Biochem. J. (1971) 121, 309-316 3 09 Printed i n Great Britain T h e Purification a n d Characterization o f Acetoacetyl-Coenzyme A Reductase from Azotobacter beijerinckii B Y G . A . F . RITCHIE,* P . SENIOR A ND E . A . DAWES Department o f Biochemistry, University o f Hull, Kinygton upon Hull HU6 7RX, U.K. (Received 1 6 June 1970) A soluble acetoacetyl-CoA reductase ( E C 1.1.1.36) w a s purified 54-fold from Azotobacter beijerinckii N.C.I.B. 9067 a nd t h e reaction product identified a s D(-)-fl-hydroxybutyryl-CoA. T h e Michaelis constants f o r acetoacetyl-CoA, NADPH a n d NADH were determined a n d t h e reaction rate w a s found t o b e some fivefold greater with NADPH than with NADH. A t neutral p H t e equilibrium greatly favours t h e formation o f t h e reduced product. Substrate specificity w a s i n t h e order: acetoacetyl-CoA> acetoacetylpantetheine > acetoacetyl- (acyl-carrier protein). Th e enzyme possesses a functional thiol group, suffers inactivation b y oxygen a n d i s inhibited b y thiol-blocking reagents. Inhibition by p-chloromercuri- benzoate i s reversed b y excess o f dithiothreitol, which also protects the enzyme from inactivation b y oxygen. Ou r interest i n the biosynthesis of t h e storage compound poly-,B-hydroxybutyrate i n bacteria ha s l e d u s to purify a n d characterize t h e enzyme acetoacetyl-CoA reductase [D(-)-fl-hydroxybuty- ryl-CoA-NADP oxidoreductase, EC 1.1.1.36] found i n beijerinckii, a n organism capable o f accumulating the polymer t o the extent o f 7 0 % o f i t s d r y weight (Stockdale, Ribbons & Dawes, 1968). Nicotinamide nucleotide-linked acetoacetyl-CoA dehydrogenases have been detected i n several micro-organisms (Schindler, 1964; Kominek & Halvorson, 1965; Carr & Lascelles, 1961; Stern, d e l Campillo & Raw, 1956; Overath, Raufuss, Stoffel & Ecker, 1967). T he reduction o f aceto- acetyl-CoA o r oxidation o f ,B-hydroxybutyryl-CoA b y crude extracts o f Hydrogenomona8 H 1 6 proceeded more rapidly with NAD thanwith NADP b u t t h e stereospecificity o f t h e product, P-hydroxybutyryl- CoA, w a s not determined (Schindler, 1964). T h e enzyme from Bacillu8 cereu8 w a s found t o b e NADH-specific a n d induced coincidently with t h e accumulation o f poly-fl-hydroxybutyrate before sporulation. T h e reaction product of this enzyme w a s n o t determined (Kominek & Halvorson, 1965). The biosynthesis o f poly-/3-hydroxybutyrate i n Rhodoapirillum rubrum hasbeen shown t o proceed by a pathway involving t h e reduction o f acetoacetyl- C oA t o L(+)-fl-hydroxybutyryl-CoA, which w a s then converted into D(-)-p-hydroxybutyryl-CoA * Present address: Department o f Bio- chemistry, Waite Agricultural Research Institute, Glen Osmond, S . Austral. 5064, Australia. vi a crotonyl-CoA, the conversion being catalysed by t w o stereospecific enoyl-CoA hydratases. There w a s n o evidence f o r t h e presence o f a speci- f i c D(-)-fl-hydroxybutyryl-CoA dehydrogenase (Moskowitz & Merrick, 1969). The present work demonstrates t h e occurrence of a specific D(-)-fl- hydroxybutyryl-CoA dehydrogenase i n A . bei- jerinckii a n d indicates a major difference between t h e routes o f poly-,B-hydroxybutyrate synthesis i n this organism a n d R . rubrum. MATERIALS AND METHODS Culture o f organism. T h e organism was Azotobacter beijerinckii N.C.I.B. 9067. T h e basal medium (nitrogen- free) wa prepared from t wo solutions o f t h e following composition (g/l). Solution A : glucose, 20.0; MgS04,7H20, 0.4; CaCl2, 0.11; FeSO4,7H20, 0.012; Na2MoO4,2H20, 0.01. Solution B : K2HPO4, 2.0; NaCl, 0.40. T h e t wo solutions were autoclaved separately (15lb/in2 f o r 20min) and combined i n equal volumes after cooling. T h e pH o f t h e complete medium was 7.7. Liquid cultures ( 1 litre) were grown i n 4-litre wide-neck Erlenmeyer flasks o n a Gyrotary shaker (New Brunswick Scientific Co., Ne w Brunswick, N.J., U.S.A.) a t 30°C b y using a 1 % inoculum o f stationary-phase cells approx. 2 4h old. Large quantities of t he organism were cultured in a 20-litre fermenter with a n aeration rate o f 2 0 litres/min and automatic pH control a t 7.4. Growth w a s determined turbidimetrically a t SOOnm i n a Unicam S P . 6 0 0 spectro- photometer. T h e d r y weight of t he cells w a s determined from curves relating turbidity and d r y weight. Harvesting o f cultures. T h e organism w a s difficult t o harvest since t h e cells possessed thick slime-capsules. Small volumes were centrifuged a t 50OOg f o r 45min a t
B i o c h e m . J . ( 1 9 7 1 ) 1 2 1 , 3 0 9 - 3 1 6 3 0 9P r i n t e d i n G r e a t B r i t a i n
The P u r i f i c a t i o n a n d C h a r a c t e r i z a t i o n o f A c e t o a c e t y l - C o e n z y m e A
R e d u c t a s e from A z o t o b a c t e r b e i j e r i n c k i i
BY G . A . F . RITCHIE,* P . J . SENIOR AND E . A . DAWESD e p a r t m e n t o f B i o c h e m i s t r y , U n i v e r s i t y o f H u l l , K i n y g t o n u p o n H u l l HU6 7RX, U . K .
( R e c e i v e d 1 6 J u n e 1 9 7 0 )
A s o l u b l e a c e t o a c e t y l - C o A r e d u c t a s e ( E C 1 . 1 . 1 . 3 6 ) was p u r i f i e d 5 4 - f o l d f r o mA z o t o b a c t e r b e i j e r i n c k i i N . C . I . B . 9 0 6 7 and t h e r e a c t i o n p r o d u c t i d e n t i f i e d a s
D ( - ) - f l - h y d r o x y b u t y r y l - C o A . The M i c h a e l i s c o n s t a n t s f o r a c e t o a c e t y l - C o A ,
NADPH and NADH w e r e d e t e r m i n e d a n d t h e r e a c t i o n r a t e wa s f o u n d t o b e somef i v e f o l d g r e a t e r w i t h NADPH t h a n w i t h NADH. At n e u t r a l pH t h e e q u i l i b r i u mg r e a t l y f a v o u r s t h e f o r m a t i o n o f t h e r e d u c e d p r o d u c t . S u b s t r a t e s p e c i f i c i t y was i n
t h e o r d e r : a c e t o a c e t y l - C o A > a c e t o a c e t y l p a n t e t h e i n e > a c e t o a c e t y l - ( a c y l - c a r r i e rp r o t e i n ) . The enzyme p o s s e s s e s a f u n c t i o n a l
t h i o l g r o u p , s u f f e r s i n a c t i v a t i o n byo x y g e n and i s i n h i b i t e d by t h i o l - b l o c k i n g r e a g e n t s . 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 i s r e v e r s e d by e x c e s s o f d i t h i o t h r e i t o l , w h i c h a l s o p r o t e c t s t h e enzymef r o m i n a c t i v a t i o n by o x y g e n .
Ou r i n t e r e s t i n t h e b i o s y n t h e si s o f t h e s t o r a g e
compound p o l y - , B - h y d r o x y b u t y r a t e i n b a c t e r i a h a sl e d u s t o p u r i f y and c h a r a c t e r i z e t h e enzymea c e t o a c e t y l - C o A r e d u c t a s e [ D ( - ) - f l - h y d r o x y b u t y -ryl-CoA-NADP o x i d o r e d u c t a s e , E C 1 . 1 . 1 . 3 6 ]f o u n d i n A z o t o b a c t e r b e i j e r i n c k i i , a n o r g a n i s mc a p a b l e o f a c c u m u l a t i n g t h e p o l y m e r t o t h e e x t e n t
o f 70% o f i t s d r y w e i g h t ( S t o c k d a l e , R i b b o n s &D a w e s , 1 9 6 8 ) .
N i c o t i n a m i d e n u c l e o t i d e - l i n k e d a c e t o a c e t y l - C o Ad e h y d r o g e n a s e s h a v e b e e n d e t e c t e d i n s e v e r a l
m i c r o - o r g a n i s m s ( S c h i n d l e r , 1 9 6 4 ; Kominek &
H a l v o r s o n , 1 9 6 5 ; C a r r & L a s c e l l e s , 1 9 6 1 ; S t e r n ,
d e l C a m p i l l o & Raw, 1 9 5 6 ; O v e r a t h , R a u f u s s ,
S t o f f e l & E c k e r , 1 9 6 7 ) . The r e d u c t i o n o f a c e t o -
a c e t y l - C o A o r o x i d a t i o n o f , B - h y d r o x y b u t y r y l - C o Aby c r u d e e x t r a c t s o f H y d r o g e n o m o n a 8 H 1 6 p r o c e e d e dmore r a p i d l y w i t h NAD t h a n w i t h NADP b u t t h e
s t e r e o s p e c i f i c i t y o f t h e p r o d u c t , P - h y d r o x y b u t y r y l -C o A , w a s n o t d e t e r m i n e d ( S c h i n d l e r , 1 9 6 4 ) .
The enzyme f r o m B a c i l l u 8 c e r e u 8 was f o u n d t o b eN A D H - s p e c i f i c and i n d u c e d c o i n c i d e n t l y w i t h t h e
a c c u m u l a t i o n o f p o l y - f l - h y d r o x y b u t y r a t e b e f o r e
s p o r u l a t i o n . The r e a c t i o n p r o d u c t o f t h i s enzymewas n o t d e t e r m i n e d ( K o m i n e k & H a l v o r s o n , 1 9 6 5 ) .
The b i o s y n t h e s i s o f p o l y - / 3 - h y d r o x y b u t y r a t e i n
R h o d o a p i r i l l u m rubrum h a s b e e n shown t o p r o c e e d
by a p a t h w a y i n v o l v i n g t h e r e d u c t i o n o f a c e t o a c e t y l -CoA t o L ( + ) - f l - h y d r o x y b u t y r y l - C o A , w h i c h wast h e n c o n v e r t e d i n t o D ( - ) - p - h y d r o x y b u t y r y l - C o A
* P r e s e n t a d d r e s s : D e p a r t m e n t o f A g r i c u l t u r a l B i o -c h e m i s t r y , W a i t e A g r i c u l t u r a l R e s e a r c h I n s t i t u t e , G l e n
O s m o n d , S . A u s t r a l . 5 0 6 4 , A u s t r a l i a .
v i a c r o t o n y l - C o A , t h e c o n v e r s i o n b e i n g c a t a l y s e d
by two s t e r e o s p e c i f i c e n o y l - C o A h y d r a t a s e s . T h e r ewas no e v i d e n c e f o r t h e p r e s e n c e o f a s p e c i -f i c D ( - ) - f l - h y d r o x y b u t y r y l - C o A d e h y d r o g e n a s e( M o s k o w i t z & M e r r i c k , 1 9 6 9 ) . The p r e s e n t workd e m o n s t r a t e s t h e o c c u r r e n c e o f a s p e c i f i c D ( - ) - f l -h y d r o x y b u t y r y l - C o A d e h y d r o g e n a s e i n A . b e i -
j e r i n c k i i and i n d i c a t e s a m a j o r d i f f e r e n c e b e t w e e nt h e r o u t e s o f p o l y - , B - h y d r o x y b u t y r a t e s y n t h e s i s i n
t h i s o r g a n i s m a n d R . r u b r u m .
MATE R IALS AND METHODS
C u l t u r e o f o r g a n i s m . The o r g a n i s m was A z o t o b a c t e r
b e i j e r i n c k i i N . C . I . B . 9 0 6 7 . T h e b a s a l medium ( n i t r o g e n -f r e e ) was p r e p a r e d f r o m two s o l u t i o n s o f t h e f o l l o w i n gc o m p o s i t i o n ( g / l ) . S o l u t i o n A : g l u c o s e , 2 0 . 0 ; M g S 0 4 , 7 H 2 0 ,0 . 4 ; C a C l 2 , 0 . 1 1 ; F e S O 4 , 7 H 2 0 , 0 . 0 1 2 ; N a 2 M o O 4 , 2 H 2 0 ,0 . 0 1 . S o l u t i o n B : K 2 H P O 4 , 2 . 0 ; N a C l , 0 . 4 0 . T h e twos o l u t i o n s w e r e a u t o c l a v e d s e p a r a t e l y ( 1 5 l b / i n 2 f o r 2 0 m i n )a n d c o m b i n e d i n e q u a l v o l u m e s a f t e r
c o o l i n g .The
pHo f
t h e c o m p l e t e medium was 7 . 7 .L i q u i d c u l t u r e s ( 1 l i t r e ) w e r e g r o w n i n 4 - l i t r e w i d e - n e c k
E r l e n m e y e r f l a s k s o n a G y r o t a r y s h a k e r ( N e w B r u n s w i c kS c i e n t i f i c C o . , Ne w B r u n s w i c k , N . J . , U . S . A . ) a t 3 0 ° C b yu s i n g a 1% i n o c u l u m o f s t a t i o n a r y - p h a s e c e l l s a p p r o x .24h o l d . L a r g e q u a n t i t i e s o f t h e o r g a n i s m w e r e c u l t u r e d
i n a 2 0 - l i t r e f e r m e n t e r w i t h a n a e r a t i o n r a t e o f 2 0 l i t r e s / m i na n d a u t o m a t i c pH c o n t r o l a t 7 . 4 . G r o w t h was d e t e r m i n e d
t u r b i d i m e t r i c a l l y a t S O O n m i n a Unicam S P . 6 0 0 s p e c t r o -
p h o t o m e t e r . T h e d r y w e i g h t o f t h e c e l l s was d e t e r m i n e df r o m c u r v e s r e l a t i n g t u r b i d i t y a n d d r y w e i g h t .
H a r v e s t i n g o f c u l t u r e s . T h e o r g a n i s m w a s d i f f i c u l t t oh a r v e s t s i n c e t h e c e l l s p o s s e s s e d t h i c k s l i m e - c a p s u l e s .S m a l l v o l u m e s were c e n t r i f u g e d a t 5 0 O O g f o r 4 5 m i n a t
G . A . F . RITCHIE, P . J . SENIOR AND E . A . DAWES
0 0 C (MSE M i s t r a l 6 L , r o t o r n o . 6 9 1 6 7 ) , a f t e r w h i c h m o s to f t h e s u p e r n a t a n t c o u l d b e d e c a n t e d . L a r g e r v o l u m e sw e r e c o l l e c t e d b y c o n t i n u o u s - f l o w c e n t r i f u g a t i o n a t
9 0 0 0 g w i t h a f l o w r a t e o f 7 0 m l / m i n ( G r i f f i n C h r i s t J u n i o r
1 5 0 0 0 , w i t h 3 0 0 m l - c a p a c i t y r o t o r ) .P r e p a r a t i o n o f c e l l e x t r a c t 8 . C e l l e x t r a c t s w e r e p r e p a r e d
b y d i sr u pt i o n o f c e l l s i n a F r e n c h p r e s s u r e c e l l ( M i l n e r ,L a w r e n c e & F r e n c h , 1 9 5 0 ) . H a r v e s t e d c e l l s w e r e r e -s u s p e n d e d i n 67mM-Na2HPO4-KH2PO4 b u f f e r , p H 7 . 2 ,a t 4 ° C a n d p a s s e d t w i c e t h r o u g h t h e p r e c o o l e d p r e s s u r e
c e l l i n 20ml p o r t i o n s . L a r g e q u a n t i t i e s o f c e l l s w e r e
d i s r u p t e d b y u l t r a s o n i c t r e a t m e n t w i t h a S o n i p r o b e t y p e
1 1 3 0 A (Dawe I n s t r u m e n t s L t d . , London W . 3 , U . K . ) i nc o m b i n a t i o n w i t h a c o o l e d a l l - g l a s s f l o w - t h r o u g h v e s s e l o f
25ml c a p a c i t y . Washed c e l l s w e r e s u s p e n d e d i n 0 . 2 M -
KH2PO4-K2HPO4 b u f f e r , p H 7 . 2 , c o n t a i n i n g 10mM-2 - m e r c a p t o e t h a n o l , a n d p a s s e d t h r o u g h t h e u l t r a s o n i c
v e s s e l a t 2 0 m l / m i n a n d a t 8A c u r r e n t . The c r u d e c e l le x t r a c t s w e r e c e n t r i f u g e d a t 3 0 0 0 0 g f o r 2 0 m i n a t 0 0 C( S o r v a l l R C - 2 B , r o t o r S S - 3 4 ) a n d t h e r e s u l t i n g b u p e r n a t a n tu s e d a s s t a r t i n g m a t e r i a l f o r e n z y m e p u r i f i c a t i o n .
P r e p a r a t i o n & . A c e t o a c e t y l - C o A w a s p r e p a r e d b yt r e a t i n g C oA w i t h d i k e t e n . CoA ( 2 0 m g ) w a s d i s s o l v e d i n2ml o f c o l d 0 . 2 M - K H C O 3 t h r o u g h w h i c h a s t r e a m o f N 2was p a s s e d . D i k e t e n ( 0 . 1 m l ) i n 1 ml o f d i e t h y l e t h e r w a sa d d e d o v e r 3 0 m i n . E x c e s s o f d i k e t e n w a s r e m o v e d i n as t r e a m o f N 2 , a n d t h e r e a c t i o n m i x t u r e w a s a d j u s t e d t o
p H 4 . 0 w i t h 1 . O M - H C I a n d e x t r a c t e d f o u r t i m e s w i t h 3mlo f d i e t h y l e t h e r . E t h e r w a s r e m o v e d f r o m t h e a q u e o u s
l a y e r i n a s t r e a m o f N 2 . T h e y i e l d b a s e d o n a d d e d C oAw a s 90-95% b y s p e c t r o p h o t o m e t r i c a s s a y a t 3 03 nm i n0 . 1 M - t r i s - H C l b u f f e r , p H 8 . 8 , c o n t a i n i n g 2 0 m M - M g C I 2 ,a n d b y u s i n g t h e v a l u e e = 2 . 2 x 1 0 7 I m o l - ' . c m - ' ( B e i n e r t ,1 9 5 3 ) ; h o w e v e r , t h e y i e l d w a s c o n s i d e r a bl y l o w e r ( 5 0 -
70%) when b a s e d o n a c e t o a c e t y l - C o A m e a s u r e d w i t h
L - , - h y d r o x y b u t y r y l - C o A d e h y d r o g e n a s e a s d e s c r i b e d
b e l o w . The d i s c r e p a n c y w a s p r e s u m a b l y d u e t o s i d er e a c t i o n s w i t h d i k e t e n , a n d t h e e n z y m i c a ll y d e t e r m i n e d
c o n c e n t r a t i o n s h a v e b e e n u s e d t h r o u g h o u t .
A c e t o a c e t y l p a n t e t h e i n e w a s s y n t h e s i z e d b y t r e a t i n g at h r e e f o l d e x c e s s o f d i k e t e n w i t h p a n t e t h e i n e a t roomt e m p e r a t u r e f o r 1 . 5 h b e f o r e e x t r a c t i o n a n d a s s a y a t3 03 nm i n t h e way d e E c r i b e d a b o v e f o r a c e t o a c e t y l - C o A .
P a n t e t h e i n e w a s p r e p a r e d b y r e d u c t i o n o f D - p a n t e t h i n e
w i t h KBH4.D ( - ) - f - H y d r o x y b u t y r y l - C o A w a s p r e p a r e d b y a
p r o c e d u r e d e s c r i b e d b y R i t c h i e & Dawes ( 1 9 6 9 ) .T h e p r e p a r a t i o n o f a c e t o a c e t y l - ( a c y l - c a r r i e r p r o t e i n ) w a s
a n a l o g o u s t o t h e m e t h o d o f A l b e r t s , M a j e r u s , T a l a m o &
V a g e l o s ( 1 9 6 4 ) . F r e e z e - d r i e d a c y l - c a r r i e r p r o t e i n ( 1 . 0 t m o l ,1 1 . 0 m g ) was d i s s o l v e d i n 2ml o f 10mM-KH2PO4-K2HPO4 b u f f e r , p H 6 . 3 , c o n t a i n i n g l 0 m M - 2 - m e r c a p t o -e t h a n o l a n d a l l o w e d t o s t a n d a t 4 ° C f o r 6 h . The p r o t e i n
s o l u t i o n was a p p l i e d t o a D E A E - c e l l u l o s e c o l u m n ( 1 cm x
4 c m ) p r e v i o u s l y e q u i l i b r a t e d w i t h l0mM-KH2PO4-K 2 H P O 0 b u f f e r , pH 6 . 3 , a t room t e m p e r a t u r e . The c o l u m nwas w a s h e d w i t h t h e s a m e b u f f e r u n t i l a l l f r e e t h i o lm a t e r i a l h a d b e e n e l u t e d . T h e r e d u c e d a c y l - c a r r i e rp r o t e i n w a s e l u t e d w i t h t h e a b o v e b u f f e r c o n t a i n i n g
0 . 5 M - L i C l a n d m o n i t o r e d b y m e a s u r e m e n t o f e x t i n c t i o n
a t 280nm b y u s i n g m i c r o c u v e t t e s ( 1 c m l i g h t - p a t h ) a n d aU n i c a m S P . 5 0 0 s p e c t r o p h o t o m e t e r . F r a c t i o n s c o n t a i n i n gp r o t e i n w e r e c o m b i n e d a n d t h e pH w a s a d j u s t e d t o 8 . 0w i t h 0 . 2 m - K H C O 3 . T h e r e d u c e d a c y l - c a r r i e r p r o t e i n was
t r e a t e d a t 2 0 C f o r 45min w i t h d i k e t e n ( 4 0 , l ) i n 1 ml o f
e t h e r i n a s t r e a m o f 0 2 - f r e e N 2 . The r e a c t i o n m i x t u r e w a s
a c i d i f i e d t o p H 3 . 5 - 4 . 0 w i t h 0 . 1 M - H C I a n d t h e a c y l - c a r r i e rp r o t e i n d e r i v a t i v e p r e c i p i t a t e d . A f t e r c e n t r i f u g a t i o n , t h e
s u p e r n a t a n t w a s r e m o v e d a n d t h e d e r i v a t i v e r e s u s p e n d e di n 0 . 1 M - H C I a n d c e n t r i f u g e d a g a i n . The w a s h i n g p r o c e d u r e
w a s r e p e a t e d t h r e e t i m e s a n d t h e a c y l - c a r r i e r p r o t e i n
d e r i v a t i v e r e d i s s o l v e d i n 2 . O m l o f 0 . 2 5 M - i m i d a z o l e - H C l
b u f f e r , p H 6 . 2 .A c e t o a c e t y l - ( a c y l - c a r r i e r p r o t e i n ) was d e t e r m i n e d a s
a c e t o a c e t a t e b y t h e method o f W a l k e r ( 1 9 5 4 ) a f t e ra l k a l i n e h y d r o l y s i s u n d e r t h e f o l l o w i n g c o n d i t i o n s .
A c e t o a c e t y l - ( a c y l - c a r r i e r p r o t e i n ) ( 5 0 n m o l o f p r o t e i n )w a s i n c u b a t e d w i t h 0 . 1 m l o f 1M-NaOH f o r 90min a t
3 0 0 C . A c yl - c a r r i e r p r o t e i n was p r e c i p i t a t e d b y a d d i t i o no f 0 . 4 0 m l o f 3 M - H C I a n d t h e s u p e r n a t a n t s o l u t i o n t a k e n
f o r d e t e r m i n a t i o n o f a c e t o a c e t a t e . S t a n d a r d s o l u t i o n s o f
l i t h i u m a c e t o a c e t a t e w e r e t r e a t e d i n t h e same way a n d
c o m p a r e d w i t h u n t r e a t e d s t a n d a r d s . A c e t o a c e t a t e was
n o t d e s t r o y e d b y t h e h y d r o l y t i c p r o c e d u r e .
P r e p a r a t i o n o f t h e S e p h a r o s e 4 B c o l u m n . S e p h a r o s e 4B( P h a r m a c i a , U p p s a l a , S w e d e n ) was o b t a i n e d a s a p r e -s w o l l e n g e l . The g e l was d i l u t e d w i t h t h e e l u t i n g b u f f e r
(5mM-KH2PO4-K2HPO4 b u f f e r , p H 7 . 4 , c o n t a i n i n g0 . 0 1 M - 2 - m e r c a p t o e t h a n o l ) a n d l o a d e d i n o n e v o l u m e on t o
a c o l u m n ( 2 . 7 c m x 4 7 c m ) . D u r i n g p r e p a r a t i o n o f t h e
c o l u m n a p r e s s u r e h e a d o f 1Ocm was m a i n t a i n e d a n d t h i sw a s i n c r e a s e d t o 25cm b e f o r e u s e . A c i r c l e o f W h a t m a nn o . 5 4 p a p e r w a s p l a c e d o n t o p o f t h e c o l u m n . The f l o w
r a t e d u r i n g c h r o m a t o g r a p h y was 4 . 7 m l / h p e r c m 2 a n d t h e
v o i d v o l u m e , d e t e r m i n e d w i t h 0 . 5 % B l u e D e x t r a n 2 0 0 0
i m m e d i a t e l y a f t e r c h r o m a t o g r a p h y , was 8 8 m l f o r t h e
c o l u m n b e d v o l u m e o f 2 7 0 m l .
E l e c t r o p h o r e s i s . E l e c t r o p h o r e s i s was c a r r i e d o u t i n a
d i s c e l e c t r o p h o r e s i s a p p a r a t u s ( S h a n d o n S c i e n t i f i c C o .
L t d . , L o n d o n N . W . 1 0 , U . K . ) b y u s i n g t h e s t a n d a r d
p r o c e d u r e s g i v e n b y t h e m a n u f a c t u r e r s . G e l s c o n t a i n e d
7 . 5 % ( w / v ) a c r y l a m i d e a n d p r o t e i n s a m p l e s w e r e r e t a i n e d
o n t h e l a r g e - p o r e g e l b e f o r e e l e c t r o p h o r e s i s w i t h a s m a l l
amount o f d r y S e p h a d e x G - 7 5 . E l e c t r o p h o r e s i s was
c o n d u c t e d a t 2 ° C f o r 6 0 m i n a t 5 m A / r u n n i n g t u b e .
P r o t e i n m e a s u r e m e n t . P r o t e i n w a s d e t e r m i n e d b y t h e
m e t h o d o f L o w r y , R o s e b r o u g h , F a r r & R a n d a l l ( 1 9 5 1 ) ,w i t h c r y s t a l l i z e d b o v i n e s e r u m a l b u m i n , d r i e d t o c o n s t a n t
w e i g h t , a s a s t a n d a r d .
A 8 s a y o f a c e t o a c e t y l - C o A r e d u c t a 8 e . F o r a s s a y o f e n z y m ea c t i v i t y t h e c o r r e c t e d l i n e a r d e c r e a s e o r i n c r e a s e i n E 3 4 0w a s m e a s u r e d i n a U n i c a m S P . 8 0 0 r e c o r d i n g s p e c t r o p h o t o -m e t e r a t 3 0 0 C . F o r t h e r e d u c t i o n r e a c t i o n e a c h a s s a ym i x t u r e c o n t a i n e d ( f i n a l c o n c e n t r a t i o n s ) 6OmM-KH2PO4-
K2HPO4 b u f f e r , p H 5 . 5 , 1 2 m M - M g C I 2 , 0 . 5 m M - d i t h i o -t h r e i t o l , 0.24mM-NADPH, 2 8 - 4 0 j u M - a c e t o a c e t y l - C o A a n d0 . I m l o f c e l l e x t r a c t i n a t o t a l v o l u m e o f 2 . 5 m l . T h eb u f f e r , M g 2 + , d i t h i o t h r e i t o l a n d e x t r a c t w e r e p r e i n c u ba t e d
f o r 5min b e f o r e a d d i t i o n o f NADPH. The d e c r e a s e i nE 3 4 0 w a s r e c o r d e d f o r 1 min b e f o r e a d d i t i o n o f a c e t o a c e t y l -
C o A . T h e r e was no l a g a n d l i n e a r i n i t i a l r a t e s w e r e
r e c o r d e d f o r 2 m i n .
At pH v a l u e s b e l o w 6 . 5 t h e r e was a s i g n i f i c a n t l i n e a rr a t e o f d e c r e a s e i n E 3 4 0 d u e t o t h e i n s t a b i l i t y o f b o t hNADH a n d NADPH a t a c i d p H . F o r e a c h enzyme a s s a yi t was n e c e s s a r y t o c o r r e c t t h e i n i t i a l r e a c t i o n r a t e s f o r t h i sd e s t r u c t i o n . The e x t i n c t i o n o f NADPH a t 3 40nm w a sm e a s u r e d a t b o t h pH 5 . 5 a n d 8 . 5 . The v a l u e s w e r e i d e n t i -
c a l , i n d i c a t i n g t h a t t h e p u b l i s h e d v a l u e o f c = 6 . 2 2 x
1 0 3 1 - m o l 1 - c m - 1 ( H o r e c k e r & K o r n b e r g , 1 9 4 8 ) w a s
a p p l i c a b l e a t l o w pH v a l u e s .
T h e a s s a y m i x t u r e f o r t h e o x i d a t i o n r e a c t i o n c o n t a i n e d
( f i n a l c o n c e n t r a t i o n s ) 3 0 m M - t r i s - H C l b u f f e r , p H 8 . 5 ,
1 2 m M - M g C l 2 , 0 . 5 m M - d i t h i o t h r e i t o l , 0.4mM-NADP+,0 . 2 m m - f i - h y d r o x y b u t y r y l - C o A a n d 0 . 1 m l o f c e l l e x t r a c t
i n a t o t a l v o l u m e o f 2 . 5 m l .
U n i t o f e n z y m e a c t i v i t y . O n e u n i t o f e n z y m e i s t h a tamount o f p r o t e i n w h i c h c a t a l y s e s t h e r e d u c t i o n o r
o x i d a t i o n o f 1 i t m o l o f s u b s t r a t e / m i n a t pH 5 . 5 o r 8 . 5 a n d
a t 3 0 ° C .
E n z y m e p u r i f i c a t i o n . A l l p r o c e d u r e s w e r e c a r r i e d o u t a t
2 ° C . The b u f f e r s u s e d t h r o u g h o u t w e r e KH2PO4-K2HPO4 m i x t u r e s a t a p p r o p r i a t e pH v a l u e s a n d , i n v i e w
o f t h e o x y g e n - s e n s i t i v i t y o f t h e e n z y m e , 2 - m e r c a p t o -
e t h a n o l was a d d e d t o a l l b u f f e r s t o g i v e a f i n a l c o n c e n t r a -
t i o n o f 0 . 0 1 M . To t h e c e l l - f r e e s u p e r n a t a n t p r e p a r e d f r o m
1 5 0 g d r y w t . o f c e l l s was a d d e d 0 . 1 v o l . o f p r o t a m i n e
s u l p h a t e s o l u t i o n ( l O m g / m l ) , p H 5 . 5 , a n d a f t e r s t a n d i n g
o n i c e f o r 2h t h e m i x t u r e was c e n t r i f u g e d a t 3 0 0 0 0 g f o r5min a n d t h e p r e c i p i t a t e d i s c a r d e d . S o l i d ( N H 4 ) 2 5 0 4 w a s
a d d e d t o t h e s u p e r n a t a n t t o 3 O % s a t u r a t i o n ( 1 6 . 4 g / 1 0 0 m l ) ,t h e s u s p e n s i o n was h e l d o v e r n i g h t a n d t h e n c e n t r i f u g e da t 3 0 0 0 0 g f o r l O m i n . The s m a l l p r e c i p i t a t e w a s d i s c a r d e d .
T h e s u p e r n a t a n t was i n c r e a s e d t o 6 0 % s a t u r a t i o n b y t h e
a d d i t i o n o f a f u r t h e r 1 8 . 1 g o f s o l i d ( N H 4 ) 2 S O 4 / l O O m l a n d
h e l d f o r 2h a t 2 0 C . The h e a v y t a n - c o l o u r e d p r e c i p i t a t ewas s e p a r a t e d b y c e n t r i f u g i n g a t l O O O O g f o r l O m i n ,
r e d i s s o l v e d i n 3 5 0 m l o f 0 . 1 4 M b u f f e r , p H 7 . 2 , a n d d i a l y s e da g a i n s t two c h a n g e s o f 5 l i t r e s o f t h e same b u f f e r . The
3 0 - 6 0 % - s a t d . - ( N H 4 ) 2 S O 4 f r a c t i o n ( 7 m g o f p r o t e i n / m l )was d i s p e n s e d i n l O m l p o r t i o n s a n d f r o z e n a t - 1 5 ° C .
S u c h f r a c t i o n s r e t a i n e d t h e i r a c t i v i t y f o r s e v e r a l m o n t h s .
F i r 8 t c h r o m a t o g r a p h y o n D E A E - c e l l u l o 8 e . A p o r t i o n o f
t h e 3 0 - 6 0 % - s a t d . - ( N H 4 ) 2 5 O 4 f r a c t i o n ( 2 0 0 m g o f p r o t e i n )
was d i a l y s e d a g a i n s t 0.5mM b u f f e r , p H 1 7 . 4 , a d s o r b e d o n aD E A E - c e l l u l o s e c o l u m n ( W h a t m a n DE 32 m i c r o g r a n u l a r ,
2.4cmx 3 0 c m ) a n d w a s h e d w i t h l O O m l o f t h e same
b u f f e r . T h e e n z y m e w a s e l u t e d w i t h a l i n e a r g r a d i e n t o f
c h l o r i d e s u p p l i e d b y m i x i n g 4 0 0 m l o f 0.5mM b u f f e r ,p H 7 . 4 , c o n t a i n i n g 0 . 3 m - K C I , i n t o 4 0 0 m l o f 0.5mMb u f f e r , p H 7 . 4 . T h e f l o w r a t e w a s 0 . 5 m l / m i n . P r o t e i n
e l u t i o n w a s m o n i t o r e d b y m e a s u r e m e n t o f E 2 8 0 . The
e n z y m e w a s e l u t e d i n t h e r a n g e 0 . 1 2 0 - 0 . 1 4 5 m M - K C l . The
m o s t a c t i v e f r a c t i o n s w e r e c o m b i n e d a n d c o n c e n t r a t e d i nd i a l y s i s t u b i n g c o v e r e d w i t h A q u a c i d e ( C a l b i o c h e m , L o s
A n g e l e s , C a l i f . , U . S . A . ) .S e c o n d c h r o m a t o g r a p h y o n D E A E - c e l l u l o 8 e . C o n c e n -
t r a t e d f r a c t i o n s c o n t a i n i n g e n z y m e w e r e d i a l y s e d o v e r -
n i g h t a g a i n s t a 2 0 0 - f o l d e x c e s s o f 12mM b u f f e r , p H 8 . 0 ,
a d s o r b e d o n a D E A E - c e l l u l o s e c o l u m n ( 1 . 3 cm x 3 0 c m ) a n dw a s h e d w i t h 5 0 m l o f t h e s a m e b u f f e r . T h e e n z y m e was
e l u t e d i n a l i n e a r c h l o r i d e g r a d i e n t s u p p l i e d b y m i x i n g
3 0 0 m l o f 12mM b u f f e r , p H 1 8 . 0 , c o n t a i n i n g 0 . 2 5 M - K C l , i n t o
3 0 0 m l o f 12mM b u f f e r , p H 1 8 . 0 . Enzyme w a s e l u t e d i n t h e
r a n g e 0 . 0 8 5 - 0 . 1 3 0 M - K C I . A c t i v e f r a c t i o n s w e r e c o m b i n e d
a n d c o n c e n t r a t e d a s b e f o r e .C h r o m a t o g r a p h y o n S e p h a r o a e 4 B . T h e c o n c e n t r a t e d
enzyme w a s d i a l y s e d a g a i n s t t w o c h a n g e s o f 5 0 0 m l o f
5 m M b u f f e r , p H 7 . 4 . A s u c r o s e s o l u t i o n , 0 . 2 5 M , was
a d d e d t o t h e e n z y m e t o g i v e a f i n a l c o n c e n t r a t i o n o f
5 0 m M , a n d t h e s o l u t i o n ( 4 . O m l ) w a s c a r e f u l l y l o a d e d o n
t h e t o p o f a c o l u m n o f S e p h a r o s e 4B p r e p a r e d a s d e s c r i b e d
a b o v e . T h e e n z y m e w a s c h r o m a t o g r a p h e d w i t h 5 mM
b u f f e r s o lu t i o n a s u s e d f o r d i a l y s i s a t a f l o w r a t e o f0 . 5 m l / m i n . A c t i v e f r a c t i o n s w e r e c o m b i n e d a n d c o n -
c e n t r a t e d t o 2 mg o f p r o t e i n / m l . V o l u m e s o f 0 . 2 m l w e r e
d i s p e n s e d a n d s t o r e d a t - 1 5 C .T a b l e 1 s u m m a r i z e s t h e p r o g r e s s o f a l a r g e - s c a l e
p u r i f i c a t i o n o f t h e e n z y m e .P r o d u c t o f t h e r e a c t i o n . An a s s a y m i x t u r e f o r t h e
r e d u c t i o n r e a c t i o n w i t h i n c r e a s e d a m o u n t s o f a c e t o a c e t y l -CoA ( 1 . 3 6 m m ) a n d NADPH ( 1 . 4 4 m M ) t o g e t h e r w i t h a
c o n t r o l m i x t u r e w i t h o u t a c e t o a c e t y l - C o A w e r e i n c u b a t e d
w i t h p u r i f i e d e n z y m e a t 3 0 ' C . On c o m p l e t i o n o f t h e r e -a c t i o n t h e d e c r e a s e o f E 3 4 0 c o n t i n u e d d u e t o d e s t r u c t i o n o f
NADPH a t pH 5 . 5 . A f t e r c o m p l e t i o n o f t h e r e a c t i o n t h e
t e s t a n d c o n t r o l i n c u b a t i o n m i x t u r e s w e r e a d j u st e d t o
pH 1 2 , b y a d d i t i o n o f 0 . 0 6 m l o f 3 . 5 M - N a O H , t o h y d r o l y s eCoA e s t e r s a n d t h e n h e a t e d a t 9 0 0 C f o r l O m i n t o d e s t r o y
r e s i d u a l NADPH. A f t e r c o o l i n g , t h e m i x t u r e s w e r e
c e n t r i f u g e d a n d t h e s u p e r n a t a n t s r e a d j u s t e d t o p H 1 8 . 5w i t h 2 M - H C I . P o r t i o n s w e r e t a k e n a s s u b s t r a t e s f o r t h e
e n z y m i c d e t e r m i n a t i o n o f D ( - ) - f i - h y d r o z y b u t y r a t e b yt h e p r o c e d u r e o f W i l l i a m s o n , M e l l a n b y & K r e b s ( 1 9 6 2 ) .
T a b l e 1 . Summary o f p u r i f i c a t i o n o f a c e t o a c e t y l - C o A r e d u c t a c e f r o m A . b e i j e r i n c k i i
O n l y t h e most a c t i v e f r a c t i o n s w e r e t a k e n a t s t e p s ( 5 ) , ( 6 ) a n d ( 7 ) .
T o t a l
v o l u m e( m l )t e p
( 1 ) C r u d e s o n i c a t e d e x t r a c t 2 7 6 0
( 1 5 0 g d r y w t . o f c e l l s )( 2 ) S u p e r n a t a n t a f t e r c e n t r i f u g i n g 2 4 1 0
a t 2 9 0 0 0 g f o r l O m i n( 3 ) P r o t a m i n e s u l p h a t e t r e a t m e n t 2 5 0 0( 4 ) ( N H 4 ) 2 S O 4 f r a c t i o n a t i o n 3 4 0
( 5 ) F i r s t c h r o m a t o g r a p h y o n
D E A E - c e l l u l o s e
( 6 ) S e c o n d c h r o m a t o g r a p h y on
D E A E - c e l l u l o s e
( 7 ) C h r o m a t o g r a p h y o n S e p h a r o s e -4B
G . A . F . R I T C H I E , P . J . SENIOR AND E . A . DAWES
C a l c i u m p h o s p h a t e g e l . T h e g e l was p r e p a r e d b y a d d i n g1 5 0 m I o f C a C I 2 s o l u t i o n ( 1 3 2 g o f C a C 1 2 , 6 H 2 0 / l ) t o 1 5 0 m Io f Na3PO4 s o l u t i o n ( 1 5 2 g o f N a 3 P O 4 , 1 2 H 2 0 / J ) . Them i x t u r e was a d j u s t e d t o pH7.4 w i t h 2 M - a c e t i c a c i d a n dt h e p r e c i p i t a t e w a s h e d b y d e c a n t a t i o n f o u r t i m e s w i t hw a t e r ( 1 5 - 2 0 l i t r e s ) . I t was s t o r e d u n d e r water i n t h ed a r k .
C a l c i u m p h o s p h a t e - g e l t r e a t m e n t . The enzyme prepara-
t i o n s u b j e c t e d t o c a l c i u m p h o s p h a t e - g e l t r e a t m e n t was onet h a t h a d b e e n t r e a t e d w i t h p r o t a m i n e s u l p h a t e f o l l o w e db y ( N H 4 ) 2 S O 4 f r a c t i o n a t i o n . The p r e c i p i t a t e f r o m30-50% s a t u r a t i o n was d i s s o l v e d i n a s m a l l v o l u m e o f
7 0 m M - p h o s p h a t e b u f f e r , p H 7 . 0 , a n d d i a l y s e d o v e r n i g h ta g a i n s t 1 0 m M - p h o s p h a t e b u f f e r , p H 6 . 0 . To t h e d i a l y s e d
enzyme w e r e a d d o d s m a l l p o r t i o n s o f g e l u n t i l no a c t i v i t yc o u l d b e d e t e c t e d i n t h e s u p e r n a t a n t . The s u s p e n s i o n wask e p t on i c e f o r 1 0 m i n , c e n t r i f u g e d a t 5 0 O O g f o r 5min a n dt h e s u p e r n a t a n t d i s c a r d e d . The g e l was w a s h e d b ys u s p e n s i o n i n a v o l u m e o f w a t e r e q u a l t o t h e v o l u m e o f
s u p e r n a t a n t a n d r e c e n t r i f u g e d . The enzyme was e l u t e df r o m t h e g e l b y two s u c c e s s i v e r e s u s p e n s i o n s i n e q u i v a l e n t
v o l u m e s o f 0 . 1 4 M - b u f f e r , p H 6 . 8 , and t h e s u p e r n a t a n t s
were c o m b i n e d . The s e v e n f o l d - p u r i f i e d enzyme r e c o r d e di n T a b l e 2 was p r e p a r e d i n t h i s w a y .
A s s a y o f a c e t o a c e t y l - C o A . A c e t o a c e t y l - C o A was
d e t e r m i n e d e n z y m i c a l l y a t 2 5 " C w i t h L ( + ) - f i - h y d r o x y -b u t y r y l - C o A d e h y d r o g e n a s e . The a s s a y m i x t u r e c o n -
t a i n e d ( f i n a l c o n c e n t r a t i o n s ) : 4 0 m M - p o t a s s i u m p h o s p h a t eb u f f e r , p H 7 . 5 ; 0 . 4 m m - d i t h i o t h r e i t o l ; 22,AM-NADPH;5 0 B , g o f L ( + ) - f i - h y d r o x y b u t y r y l - C o A d e h y d r o g e n a s e( a p p r o x . 4 u n i t s ) ; 1 0 - 5 0 , u l o f a c e t o a c e t y l - C o A s o l u t i o n
i n a t o t a l v o l u m e o f 2 . 5 m l . The r e a c t i o n was s t a r t e d b yt h e a d d i t i o n o f t h e enzyme a n d m o n i t o r e d s p e c t r o p h o t o -m e t r i c a l l y u n t i l e q u i l i b r i u m was r e a c h e d . The d e c r e a s e
i n E 3 4 0 was m e a s u r e d a n d t h i s v a l u e u s e d t o c a lc u la t e t h ei n i t i a l c o n c e n t r a t i o n o f a c e t o a c e t y l - C o A .
C h e m i c a l s . NADPH, C o A , d i k e t e n a n d D - p a n t e t h i n ewere o b t a i n e d f r o m S i g m a C h e m i c a l C o . ( S t L o u i s , M o . ,
U . S . A ) ; NAD+, NADH, L ( + ) - f l - h y d r o x y b u t y r y l - C o Ad e h y d r o g e n a s e a n d D ( - ) - p - h y d r o x y b u t y r a t e d e h y d r o g e n -a s e were f r o m C . F . B o e h r i n g e r u n d S o e h n e G . m . b . H .( M a n n h e i m , G e r m a n y ) ; NAD P+ and c r y s t a l l i n e b o v i n ea l b u m i n w e r e from B r i t i s h Drug H o u s e s L t d . ( P o o l e ,D o r s e t , U . K . ) ; d i t h i o t h r e i t o l was f r o m C a l b i o c h e m ;2 - m e r c a p t o e t h a n o l a n d p r o t a m i n e s u l p h a t e ( p u r e , f r o mh e r r i n g ) were f r o m K o c h - L i g h t L a b o r a t o r i e s L t d . ,( C o l n b r o o k , B u c k s . , U . K . ) ; p a l m i t o y l - C o A was fromP-L B i o c h e m i c a l s I n c . ( M i l w a u k e e , W i s . , U . S . A . )
RESULTS
R e l a t i o n s h i p b e t w e e n g r o w t h and s p e c i f i c a c t i v i t yo f a c e t 4 a c e t y l - C o A r e d u c t a s e . The v a r i a t i o n o f
s p e c i f i c a c t i v i t y o f a c e t o a c e t y l - C o A reductase a s a
f u n c t i o n o f t h e growth c y c l e i s recorded i n F i g . 1 .
A 4 . 5 - f o l d i n c r e a s e i n the s p e c i f i c a c t i v i t y was
observed d u r i n g e x p o n e n t i a l g r o w t h . From the
time ( I l h ) c o r r e s p o n d i n g t o the t r a n s i t i o n t o a
lower growth r a t e t h e r e was an a b r u p t f a l l w h i c h
continued u n t i l the c e l l mass h a d rea ched a m a x i -
mum i n the s t a t i o n a r y p h a s e , when constant
s p e c i f i c a c t i v i t i e s were r e c o r d e d . Cultures u s e d f o r
r-
b D
4~
0 c
-4e )0 4
1 9 7 1
5
p 404
0
0
pq
*
; -0
P s L
. P
C a
T i m e ( h )
F i g . 1 . S p e c i f i c a c t i v i t y o f a c e t o a c e t y l - C o A r e d u c t a s ed u r i n g b a t c h c u l t u r e g r o w t h i n n i t r o g e n - f r e e 1% g l u c o s emedium ( 1 l i t r e ) . T h e i n o c u l u m was l O O m l o f a 1 6 hc u l t u r e a n d s a m p l e s f o r enzyme a s s a y s were w i t h d r a w nw i t h a s e p t i c p r e c a u t i o n s d u r i n g g r o w t h . C e l l s were
c e n t r i f u g e d , w a s h e d a n d r e s u s p e n d e d i n 5ml o f O . 1 M -KH2PO4-K2HPO4 b u f f e r , pH 7 . 5 , c o n t a i n i n g 0.1 M - 2 -m e r c a p t o e t h a n o l . E x t r a c t s were p r e p a r e d b y s o n i c a t i o na t 0 ° C f o r 1 min a t 1 A . C e l l d e b r i s was removed b y c e n t r i -f u g i n g a t 6 2 0 0 g f o r 20min a t 0 ° C a n d t h e s u p e r n a t a n t sw e r e a s s a y e d u n d e r t h e s t a n d a r d c o n d i t i o n s d e s c r i b e d i nt h e M a t e r i a l s a n d M e t h o d s s e c t i o n . o , C e l l d e n s i t y ;
* , enzyme s p e c i f i c a c t i v i t y .
enzyme p u r i f i c a t i o n were h a r v e s t e d a t t h e end o f t h ee x p o n e n t i a l p h a s e .
P u r i f i c a t i o n o f a c e t o a c e t y l - C o A r e d u c t a s e . E n z y m eo b t a i n e d a f t e r t h e s e c o n d D E A E - c e l l u l o s e c h r o m a t o -g r a p h i c s t e p was 3 9 - f o l d p u r i f i e d . S a m p l e s were
c h e c k e d f o r p u r i t y by d i s c g e l e l e c t r o p h o r e s i s a t
pH 9 . 5 . A f t e r s t a i n i n g , t wo i n t e n s e bands o f p r o t e i nwere s e e n very c l o s e t o g e t h e r a n d , i n a d d i t i o n ,s e v e r a l s m a l l f a i n t l y s t a i n e d b a n d s m i g r a t i n g a t a
f a s t e r r a t e .
A f t e r c h r o m a t o g r a p h y on S e p h a r o s e 4B t h ef r a c t i o n c o n t a i n i n g enzyme w i t h h i g h e s t s p e c i f i ca c t i v i t y r e p r e s e n t e d a 5 4 - f o l d p u r i f i c a t i o n . T hed i s c - g e l - e l e c t r o p h o r e t i c p a t t e r n a t pH 9 . 5 was
e s s e n t i a l l y t h e same a s t h a t f o r enzyme a f t e r
D E A E - c e l l u l o s e c h r o m a t o g r a p h y ; t h e enzyme
a c t i v i t y c o i n c i d e d w i t h b o t h o f t h e main b a n d s .E f f e c t o f pH on enzyme a c t i v i t y . The pH o p t i m a
f o r t h e r e d u c t i o n a nd o x i d a t i o n r e a c t i o n s w i t h
p u r i f i e d enzyme a r e shown i n F i g . 2 . The optimumpH f o r r e d u c t i o n was 5 . 5 f o r both p u r i f i e d enzymean d c r u d e e x t r a c t s . The a c t i v i t y o f t h e p u r i f i e d
enzyme i n p o t a s s i u m p h t h a l a t e - s o d i u m h y d r o x i d e
b u f f e r was 80% o f t h e a c t i v i t y i n p o t a s s i u mp h o s p h a t e b u f f e r . The o x i d a t i o n r e a c t i o n showed
a r a t h e r b r o a d optimum a c t i v i t y a t pH 8 . 0 - 9 . 0 ,b u t a b o v e pH 9 . 0 t h e enzyme was d e n a t u r e d .
A c t i v i t i e s w e r e i d e n t i c a l i n p o t a s s i u m p h o s p h a t e
b u f f e r a n d t r i s - h y d r o c h l o r i c a c i d b u f f e r a t pH 7 . 0and 7 . 5 .
E f f e c t o f t e m p e r a t u r e o n e n z y m e a c t i v i t y . I n -c r e a s i n g t h e t e m p e r a t u r e f r o m 2 0 0 C t o 4 5 ' C
p r o d u c e d a l i n e a r f o u r f o l d i n c r e a s e i n t h e i n i t i a lr e a c t i o n r a t e a t pH 5 . 5 . At 5 0 0 C t h e r e was a d e -
c r e a s e i n a c t i v i t y a l t h o u g h no enzyme p r e c i p i t a t i o n
was o b s e r v e d ; t h i s m a y r e f l e c t t h e i n s t a b i l i t y o f
NADPH a t t h i s t e m p e r a t u r e and a t a c i d p H .C o e n z y m e s p e c i f i c i t y . The r e a c t i o n r a t e s w i t h
NADPH w e r e c o n s i s t e n t l y g r e a t e r t h a n w i t h
NADH a t a l l s t a g e s o f p u r i f i c a t i o n ( T a b l e 2 ) . With5 4 - f o l d p u r i f i e d enzyme t h e NADPH/NADHr e a c t i o n - r a t e r a t i o was 5 a t f o u r d i f f e r e n t s u b s t r a t e
c o n c e n t r a t i o n s .
1 6 r
* 1 20-0
0 8
- f 4: 0
4 5 6 7
pH
N8
F i g . 2 . pH o p t i m a o f a c e t o a c e t y l - C o A r e d u
s o l u t i o n s ( 0 . 1 M ) w e r e : p H 5 . 0 - 6 . 0 , p o t a s s i uNaOH; p H 5 . 5 - 7 . 5 , K H 2 P O 4 - K 2 H P O 4 ; pBH C l . A s s a y c o n d i t i o n s w e r e a s d e s c r i b e d i na n d M e t h o d s s e c t i o n a n d r e a c t i o n r a t e s w e r 4b u f f e r c h a n g e s w h e r e a p p r o p r i a t e . 0 , R e d u (* , o x i d a t i o n r e a c t i o n .
M i c h a e l i s c o n s t a n t s . T he e nz ym e s h o w e d n o r m a l
M i c h a e l i s - M e n t e n k i n e t i c s . Some d i f f i c u l t y was
e x p e r i e n c e d i n o b t a i n i n g r e p r o d u c i b l e v a l u e s o f
Km and v a l u e s w e r e t h e r e f o r e c a l c u l a t e d b o t h byL i n e w e a v e r & Burk ( 1 9 3 4 ) p l o t s and by p l o t s o f
s u b s t r a t e c o n c e n t r a t i o n v e r s u s t h e v a l u e o b t a i n e d
by d i v i d i n g t h e s u b s t r a t e c o n c e n t r a t i o n by t h e
r e a c t i o n r a t e . The s l o p e s o f t h e s t r a i g h t l i n e s w e r eo b t a i n e d by t h e method o f l e a s t s q u a r e s . F o r p u r i -
f i e d a c e t o a c e t y l - C o A r e d u c t a s e w i t h a c e t o a c e t y l -
CoA and e i t h e r NADH o r NADPH a s coenzyme
Km v a l u e s f o r a c e t o a c e t y l - C o A w e r e i n t h e r a n g e
2 . 9 2 x 1 0 - 6 - 2 1 . 1 x 1 O - 6 M w h e r e a s K m v a l u e s f o r
NADPH a n d NADH w e r e 1 . 1 9 x 1 0 - 4 - 7 . 6 5 x 1 0 - 4 M .
The m a x i m u m i n i t i a l v e l o c i t i e s f o r t h e same b a t c h
o f p u r i f i e d enzyme w e r e w i t h i n a p p r o x i m a t e l y t h e
same r a n g e f o r a c e t o a c e t y l - C o A ( 1 4 0 - 2 2 0 , u m o l /min p e r m g o f p r o t e i n ) and f o r c o e n z y m e ( 1 4 4 -
1 6 0 , u m o l / m i n p e r m g o f p r o t e i n ) .E q u i l i b r i u m c o n s t a n t . The e q u i l i b r i u m o f t h e
r e a c t i o n : A c e t o a c e t y l - C o A + NADPH + H + =D ( - ) - f l - h y d r o x y b u t y r y l - C o A + N A D P + w a s a p -
p r o a c h e d f r o m t h e l e f t - h a n d s i d e a t pH 7 . 5 ( T a b l e
3 ) . At e q u i l i b r i u m t h e r a t e s o f e x t i n c t i o n d e c r e a s e
i n b o t h t e s t and c o n t r o l i n c u b a t i o n s w e r e e q u a l . Thee q u i l i b r i u m c o n s t a n t , K, was d e t e r m i n e d f r o m t h e
e q u a t i o n :
[ A c e t o a c e t y l - C o A ] [NADPH] [ H + ]
[ D ( - ) - f - H y d r o x y b u t y r y l - C o A ] [NADP+]
The a v e r a g e v a l u e f o r K= 3 . 4 5 ± 0 . 1 0 x 1 0 - 1 0 M ( 5 ) .
J - x - , P r o d u c t o f t h e r e a c t i o n . I n t h r e e e x p e r i m e n t s w i t h
9 1 0 0 . 3 9 4 , u m o l o f a c e t o a c e t y l - C o A r e d u c e d i n t h e r e -a c t i o n medium a t pH 7 . 5 , t h e y i e l d s o f D ( - ) - . -
h y d r o x y b u t y r a t e i n t h e h y d r o l y s e d i n c u b a t i o n
i c t a s e . B u f f e r m i x t u r e on t h e c o m p l e t i o n o f t h e r e a c t i o n w e r eI 7 5 p h 9 h a l 5 a t e - r e s p e c t i v e l y 0 . 3 6 3 , 0 . 3 3 5 and 0 . 3 7 9 , u m o l r e p r e s e n t -
l t h e M a t e r i a l s i n g r e c o v e r i e s o f 9 2 . 2 , 8 5 . 1 a n d 96.2%. The r e -e c o r r e c t e d f o r a c t i o n product w a s t h e r e f o r e i d e n t i f i e d a s D ( - ) - -c t i o n r e a c t i o n ; h y d r o x y b u t y r y l - C o A .
S u b s t r a t e s p e c i f i c i t y o f t h e e n z y m e . The enzyme
T a b l e 2 . C o e n z y m e s p e c i f i c i t y f o r a c e t o a c e t y l - C o A r e d u c t a s e
A s s a y c o n d i t i o n s w e r e s t a n d a r d a n d 0 . 2 4 m M - c o e n z y m e s w e r e u s e d . Enzyme p r e p a r a t i o n s : 1 , s e v e n f o l d
p u r i f i e d a f t e r c a l c i u m p h o s p h a t e - g e l t r e a t m e n t ( 3 . 6 , u g o f p r o t e i n ) ; 2 , 2 0 - f o l d p u r i f i e d a f t e r D E A E - c e l l u l o s e
c h r o m a t o g r a p h y a t p H 1 7 . 4 ( 5 . 7 , u g o f p r o t e i n ) ; 3 , 5 4 - f o l d p u r i f i e d a f t e r S e p h a r o s e 4B c h r o m a t o g r a p h y ( 0 . 8 2 , u go f p r o t e i n ) .
G . A . F . R I T C H I E , P . J . SENIOR AND E . A . DAWES
T a b l e 3 . E q u i l i b r i u m c o m s t a n t o f t h e a c e t o a c e t y l - C o A r e d u c t a s e
T h e 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 ( f i n a l c o n c e n t r a t i o n s ) 4 0 m 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 . 5 , 1 3 1 L i m -NADPH, 1 2 7 p M - a c e t o a c e t y l - C o A , 2 m M - d i t h i o t h r e i t o l a n d 1 5 . & g o f p a r t i a l l y p u r i f i e d enzyme ( s p . a c t i v i t y6 7 . 0 , u m o l / m i n p e r m g o f p r o t e i n ) i n a t o t a l v o l u m e o f 2 . 5 m l . T h e t e m p e r a t u r e was 2 5 0 C . T h e E 3 4 0 w a sm e a s u r e d a g a i n s t a d i s t i l l e d - w a t e r r e f e r e n c e . The e n z y m e s o l u t i o n was a d d e d a n d , a f t e r m i x i n g , e x t i n c t i o n s
w e r e r e c o r d e d c o n t i n u o u s l y u n t i l e q u i l i b r i u m was r e a c h e d . T h e pH o f t h e r e a c t i o n m i x t u r e was t h e n m e a s u r e d .
The c o n c e n t r a t i o n s o f a l l c o m p o n e n t s a t e q u i l i b r i u m w e r e c a l c u l a t e d f r o m t h e c h a n g e i n e x t i n c t i o n a t 3 4 0 n m .
C o n c e n t r a t i o n a t e q u i l i b r i u m
A c e t o a c e t y l - C o A
( J M )
1 0 . 5 0
1 0 . 5 0
9 . 6 0
NADPH
( J M )
1 4 . 4 5
1 4 . 4 5
1 3 . 6 5
D - ( - ) - p - H y d r o x y b u t y r y l -CoA ( j A M )
1 1 6 . 5
1 1 6 . 5
1 1 7 . 4
Mean o f K: 3 . 4 5 ( s . D .± . 1 0 ) X 1 0 - 1 0 M
T a b l e 4 . I n h i bi t i o n o f a c e t o a c e t y U - C o A r e d u c t a . e
Each i n h i b i t o r was p r e i n c u b a t e d f o r 5min a t 3 0 0 C w i t h t h e e n z y m e b e f o r e a d d i t i o n o f c o e n z y m e and s u b -
s t r a t e . A s s a y c o n d i t i o n s were s t a n d a r d e x c e p t f o r t h e o m i s si o n o f d i t h i o t h r e i t o l . En zyme p r e p a r a t i o n s : A ,
s e v e n f o l d - p u r i f i e d e n z y m e , 20,g o f p r o t e i n / a s s a y ; B , 5 4 - f o l d p u r i f i e d e n z y m e , 0 . 3 3 , u g o f p r o t e i n / a s s a y .
The r a t e s i n t h e a b s e n c e o f i n h i b i t o r w e r e: A , 5 0 n m o l / m i n ; B , 1 4 . 1 n m o l / m i n .
I n h i b i t o r
I o d o a c e t a m i d e
I o d o a c e t a t e
N - E t h y l m a l e i m i d e
Sodium a r s e n i t e
p - C h l o r o m e r c u r i b e n z o a t e
u t i l i z e d a c e t o a c e t y l p a n t e t h e i n e a s a s u b s t r a t e ,
b u t t h e M i c h a e l i s c o n s t a n t was 2 . 6 x 1 0 - 4 M i n d i c a t -
i n g a l o w e r a f f i n i t y o f t h e enzyme f o r t h i s c o m p o u n d .
H o w e v e r , t h e m a x i m u m v e l o c i t y ( V m a x . 1 1 3 , u m o lr e d u c e d / m i n pe r mg o f p r o t e i n ) was s i m i l a r t o t h a t
w i t h a c e t o a c e t y l - C o A .A c e t o a c e t y l - ( a c y l - c a r r i e r p r o t e i n ) p r e p a r e d f o r
t h i s i n v e s t i g a t i o n c o n t a i n e d 2mol o f a c e t o a c e t a t e /
mol o f a c y l - c a r r i e r p r o t e i n but t h e a s s a y s were
b a s e d on t h e a s s u m p t i o n t h a t o n l y one r e s i d u e o f
a c e t o a c e t a t e was r e a c t i v e i n t h e e n z y m e - c a t a l y s e dr e a c t i o n . With p u r i f i e d enzyme, a c t i v i t y was
d e t e c t e d o n l y a t very h i g h enzyme c o n c e n t r a t i o n s
an d a c t i v i t y w i t h a c e t o a c e t y l - C o A was some
4 0 - f o l d g r e a t e r than w i t h a c e t o a c e t y l - ( a c y l - c a r r i e r
p r o t e i n ) . E x p e r i m e n t s t o c h e c k w h e t h e r t h e a c y l -c a r r i e r p r o t e i n e s t e r m i g h t b e r e d u c e d more r a p i d l y
by a c r u d e c e l l e x t r a c t r e v e a l e d t h a t t h i s was not
I n h i b i t i o n ( % )C o n e n . , _i( m m ) P r e p . A P r e p . B
1 . 0 0
2 . 0 0
8 . 0 0
0 . 5 0
1 . 0 0
4 . 0 0
1 . 0 02 . 0 0
0 . 0 3
0 . 0 5
1 . 0 0
4 5
9 4
1 0 0
9 5
0
3 6
4 0
8
7 3
1 0 0
8 5 1 0 0
t h e c a se: e . g . i n i t i a l r a t e s o f 0 . 1 8 , 0 . 2 5 and 0 . 3 3 , u m o l /min pe r mg o f p r o t e i n were r e c o r d e d w i t h a c e t o -
a c e t y l - ( a c y l - c a r r i e r p r o t e i n ) c o n c e n t r a t i o n s o f 5 ,1 0 and 1 5 , U M r e s p e c t i v e l y compared w i t h 6 . 2 5 , l m o l /min pe r mg o f p r o t e i n f o r a c e t o a c e t y l - C o A a t t h e
h i g h e s t c o n c e n t r a t i o n ( 1 5 , U M ) .The enzyme was i n a c t i v e w i t h a c e t o a c e t a t e o r
p a l m i t o y l - C o A a s s u b s t r a t e . No i n h i b i t i o n was
r e c o r d e d i n t h e pr e s e n c e o f a d d e d p a l m i t o y l - C o A
a t 4 0 and 8 0 , U M .E f f e c t o f t h i o t c o m p o u n d s . A s e v e n f o l d - p u r i f i e d
enzyme was p r o t e c t e d by d i t h i o t h r e i t o l , c y s t e i n e ,
GSH, t h i o g l y c o l l a t e , 2 - m e r c a p t o e t h a n o l and 2 , 3 -d i m e r c a p t o p r o p a n - 1 - o l , a l l a t mM. I n t h e a b s e n c e
o f a t h i o l compound a c t i v i t y was r a p i d l y l o s t b u tc o u l d b e f u l l y r e s t o r e d by i n c u b a t i o n f o r 2min a t
3 0 0 C w i t h any o f t h e s e t h i o l compounds a t 1mm.
The 5 4 - f o l d - p u r i f i e d enzyme was not i n a c t i v a t e d
V o l . 1 2 1 ACETOACETYL-CoA R ED U C T AS E FROM AZOTOBACT ER 3 1 5
s o r a p i d l y u n d e r t h e s e c o n d i t i o n s and 0.5mM-d i t h i o t h r e i t o l e i t h e r p r o t e c t e d c o m p l e t e l y o r s l i g h t l ys t i m u l a t e d t h e e n z y m e .
I n h i b i t i o n 8 t u d i m e . T he e nz ym e w a s i n h i b i t e d byv a r i o u s t h i o l r e a g e n t s ( T a b l e 4 ) . At t h e c o n c e n -
t r a t i o n s t e s t e d , N - e t h y l m a l e i m i d e and p - c h l o r o -
m e r c u r i b e n z o a t e b r o u g h t a b o u t s i g n i f i c a n t i n h i b i -t i o n o f t h e p u r i f i e d enzyme ( i o d o a c e t a t e was n o tt e s t e d ) . R e v e r s a l o f 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 was a c h i e v e d by a n e x c e s s o f d i -t h i o t h r e i t o l , e . g . 0 . 0 5 m M - p - c h l o r o m e r c u r i b e n z o a t ee f f e c t e d 100% i n h i b i t i o n on enzyme w i t h an i n i t i a lr e a c t i o n r a t e o f 5 0 , t m o l / m i n p e r m g o f p r o t e i n a n dI . O m m - d i t h i o t h r e i t o l r e s t o r e d a b o u t h a l f t h e i n i t i a la c t i v i t y .
DISCUSSION
A l t h o u g h a c e t o a c e t y l - C o A r e d u c t a s e a p p e a r s t o
b e a c o n s t i t u t i v e enzyme i n A . b e i j e r i n c k i i , a markedv a r i a t i o n i n s p e c i f i c a c t i v i t y i s o b s e r v e d d u r i n gg r o w t h i n b a t c h c u l t u r e , a p e a k b e i n g r e a c h e d b e f o r e
t h e o n s e t o f t h e m a s s i v e a c c u m u l a t i o n o f p o l y - / -h y d r o x y b u t y r a t e t h a t o c c u r s t o w a r d s t h e e n d o f
e x p o n e n t i a l g r o w t h . At t h i s t i m e t h e oxygenc o n c e n t r a t i o n o f t h e medi um f a l l s t o z e r o ( R i t c h i e ,S e n i o r & D a w e s , 1 9 6 9 ) . H i g h s p e c i f i c a c t i v i t i e s were
d e t e c t e d i n c e l l e x t r a c t s and t h e enzyme was f o u n dt o r e a c t w i t h NADPH a t f i v e t i m e s t h e i n i t i a l r a t e
w i t h NADH. T h i s i n c r e a s e d r a t e was a l s o o b s e r v e d
w i t h t h e p u r i f i e d enzyme and i s n o t a t t r i b u t a b l e t o
t h e a c t i v i t y o f a t r a n s h y d r o g e n a s e .
The p r o p e r t i e s o f t h i s r e d u c t a s e a r e s i m i l a r t ot h o s e o f a c e t o a c e t y l C o A r e d u c t a s e p u r i f i e d f r o mp i g e o n l i v e r ( W a k i l & B r e s s l e r , 1 9 6 2 ) . The pHo p t i m u m , coenzyme s p e c i f i c i t y , r e a c t i o n p r o d u c tand p h y s i o l o g i c a l e q u i l i b r i u m a r e a l l a n a l o g o u s t o
t h e b a c t e r i a l r e d u c t a s e . D i f f e r e n c e s were e v i d e n t ,
h o w e v e r , f o r v a l u e s o f t h e M i c h a e l i s c o n s t a n t s andi n h i b i t o r c h a r a c t e r i s t i c s . The b a c t e r i a l r e d u c t a s e
h a s an a f f i n i t y f o r a c e t o a c e t y l - C o A 1 0 0 - f o l d g r e a t e r
( K m 2 . 9 x 1 0 - 6 - 2 1 . 0 X1o-6 M ) t h a n t h a t o f t h e a v i a n
e n z y m e . The b a c t e r i a l enzyme i s p r o t e c t e d f r o moxygen i n a c t i v a t i o n by t h i o l compounds andi n h i b i t e d by t h e t h i o l - b l o c k i n g r e a g e n t s N - e t h y l -
m a l e i m i d e and p - c h l o r o m e r c u r i b e n z o a t e , w h e r e a st h e a v i a n enzyme i s u n a f f e c t e d by t h e s e c o m p o u n d s .
The p r o d u c t o f e n z y m i c r e d u c t i o n i s D ( - ) - f -h y d r o x y b u t y r y l - C o A , t h e i m m e d i a t e p r e c u r s o r o f
p o l y - , - h y d r o x y b u t y r a t e . The r e a c t i o n i s r e a d i l yr e v e r s i b l e a t a l k a l i n e pH w i t h a u t h e n t i c D ( - ) - f -h y d r o x y b u t y r y l - C o A .
O v e r t h e r a n g e o f pH i n v e s t i g a t e d ( 6 . 3 - 7 . 1 ) t h e
e q u i l i b r i u m o f t h e r e a c t i o n f a v o u r s t h e f o r m a t i o no f f l - h y d r o x y b u t y r y l - C o A , t h e s u b s t r a t e f o r p o l y - , B -h y d r o x y b u t y r a t e s y n t h e t a s e , and h e n c e f a c i l i t a t e ss y n t h e s i s o f t h e p o l y m e r . At pH 7 . 5 , K = 3 . 4 5 x
1 0 - 1 0 M . C o n s e q u e n t l y , i n t h e p r e s e n c e o f a c e t o -
a c e t y l - C o A r e d u c t a s e and NADPH a t t h i s pH,
a c e t y l - C o A i s r a p i d l y c o n v e r t e d i n t o a c e t o a c e t y l -
C oA by t h e a c t i o n o f p - k e t o t h i o l a s e ( R i t c h i e , 1 9 6 8 ) ,
t h e s p e c i f i c a c t i v i t y o f w h i c h enzyme u n d e r g o e s a
s e q u e n c e o f c h a n g e d u r i n g b a t c h g r o w t h s i m i l a r t o
t h a t o b s e r v e d w i t h a c e t o a c e t y l - C o A r e d u c t a s e
( R i t c h i e e t a l . 1 9 6 9 ) .The p o s s i b i l i t y t h a t t h e r e d u c t a s e m a y b e i n -
v o l v e d i n f a t t y a c i d b i o s y n t h e s i s was i n v e s t i g a t e d
by c o m p a r i n g t h e r e l a t i v e a c t i v i t i e s o f a c e t o a c e t y l
e s t e r s o f C o A , p a n t e t h e i n e and a c y l - c a r r i e r p r o t e i n
i n t h e e n z y m i c r e a c t i o n . The a f f i n i t y o f a c e t o a c e t yl -
p a n t e t h e i n e f o r t h e enzyme i s s i g n i f i c a n t l y l e s s t h a n
t h a t o f t h e C oA e s t e r and t h e r e l a t i v e r a t e s o f
r e d u c t i o n o f a c y l - c a r r i e r p r o t e i n e s t e r and CoAe s t e r a r e a b o u t 1 : 4 0 . I t i s c o n c l u d e d t h e r e f o r e t h a t
t h e enzyme r e a c t s p r i m a r i l y w i t h t h e CoA e s t e r and
a l a c k o f s p e c i f i c i t y t o w a r d s a c e t o a c e t y l t h i o le s t e r s p e r m i t s t h e a c y l - c a r r i e r p r o t e i n and p a n t e -
t h e i n ee s t e r s t o
s e r v e a s s u b s t r a t e si n
t h i sr e a c t i o n .
A c e t o a c e t y l - C o A r e d u c t a s e i s u n a f f e c t e d by t h e
p r e s e n c e o f p a l m i t o y l - C o A , a l t h o u g h t h i s e s t e ri n h i b i t s t h e i n d i v i d u a l enzyme a c t i v i t i e s o f f a t t y
a c i d b i o s y n t h e s i s i n E 8 c h e r i c h i a c o l i ( M a j e r u s &V a g e l o s , 1 9 6 7 ) . T h e s e f i n d i n g s s u g g e s t t h a t t h e
enzyme i s n o t i n v o l v e d i n f a t t y a c i d b i o s yn t h e s i s .
The a v a i l a b l e e v i d e n c e t h u s i n d i c a t e s t h a t p o l y -
, - h y d r o x y b u t y r a t e b i o s y n t h e s i s p r o c e e d s v i a a
d i f f e r e n t s e q u e n c e o f r e a c t i o n s i n A . b e i j e r i n c k i i and
R . r u b r u m , p o s s i b l y r e f l e c t i n g t h e d i f f e r e n t modeso f e x i s t e n c e o f t h e s e b a c t e r i a i n n a t u r e .
We a r e g r a t e f u l f o r t h e a w a r d o f S c i e n c e R e s e a r c h
C o u n c i l S t u d e n t s h i p s t o G . A . F . R . a n d P . J . S .
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A l b e r t s , A . W . , M a j e r u s , P . W . , T a l a m o , B . & V a g e l o s ,P . R . ( 1 9 6 4 ) . B i o c h e m i 8 t r y , E a s t o n , 3 , 1 5 6 3 .
B e i n e r t , H . ( 1 9 5 3 ) . J . b i o l . C h e m . 2 0 5 , 5 7 5 .
C a r r , N . G . & L a s c e l l e s , J . ( 1 9 6 1 ) . B i o c h e m . J . 8 0 , 7 0 .E l l m a n , G . L . ( 1 9 5 9 ) . A r c h 8 B i o c h e m . B i o p h y s . 8 2 , 7 0 .H o r e c k e r , B . L . & K o r n b e r g , A . ( 1 9 4 8 ) . J . b i o l . C h e m .
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