h e n t h e s t a n d a r d a s s a y was i n i t i a t e d by a d d i t i o n o f t h e e l e c t r o n d o n or. A d d i t i o n s I n h i b i t i o n a: N i S 0 4 37 N a 2 C r O ? 61 FeS04 21 C o C l 2 40 AgN03 89 CUSO4 100 Na 2W0 4 35 N a 2 V 0 4 100 N a A s 0 2 68 Z n S 0 4 71 MnS04 45 * A t a f i n a l c o n c e n t r a t i o n o f 1 x 10 M. 96. T a b l e XV The E f f e c t o f V a r i o u s M e t a l - c h e l a t i n g A g e nts on t h e A c t i v i t y o f NRase R e a c t i o n m i x t u r e c o n t a i n e d t h e i n h i b i t o r s i n d i c a t e d , F r a c t i o n V, and a l l t h e c o n s t i t u e n t s o f t h e s t a n d a r d a s s a y m i x t u r e w i t h t h e e x c e p t i o n o f e l e c t r o n donor. These were p r e i n c u b a t e d f o r 10 m i n u t e s a t 4°C; t h e n t h e s t a n d a r d a s s a y was i n i t i a t e d by t h e a d d i t i o n o f DPNH. A d d i t i o n F i n a l c o n c e n t r a t i o n I n h i b i t i o n — _ P o t a s s i u m c y a n i d e 1 X I O " 3 100 1 X I O " 4 94 1 X 10-5 65 Sodium a z i d e 1 X I O " 3 100 1 X I O " 4 91 1 X I O " 5 52 T i r o n 1 X I O " 4 45 T h i o u r e a 1 X I O " 3 40 a, a - D i p y r i d y l 1 X I O " 4 71 8 - H y d r o x y q u i n o l i n e 1 X i o - 4 64 P h e n y l h y d r a z i n e 1 X 10" 4 42 o - P h e n a n t h r o l i n e 1 X 10~4 38 Sodium d i e t h y l -d i t h i o c a r b a m a t e 1 X 10" 3 35 Sodium c i t r a t e 1 X I O " 3 37 EDTA 1 X I O " 3 0 Sodium f l u o r i d e 1 X 10~3 0 T a b l e XVI The E f f e c t o f N o n - s p e c i f i c I n h i b i t o r s on t h e A c t i v i t y o f NRase R e a c t i o n m i x t u r e s c o n t a i n e d t h e i n h i b i t o r s i n d i c a t e d , F r a c t i o n V, and a l l c o n s t i t u e n t s o f t h e s t a n d a r d a s s a y m i x t u r e w i t h t h e e x c e p t i o n o f e l e c t r o n donor. These were p r e i n c u b a t e d f o r 10 m i n u t e s a t 4° C; t h e n t h e s t a n d a r d a s s a y was i n i t i a t e d by a d d i t i o n o f e l e c t r o n donor. A d d i t i o n F i n a l c o n c e n t r a t i o n I n h i b i t i o n (M) <%) E t h y l u r e t h a n e 1 0 " 4 24 H y d r o x y l a m i n e h y d r o c h l o r i d e 1 0 " 4 41 Sodium a m y t a l 1 0 " 4 48 A t e b r i n l O " 4 73 A t e b r i n l O " 5 51 A t e b r i n , FAD 1 0 " 5 , 1 0 " 4 0 2 , 4 - D i n i t r o p h e n o l l O " 4 30 Sodium s u l f i t e l O " 4 0 R i b o f l a v i n 1 0 ~ 4 41 I o d o a c e t a t e l O " 4 54 98. T a b l e X V I I The E f f e c t o f P h e n o l i c Compounds on t h e A c t i v i t y o f NRase R e a c t i o n m i x t u r e s c o n t a i n e d t h e p h e n o l i c compound i n d i c a t e d , F r a c t i o n V enzyme, and a l l c o n s t i t u e n t s o f t h e s t a n d a r d a s s a y m i x t u r e w i t h t h e e x c e p t i o n o f e l e c t r o n donor. These were p r e -i n c u b a t e d f o r 10 m i n u t e s a t 4° C; t h e n t h e s t a n d a r d a s s a y was i n i t i a t e d by a d d i t i o n o f DPNH. A d d i t i o n F i n a l c o n c e n t r a t i o n I n h i b i t i o n (M) a: p-Ben zo q u i n o n e 1 X 1 0 " 4 100 1 X io'5 94 1 X io"6 58 P y r o c a t e c h o l 1 X io'4 100 1 X io'5 85 1 X io"6 41 Menadione 1 X io" 4 48 1 X io"5 20 H y d r o x y q u i n o n e 1 X io"4 52 4-Carb oxyc a t echo1 1 X io"4 45 P h l o r o g l u c i n o l 1 X io"4 38 P y r o g a l l o l 1 X io'4 48 99. 13. S e p a r a t i o n o f D P N H - n i t r a t e R e d u c t a s e from T P N H - n i t r a t e  R e d u c t a s e D u r i n g t h e c o u r s e o f t h i s i n v e s t i g a t i o n each f r a c t i o n was examined f o r p y r i d i n e n u c l e o t i d e s p e c i f i c i t y . I t was fou n d t h a t F r a c t i o n I , I I , and I I I enzyme p r e p a r a t i o n c o u l d use e i t h e r DPNH o r TPNH as e l e c t r o n donor. However, r a t e s w i t h DPNH were g r e a t e r t h a n w i t h TPNH i n t h e range o f two t o f i v e t i m e s . When t h e enzyme was e l u t e d from c a l c i u m phosphate g e l and t h e n p r e c i p i t a t e d by ammonium s u l f a t e (0-507. s a t u r a t i o n ) , o n l y DPNH c o u l d be us e d as e l e c t r o n donor ( T a b l e X V I I I ) . The r a t i o s o f a c t i v i t y between DPNH and TPNH f o r t h e F r a c t i o n I , I I , and I I I were 5.1, 2,4, and 3.7 r e s p e c t i v e l y . Due t o t h e f a c t t h a t t h e r a t i o s i n t h e t h r e e f r a c t i o n s d i f f e r e d , i t seems l o g i c a l t o p o s t u l a t e t h a t more t h a n one enzyme was i n v o l v e d . 14. Other E n z y m a t i c A c t i v i t y P r e s e n t i n P a r t i a l l y P u r i f i e d Enzyme The p a r t i a l l y p u r i f i e d n i t r a t e r e d u c t a s e p r e p a r a t i o n s c o n t a i n o t h e r e n z y m a t i c a c t i v i t y . T h i s i n c l u d e d DPNH-quinone r e d u c t a s e , DPNH-manadione r e d u c t a s e , DPNH-cytochrome c r e d u c t a s e and DPNH-diaphorase. Other p y r i d i n e n u c l e o t i d e enzymes such as D P N H - g l u t a t h i o n e r e d u c t a s e ( R a c k e r , 1955) and n i t r i t e r e d u c t a s e (Roussos and Nason, 1960) a r e n o t p r e s e n t i n t h e enzyme p r e -p a r a t i o n F r a c t i o n V, As i n d i c a t e d i n T a b l e X I X , t h e f i r s t two enzymes, DPNH-quinone r e d u c t a s e and DPNH-menadione r e d u c t a s e , seem t o have been p u r i f i e d t o g e t h e r w i t h n i t r a t e r e d u c t a s e . Each s t e p i n t h e p u r i f i c a t i o n o f NRase r e s u l t e d i n a p u r i f i c a t i o n o f o t h e r enzymes, a l t h o u g h t h e degree o f p u r i f i c a t i o n i n each c a s e i s l e s s t h a n t h a t a c h i e v e d f o r t h e NRase. 15. P r o d u c t s and S t o i c h i o m e t r y o f R e a c t i o n The s t o i c h i o m e t r y o f su g a r b e e t NRase was d e t e r m i n e d by m e a s u r i n g t h e c o n c o m i t a n t o x i d a t i o n o f DPNH and t h e f o r -m a t i o n o f n i t r i t e i n r e a c t i o n m i x t u r e s w i t h e x a c t l y t h e same c o m p o s i t i o n as t h o s e employed i n t h e s t a n d a r d a s s a y . DPNH o x i d a t i o n was o b s e r v e d a t v a r i o u s i n t e r v a l s d u r i n g t h e r e a c t i o n p e r i o d by m e a s u r i n g t h e d e c r e a s e i n a b s o r p t i o n , a t 340 mu, o f a l i q u o t s o f r e a c t i o n m i x t u r e m a i n t a i n e d i n s p e c t r o -p h o t o m e t r i c c u v e t t e s . The e x t i n c t i o n c o e f f i c i e n t 6.22 x 10 s q . cm. x mole ^ a t 340 mu was u s e d t o compute DPNH o x i d a t i o n a f t e r f i r s t s u b t r a c t i n g t h e endogenous a c t i v i t y o b s e r v e d i n t h e absence o f n i t r a t e . A t t h e same t i m e , o t h e r a l i q u o t s o f the r e a c t i o n m i x t u r e s were removed and a s s a y e d . The t i m e c o u r s e o f t h e su g a r b e e t NRase i s p r e s e n t e d i n F i g . 10. T h e r e was a v e r y r a p i d d e c r e a s e i n a b s o r p t i o n i n t h e p r e s e n c e o f t h e c o m p l e t e s y s t e m , and a moderate endogenous r a t e when a l l cxmasfcitfcuaeitlss e x c e p t n i t r a t e were 101 T a b l e X V I I I S e p a r a t i o n o f DPNH-NRase from TPNH-NRase The s t a n d a r d a s s a y , w i t h t h e e x c e p t i o n o f t h e f i n a l c o n c e n t r a t i o n o f e l e c t r o n donors b e i n g 1 uM, was u s e d . F r a c t i o n Enzyme A c t i v i t y DPNH TPNH R a t i o o f A c t i v i t y DPNH/TPNH ( u n i t s / m g p r o t e i n ) I . Crude e x t r a c t 15.3 3.0 5.1 I I . 0-457. Ammonium s u l f a t e pp. 36.0 15.0 2.4 I I I . 22-357. Ammonium s u l f a t e p p t . 81.0 21.9 3.7 IV. C a l c i u m p h o s p h a t e g e l e l u a t e 324.0 - -V. 0-507. Ammonium s u l f a t e p p t . 975.0 102. T a b l e X I X Ot h e r E n z y m a t i c A c t i v i t i e s P r e s e n t i n t h e v a r i o u s NRase f r a c t i o n s Enzyme I Enzyme f r a c t i o n I I V N i t r a t e r e d u c t a s e S p e c i f i c a c t i v i t y 21 125 1250 P u r i f i c a t i o n 6 60 Quinone r e d u c t a s e S p e c i f i c a c t i v i t y 175 1245 3750 P u r i f i c a t i o n 7.1 21.3 Menadione r e d u c t a s e S p e c i f i c a c t i v i t y 158 1016 2530 P u r i f i c a t i o n 6.4 16.0 Cytochrome c r e d u c t a s e S p e c i f i c a c t i v i t y 0.75 1.75 4.62 P u r i f i c a t i o n 2.3 6.1 D i a p h o r a s e S p e c i f i c a c t i v i t y 1.24 1.53 5.75 P u r i f i c a t i o n 1.20 4.79 p r e s e n t . The a d d i t i o n o f t h e D P N H - r e g e n e r a t i n g s y s t e m c o n t a i n i n g sodium l a c t a t e and y e a s t l a c t a t e dehydrogenase r e s t o r e d t h e a b s o r p t i o n o f DPNH t o t h e o r i g i n a l l e v e l . Thus, DPN + was shown t o be one o f t h e p r o d u c t s o f sugar beet NRase, T a b l e XX shows a mole f o r mole r e l a t i o n s h i p between DPNH o x i d a t i o n and n i t r i t e f o r m a t i o n . These r e s u l t s a r e i n agreement w i t h t h e c o n c l u s i o n t h a t n i t r a t e r e d u c t i o n by t h i s s y s t e m p r o c e e d s a c c o r d i n g t o t h e f o l l o w -i n g e q u a t i o n : N0~ + DPNH + H + > NO" + DPN + + H, 104. T a b l e XX S t o i c h i o m e t r y o f NRase R e a c t i o n The e n z y m a t i c r a t e o f DPNH o x i d a t i o n was c a l c u l a t e d from Curves A and B o f F i g . 10. N i t r i t e formed was d e t e r m i n e d i n 0.5 m l a l i q u o t s o f t h e c o m p l e t e r e a c t i o n m i x t u r e under t h e c o n d i t i o n o f s t a n d a r d a s s a y . Time o f DPNH N i t r i t e R a t i o o f i n c u b a t i o n O x i d a t i o n formed DPNH N i t r i t e o x i d i z e d formed (min.) (mu moles/ml) (mu moles/ml) 0 0.0 0.0 0,00 2 11.2 10.8 1.03 4 24.1 21.5 1.12 5 36.9 34.5 1.07 8 50.6 45.6 1.10 10 61.0 56.7 1.07 12 ; 70.7 65.5 1.03 14 78.7 70.7 1,10 16 85.2 75.4 1.11 18 86.0 76.9 1.10 105. 4 8 12 16 2 0 T I M E ( M I N U T E S ) F i g 1 . 10. Time c o u r s e o f n i t r a t e r e d u c t a s e a c t i v i t y as f o l l o w e d by t h e o x i d a t i o n o f DPNH. R e a c t i o n m i x t u r e s con-t a i n e d the f o l l o w i n g i n a f i n a l volume o f 3.0 m l : Curve A r e p r e s e n t s the complete r e a c t i o n m i x t u r e c o n t a i n i n g 220 u n i t s o f t h e enzyme ( F r a c t i o n V ) , 60 uM KN0 3, 0.6 uM DPNH, 60 muM FAD and 600 uM phosphate b u f f e r a t pH 7 . 0 ; c u r v e B r e p r e s e n t s the same as Curve A, e x c e p t t h a t KNO3 was o m i t t e d . Curve C r e p r e s e n t s the same as c u r v e A, e x c e p t t h a t the enzyme was o m i t t e d . A f t e r 18 m i n u t e s , the complete r e a c t i o n m i x t u r e was poured i n t o a s m a l l b eaker c o n t a i n i n g 2.5 ml o f 0.1 M g l y c i n e b u f f e r , pH 10.2, 0.3 m l o f 15% sodium D L - l a c t a t e and 0.2 m l o f l a c t i c dehydrogenase and the r e a c t i o n m i x t u r e r e a d a g a i n a t 340 mu. S u b s e q u e n t l y i n d i ~ c a t e d A 340 r e a d i n g r e p r e s e n t s the o r i g i n a l r e a d i n g s x 2. 106. I I I . EFFECT OF AGE OF PLANT ON NITRATE REDUCTASE ACTIVITY The e f f e c t o f age o f p l a n t on t h e l e v e l o f e x t r a c t a b l e NRase i s shown i n T a b l e X X I . The r e s u l t s - i n d i c a t e t h a t t h e c r u d e e x t r a c t from t h e l e a f o f two-weeks o l d s u g a r b e e t s e e d l i n g s c o n t a i n e d a v e r y low enzyme a c t i v i t y . The l e v e l o f enzyme a c t i v i t y i n c r e a s e d r a p i d l y between f o u r and s i x weeks o f g r o w i n g . D u r i n g t h i s t i m e a c o n c o m i t a n t i n c r e a s e i n l e a f a r e a was o b s e r v e d . The a c t i v i t y became c o n s t a n t from t h e age o f 8 weeks. T a b l e X X I E f f e c t o f Age o f P l a n t on NRase A c t i v i t y i n t h e Crude E x t r a c t o f Sugar Beet L e a v e s . Age o f P l a n t Enzyme P r o t e i n S p e c i f i c A c t i v i t y C o n t e n t A c t i v i t y (weeks) ( u n i t s / g f.wt.) (mg/g f.wt.) 2 71 9.5 7.4 4 212 14.4 14.7 6 457 18.3 24.4 8 504 22.7 21.3 10 465 21.2 21.4 12 495 24.3 20.3 107. IV. DISTRIBUTION OF NITRATE REDUCTASE IN THE SUGAR BEET PLANT The d i s t r i b u t i o n o f n i t r a t e and NRase a c t i v i t y i n t h e s u g a r b e e t p l a n t i s g i v e n i n T a b l e X X I I . I n g e n e r a l , t h e c o n c e n t r a t i o n o f n i t r a t e i n t h e p e t i o l e was f i v e t o t e n t i m e s h i g h e r t h a n t h a t i n t h e l a m i n a . A v e r y low c o n t e n t o f n i t r a t e was found i n t h e r o o t s . T a b l e X X I I a l s o shows t h a t t h e a c t i v i t y o f NRase o f t h e l a m i n a was r o u g h l y t r i p l e t h a t o f t h e p e t i o l e . No a p p r e c i a b l e enzyme a c t i v i t y c o u l d be d e t e c t e d i n k t h e e x t r a c t s o f r o o t s . T a b l e X X I I D i s t r i b u t i o n o f NRase A c t i v i t y i n B e t a v u l g a r i s S e e d l i n g s R e g i o n s Dry wt. C h l o r o p h y l l N i t r a t e P r o t e i n NRase mg/g f r e s h ug/mg d r y ug NO* ug/mg u n i t s / m g wt. w t . N/mg d r y wt. d r y wt. d r y wt. Lamina 98.5 9.71 3.55 167.51 3.60 P e t i o l e 76.0 1.58 16.44 63.15 1.32 Root 185.0 0.00 1.02 29.12 0.00 108. V. SUB-CELLULAR LOCALIZATION OF SUGAR BEET NITRATE REDUCTASE The s u b - c e l l u l a r d i s t r i b u t i o n o f NRase from s u g a r b e e t l e a v e s ( l a m i n a ) i s summarized i n T a b l e X X I I I . An a v e r a g e o f 607o o f t h e t o t a l NRase was r e c o v e r e d a f t e r f r a c t i o n a t i o n . N e a r l y 907. o f t h e r e c o v e r e d enzyme a c t i v i t y was found i n t h e m i c r o s o m e - f r e e s u p e r n a t a n t . Only a s l i g h t e n z y m a t i c a c t i v i t y r e s i d e d i n c h l o r o p l a s t and i n m i t o -c h o n d r i a l f r a c t i o n s . C h l o r o p h y l l was p r e s e n t i n a l l c e l l u l a r f r a c t i o n s . A c o n c o m i t a n t r e l e a s e o f NRase from r u p t u r e d c h l o r o p l a s t s c o u l d have o c c u r r e d i f t h e enzyme were i n d e e d p r e s e n t w i t h i n t h e c h l o r o p l a s t . The method o f S t o c k i n g (1959) w h i c h employed a non-aqueous medium was u s e d t o c l a r i f y t h i s p o i n t . F o l l o w i n g S t o c k i n g ' s p r o c e d u r e , 5 g o f s u g a r b e e t l e a v e s was removed and p l a c e d i n a c e l l o p h a n e bag. T h i s was s t o r e d a t -15° C f o r 30 m i n u t e s . The m i d v e i n s and t h e major l a t e r a l v e i n s were t h e n removed from t h e s u g a r b e e t l e a v e s and d i s c a r d e d . The m a t e r i a l . w a s t h e n ground i n a homogenizer f o r 2 m i n u t e s w i t h a c o l d h e x ane-carbon t e t r a c h l o r i d e m i x t u r e o f s p e c i f i c g r a v i t y 1.3, u s i n g 20 ml o f h e x a n e - C C l ^ p e r gram o f t i s s u e . T h i s s u s p e n s i o n was f i l t e r e d t h r o u g h a pad o f g l a s s w o o l and c h e e s e c l o t h and c e n t r i f u g e d f o r 15 m i n u t e s a t 12,000 x g. The g r e e n p e l l e t , w h i c h a c c u m u l a t e d n e a r t h e s u r f a c e 109. o f t h e l i q u i d , was c o l l e c t e d . The g r e e n p e l l e t was d i s s o l v e d i n 100 ml o f hexane, s t i r r e d w i t h a g l a s s r o d , and c e n t r i -f u g e d f o r 5 m i n u t e s a t 1000 x g. The sediment o b t a i n e d from t h i s c e n t r i f u g a t i o n was d i s s o l v e d i n 10 m l o f 0.01 M phosphate -3 b u f f e r c o n t a i n i n g 10 M g l u t a t h i o n e . A f t e r s t a n d i n g a t a t e m p e r a t u r e o f -4° C f o r 10 m i n u t e s , t h e s u s p e n s i o n was c e n t r i f u g e d f o r 10 m i n u t e s a t 1000 x g. The s u p e r n a t a n t was c o l l e c t e d and t e s t e d f o r NRase a c t i v i t y . No e n z y m a t i c a c t i v i t y c o u l d be d e t e c t e d i n t h e e x t r a c t Of c h l o r o p l a s t s . T a b l e X X I I I S u b - C e l l u l a r L o c a l i z a t i o n o f Sugar Beet NRase G r a v i t y Time F r a c t i o n T o t a l T o t a l T o t a l c h l o r o p h y l l p r o t e i n NRase (xg) (min.) (mg) (mg) ( u n i t ] homogenate* 13.00 265.00 1240 200 2 C e l l u l a r d e b r i s and n u c l e i 1.04 10.43 ** 1,000 7 C h l o r o p l a s t s 8.12 79.26 45 10,000 30 M i t o c h o n d r i a 3.49 6.40 27 105,000 30 "Microsome" 0.22 15.80 ** S u p e r n a t a n t 0.13 114.75 839 * S t a r t i n g m a t e r i a l : 5 g o f s u g a r b e e t l a m i n a ** No d e t e c t a b l e enzyme a c t i v i t y 110. V I . FLAVIN NUCLEOTIDE-CATALYZED ENZYMATIC PHOTOREDUCTION OF NITRATE 1. P h o t o r e d u c t i o n o f F l a v i n N u c l e o t i d e s The t i m e c o u r s e o f b o t h FAD and FMN p h o t o r e d u c t i o n under a n a e r o b i c c o n d i t i o n s i s p r e s e n t e d i n F i g . 11 and F i g . 12. From t h e d a t a g i v e n i t i s n o t e d t h a t t h e FMN was p h o t o r e d u c e d a t a moderate r a t e i n t h e absence o f EDTA o r m e t h i o n i n e b u t t h a t t h e a d d i t i o n o f t h e s e a c t i v a t o r s m a r k e d l y s t i m u l a t e d t h e r a t e o f p h o t o r e d u c t i o n o f f l a v i n n u c l e o t i d e . S i m i l a r l y , t h e p r e s e n c e o f EDTA o r m e t h i o n i n e was r e q u i r e d b e f o r e any p h o t o -r e d u c t i o n o f FAD was o b s e r v e d . 2. C h a r a c t e r i z a t i o n o f F l a v i n N u c l e o t i d e - C a t a l y z e d E n z y m a t i c  P h o t o r e d u c t i o n o f N i t r a t e T a b l e XXIV shows t h a t f l a v i n n u c l e o t i d e - c a t a l y z e d enzymic p h o t o r e d u c t i o n o f n i t r a t e can c o u p l e w i t h t h e f l a v i n n u c l e o t i d e p h o t o c h e m i c a l r e d u c t i o n i n w h i c h EDTA a c t s as an e l e c t r o n donor i n an a n a e r o b i c p h o t o c h e m i c a l r e d u c t i o n o f f l a v i n n u c l e o t i d e s . The r e a c t i o n d i d n o t p r o c e e d i n t h e d a r k and i n t h e absence o f n i t r a t e o r s u g a r b e e t NRase. FAD c o u l d be r e p l a c e d by FMN a t t h e same c o n c e n t r a t i o n . I n t h e absence o f EDTA or under a e r o b i c c o n d i t i o n , a s l i g h t p h o t o r e d u c t i o n was o b s e r v e d . The h i g h e s t a c t i v i t y was o b t a i n e d by t h e a d d i t i o n o f DPNH. 1U. TTME IN MINUTES F i g . 11. P h o t o r e d u c t i o n o f FAD and FMN w i t h EDTA under a n a e r o b i c c o n d i t i o n s . The r e a c t i o n m i x t u r e c o n s i s t e d o f 40 uM phosphate b u f f e r , pH 7.5, 0.1 uM FAD of FMN, and 7.5 uM EDTA i n a f i n a l volume o f 2.0 m l . The p r o c e d u r e f o r o b t a i n i n g a n a e r o b i c i t y and t h e ad a p t e d Thunberg tubes used a r e d e s c r i b e d i n " M a t e r i a l s and Methods". m e t h i o n i n e under a n a e r o b i c c o n d i t i o n s . The r e a c t i o n m i x t u r e c o n s i s t e d o f 40 uM phosphate b u f f e r , pH 7.5, 0.1 uM FAD o r FMN, and 7.5 uM m e t h i o n i n e i n a f i n a l volume o f 2.0 m l . The p r o -cedure f o r o b t a i n i n g a n a e r o b i c i t y and t h e adapted Thunberg tubes used a r e d e s c r i b e d i n " M a t e r i a l s and Methods". 113. T a b l e XXIV C h a r a c t e r i z a t i o n o f F l a v i n N u c l e o t i d e - C a t a l y z e d Enzymic P h o t o r e d u c t i o n o f N i t r a t e The c o m p l e t e r e a c t i o n m i x t u r e c o n s i s t e d o f 0.2 m l 0.1 M KNO,, 0.3 m l -4 10 M FAD (FMN), 0.3 m l s u g a r b e e t NRase ( F r a c t i o n I V ) , and 1.7 ml -2 0.1 M phosphate b u f f e r , pH 7.5, c o n t a i n i n g 10 M EDTA ( o r m e t h i o n i n e ) i n a f i n a l volume of 2.5 m l . The a d a p t e d Thunberg tub e s and t h e p r o -c e d u r e f o r o b t a i n i n g a n a e r o b i c i t y a r e d e s c r i b e d under "Methods". L i g h t i n t e n s i t y 375 f o o t c a n d l e s , b l u e f i l t e r , t e m p e r a t u r e 30° C. System N i t r i t e Formed (muM) 56 0 0 6 0 60 24 5 0 70 Complete NRase o m i t t e d N i t r a t e o m i t t e d EDTA o m i t t e d , m e t h i o n i n e added FAD o m i t t e d FAD o m i t t e d , FMN added FAD o m i t t e d , R i b o f l a v i n added C o m p l e t e , a e r o b i c C o m p l e t e , d a r k Complete, DPNH (1 mM) added 114. V I I . DIURNAL CHAN1SES IN NITRATE CONTENT AND NITRATE REDUCTASE ACTIVITY IN SUGAR BEET LEAVES I t was found t h a t t h e s u g a r b e e t l e a v e s h a r v e s t e d a t the end o f t h e d a r k p e r i o d always c o n t a i n e d a l o w e r NRase a c t i v i t y t h a n t h o s e h a r v e s t e d a t t h e end o f t h e l i g h t p e r i o d . I n o r d e r t o d e t e r m i n e t h e e f f e c t o f t h e l i g h t and d a r k on t h e a c t i v i t y o f NRase and t h e c o n t e n t o f n i t r a t e , t h e f o l l o w i n g e x p e r i m e n t was c a r r i e d o u t . The p l a n t s were grown i n a c o n t r o l l e d room a t a day (12 h o u r s ) t e m p e r a t u r e o f 22-25° C and a n i g h t (12 Hours) t e m p e r a t u r e o f 20-22° C. The r e l a t i v e h u m i d i t y was k e p t 62-70% a t day and 65-807. a t n i g h t . I l l u m i n a t i o n was by s l i m l i n e f l u o r e s c e n t tubes supplemented w i t h 60 w a t t i n c a n -d e s c e n t b u l b s i n o r d e r t o g i v e a l i g h t i n t e n s i t y o f 1800 f o o t c a n d l e s a t t h e t o p o f t h e p l a n t s . When t h e p l a n t s were 50 days o l d , t h e l a m i n a p o r t i o n o f t h e l e a v e s were removed a t 3, 6 and 12 hour i n t e r v a l s i n l i g h t and i n d a r k n e s s . T w e n t y - f i v e g o f l a m i n a from each h a r v e s t was u s e d f o r t h e d e t e r m i n a t i o n o f n i t r a t e c o n t e n t and NRase a c t i v i t y . The r e m a i n d e r (60 - 80g) a f t e r w e i g h i n g , was d r i e d i n an oven a t 75° t o 80° C f o r 24 hours and t h e d r y w e i g h t d e t e r m i n e d . 115 The r e s u l t s ( T a b l e XXV) i n d i c a t e d t h a t NRase a c t i v i t y o f s u g a r b e e t l a m i n a v a r i e d d i u r n a l l y w i t h a minimum a t end o f dark p e r i o d and maximum a t end o f l i g h t p e r i o d . Maximum a c t i -v i t y was a l m o s t t h r e e t i m e s minimum. The same degree o f v a r i a t i o n p e r s i s t e d when t h e a c t i v i t y was c a l c u l a t e d on t h e b a s i s o f d r y w e i g h t . T h e r e was a l s o a rhythm i n n i t r a t e c o n t e n t , w h i c h was c o n t r a r y t o t h a t o f t h e NRase a c t i v i t y , b e i n g h i g h e s t a t t h e end o f t h e da r k and l o w e s t a t t h e end o f t h e l i g h t p e r i o d . When e q u a l volumes o f t h e homogenate from m a t e r i a l c o l l e c t e d a t t h e end o f t h e l i g h t and da r k p e r i o d were m i x e d , i t was f o u n d t h a t e n z y m a t i c a c t i v i t y o f t h e m i x e d homogenate was c l o s e t o t h e a r i t h m e t i c mean o f t h e i n d i v i d u a l a c t i v i t y o f t h e two sa m p l e s . T h i s i n d i c a t e d t h a t an a c c u m u l a t i o n o f an i n h i b i t o r i n t h e l e a v e s had n o t o c c u r r e d d u r i n g t h e da r k p e r i o d . The a c i d i t y o f t h e j u i c e o f s u g a r b e e t l e a v e s was f o l l o w e d d u r i n g a 24-hour p e r i o d . The a c i d i t y was h i g h e r a t t h e end o f t h e da r k p e r i o d (pH 5.7-6.0) and l o w e r a t t h e end o f t h e l i g h t p e r i o d (pH 6.5-6.7). 116. Table XXV Dirurnal Change in Nitrate Content and NRase Activity in Sugar Beet Leaves Time of collection of sample Dry Wt. Nitrate Content NRase per units gram mg/g fresh wt. ug NO"-fresh •N/g. wt. ug NO^ N/mg dry wt. fresh wt. dry wt. Light 3 hours 96.2 598 6.21 158 1.6 6 hours 98.5 399 4.03 310 3.1 12 hours 103.4 345 3.33 384 3.7 Dark 3 hours 94.8 474 5.01 245 2.6 6 hours 90.5 522 5.76 165 1.8 12 hours 91.1 786 8.26 125 1.3 117. V I I I . OTHER METHODS OF ENZYME PURIFICATION 1. E x t r a c t i o n o f NRase from A c e t o n e Powder A c e t o n e - d r i e d powders were p r e p a r e d by t h e method d e s c r i b e d by Nason (1955) f o r s o l u b l e enzyme from h i g h e r p l a n t s . A l l s t e p s i n t h e f o l l o w i n g p r o c e d u r e were c a r r i e d out a t -10° C. Twenty grams o f su g a r b e e t l e a v e s was p l a c e d i n a Wa r i n g B l e n d o r w i t h 200 ml o f a c e t o n e and b l e n d e d f o r one m i n u t e . The r e s u l t i n g s l u r r y was f i l t e r e d t h r o u g h a Buchner f u n n e l and washed w i t h an e x c e s s o f a c e t o n e . The r e s i d u e was s p r e a d out on f i l t e r p a p e r , a l l o w e d t o d r y and was s t o r e d i n an e v a c u a t e d f l a s k . A c r u d e e x t r a c t o f t h i s l e a f a c e t o n e powder was p r e -p a r e d by s t i r r i n g t h e powder w i t h 100 ml o f 0.1 M KgHPO^ c o n t a i n i n g 10 3 M g l u t a t h i o n e f o r 5 m i n u t e s a t 0° C. A sub-sequent c e n t r i f u g a t i o n was c a r r i e d o u t a t 4° C a t a f o r c e o f 20,000 x g f o r 15 m i n u t e s . The r e s u l t s a r e g i v e n i n T a b l e XXVI. They i n d i c a t e t h a t t h e enzyme e x t r a c t e d f r o m a c e t o n e powder had a low s p e c i f i c a c t i v i t y . 118 T a b l e XXVI E x t r a c t i o n o f NRase from A c e t o n e Powder E x p e r i m e n t T o t a l A c t i v i t y T o t a l P r o t e i n S p e c i f i c A c t i v i t y No. U n i t s mg U n i t s / m g p r o t e i n 1 1526 415 3.6 2 1780 323 5.5 2. Removal o f N u c l e o p r o t e i n from Enzyme P r e p a r a t i o n The 0-457« ammonium s u l f a t e p r e c i p i t a t e was d i a l y z e d -3 a g a i n s t 0.01 M pho s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 10 M g l u t a t h i o n e f o r one hou r a t 4° C. To t h e d i a l y z e d enzyme p r e p a r a t i o n a 0.2% aqueous s o l u t i o n o f p r o t a m i n e s u l f a t e , pH 4.5, was s l o w l y added u n t i l no f u r t h e r p r e c i p i t a t i o n o c c u r r e d . The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t i o n a t 5000 x g f o r 10 m i n u t e s a t 0° C and d i s c a r d e d . Enzyme a c t i v i t y and p r o t e i n c o n t e n t o f s u p e r n a t a n t was d e t e r m i n e d by t h e u s u a l methods. The r e s u l t s a r e g i v e n i n T a b l e X X V I I . They show t h a t t h e r e m o v a l o f n u c l o e p r o t e i n i n c r e a s e d t h e s p e c i f i c a c t i v i t y o f t h e enzyme s l i g h t l y . 119. T a b l e X X V I I Removal o f N u c l e o p r o t e i n from Enzyme P r e p a r a t i o n F r a c t i o n T o t a l volume T o t a l a c t i v i t y T o t a l p r o t e i n S p e c i f i c a c t i v i t y R e c o v e r y (ml) ( u n i t s ) (mg) (%) 0-45% Ammonium S u l f a t e p p t . 50 5200 155 33 P r o t a m i n e S u p e r n a t a n t 58 3650 82 44 70 3. A b s o r p t i o n o f NRase on A l u m i n a C r G e l T h i r t y m l o f d i a l y z e d 0-45% ammonium s u l f a t e p r e c i p i -t a t e d enzyme was t r e a t e d w i t h 6 m l o f a l u m i n a C gamma ( C r ) g e l (15 mg d r y w e i g h t p e r ml) and s t i r r e d i n t e r m i t t e n t l y f o r 15 mi n u t e s a t 0° C. The s u p e r n a t a n t and g e l p r e c i p i t a t e was washed by r e s u s p e n d i n g i t i n 5 m l o f 0.01 M p o t a s s i u m p h o s p h a t e b u f f e r -3 pH 7.0, c o n t a i n i n g 10 M g l u t a t h i o n e . A f t e r 5 m i n u t e s t h e p r e c i p i t a t e was c e n t r i f u g e d down and washed i n 0.5 M p o t a s s i u m -3 ph o s p h a t e b u f f e r , pH 7.0, c o n t a i n i n g 10 M g l u t a t h i o n e . The s u p e r n a t a n t s o l u t i o n o b t a i n e d by c e n t r i f u g a t i o n a f t e r each o f two w a s h i n g s i s r e f e r r e d t o as S u p e r n a t a n t 2 and S u p e r n a t a n t 3, r e s p e c t i v e l y . The enzyme a c t i v i t y and p r o t e i n c o n t e n t were d e t e r m i n e d by t h e u s u a l methods. The r e s u l t s a r e g i v e n i n T a b l e X X V I I I . 120. T a b l e X X V I I I A b s o r p t i o n o f NRase on A l u m i n a C r G e l F r a c t i o n s Volume T o t a l a c t i v i t y T o t a l p r o t e i n S p e c i f i c a c t i v i t y R e c o v e r y (ml) ( U n i t s ) (mg) (%) 0-45% Ammonium S u l f a t e p p t . 30 2750 75 36 S u p e r n a t a n t I 36 1850 12 152 67 S u p e r n a t a n t 2 5 0 . 0.80 - -S u p e r n a t a n t 3 5 0 1.25 - -121. DISCUSSION E x p e r i m e n t a l r e s u l t s r e p o r t e d h e r e p r o v i d e e v i d e n c e t h a t a s o l u b l e s u l f h y d r y l NRase w i t h a m e t a l l o f l a v o p r o t e i n n a t u r e i s p r e s e n t i n sugar b e e t l e a v e s . A l m o s t a l l NRase a c t i v i t y o f the homogenate o f t h e l e a v e s was r e c o v e r e d i n t h e 20,000 x g s u p e r n a t a n t . F r a c t i o n a t i o n by d i f f e r e n t speeds i n 0.1 M s u c r o s e - p h o s p h a t e b u f f e r c o n t a i n i n g -3 10 M g l u t a t h i o n e showed t h a t no p a r t i c u l a t e p o r t i o n c o n t a i n e d s i g n i f i c a n t e n z y m a t i c a c t i v i t y . T h i s i n t r a c e l l u l a r d i s t r i b u t i o n o f s u g a r b e e t NRase i s s i m i l a r t o t h a t o f t h e a s s i m i l a t o r y NRases o f most h i g h e r p l a n t s ( T a b l e I V ) . The NRase was s h a r p l y i n h i b i t e d by p - c h l o r o m e r c u r i -b e n z o a t e . The i n h i b i t i o n c o u l d be r e v e r s e d by s u l f h y d r y l r e a g e n t s s u c h as g l u t a t h i o n e and c y s t e i n e . I n h i b i t i o n a l s o r e s u l t e d from th e u s e o f t h e a l k y l a t i n g r e a g e n t i o d o a c e t a t e and t h e o x i d i z i n g r e a g e n t c u p r i c s u l f a t e . These r e s u l t s i n d i c a t e t h a t t h e NRase i s a s u l f h y d r y l enzyme w h i c h l o s e s i t s c a t a l y t i c a c t i v i t y when some o r a l l o f i t s -SH groups undergo c h e m i c a l m o d i f i c a t i o n . The e x p e r i m e n t s d e m o n s t r a t i n g t h a t c h e m i c a l l y r e d u c e d molybdenum c a n s e r v e as an e l e c t r o n donor s u g g e s t e d t h a t enzyme systems w h i c h y i e l d r e d u c e d m o l y b d a t e may be c a p a b l e o f r e p l a c i n g p y r i d i n e n u c l e o t i d e as t h e e l e c t r o n donor i n t h e NRase sequence. I t was n o t p o s s i b l e t o e s t a b l i s h t h a t Mo was t h e m e t a l p r o s t h e t i c group as d i d N i c h o l a s and Nason (1954) f o r N e u r o s p o r a NRase. 122. T h r e e l i n e s o f e v i d e n c e s u g g e s t t h a t FAD i s . t h e f l a v i n n u c l e o t i d e p r o s t h e t i c group o f t h e enzyme - t h e marked r e s p o n s e o f t h e p a r t i a l l y p u r i f i e d enzyme t o FAD a d d i t i o n , t h e low Km f o r FAD compared w i t h t h a t f o r FMN, and t h e i s o l a t i o n and t e n -t a t i v e i d e n t i f i c a t i o n o f t h e enzyme-bound f l a v i n as FAD. Because o f t h e p r e s e n c e o f some o t h e r f l a v o p r o t e i n enzymes, s u c h as q u i n o n e r e d u c t a s e , i n t h e enzyme p r e p a r a t i o n p o s i t i v e i d e n t i f i c a t i o n o f t h e enzyme-bound f l a v i n n u c l e o t i d e w i l l r e q u i r e f u r t h e r p u r i f i c a t i o n o f t h e NRase. T a b l e IV i n d i c a t e s t h e FAD s p e c i f i c i t y o f t h e a s s i m i l a t o r y NRases from s e v e r a l p l a n t s p e c i e s . The optimum pH o f t h e s u g a r b e e t l e a f enzyme F r a c t i o n V i s 7.0 w h i c h i s i d e n t i c a l w i t h t h a t o f t h e NRase from N e u r o s p o r a (Nason and E v a n s , 1953). I n c o n t r a s t t h e j5H optimum f o r c r u d e NRase ( F r a c t i o n I ) was about 8.0. A p p a r e n t l y some p h y s i c a l changes t o o k p l a c e d u r i n g t h e p u r i f i c a t i o n p r o c e d u r e . The i n h i b i t i o n by c y a n i d e , a z i d e , d i p y r i d y l , vanadate and c u p r i c s u l f a t e , shown t o o c c u r w i t h sugar b e e t NRase, has a l s o been r e p o r t e d f o r t h e NRase o f soybean l e a v e s (Evans and Nason, 1953; N i c h o l a s and Nason, 1955) and wheat embryo ( S p e n c e r , 1959). The end p r o d u c t s , n i t r i t e and DPN +, a r e a l s o t h e same as t h o s e o f o t h e r a s s i m i l a t o r y NRases and i n d i c a t e i t s r o l e as an A s s i m i l a t o r y NRase r a t h e r t h a n i t s i n v o l v e m e n t i n n i t r a t e r e s p i r a t i o n . 123. I t i s l i k e l y t h a t t h e mechanism o f e l e c t r o n t r a n s f e r i s s i m i l a r t o t h a t e s t a b l i s h e d f o r t h e enzyme i n N e u r o s p o r a and soybean l e a v e s : DPNH x / FAD K •* 2 M o 5 + ^ j NO +2H+ 3 DPN ^ FADH ' v 2Mo »S ^ NO - + H 0 1 2 2 I t i s e v i d e n t t h a t t h e f i n a l s t e p o f t h e s y s t e m 5+ i n v o l v e d a o n e - e q u i v a l e n t donor Mo and a t w o - e q u i v a l e n t a c c e p t o r N"*+ . T h i s t y p e o f e l e c t r o n t r a n s f e r i s uncommon. G e n e r a l l y s p e a k i n g , m e t a l i s n e c e s s a r y f o r t h e r e a c t i o n w i t h t h o s e a c c e p t o r s w h i c h r e q u i r e o n l y one e q u i v a l e n t o f hydrogen f o r t h e i r r e d u c t i o n and i s n o t n e c e s s a r y f o r t h o s e r e q u i r i n g t w o - e q u i v a l e n t s ( M a h l e r and G l e n n , 1956). The r e d u c t i o n o f n i t r a t e t o n i t r i t e r e q u i r e s two e q u i v a l e n t s , and y e t t h e r e d u c t i o n i n v a r i a b l y needs molybdenum. The q u e s t i o n whether t h e r e d u c t i o n o f n i t r a t e may p r o c e e d i n two o n e - e q u i v a l e n t s t e p s , o r n i t r a t e as an a c c e p t o r may r e q u i r e a c t i v a t i o n by c o m p l e x i n g w i t h t h e m e t a l molybdenum, i s s t i l l n o t e s t a b l i s h e d s i n c e an a d d i t i o n a l mechanism must be p o s t u l a t e d t o e x p l a i n t h i s s p e c i a l r e a c t i o n . I n i t s p y r i d i n e n u c l e o t i d e s p e c i f i c i t y p u r i f i e d NRase o f t h e s u g a r b e e t i s s i m i l a r t o t h e a s s i m i l a t o r y NRase from E s c h e r i c h i a c o l i ( N i c h o l a s and Nason, 1955b) , wheat embryo ( S p e n c e r , 1 9 5 9 ) , tomato l e a v e s ( S a n d e r s o n and C o c k i n g , 1 9 6 4 ) , 124. i n t h a t i t i s s p e c i f i c a l l y l i n k e d t o DPNH. The enzyme from N e u r o s p o r a (Nason and E v a n s , 1954) i s T P N H - s p e c i f i c . Impure NRase o f t h e s u g a r b e e t r e s e m b l e s t h a t from soybean l e a v e s (Evans and Nason, 1 9 5 3 ) , y e a s t ( S i l v e r , 1 9 5 6 ) , wheat l e a v e s (Anacker and S t o y , 1 9 5 8 ) , and t h e b a c t e r i u m A z o t o b a c t e r  v i n e l a n d i i ( T a n i g u c h i and Ohmachi, 1960) w h i c h c a n be l i n k e d t o e i t h e r DPNH o r TPNH w i t h e q u a l e f f e c t i v e n e s s . Spencer (1959) s u g g e s t e d t h a t i n h i g h e r p l a n t s DPNH-s p e c i f i c i t y m i g h t be a c h a r a c t e r i s t i c o f t h e NRase o f t h e embryo. He fo u n d t h a t g e r m i n a t i n g soybean seeds appear t o c o n -t a i n o n l y a D P N H - s p e c i f i c NRase w h i l e o l d e r t i s s u e o f soybean (Evans and Nason, 1953) and wheat ( S p e n c e r , 1959) c o n t a i n t h e non-s p e c i f i c NRase. From t h e e v i d e n c e p r e s e n t e d above i t i s p o s s i b l e t h a t two t y p e s o f NRase o c c u r i n su g a r b e e t l e a v e s . One enzyme i s s p e c i -f i c a l l y l i n k e d t o DPNH and t h e o t h e r t o TPNH. D u r i n g t h e c o u r s e o f p u r i f i c a t i o n o f t h e c r u d e "enzyme" t h e two enzymes can be s e p a r a t e d . I t i s a l s o p o s s i b l e t h a t t h e l e a v e s o f su g a r beet may c o n t a i n a D P N H - s p e c i f i c NRase and an enzyme w h i c h c a t a l y z e s a r e a c t i o n between TPNH and endogenous DPN t o prod u c e DPNH and TPN. K e i s t e r e t a l . (1960) d e s c r i b e d s u c h an enzyme i n s p i n a c h , p y r i d i n e n u c l e o t i d e t r a n s h y d r o g e n a s e , w h i c h c a t a l y z e s t h e f o l l o w i n g r e a c t i o n : TPNH + DPN > DPNH + T P N + 125. S h i n e t a l . (1963) o b t a i n e d a c r y s t a l l i n e f e r r e d o x i n - T P N r e d u c t a s e from s p i n a c h c h l o r o p l a s t s , w h i c h c a t a l y z e d t h e t r a n s -f e r o f t h e hydrogen from TPNH t o a number o f h y d r o g e n o r e l e c t r o n a c c e p t o r s such as DPN, menadione and FAD. A c o m p a r i s o n o f some o f t h e p r o p e r t i e s o f t h e NRase from sugar b e e t l e a v e s w i t h t h a t from soybean, c a u l i f l o w e r , w heat, and tomato, i s g i v e n i n T a b l e XXIX. The p a r t i a l l y p u r i f i e d s u g a r b e e t NRase p r e p a r a t i o n c o n -t a i n e d a number o f o t h e r e n z y m a t i c a c t i v i t i e s . These i n c l u d e d DPNH-cytochrome c r e d u c t a s e , D P N - d i a p h o r a s e , DPNH-manadione r e d u c t a s e , and DPNH-quinone r e d u c t a s e . The l a t t e r t h r e e enzymes have been d e s i g n a t e d "quinone r e d u c t a s e s " w h i c h a r e c h a r a c t e r i z e d by t h e i r a b i l i t y t o t r a n s f e r h ydrogen from r e d u c e d p y r i d i n e nuc-l e o t i d e t o s u i t a b l e q u i n o n e s ( M a r t i u s , 1963). A l t h o u g h t h e p o s s i b l e r e l a t i o n s h i p between t h e DPNH-NRase and q u i n o n e r e d u c t a s e a c t i v i t i e s p r e s e n t i n p u r i f i e d s u g a r b e e t NRase i s o b s c u r e i t i s a p p a r e n t t h a t t h e y a r e n o t i d e n t i c a l . U n l i k e t h e NRase q u i n o n e r e d u c t a s e s a r e n o t i n h i b i t e d by m e t a l -c h e l a t i n g a g e n t s such as c y a n i d e , a z i d e and o - p h e n o t h i o l i n e , i n d i c a t i n g t h e absence o f m e t a l i n t h e e n z y m a t i c s y s t e m , and t h e y a r e i n s e n s i t i v e t o s u l f h y d r y l a g e n t s , such as p - c h l o r o m e r c u r i -b e n z o a t e and i o d o a c e t a t e ( M a r t i u s , 1963). The e x p e r i m e n t s d e s c r i b e d above have r e v e a l e d t h a t i n t h e p r e s e n c e o f EDTA f l a v i n n u c l e o t i d e s can e f f e c t t h e t r a n s f e r 126. Table XXIX Comparison of Properties of Assimilatory NRase from Beta vulgaris and Other Higher Plants Source of pH Flavin enzyme Optimum Rm Enzyme specificity Substrate Reference and Km Km Sugar beet 7,0 leaves Soybean leaves 6.0 FAD: 1 x 10**7 M FMN: 5 x 10~7 M FAD: 1 x 10 M FMN: v-6. DPNH-specif i c . 5 x IO"5 M Non-specific DPNH: -5 3.7 x 10 M 3.2 x 10 ' M TPNH: 2.3 x 10 M 4.5 x 10~ 4 M -3 7.5 x 10 M Evans and Nason (1953) Wheat leaves Wheat embryo Tomato leaves 7.0 7.4 7.5 FAD: 2 x FMN FAD: -7 1 x 10 M Non-specific. DPNH-TPNH DPNH-specif ic DPNH-specif ic , 2.3 x 10 M 3.8 x 10~4 M 4 x 10 " 4 M Anacker and Stoy (1958) Spencer (1959) Sanderson and Cocking (1964) 127. o f e l e c t r o n s from some r e d u c i n g agent t o n i t r a t e w i t h t h e a i d o f NRase. I n t h i s s ystem f l a v i n n u c l e o t i d e s may a c t as l i g h t -a b s o r b i n g c a t a l y s t s . The p a r t i c i p a t i o n o f NRase and f l a v i n n u c l e o t i d e s i n a new t y p e of n o n - c y c l i c p h o t o s y n t h e t i c e l e c t r o n f l o w has been d e s c r i b e d by R a m i r e x e t a l . ( 1 9 6 4 ) . They s u g g e s t t h a t p y r i d i n e n u c l e o t i d e NRase may be i n f a c t a m i x t u r e o f T P N - r e d u c t a s e and NRase. The f l a v i n n u c l e o t i d e - c a t a l y z e d e n z y m a t i c p h o t o c h e m i c a l r e d u c t i o n o f n i t r a t e may be r e p r e s e n t e d by t h e f o l l o w i n g : P h o t o r e d u c t i o n DPNH DPN Reduced F l a v i n N u c l e o t i d e *• F l a v i n A H 2 A ^ N u c l e o t i d e N i t r a t e R e d u c t a s e -> Mo > NO" AH^ = R e d u c t i n g agent J u s t what p h y s i o l o g i c a l s i g n i f i c a n c e t h i s r e a c t i o n may have i n p l a n t t i s s u e r e m a i n s t o be shown. C e r t a i n l y i t seems t h a t f l a v i n n u c l e o t i d e s s h o u l d n o t be i g n o r e d when c o n s i d e r i n g p h o t o -c h e m i c a l r e a c t i o n s . The o v e r a l l abundance o f f l a v i n n u c l e o t i d e s i n t h e p l a n t t i s s u e and t h e p r e s e n c e o f n a t u r a l r e d u c i n g a g e n t s w h i c h c o u l d s e r v e t o c o n t r o l t h e s e r e a c t i o n s , speak s t r o n g l y i n f a v o r o f t h e t h e o r y t h a t f l a v i n n u c l e o t i d e s , enzyme-bound f l a v i n s , p l a y some r o l e i n l i g h t a c t i v a t e d r e a c t i o n s . 128. I t was found t h a t s u g a r b e e t l e a f NRase a c t i v i t y was h i g h d u r i n g t h e l i g h t p e r i o d and low d u r i n g d a r k n e s s . A s i m i l a r v a r i a t i o n has been found t o o c c u r i n m a i z e (Hagemen and F l e t c h e r , 1960) and tomato (Sanderson and C o c k i n g , 1964). The f o r m e r i n v e s t i g a t o r s s u g g e s t e d two p o s s i b l e r e a s o n s f o r t h e l o s s o f a c t i v i t y d u r i n g d a r k n e s s - t h e a c c u m u l a t i o n o f an i n h i b i t o r y n i t r o g e n o u s m e t a b o l i t e and t h e o x i d a t i o n o f t h e a c t i v e s u l f h y d r y l g r o u p s . However t h e o c c u r r e n c e o f e i t h e r was n o t p r o v e n . I n t h e e x p e r i m e n t s w i t h sugar b e e t NRase t h e a c c u m u l a t i o n o f an i n -h i b i t o r i n t h e l e a v e s d u r i n g t h e d a r k p e r i o d was n o t d e t e c t e d . I n common w i t h t h e o b s e r v a t i o n s o f C l e v e n g e r (1919)', I n g a l l s and S h r i v e ( 1 9 3 1 ) , B e n n e t - C l a r k (1953) and o t h e r s , who u s e d v a r i o u s s p e c i e s o f p l a n t s , i t was f o u n d t h a t growth i n d a r k n e s s and i n l i g h t was a s s o c i a t e d w i t h r e l a t i v e l y low and h i g h pH v a l u e s o f t h e e x p r e s s e d l e a f s a p . The s h a r p optimum o f s u g a r b e e t NRase (pH 7.0) and i t s s e n s i t i v i t y t o pH change c o u p l e d w i t h t h e f a l l i n l e a f pH v a l u e s i n t h e d a r k (pH 5.7 - 6.0) and t h e i r r i s e i n t h e l i g h t (pH 6.5 - 6.7) s u g g e s t a c a u s a l r e l a t i o n s h i p between t h i s rhythm i n hydrogen i o n c o n c e n t r a t i o n and t h e d i u r n a l a l t e r n a t i o n i n a c t i v i t y o f NRase. 129. BIBLIOGRAPHY (1) A f r i d i , M.M.R., and E . J . H e w i t t . 1962. I n d u c t i o n and s t a b i l i t y o f n i t r a t e r e d u c t a s e i n t i s s u e s o f h i g h e r p l a n t s . L i f e S c i e n c e 7:287-295. (2) A n a c k e r , W. F. , and S. V. S t o y . 1958. P r o t e i n c h r o m a t o g r a p h i c anc C a l c i u m p h o s p h a t e 1. R e i n i g u n g von n i t r a t e - R e d u z t a s e aus W e i z e n b l a t t e r n . Biochem. Z. 330:141-159. (3) A n d e r s o n , V. L. 1924. Some o b s e r v a t i o n s on t h e n i t r a t e - r e d u c i n g p r o p e r t i e s o f p l a n t s . Ann. B o t . 38:699-706. (4) A r n o n , D. I . 1949. Copper enzymes i n i s o l a t e d c h l o r o p l a s t p o l y p h e n o l o x i d a s e *-n B e t a v u l g a r i s . P l a n t P h y s i o l . 24:1-15. (5) A r n o n , M., and A. T. J a g e n d o r d . 1956. A TPNH-diaphorase from c h l o r o p l a s t s . A r c h . Biochem. B i o p h y s . 65:475-490. (6) B e c k i n g , J . H. 1962 N i t r a t e r e d u c t a s e i n c e l l - f r e e e x t r a c t s o f A z o t o b a c t e r . P l a n t and S o i l 16:202-213. (7) B e n n e t - C l a r k , T. A. 1933. O r g a n i c a c i d s i n p l a n t s . Ann. Rev. Biochem. 6:579-597. (8) B e n n e t - C l a r k , T. A. 1949. O r g a n i c a c i d s o f p l a n t s . Ann. Rev. Biochem. 18:639-655. (9) Bonner, J . 1952. P l a n t B i o c h e m i s t r y . Academic P r e s s , New Y o r k . (10) B u r s t r o m , H. 1946. The n i t r a t e n u t r i t i o n o f p l a n t s . Ann. Roy. A g r i c u l t . C o l l . Sweden. 13:1-86. (11) C a n d e l a , M. I . , E. G. F i s h e r , and E. J . H e w i t t , 1957. Molybdenum as a p l a n t n u t r i e n t . V. Some f a c t o r s a f f e c t i n g t h e a c t i v i t y o f r e d u c t a s e i n c a u l i f l o w e r p l a n t s grown w i t h d i f f e r e n t n i t r o g e n s o u r c e and molybdenum l e v e l s i n sand c u l t u r e . P l a n t P h y s i o l . 32:280-288. (12) Champigmy, M. L. 1963. A c t i v i t y and i n d u c t i o n o f n i t r a t e r e d u c t a s e i n m a i z e p l a n t s . P h y s i o l . V e g e t a b l e 1(2):139-169. 130. (13) C h e n i a e , G. M., and H. J . E v a n s . 1955. N i t r a t e r e d u c t a s e from t h e n o d u l e s o f Leguminous P l a n t s . I n W. D. M c E l r o y and B, H. G l a s s , ( E d ) , Symposium on i n o r g a n i c n i t r o g e n m e t a b o l i s m , Johns Hopkins P r e s s , B a l t i m o r e , p, 184-188. (14) C h e n i a e , G. M. and H. J . Evans. 1956. S t u d i e s on " n o d u l e n i t r a t e r e d u c t a s e " . P l a n t P h y s i o l . 31, x. (15) C h e n i a e , G. M., and H. J . E v a n s . 1957. On t h e r e l a t i o n between n i t r o g e n f i x a t i o n and n o d u l e n i t r a t e r e d u c t a s e o f soybean r o o t n o d u l e s . Biochem. B i o p h y s . A c t a 26:654-655. (16) C h e n i a e , G. M,, and H. J . E v a n s , 1959. P r o p e r t i e s o f a p a r t i c u l a t e n i t r a t e r e d u c t a s e from t h e n o d u l e s o f t h e soybean p l a n t . Biochem. B i o p h y s . A c t a 35:140-153. (17) C h e n i a e , G. M., and H. J . Evans. 1960 P h y s i o l o g i c a l s t u d i e s on n o d u l e - n i t r a t e r e d u c t a s e . P l a n t P h y s i o l . 35:454-462. (18) C l e v e n g e r , C. B. 1919. H y d r o g e n - i o n c o n c e n t r a t i o n o f p l a n t j u i c e . I I . F a c t o r s a f f e c t i n g t h e a c t i v i t y o r h y d r o g e n - i o n c o n c e n t r a t i o n o f p l a n t j u i c e s . S o i l S c i . 8:227-235. (19) C o l o w i c h , S. P. 1955. S e p a r a t i o n o f p r o t e i n s by use o f a d s o r b e n t s . I n S. P. C o l o w i c k and N. 0. K a p l a n (Ed.) Methods i n Enzymology, V o l . I . Academic P r e s s , New Y o r k , p. 90-98. (20) C r e s s w e l l , C. F., R. H. Hageman, and E. J . H e w i t t . 1962. The r e d u c t i o n o f n i t r i t e and h y d r o x y l a m i n e t o ammonia by enzymes o f v e g e t a b l e marrow. Biochem. J . 83:38P-39P. (21) C y z a n , F. C. 1963. The r e d u c t i o n o f n i t r a t e by t h e gr e e n a l g a e A n k i s t r o d e s m u s . P l a n t a . 6 0 (3):225-242. (22) D e l Campo, F. F., A. Paneque, J . M. R a m i r e z , and M. L o s a d a . 1963. N i t r a t e r e d u c t i o n i n t h e l i g h t by i s o l a t e d c h l o r o p l a s t s . Biochem. B i o p h y s . A c t a 66:450-452. (23) D e l w i c h e , C. C. 1951. The a s s i m i l a t i o n o f ammonium and n i t r a t e i o n s by t o b a c c o p l a n t s . J . B i o l . Chem. 189:167-175. 131. (24) D e l w i c h e , C. C. 1952. R e d u c t i o n o f n i t r i t e and n i t r a t e i o n s by p r e p a r a t i o n s from h i g h e r p l a n t s . F e d e r a t . P r o c . 11:201-208. (25) E c k e r s o n , S. H. 1924. P r o t e i n S y n t h e s i s by p l a n t s . 1. N i t r a t e r e d u c t i o n . B o t . Gaz. 77:377 (26) E c k e r s o n , S. H. 1931. S e a s o n a l d i s t r i b u t i o n o f r e d u c t a s e i n t h e v a r i o u s organs o f an a p p l e t r e e . C o n t r . Boyce. Thompson I n s t . 3:405. (27) Egami, F., K. Ohmachi, and S. T a n i g u c h i . 1957. N i t r a t e r e d u c i n g s y s t e m and s e e d l i n g s o f bean s e e d embryo, V i g n a s e s q u i p e d a t i s , d u r i n g t h e g e r m i n a t i n g s t a g e . B i o k h i m i a 22 ( 2 2 - 1 2 ) . (28) E v a n s , H. J . 1954. D i p h o s p h o p y r i d i n e n u c l e o t i d e - n i t r a t e r e d u c t a s e f r o m soybean n o d u l e s . P l a n t P h y s i o l . 29:298-301. (29) E v a n s , H. J . 1956. R o l e o f molybdenum i n p l a n t n u t r i t i o n . S o i l S c i . 81: 199-209. (30) E v a n s , H. J . , and N. S. H a l l , 1955. A s s o c i a t i o n o f molybdenum w i t h n i t r a t e r e d u c t a s e from soybean l e a v e s . S c i e n c e 122:922-923. (31) E v a n s , H. J . , and A. Nason. 1952. The e f f e c t o f r e d u c e d t r i p h o s p h o p y r i d i n e n u c l e o t i d e on n i t r a t e r e d u c t i o n by p u r i f i e d n i t r a t e r e d u c t a s e . A r c h . Biochem. B i o p h y s . 39:234-235. (32) E v a n s , H. J . , and A. Nason. 1953. P y r i d i n e n u c l e o t i d e - n i t r a t e r e d u c t a s e from e x t r a c t s o f h i g h e r p l a n t s . P l a n t P h y s i o l , 28:233-254. (33) F a r k a s - H i m s l e y , H., and M. Artman. 1957. S t u d i e s on n i t r a t e r e d u c t i o n by E s c h e r i c h i a c o l i . J . B a c t e r i o l . 74:690-692. (34) Fewson, C. A., and D. J . D. N i c h o l a s , 1961. N i t r a t e r e d u c t a s e from Pseudomonas a e r u g i n o s a . B i o c h i m . B i o p h y s . A c t a 49:335-349. (35) Fukuzumi, T., and E. Tamaki. 1962. N i t r o g e n m e t a b o l i s m i n t o b a c c o p l a n t s . A g r . B i o l . Chem. (Tokyo) 26:809-815. 132. (36) Haas, A. R. C. 1920. S t u d i e s on t h e r e a c t i o n o f p l a n t j u i c e s . S o i l S c i . 9:341-370. (37) Hageman, R. H., C. F. C r e s s w e l l , and E. J . H e w i t t . 1962. R e d u c t i o n o f n i t r a t e , n i t r i t e and h y d r o x y l a m i n e t o ammonia by enzyme e x t r a c t s from h i g h e r p l a n t s . N a t u r e 193:247-257. (38) Hageman, R. H., and D. F l e s h e r . 1960. N i t r a t e r e d u c t a s e a c t i v i t y i n c o r n s e e d l i n g s as a f f e c t e d by l i g h t and n i t r a t e c o n t e n t o f n u t r i e n t m e d ia. P l a n t P h y s i o l . 35:700-708. (39) H ass, E. 1944. The e f f e c t o f a t a b r i n e and q u i n o n e on i s o l a t e d r e s p i r a t o r y enzymes. J . B i o l . Chem. 155:321-331. (40) H a t t o r i , A. 1962. A d a p t i v e f o r m a t i o n o f n i t r a t e r e d u c i n g s y s t e m i n Anabaena c y l i n d r i c a . P l a n t C e l l P h y s i o l . 3 ( 4 ) : 371. (41) H e l l e r m a n , L., A. L i n d s a y , and M. Bavasmich. 1946. Flavoenzyme c a t a l y s i s . J . B i o l . Chem. 163:553-557. (42) H e r e d i a , C. F., and A. Medina. 1960. N i t r a t e r e d u c t a s e and r e l a t e d enzyme i n E s c h e r i c h i a c o l i . Biochem. J . 77:24-30. (43) H e w i t t , D. J . , and M.M.R.K. A f r i d i . 1959. A d a p t i v e s y n t h e s i s o f n i t r a t e r e d u c t a s e i n h i g h e r p l a n t s . N a t u r e 183:57-58. (44) H o r e c k e r , B. L., and A. R o m b e r g . 1948. The e x t i n c t i o n c o e f f i c i e n t o f t h e r e d u c e d band o f p y r i d i n e n u c l e o t i d e . J , B i o l . Chem. 175:385-390. (45) I i d a , R., and S. T a n i g u c h i . 1959. S t u d i e s on n i t r a t e r e d u c t a s e s y s t e m o f E s c h e r i c h i a  c o l i . I . P a r t i c u l a t e e l e c t r o n t r a n s p o r t s y s t e m t o n i t r a t e and i t s s o l u b i l i z a t i o n . J . Biochem. (Tokyo) 46:1041-1055. (46) I i d a , R., and K. Y a m a s a k i . 1960. S p e c t r o g r a p h i c d e t e r m i n a t i o n o f molybdenum i n t h e n i t r a t e r e d u c t a s e from E s c h e r i c h i a c o l i . Biochem. B i o p h y s . A c t a 44:352-357. 133. (47) I n g a l l s , R. A., and J . W. S h i v e . 1931. R e l a t i o n o f H - i o n c o n c e n t r a t i o n o f t i s s u e f l u i d t o t h e d i s t r i b u t i o n o f i r o n i n p l a n t s . P l a n t P h y s i o l . 6:103. (48) I r v i n g , A. A., and R. H a n k i n s o n . 1908. The p r e s e n c e o f a n i t r a t e r e d u c i n g enzyme i n gre e n p l a n t s . Biochem. J . 3:87-96. (49) I t a g a k i , E., and S. T a n i g u c h i . 1959. S t u d i e s on n i t r a t e r e d u c t a s e s y s t e m o f E. c o l i . I I . S o l u b l e n i t r a t e r e d u c t a s e s y s t e m o f a e r o -b i c a l l y grown c e l l s i n a s y n t h e t i c medium. J . Biochem. (Tokyo) 46:1419-1436. (50) J a g e n d o r f , A. T. 1956. O x i d a t i o n and r e d u c t i o n o f p y r i d i n e n u c l e o t i d e s by p u r i f i e d c h l o r o p l a s t s . A r c h . Biochem. B i o p h y s . 62:141-150. (51) K a s t l e , J . H., and E. E l v o v e . 1904. On t h e r e d u c t i o n o f n i t r a t e s by c e r t a i n p l a n t e x t r a c t s and m e t a l s and t h e a c c e l e r a t i n g e f f e c t o f c e r t a i n s u b s t a n c e s on t h e p r o g r e s s o f t h e r e d u c t i o n . J . Amer. Chem. Soc. 31:606-641. (52) K a t o , T. 1963. N i t r a t e r e d u c t i o n i n t h e p h o t o s y n t h e t i c b a c t e r i u m R h o d o s p i r i l i u m rubrum. P l a n t C e l l P h y s i o l . (Tokyo) 4:199-215. (53) K e i l i n , D., and E. F. H a r t r e e . 1938. On t h e mechanism o f t h e d e c o m p o s i t i o n o f hyd r o g e n pero x i d e ; : by c a t a l a s e . P r o c . Roy. Soc. B. 124: 397-410. (54) K e i s t e r , D. L., A. San P i e t r o , and F. E. S t o l z e n b a c h . I960, P y r i d i n e n u c l e o t i d e t r a n s h y d r o g e n a s e from s p i n a c h . J . B i o l . Chem. 235:2989-2996. (55) K e s s l e r , E. 1964. N i t r a t e a s s i m i l a t i o n by p l a n t s . Ann. Rev. o f P l a n t P h y s i o l . 15:57-72. (56) K i n s k y , S. C. 1961. I n d u c t i o n and r e p r e s s i o n o f n i t r a t e r e d u c t a s e i n N e u r o s p o r a c r a s s a . J . B a c t . 82:898-904. 134. (57) K i n s k y , S. C., and W. D. M c E l r o y . 1958. N e u r o s p o r a n i t r a t e r e d u c t a s e : The r o l e o f p h o s p h a t e , f l a v i n and cytochrome c r e d u c t a s e . A r c h . Biochem. B i o p h y s . 73:466-483. (58) L i n e w e a v e r , H. and D. Bruk. 1934. The d e t e r m i n a t i o n o f enzyme d i s s o c i a t i o n c o n s t a n t s . J . Amer. Chem. Soc. 56:658-662 (59) Lowe, R. H., and H. J . E v a n s , 1964. P r e p a r a t i o n and some p r o p e r t i e s o f a s o l u b l e n i t r a t e r e d u c t a s e from R h i z o b i u m j a p o n i c a . Biochem. B i o p h y s . A c t a 85:377-389. (60) Lowery, 0. H., N. J . Rosebrough, A. L. F a r r , and R. J . R a n d a l l . 1951. P r o t e i n measurement w i t h t h e F o l i n p h e n o l r e a g e n t . J . B i o l . Chem. 193:265-275. (61) M a h l e r , H. R., and J . L. G l e n n . 1956. G e n e r a l s i g n i f i c a n c e o f m e t a l l o f l a v o p r o t e i n s i n e l e c t r o n t r a n s p o r t . I n W. D. M c E l r o y and B. H. G l a s s , ( E d . ) . Symposium i n i n o r g a n i c n i t r o g e n m e t a b o l i s m . Johns Hopkins P r e s s , B a l t i m o r e , p. 575-597. (62) M a h l e r , H. R., B. M a c k l e r , D. E. Gr e e n , and R. Bock. 1954. S t u d i e s on m e t a l l o f l a v o p r o t e i n s I I I . A l d e h y d e o x i d a s e : a m o l y b d o f l a v o p r o t e i n , J . B i o l . Chem. 210:465-480. (63) M a r t i n s , C. 1963. Quinone r e d u c t a s e s . I n P. D. B o y e r , H. L a r d y and K. Myrback, ( E d . ) , The Enzymes. Academic P r e s s , New Y o r k . V o l . V I I . p.517-532. (64) M e d i n a , A. and C. F. H e r e d i a . 1958. V i t a m i n K-dependent n i t r a t e r e d u c t a s e i n E. c o l i B i o c h i m . B i o p h y s . A c t a 28:452-453. (65) M e n d e l , J . L., and D. W. V i s s e r . 1951. N i t r a t e r e d u c t i o n i n h i g h e r p l a n t s . A r c h . Biochem. B i o p h y s . 32:158-169. (66) M e u s e l , E. 1875. N i t i l b i l d u n g d u r c h B a c t e r i e n . C i t e d by M. S. McKee i n " N i t r o g e n m e t a b o l i s m i n p l a n t s " . C l a r e n d o n P r e s s . 1962. (67) M o r t o n , A. G. 1956. A S t u d y o f n i t r a t e r e d u c t i o n i n f u n g i . J o u r . E x p t l . B o t . 7:97-112. 135. (68) Nason, A. 1955. E x t r a c t i o n o f s o l u b l e enzymes from h i g h e r p l a n t s . I n S. P. C o l o w i c h and N. 0. K a p l a n , ( E d . ) , Methods i n Enzymology, Academic P r e s s , New Y o r k , p. 62-63. (69) Nason, A. 1956. E n z y m a t i c s t e p s i n t h e a s s i m i l a t i o n o f n i t r a t e and n i t r i t e i n f u n g i and g r e e n p l a n t s . I n W. D. M c E l r o y and B. H. G l a s s , ( E d . ) , Symposium i n i n o r g a n i c n i t r o g e n m e t a b o l i s m . _Johns Hopkins P r e s s , B a l t i m o r e , p. 109-136. (70) Nason, A. 1962. E n z y m a t i c pathways o f n i t r a t e , n i t r i t e and h y d r o x y l a m i n e m e t a b o l i s m . B a c t e r . Rev. 26:16-41. (71) Nason, A. 1963. N i t r a t e r e d u c t a s e . I n P. D. B o y e r , H. L a r d y and K. Myrback (Ed.) The Enzymes. Academic P r e s s , New Y o r k , p. 587-607. (72) Nason, A., and J . H. Evans. 1953. T r i p h o s p h o p y r i d i n e n u c l e o t i d e - n i t r a t e r e d u c t a s e i n N e u r o s p o r a . J . B i o l . Chem. 202:656-673. (73) Nason, A., and H. T a k a h a s h i . 1958. I n o r g a n i c n i t r o g e n m e t a b o l i s m . Ann, Rev. M i c r o b i o l . 12:203-246. (74) N i c h o l a s , D. J . D. 1952. The use o f f u n g i f o r d e t e r m i n i n g t r a c e m e t a l s i n b i o l o g i c a l m a t e r i a l s . A n a l y s t 77:920-923 (1952) (75) N i c h o l a s , D. J . D. 1957. The f u n c t i o n o f t r a c e m e t a l s i n t h e n i t r o g e n m e t a b o l i s m . Ann. Botany 21:587-589. (76) N i c h o l a s , D. J . D., and A. Nason. 1954a Molybdenum and n i t r a t e r e d u c t a s e . I I . Molybdenum as a c o n s t i t u e n t o f n i t r a t e r e d u c t a s e . J . B i o l . Chem. 207:353-360. (77) N i c h o l a s , D. J . D., and A. Nason, 1954b Mechanism o f a c t i o n o f n i t r a t e r e d u c t a s e from N e u r o s p o r a . J . B i o l . Chem. 211:183-197. (78) N i c h o l a s , D. J . D., and A. Nason. 1955a R o l e o f molybdenum as a c o n s t i t u e n t o f n i t r a t e r e d u c t a s e from soybean l e a v e s . P l a n t P h y s i o l . 30:135-138. 136. (79) N i c h o l a s , D. J . D., and A. Nason. 1955b. D i p h o s p h o p y r i d i n e n u c l e o t i d e - n i t r a t e r e d u c t a s e from E s c h e r i c h i a c o l i . J . B a c t . 69:580-583. (80) N i c h o l a s , D. J . D., A. Nason and W. D. M c E l r o y . 1954. Molybdenum and n i t r a t e r e d u c t i o n 1. E f f e c t o f molybdenum d e f i c i e n c y on t h e N e u r o s p o r a enzyme. J . B i o l . Chem. 207:341-351. (81) N i c h o l a s , D. J . D., and J . H. S c a w i n . 1956. A phosphate r e q u i r e m e n t f o r n i t r a t e r e d u c t a s e from N e u r o s p o r a c r a s s a . N a t u r e 78:1474-1475. (82) N i c h o l a s , D. J . D., and H. M. S t e v e n s . 1955. V a l e n c y changes o f molybdenum d u r i n g t h e e n z y m a t i c r e d u c t i o n o f n i t r a t e i n N e u r o s p o r a . N a t u r e 176: 1066-1067. (83) N i c h o l a s , D. J . D., and H. M. S t e v e n s . 1956. The r o l e o f molybdenum i n o x i d a t i o n - r e d u c t i o n p r o c e s s i n N e u r o s p o r a and A z o t o b a c t e r . I n W. D. M c E l r o y and B. H. G l a s s ( E d . ) , Symposium on i n o r g a n i c n i t r o g e n m e t a b o l i s m . Johns Hopkins P r e s s , B a l t i m o r e , p. 178-183. (84) N i e l , C. B. v a n , , M. B. A l l e n and B. E. W r i g h t . 1953. On t h e p h o t o c h e m i c a l r e d u c t i o n o f n i t r a t e by a l g a e . B i o c h i m . B i o p h y s . A c t a 12:67-74. (85) Normura, I . 1960. A s u r v e y o f n i t r a t e r e d u c t a s e s y s t e m i n h i g h e r p l a n t s . S c i . R e p t s . Tokyo K y s i k u D a i g u k u S e c t . B. 9:168-180. (86) Ohmachi, K., S. T a n i g u c h i , and F. Egami. 1959. The s o l u b l e and c y t o c h r o m e - l a c k i n g n i t r a t e - r e d u c i n g s y s t e m i n g e r m i n a t i n g c o t y l e d o n s o f bean s e e d embryo. V i g n a s e s q u i p e d a l i s . J , Biochem. (Tokyo) 46: 911-915. (87) P i e r p o i n t , W. S. 1960. M i t r o c h o n d r i a l p r e p a r a t i o n from t h e l e a v e s o f t o b a c c o . Biochem. J . 75:511-515. (88) P o z z i - E s c o t , E. 1903. The r e d u c i n g enzyme. Amer. Chem. J . 29:517-563. (89) R a c k e r , E. 1955. G l u t a t h i o n e r e d u c t a s e . I n S. P. C o l o w i c h and N. 0. K a p l a n , ( E d . ) , Methods i n Enzymology. V o l . I I . Academic P r e s s , New Y o r k . p. 722-725. 137. (90) Rada, G. K., and M. C a l w i n . 1963. C h e m i c a l and p h o t o c h e m i c a l r e d u c t i o n s o f f l a v i n n u c l e o t i d e s and a n a l o g s . Biochem. 3:383-393. (91) R a m i r e z , J . M., F. F. d e l Campo, A. Paneque and M. L o s a d a . 1964. Mechanism o f n i t r a t e r e d u c t i o n s i n c h l o r o p l a s t s . Biochem. B i o p h y s . Res. Comm. 15:297-302. (92) R i j v e n , A. H. G. C. 1958. E f f e c t s o f some i n o r g a n i c n i t r o g e n s u b s t a n c e s on growth and n i t r o g e n a s s i m i l a t i o n o f young p l a n t embryo i n v i t r o . A u s t r a l . J . B i o l . S c i . 11: 142-154. (93) R o b i n s o n , J . A Manometric d e t e r m i n a t i o n o f n i t r a t e r e d u c t a s e a c t i v i t y . A r c h . Biochem. 52:148-155. (94) R o u s s o s , G. C., and A, Nason. 1960. P y r i d i n e n u c l e o t i d e - n i t r i t e and h y d r o x y l a m i n e enzymes from soybean l e a v e s . B i o l . Chem. 235:2997-3007. (95) Sadana, J . C., and W. D. M c E l r o y . 1957. N i t r a t e r e d u c t a s e from Achromobacter f i s c h e r i , p u r i f i c a t i o n and p r o p e r t i e s : f u n c t i o n o f f l a v i n and cytochrome. A r c h . Biochem. B i o p h y s . 67:16-34. (96) S a n d e r s o n , G. W., and E. C. C o c k i n g . 1964. Enzymic a s s i m i l a t i o n o f n i t r a t e i n tomato p l a n t . I . R e d u c t i o n o f n i t r a t e t o n i t r i t e . P l a n t P h y s i o l . 39:416. (97) S a t o , R. 1956. The cytochrome s y s t e m and m i c r o b i a l r e d u c t i o n o f n i t r a t e . I n W. D. M c E l r o y and B. H. G l a s s , ( E d . ) , Symposium on i n o r g a n i c n i t r o g e n m e t a b o l i s m . Johns H o p k i n s P r e s s , B a l t i m o r e , p. 163-175. (98) S c h o e n b e i n , C. F. 1863. Uber das Vorkommen S a l p e t r i c h t und S a l p e t e r s a u r e r S a l z e i n der P f l a n z e n w e l t C i t e d by H. S. McKee, " N i t r o g e n m e t a b o l i s m i n p l a n t s " . C l a r e n d o n P r e s s O x f o r d , 1962. (99) S h a f e r , J . J . , J . E. B a k e r , and J . F. Thompson, 1961. A c h l o r e l l a mutant l a c k i n g n i t r a t e r e d u c t a s e . Amer. J . B o t . 48(10):896-899. 138. (100) S h i n , M., K. Tagawa, and D. I . Arnon. 1963. C r y s t a l l i z a t i o n o f f e r r e d o x i n - T P N r e d u c t a s e and i t s r o l e i n t h e p h o t o s y n t h e t i c a p p a r a t u s o f c h l o r o p l a s t s . Biochem. Z. 338:84-96. (101) S i l v e r , W. S. 1957. P y r i d i n e n u c l e o t i d e - n i t r a t e r e d u c t a s e from H a n s e n u l a anomula. J . B a c t . 73:241-246. (102) S n e l l , F. D., and C. T. S n e l l . 1949. C o l o r i m e t r i c methods o f a n a l y s i s . 3 r d Ed. V o l . I I . Van N o s t r a n d P r e s s , New Y o r k . p. 804. (103) S p e n c e r , D. 1959. A D P N H - s p e c i f i c n i t r a t e r e d u c t a s e from g e r m i n a t i n g wheat. A u s t r a l . J . B i o l . S c i . 12:181-191. (104) S t o c k i n g , C. R. 1959. C h l o r o p l a s t i s o l a t i o n i n nonaqueous media. P l a n t P h y s i o l . 34:56-58. (105) S t o y , V. 1956. R i b o f l a v i n - c a t a l y z e d enzymic p h o t o r e d u c t i o n o f n i t r a t e . Biochem. B i o p h y s . A c t a 21:395-396. (106) S t r a u s s , G., and W. J . N i c k e r s o n . 1960. P h o t o c h e m i c a l c l e a v a g e o f w a t e r by r i b o f l a v i n . I I . R o l e o f a c t i v a t o r s . J . Amer. Chem. Soc. 83:3187-3189. (107) S t r e e t , H. P. E. 1949. N i t r o g e n m e t a b o l i s m o f h i g h e r p l a n t s . Adv. i n Enzymology 9:391-454. (108) S y r e t t , P. J . , and I . M o r r i s . 1963. The i n h i b i t i o n o f n i t r a t e a s s i m i l a t i o n by ammonium i n c h l o r e l l a . Biochem. B i o p h y s . A c t a 67:566-575. (109) Tang, P. and H. Wu. 1957. A d a p t i v e f o r m a t i o n o f n i t r a t e r e d u c t a s e i n r i c e s e e d l i n g s . N a t u r e 179:1355-1356. (110) T a n i g u c h i , S. and E. I t a g a k i . 1959. S o l u b i l i z a t i o n and p u r i f i c a t i o n o f p a r t i c u l a t e n i t r a t e r e d u c t a s e o f a n a e r o b i c a l l y grown E s c h e r i c h i a c o l i . B i o c h i m . B i o p h y s . A c t a 31:294-295. 139. (111) T a n i g u c h i , S., and E. I t a g a k i . 1960. N i t r a t e r e d u c t a s e o f n i t r i t e r e s p i r a t i o n t y p e from E» c o l i . I . S o l u b i l i z a t i o n and p u r i f i c a t i o n from t h e p a r t i c u l a t e s y s t e m w i t h m o l e c u l a r c h a r a c t e r i z a t i o n as a m e t a l l o p r o t e i n . B i o c h i m . B i o p h y s . A c t a 44: 263-279. (112) T a n i g u c h i , S., and K. Ohmachi. 1960. P a r t i c u l a t e n i t r a t e r e d u c t a s e o f A z o t o b a c t e r  y i n l a n d i i . J . Biochem. (Tokyo) 48:50-62. (113) T a n i g u c h i , S., R. S a t o , and F. Egami 1956. The e n z y m a t i c mechanism o f n i t r a t e and n i t r i t e m e t a b o l i s m i n b a c t e r i a . I n W. D. M c E l r o y and B. H. G l a s s , ( E d . ) , Symposium on i n o r g a n i c n i t r o g e n m e t a b o l i s m . . Johns H o p k i n s P r e s s , B a l t i m o r e , p. 87-107. (114) V a i d y a n a t h a n , C. S., and H. E. S t r e e t . 1959. N i t r a t e r e d u c t i o n by aqueous e x t r a c t s o f e x c i s e d tomato r o o t s . N a t u r e 184:531-533. (115) V e r h o e v e n , W. 1956. Some remarks on n i t r a t e and n i t r i t e m e t a b o l i s m i n m i c r o o r g a n i s m s . I n W. D. M c E l r o y and B. H. G l a s s (Ed.} Symposium on i n o r g a n i c n i t r o g e n m e t a b o l i s m . Johns H o p k i n s P r e s s , B a l t i m o r e , p. 61-86. (116) V e r n o n , L. P. 1959. P h o t o c h e m i c a l o x i d a t i o n and r e d u c t i o n r e a c t i o n s c a t a l y z e d by f l a v i n n u c l e o t i d e s . B i o c h i m , B i o p h y s . A c t a 36:177-182. (117) V i r t a n e n , A. I . , and N. R a u t a v e n . 1952. N i t r o g e n m e t a b o l i s m . I n J . B. Summer and K. Myrback, E d . ) , The enzymes. Academic P r e s s , I n c . , New Y o r k , p. 1089-1130. (118) W a l k e r , G. G., and D. J . D. N i c h o l a s . 1961. N i t r a t e r e d u c t a s e from Pseudomonas a e r u g i n o s a . B i o c h i m . B i o p h y s . A c t a 49:350-360. (119) W o o l l e y , J . T., G. P. H i c k s and R. H. Hageman. 1960. R a p i d d e t e r m i n a t i o n o f n i t r a t e and n i t r i t e i n p l a n t m a t e r i a l . J o u r . A g r . and Food Chem. 8:260-261. 140. (120) W o s i l a i t , W. D. and Nason. 1954. P y r i d i n e n u c l e o t i d e - m e n a d i o n e r e d u c t a s e from E. c o l i . J . B i o l . Chem. 208:785-798. (121) W o s i l a i t , W. D., and A. Nason. 1955. P y r i d i n e n u c l e o t i d e - q u i n o n e r e d u c t a s e . I . P u r i f i c a t i o n and p r o p e r t i e s o f t h e enzyme. J . B i o l . Chem. 206:255-271. (122) W o s i l a i t , W. D., A. Nason and A. J . T e r r e l . 1955. P y r i d i n e n u c l e o t i d e - q u i n o n e r e d u c t a s e . I I . R o l e i n e l e c t r o n t r a n s p o r t . J . B i o l . Chem. 206:271-283. (123) W r i g h t , C. I , , and J . C. S a b i n e . 1944. The e f f e c t o f a t a b r i n e on t h e oxygen c o n s u m p t i o n o f t i s s u e . J . B i o l . Chem. 155:315-320. (124) Wu, H. Y., and S. W. L o o . 1950. R e d u c t a s e i n p l a n t t i s s u e s . S c i e n c e R e c o r d ( C h i n a ) 3:11-19. 

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