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Solubilization and assay of the “insoluble” α-glycerophosphate dehydrogenase of animal tissues
VOL. 2~) (Z958)
PRELIMINARY NOTES
66I
Solubilization and assay of the "insoluble" a-glycerophosphate dehydrogenaseof animal tissues* W h i l e t h e D P N - l i n k e d a - g l y c e r o p h o s p h a t e d e h y d r o g e n a s e (a-GPD) is r e a d i l y solubilized a n d purified, its m i t o c h o n d r i a l c o u n t e r p a r t , w h i c h r e d u c e s c y t o e h r o m e c w i t h o u t t h e m e d i a t i o n of D P N 1, h a s n o t been p r e v i o u s l y o b t a i n e d in t r u e solution, e x t e n s i v e l y purified, or characterized. E x t r a c t i o n of v a r i o u s m i t o c h o n d r i a l p r e p a r a t i o n s -with bile salts 2, w i t h or w i t h o u t p r e v i o u s digestion w i t h p r o t e o l y t i c e n z y m e s 3, a n d d i g i t o n i n e x t r a c t i o n 4 yield stable dispersions w h i c h do n o t s e d i m e n t readily in t h e u l t r a c e n t r i f u g e . Such " t e m p o r a r i l y solubilized ''2 p r e p a r a t i o n s do n o t lend t h e m s e l v e s to e x t e n s i v e purification b y classical m e t h o d s a n d u p o n c o m p l e t e r e m o v a l of t h e solubilizing a g e n t a n d on r e p e a t e d freezing a n d t h a w i n g t h e y u s u a l l y r e v e r t to t h e p a r t i c u l a t e form. I n c o n t r a s t , digestion of m i t o c h o n d r i a l a c e t o n e p o w d e r s w i t h lecithinase A a t n e u t r a l p H releases t h e d e h y d r o g e n a s e in soluble form, as j u d g e d b y t h e following facts: (i) it r e m a i n s in solution a f t e r e x t e n s i v e dialysis a n d r e p e a t e d freezing a n d t h a w i n g ; (2) it does n o t s e d i m e n t at i44,ooo × g in I h; a n d (3) it m a y be readily f r a c t i o n a t e d with (NH4)2SO , a n d a h i g h salt conc e n t r a t i o n is required for precipitation, while dispersions p r e p a r e d with d i g i t o n i n precipitate at a relatively low (NH4)~SO 4 c o n c e n t r a t i o n . I n t h e initial e x p e r i m e n t s a c e t o n e p o w d e r s of p i g - b r a i n m i t o c h o n d r i a were t r e a t e d with cobra ( N a j a naja) or r a t t l e s n a k e (Crotalus terrificus) v e n o m s as sources of lecithinase A. E s s e n t i a l l y c o m p l e t e e x t r a c t i o n w a s o b t a i n e d w i t h relatively little loss of a c t i v i t y . T h e fact t h a t t h e p h o s pholipase w a s t h e a g e n t responsible for t h e solubilization was confirmed b y t h e use of crystalline c r o t o x i n (the kind gift of Dr. FRAENKEL-CONRAT).T h e clear, yellow e x t r a c t o b t a i n e d was s u b j e c t e d to t w o cycles of (NH4)2SO 4 fractionation, followed b y differential u l t r a c e n t r i f u g a t i o n , r e s u l t i n g in a n a m b e r - c o l o r e d solution of t h e purified e n z y m e . T h e a b s o r p t i o n s p e c t r u m showed a p r o m i n e n t p e a k a t 412 m/~, w h i c h was n o t a Soret b a n d , since h e i n e c o m p o u n d s were a b s e n t . T h i s p e a k is c h a r a c t e r i s t i c of a - G P D , since a - g l y c e r o p h o s p h a t e (a-GP) bleached it r a p i d l y a n d completely, yielding a difference s p e c t r u m w i t h a p e a k at 412 m/~ a n d a s h o u l d e r in t h e flavin region (455 rap). As s h o w n in T a b l e I, p h e n a z i n e m e t h o s u l f a t e is t h e preferred assay, as w i t h o t h e r " c y t o c h r o m e r e d u c i n g d e h y d r o g e n a s e s " , a n d gives t h e h i g h e s t a c t i v i t y in purified soluble p r e p a r a t i o n s , in dispersed p r e p a r a t i o n s , a n d in i n t a c t m i t o c h o n d r i a l p r e p a r a t i o n s f r o m brain, yeast, as well as skeletal m u s c l O . W i t h b r a i n m i t o c h o n d r i a t h e a c t i v i t y in t h e m a n o m e t r i c p h e n a z i n e a s s a y s in t h e presence of c y a n i d e is significantly h i g h e r t h a n t h r o u g h t h e c y t o c h r o m e chain, a l t h o u g h t h e t u r n o v e r r a t e of t h e c y t o c h r o m e s y s t e m is far in excess of t h e r e q u i r e m e n t of a - G P D . T h u s in t h e oxidase t e s t t h e linkage of a - G P D to t h e r e s p i r a t o r y c h a i n m a y be t h e r a t e - l i m i t i n g step. W h i l e m e t h y l e n e blue is a relatively good a c c e p t o r Ior a - G P D (provided a c t i v i t y at infinite dye concent r a t i o n is m e a s u r e d ) a n d t h i s a c t i v i t y is r e t a i n e d o n solubilization, this d y e is a poor acceptor for p a r t i c u l a t e succinic d e h y d r o g e n a s e f r o m a n i m a l t i s s u e s a n d t h e a c t i v i t y is lost on solubilization. a - G P D h a s a s h a r p o p t i m u m a t p H 7.7 (38°) in t h e m a n o m e t r i c p h e n a z i n e m e t h o s u l f a t e a s s a y a n d at p H 7.9 in t h e m e t h y l e n e blue assay. T h e K m for a-L-GP is 6. 7" Io -3 M (pH 7.7, 3 8°) a n d t h e p r o d u c t 2, d i h y d r o x y a c e t o n e p h o s p h a t e , is a p o t e n t c o m p e t i t i v e inhibitor. A n i n t e r e s t i n g p r o p e r t y of t h e e n z y m e a n d one of i m p o r t a n c e in its a s s a y is t h a t it u n d e r g o e s a n a c t i v a t i o n a n a l o g o u s to t h a t of succinic d e h y d r o g e n a s e 6, i n v o l v i n g a conversion of a less a c t i v e TABLE REACTION
RATES OF a-GPD
I WITH VARIOUS ACCEPTORS
(Vmax v a l u e s at 38°) Preparation
Brain mitochondria A c e t o n e p o w d e r of b r a i n mitochondria Digitonin-extr., purified Soluble p r e p a r a t i o n Yeast granules
Phenazi.e methosul/ate
02 (with excess cyt. c)
Cyt. c
Methylene blue
ioo
64-73
65
54
i oo ioo ioo ioo
o o o 64
o o o
58 68 ,66 58
Ferricyanide
88
* S u p p o r t e d b y g r a n t s f r o m t h e N a t i o n a l H e a r t I n s t i t u t e , U.S. Public H e a l t h Service, a n d t h e A m e r i c a n H e a r t Association, a n d b y c o n t r a c t No. N O N R z656 (oo) b e t w e e n t h e Office of N a v a l R e s e a r c h , U. S. N a v y , a n d t h e E. B. Ford I n s t i t u t e .
662
PRELIMINARY NOTES
VOL. 29 (1958)
form to a m o r e a c t i v e one u p o n brief i n c u b a t i o n w i t h t h e s u b s t r a t e in t h e a b s e n c e of c a t a l y t i c a c t i v i t y . T h e lag p eriod r e g u l a r l y o b s e r v e d in a - G P D or a - G P o x i d a s e a s s a y s is c o m p l e t e l y a b o l i s h e d b y prio r i n c u b a t i o n of t h e e n z y m e w i t h its s u b s t r a t e a n d is in all p r o b a b i l i t y a n e x p r e s s i o n of t h e a c t i v a t i o n process.
Edsel B. Ford Institute [or Medical Research, Henry Ford Hospital, Detroit, Mich. (U.S.A.)
ROBERT L. RINGLER THOMAS 19. GINGER *
I D. E. GREEN, Biochem. J., 3 ° (I936) 629. T. TUNG, L. ANDERSON AND A. LARDY, Arch. Biochem. Biophys., 40 (1952) z94. 3 K. LING, S. WU, S. TING AND T. TUNG, Intern. Symposium Enzyme Chemistry, Tokyo and Kyoto,
Japan, Oct. ~957. 4 R. L. RINGLER AND T. P. SINGER, Federation Proc., 17 (1958) 297. ii T. P. SINGER AND E. B. KEARNEY, Methods of Bioehem. Anal., 4 (1957) 307 • o E. ]3. I~EARNEY, J. Biol. Chem., 229 (1957) 363 . R e c e i v e d M a y 28th, 1958 *
E s t a b l i s h e d I n v e s t i g a t o r of the A m e r i c a n H e a r t Association.
Competitive inhibition of nitrogen fixation by oxygen T h e effect of O 3 on t h e a s s i m i l a t i o n of m o l e c u l a r N 2 h a s been r e - e x a m i n e d . Cells of Azotobacter vinelandii were g r o w n in t h e vessels of W a r b u r g r e s p i r o m e t e r s s h a k e n in a b a t h a t 3 o°. A t m o s p h e r e s used c o n t a i n e d : 03 , IO a n d 2 0 % ; N 2, 2, 4, 8 a n d 1 6 % ; t h e r e s i d u e w a s m a d e up w i t h a r g o n to a t m o s p h e r i c pressure. I n each e x p e r i m e n t o r g a n i s m s f r o m t h e s a m e s u b c u l t u r e were g r o w n a t all 03 a n d N , c o n c e n t r a t i o n s to e l i m i n a t e v a r i a t i o n s from one s u b c u l t u r e to a n o t h e r . T o t a l N w a s d e t e r m i n e d b y an u l t r a m i c r o K j e l d a h l m e t h o d I a n d t h e fi rs t -orde r v e l o c i t y c o n s t a n t , k, d e t e r m i n e d . T h e r e s u l t s were p l o t t e d a c c o r d i n g to DIXON'S g r a p h i c a l e x t e n s i o n s of LINEWEAVER AND BURK'S m e t h o d 3. T h is p l o t s h o w e d an i n h i b i t i o n of n i t r o g e n f i x a t i o n b y O 3 c o m p e t i t i v e w i t h r e s p e c t to Nz. The Michaelis c o n s t a n t s for n i t r o g e n f i x a t i o n a v e r a g e d a b o u t i % a t i o % O 3 a n d a b o u t 2 % a t 2 0% 03, t h e difference being s t a t i s t i c a l l y s i g n i f i c a n t ( P ~ o.oi). The v a l u e for 20% o x y g e n is in a g r e e m e n t w i t h t h a t found b y WILSON, ]3URRIS AND LIND in 19424 a t t h e s a m e 02 c o n c e n t r a t i o n . A t 3 0 % 02, WILSON AND ROBERTS 5 found a Michaelis c o n s t a n t of 6 % , b u t w i t h our o r g a n i s m i t w a s n o t possible to d e t e r m i n e t h e c o n s t a n t a t this 02 c o n c e n t r a t i o n b e c a u s e n i t r o g e n f i x a t i o n w a s no longer t h e l i m i t i n g f a c t o r in g r o w t h . This is a n effect of t h e c o m p l e x r e l a t i o n b e t w e e n 02 c o n c e n t r a t i o n , n i t r o g e n f i x a t i o n a n d g r o w t h in ,q zotobacter. BURK 6 showed, a n d we h a v e confirmed 7, t h a t i n c r e a s i n g O 3 c o n c e n t r a t i o n can i n h i b i t g r o w t h w h e t h e r n i t r o g e n - f i x a t i o n is p r o c e e d i n g or not. H o w e v e r our finding i n d i c a t e s t h a t , c o n t r a r y to BURK'S v i e w s 6, t h e r e is also specific i n h i b i t i o n b y O~ of t h e n i t r o g e n - f i x a t i o n process, a n d further, t h a t t h e i n h i b i t i o n is c o m p e t i t i v e . Thi s is in a c c o r d a n c e w i t h PARKER'S h y p o t h e s i s s t h a t O~ a n d N~ c a n c o m p e t e as a l t e r n a t i v 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 s in t h e r e s p i r a t i o n of A~otobacter. ]3AYLISS9 ha s s h o w n t h a t t h i s h y p o t h e s i s is t h e r m o d y n a m i c a l l y possible. C. A. PARKER Institute O/A griculture, University o/ Western A ustralia, Nedlands, Perth (d uslralia) P . B . SCUTT 1 C. A. PARKER, (unpublished). 2 M. DIXON, Biochem. J., 55 (1953) ~ 7°. 3 H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 4 p. W. WILSON, R. H. BURRIS AND C. J. LIND, Proc. Natl. Acad. Sci., U.S., 28 (1942) 243. 5 T. G. G. WILSON AND E. R. ROBERTS, Biochim. Biophys. dcta, 15 (I954) 390. e D. BURK, J. Phys. Chem., 34 (193 o) 1195. 7 C. A. PARKER AND P. B. SCUTT (unpublished). 8 C. A. PARKER, Nature, 173 (I954) 870. 9 N. S, BAYLISS, Australian J. Biol. Sci., 9 (1956) 364 • R e c e i v e d M a y 29th, 1958
PRELIMINARY NOTES
66I
Solubilization and assay of the "insoluble" a-glycerophosphate dehydrogenaseof animal tissues* W h i l e t h e D P N - l i n k e d a - g l y c e r o p h o s p h a t e d e h y d r o g e n a s e (a-GPD) is r e a d i l y solubilized a n d purified, its m i t o c h o n d r i a l c o u n t e r p a r t , w h i c h r e d u c e s c y t o e h r o m e c w i t h o u t t h e m e d i a t i o n of D P N 1, h a s n o t been p r e v i o u s l y o b t a i n e d in t r u e solution, e x t e n s i v e l y purified, or characterized. E x t r a c t i o n of v a r i o u s m i t o c h o n d r i a l p r e p a r a t i o n s -with bile salts 2, w i t h or w i t h o u t p r e v i o u s digestion w i t h p r o t e o l y t i c e n z y m e s 3, a n d d i g i t o n i n e x t r a c t i o n 4 yield stable dispersions w h i c h do n o t s e d i m e n t readily in t h e u l t r a c e n t r i f u g e . Such " t e m p o r a r i l y solubilized ''2 p r e p a r a t i o n s do n o t lend t h e m s e l v e s to e x t e n s i v e purification b y classical m e t h o d s a n d u p o n c o m p l e t e r e m o v a l of t h e solubilizing a g e n t a n d on r e p e a t e d freezing a n d t h a w i n g t h e y u s u a l l y r e v e r t to t h e p a r t i c u l a t e form. I n c o n t r a s t , digestion of m i t o c h o n d r i a l a c e t o n e p o w d e r s w i t h lecithinase A a t n e u t r a l p H releases t h e d e h y d r o g e n a s e in soluble form, as j u d g e d b y t h e following facts: (i) it r e m a i n s in solution a f t e r e x t e n s i v e dialysis a n d r e p e a t e d freezing a n d t h a w i n g ; (2) it does n o t s e d i m e n t at i44,ooo × g in I h; a n d (3) it m a y be readily f r a c t i o n a t e d with (NH4)2SO , a n d a h i g h salt conc e n t r a t i o n is required for precipitation, while dispersions p r e p a r e d with d i g i t o n i n precipitate at a relatively low (NH4)~SO 4 c o n c e n t r a t i o n . I n t h e initial e x p e r i m e n t s a c e t o n e p o w d e r s of p i g - b r a i n m i t o c h o n d r i a were t r e a t e d with cobra ( N a j a naja) or r a t t l e s n a k e (Crotalus terrificus) v e n o m s as sources of lecithinase A. E s s e n t i a l l y c o m p l e t e e x t r a c t i o n w a s o b t a i n e d w i t h relatively little loss of a c t i v i t y . T h e fact t h a t t h e p h o s pholipase w a s t h e a g e n t responsible for t h e solubilization was confirmed b y t h e use of crystalline c r o t o x i n (the kind gift of Dr. FRAENKEL-CONRAT).T h e clear, yellow e x t r a c t o b t a i n e d was s u b j e c t e d to t w o cycles of (NH4)2SO 4 fractionation, followed b y differential u l t r a c e n t r i f u g a t i o n , r e s u l t i n g in a n a m b e r - c o l o r e d solution of t h e purified e n z y m e . T h e a b s o r p t i o n s p e c t r u m showed a p r o m i n e n t p e a k a t 412 m/~, w h i c h was n o t a Soret b a n d , since h e i n e c o m p o u n d s were a b s e n t . T h i s p e a k is c h a r a c t e r i s t i c of a - G P D , since a - g l y c e r o p h o s p h a t e (a-GP) bleached it r a p i d l y a n d completely, yielding a difference s p e c t r u m w i t h a p e a k at 412 m/~ a n d a s h o u l d e r in t h e flavin region (455 rap). As s h o w n in T a b l e I, p h e n a z i n e m e t h o s u l f a t e is t h e preferred assay, as w i t h o t h e r " c y t o c h r o m e r e d u c i n g d e h y d r o g e n a s e s " , a n d gives t h e h i g h e s t a c t i v i t y in purified soluble p r e p a r a t i o n s , in dispersed p r e p a r a t i o n s , a n d in i n t a c t m i t o c h o n d r i a l p r e p a r a t i o n s f r o m brain, yeast, as well as skeletal m u s c l O . W i t h b r a i n m i t o c h o n d r i a t h e a c t i v i t y in t h e m a n o m e t r i c p h e n a z i n e a s s a y s in t h e presence of c y a n i d e is significantly h i g h e r t h a n t h r o u g h t h e c y t o c h r o m e chain, a l t h o u g h t h e t u r n o v e r r a t e of t h e c y t o c h r o m e s y s t e m is far in excess of t h e r e q u i r e m e n t of a - G P D . T h u s in t h e oxidase t e s t t h e linkage of a - G P D to t h e r e s p i r a t o r y c h a i n m a y be t h e r a t e - l i m i t i n g step. W h i l e m e t h y l e n e blue is a relatively good a c c e p t o r Ior a - G P D (provided a c t i v i t y at infinite dye concent r a t i o n is m e a s u r e d ) a n d t h i s a c t i v i t y is r e t a i n e d o n solubilization, this d y e is a poor acceptor for p a r t i c u l a t e succinic d e h y d r o g e n a s e f r o m a n i m a l t i s s u e s a n d t h e a c t i v i t y is lost on solubilization. a - G P D h a s a s h a r p o p t i m u m a t p H 7.7 (38°) in t h e m a n o m e t r i c p h e n a z i n e m e t h o s u l f a t e a s s a y a n d at p H 7.9 in t h e m e t h y l e n e blue assay. T h e K m for a-L-GP is 6. 7" Io -3 M (pH 7.7, 3 8°) a n d t h e p r o d u c t 2, d i h y d r o x y a c e t o n e p h o s p h a t e , is a p o t e n t c o m p e t i t i v e inhibitor. A n i n t e r e s t i n g p r o p e r t y of t h e e n z y m e a n d one of i m p o r t a n c e in its a s s a y is t h a t it u n d e r g o e s a n a c t i v a t i o n a n a l o g o u s to t h a t of succinic d e h y d r o g e n a s e 6, i n v o l v i n g a conversion of a less a c t i v e TABLE REACTION
RATES OF a-GPD
I WITH VARIOUS ACCEPTORS
(Vmax v a l u e s at 38°) Preparation
Brain mitochondria A c e t o n e p o w d e r of b r a i n mitochondria Digitonin-extr., purified Soluble p r e p a r a t i o n Yeast granules
Phenazi.e methosul/ate
02 (with excess cyt. c)
Cyt. c
Methylene blue
ioo
64-73
65
54
i oo ioo ioo ioo
o o o 64
o o o
58 68 ,66 58
Ferricyanide
88
* S u p p o r t e d b y g r a n t s f r o m t h e N a t i o n a l H e a r t I n s t i t u t e , U.S. Public H e a l t h Service, a n d t h e A m e r i c a n H e a r t Association, a n d b y c o n t r a c t No. N O N R z656 (oo) b e t w e e n t h e Office of N a v a l R e s e a r c h , U. S. N a v y , a n d t h e E. B. Ford I n s t i t u t e .
662
PRELIMINARY NOTES
VOL. 29 (1958)
form to a m o r e a c t i v e one u p o n brief i n c u b a t i o n w i t h t h e s u b s t r a t e in t h e a b s e n c e of c a t a l y t i c a c t i v i t y . T h e lag p eriod r e g u l a r l y o b s e r v e d in a - G P D or a - G P o x i d a s e a s s a y s is c o m p l e t e l y a b o l i s h e d b y prio r i n c u b a t i o n of t h e e n z y m e w i t h its s u b s t r a t e a n d is in all p r o b a b i l i t y a n e x p r e s s i o n of t h e a c t i v a t i o n process.
Edsel B. Ford Institute [or Medical Research, Henry Ford Hospital, Detroit, Mich. (U.S.A.)
ROBERT L. RINGLER THOMAS 19. GINGER *
I D. E. GREEN, Biochem. J., 3 ° (I936) 629. T. TUNG, L. ANDERSON AND A. LARDY, Arch. Biochem. Biophys., 40 (1952) z94. 3 K. LING, S. WU, S. TING AND T. TUNG, Intern. Symposium Enzyme Chemistry, Tokyo and Kyoto,
Japan, Oct. ~957. 4 R. L. RINGLER AND T. P. SINGER, Federation Proc., 17 (1958) 297. ii T. P. SINGER AND E. B. KEARNEY, Methods of Bioehem. Anal., 4 (1957) 307 • o E. ]3. I~EARNEY, J. Biol. Chem., 229 (1957) 363 . R e c e i v e d M a y 28th, 1958 *
E s t a b l i s h e d I n v e s t i g a t o r of the A m e r i c a n H e a r t Association.
Competitive inhibition of nitrogen fixation by oxygen T h e effect of O 3 on t h e a s s i m i l a t i o n of m o l e c u l a r N 2 h a s been r e - e x a m i n e d . Cells of Azotobacter vinelandii were g r o w n in t h e vessels of W a r b u r g r e s p i r o m e t e r s s h a k e n in a b a t h a t 3 o°. A t m o s p h e r e s used c o n t a i n e d : 03 , IO a n d 2 0 % ; N 2, 2, 4, 8 a n d 1 6 % ; t h e r e s i d u e w a s m a d e up w i t h a r g o n to a t m o s p h e r i c pressure. I n each e x p e r i m e n t o r g a n i s m s f r o m t h e s a m e s u b c u l t u r e were g r o w n a t all 03 a n d N , c o n c e n t r a t i o n s to e l i m i n a t e v a r i a t i o n s from one s u b c u l t u r e to a n o t h e r . T o t a l N w a s d e t e r m i n e d b y an u l t r a m i c r o K j e l d a h l m e t h o d I a n d t h e fi rs t -orde r v e l o c i t y c o n s t a n t , k, d e t e r m i n e d . T h e r e s u l t s were p l o t t e d a c c o r d i n g to DIXON'S g r a p h i c a l e x t e n s i o n s of LINEWEAVER AND BURK'S m e t h o d 3. T h is p l o t s h o w e d an i n h i b i t i o n of n i t r o g e n f i x a t i o n b y O 3 c o m p e t i t i v e w i t h r e s p e c t to Nz. The Michaelis c o n s t a n t s for n i t r o g e n f i x a t i o n a v e r a g e d a b o u t i % a t i o % O 3 a n d a b o u t 2 % a t 2 0% 03, t h e difference being s t a t i s t i c a l l y s i g n i f i c a n t ( P ~ o.oi). The v a l u e for 20% o x y g e n is in a g r e e m e n t w i t h t h a t found b y WILSON, ]3URRIS AND LIND in 19424 a t t h e s a m e 02 c o n c e n t r a t i o n . A t 3 0 % 02, WILSON AND ROBERTS 5 found a Michaelis c o n s t a n t of 6 % , b u t w i t h our o r g a n i s m i t w a s n o t possible to d e t e r m i n e t h e c o n s t a n t a t this 02 c o n c e n t r a t i o n b e c a u s e n i t r o g e n f i x a t i o n w a s no longer t h e l i m i t i n g f a c t o r in g r o w t h . This is a n effect of t h e c o m p l e x r e l a t i o n b e t w e e n 02 c o n c e n t r a t i o n , n i t r o g e n f i x a t i o n a n d g r o w t h in ,q zotobacter. BURK 6 showed, a n d we h a v e confirmed 7, t h a t i n c r e a s i n g O 3 c o n c e n t r a t i o n can i n h i b i t g r o w t h w h e t h e r n i t r o g e n - f i x a t i o n is p r o c e e d i n g or not. H o w e v e r our finding i n d i c a t e s t h a t , c o n t r a r y to BURK'S v i e w s 6, t h e r e is also specific i n h i b i t i o n b y O~ of t h e n i t r o g e n - f i x a t i o n process, a n d further, t h a t t h e i n h i b i t i o n is c o m p e t i t i v e . Thi s is in a c c o r d a n c e w i t h PARKER'S h y p o t h e s i s s t h a t O~ a n d N~ c a n c o m p e t e as a l t e r n a t i v 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 s in t h e r e s p i r a t i o n of A~otobacter. ]3AYLISS9 ha s s h o w n t h a t t h i s h y p o t h e s i s is t h e r m o d y n a m i c a l l y possible. C. A. PARKER Institute O/A griculture, University o/ Western A ustralia, Nedlands, Perth (d uslralia) P . B . SCUTT 1 C. A. PARKER, (unpublished). 2 M. DIXON, Biochem. J., 55 (1953) ~ 7°. 3 H. LINEWEAVER AND D. BURK, J. Am. Chem. Soc., 56 (1934) 658. 4 p. W. WILSON, R. H. BURRIS AND C. J. LIND, Proc. Natl. Acad. Sci., U.S., 28 (1942) 243. 5 T. G. G. WILSON AND E. R. ROBERTS, Biochim. Biophys. dcta, 15 (I954) 390. e D. BURK, J. Phys. Chem., 34 (193 o) 1195. 7 C. A. PARKER AND P. B. SCUTT (unpublished). 8 C. A. PARKER, Nature, 173 (I954) 870. 9 N. S, BAYLISS, Australian J. Biol. Sci., 9 (1956) 364 • R e c e i v e d M a y 29th, 1958