1996-detection, assay, and isolation of allene oxide synthase

8/10/2019 1996-Detection, Assay, And Isolation of Allene Oxide Synthase

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25 0 PLANT P450S [28]

h o m o g e n i z e r i n a b u f f e r c o ns is ti ng o f 25 m M H E P E S , p H 7 .0 - 7 .5 , 1 m M

E G T A , 10 m M M gC 12, 2 5 m M K C1 , 5 m M D T T , 1 / z g / m l l e u p e p ti n , a n d

2 0 % g l y c e r o l. G e n e r a l l y , t h e s u s p e n s i o n c a n b e s t o r e d a t - 8 0 f o r a t l e a s t

2 w e e k s w i t h o u t s i g n i f i c a n t l o s s o f e n z y m a t i c a c t i v i t y .

A c k n o w l e d g m e n t s

This research was supported by Grant CA55254 from the National Institutes of Health

and Gr ant 94-37302-0614 from the U.S. Dep art men t of Agriculture. We than k C hristop her

Steele for helpful discussions.

[ 2 8 ] D e t e c t i o n , A s s a y , a n d I s o l a t i o n

o f A l l e n e O x i d e S y n t h a s e

B y A L A N R . B R A SH a n d W E N C HA O S O NG

I n t r o d u c t i o n

T h e e n z y m e ( s ) n o w c o m m o n l y r e f e r r e d to a s a l l e n e o x id e s y n th a s e

( A O S ) ,

C Y P 7 4 1

w a s r e p o r t e d i n 1 96 6 as a n a c t iv i ty i n f l a x se e d t h a t m e t a b o -

l iz e d l i n o l e i c a c i d h y d r o p e r o x i d e t o a n o ~ -k et ol d e r i v a t i v e . 2 E a r l y p u b l i c a -

t io n s e s ta b l i sh e d t h a t o t h e r a s s o c i a te d p r o d u c t s f r o m u n s a t u r a t e d f a t ty

a c id h y d r o p e r o x i d e s a r e y - k e t o l s a n d c y c l o p e n t e n o n e f a t t y a c id s . 3,4 T h e s e

d e r i v a ti v e s a re n o w r e c o g n i z e d t o a r i se b y h y d r o ly s i s an d r e a r r a n g e m e n t s

o f t h e i n it ia l e n z y m i c p r o d u c t , t h e v e r y u n s t a b l e a l l e n e o x i d e ( F ig . 1 ) . 5,6

T h e e a r l y l i t e r a tu r e r e f e r s t o A O S a s h y d r o p e r o x i d e i s o m e r a s e '2,3 o r

h y d r o p e r o x i d e c y c la s e, '4 a n d h y d r o p e r o x i d e d e h y d r a s e h a s a ls o b e e n

u s e d . 7 I n p l a n t s , t h e 1 2 , 1 3 - e p o x y a l l e n e o x i d e s a r e s u b s t r a t e s f o r a s p e c i fi c

c y c la s e t h a t p r o m o t e s a l m o s t c o m p l e t e c o n v e r s i o n t o a ch i ra l c y c l o p e n t e n -

o n e , 1 2 - 0 x o p h y t o d i e n o i c a c i d , a m e t a b o l i c p r e c u r s o r o f j a s m o n i c a c id . 8

1W .-C. Song, C. D. Fu nk , and A . R. B rash, Proc . Nat l . Acad. Sc i . U.S.A. 90, 85 19 (1993).

2 D. C. Zimm erman, B i o c h e m . B i o p h y s . R e s . C o m m u n . 23, 398 (1966).

3 H. W. Gardner, J. L i p i d R e s . 11, 311 (1970).

4 D. C. Zimm erman and P. Feng, L i p i d s 13, 602 (1975).

5 M. Ham berg, B i o c h i m . B i o p h y s . A c t a 920 , 76 (1987).

6 A. R. B rash, S. W. B aertschi, C. D. Ingram , and T. M . Harris, Proc . Nat l . Acad. Sc i . U.S.A.

85, 3382 (1988).

7 M. Ham berg and H . W. G ardner, B i o c h i m . B i o p h y s . A c t a 1165, 1 (1992).

8 M. Ham berg, B i o c h e m . B i o p h y s . R e s . C o m m u n . 156, 543 (1988).

Copyright 1996 by Academic Press Inc.

METHODS IN ENZYMOLOGY VOL. 272 All rights of reproduction in any form reserved.


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[281 ALLENE OXIDE SYNTHASE 25

13S-H ydroperoxylinolenic cid

H O 2 c / ( C H a ) ~ C H a

l lene O xide

Synthase H20

I ~ - - - J ~ , ~ J ~ ~ A l le n e

Ox ide

H20 ..... Ha ~ ''..

~-ketol cyclopentenone

(minor) ~-ketol (m in or on-

(m ajo r) enzymic product)

FIG. 1. Pathways of allen e oxide synthesis and degrad ation. Th e m ajor allene oxide-d erived

prod ucts starting from 13S-hydroperoxy linolenic cid are a y-ketol (12-keto-9-hydroxyoctadec-

10E,15Z -dienoic acid), an c~-ketol (12-keto-13-hydroxyo ctadec-9Z,15Z-dienoic cid), and a

cyclop enteno ne (12-oxophyto-10,15Z-dienoic cid).

P r e p a r a t i o n o f F a t t y A c i d H y d r o p e r o x i d e S u b s t r a t e f or A O S A s s a y

1 3 - H y d r o p e r o x y l i n o l e i c a c id , 1 3 - h y d r o p e r o x y l i n o l e n i c ac id , o r 1 5 -h y -

d r o p e r o x y e i c o s a t e t r a e n o i c a ci d ( 1 5 - H P E T E ) ( 1 5 - h y d r o p e r o x y a r a c h i d o n i c

a c id ) c a n b e p u r c h a s e d ( e. g. , C a y m a n C h e m i c a l , A n n A r b o r ) , b u t r e p e t i t i v e

a s sa y s r e q u i r e m u l t im i l l i g ra m s u p p li e s o f s u b s t r a t e t h a t c a n b e p r e p a r e d

f o r a s m a l l f r a c t i o n o f t h e c o m m e r c i a l c o s t .

Materials

Soybea n L ipoxyg enase (S igma Type V , Cat . No . L 6632; S igma Uses the

O l d N a m e L i pox idas e ) . T h e t y p e V is t h e m o s t c o n v e n i e n t f o r m o f

t h e e n z y m e b e c a u s e i t is a n a m m o n i u m s u l fa t e m i lk y s lu r r y t h a t c a n b e

p i p e t t e d ( i n s te a d o f h a v i n g t o w e ig h o u t s m a ll a m o u n t s o f t h e s o l id e n z y m e ) .

T h i s p r e p a r a t i o n is s t a b le f o r y e a r s a t 4 F i v e m i l li o n S i g m a u n i t s i s s u ff i ci e n t

t o p r e p a r e > 1 g o f h y d r o p e r o x i d e .

Linolen ic A c id [e .g . , S igma Cat . No . L 2376 , or Nu Chec k Prep , Inc .

(Elysian, M N ) Cat. No . U -62-A]. L i n o l e i c ac i d o r a r a c h i d o n i c a c i d i s e q u a l l y

a c c e p t a b le . P r e p a r e a s t o ck s o l u t i o n o f 10 o r 2 0 m g / m l i n e t h a n o l a n d s t o r e

a t - 2 0 o r b e l o w u n d e r n i t r o g e n o r a r g o n .

Buffer . T h e s o y b e a n li p o x y g e n a s e L - 1 ( t h e i s o z y m e i n t h e S i g m a t y p e

V p r e p a r a t i o n ) c a t al y z e s s p ec if ic r e a c t io n s a t h i g h p H , a n d t y p i c a ll y p H

8 . 5 - 1 0 . 5 b u f f e r s a r e u s e d . W e r o u t i n e l y u s e 0.1 M K 2 H P O 4 ( p H n o t a d j u s t e d

a n d u s u a l l y o b s e r v e d a t p H 8 . 5 - 9 ) .


3/10

2 5 2 P LA N T P 4 5 0 s [ 2 8 ]

2 .0 . . . . . . . . . . j

I

Absorbance [

1.0

3 . 2 1 ~ - [ l l l i

q

0

200 220 240 260 280 300 200 220 240 260 280 300

W ave leng th nm) W ave leng th nm)

FIG. 2. Preparation of 13S-hydrop eroxylinolenic acid using soybean lipoxygenase. Left)

Analytical scale reaction. U V reco rdings o f substrate 25 /zg c~-linolenic acid in 1 ml 0.1 M

K2HPO 4) and prod uct formation on addition o f 0.25/zg/ml soybean l ipoxygenase type V,

Sigma). Reaction is at roo m tem perature. Right) Preparative reaction. UV recordings of

substrate 10 mg c~-linolenic acid added in 1 ml E tO H to 50 ml of oxygen-saturated 0.1 M

K2HPO 4) and the formation of a hydroperoxide prod uct fol lowing the addit ion of 2/xg/m l

lipoxygenase. Although the UV is saturated, product formation can be quantified accurately

in diluted aliquots. The absence o f distinct chrom opho res in the 280-nm region is a reflection

of the spec ific oxyg enation and minim al formation of oxodiene by-products.

Soybean Lipoxygenase Reaction

T h i s is v e r y s t r a i g h t f o r w a r d a n d h a s b e e n u s e d t o p r e p a r e g r a m s o f f a t t y

a c i d h y d r o p e r o x i d e s . I n i t ia l l y i t is u s e f u l t o c a r r y o u t a n a n a l y t i c a l s c a l e

i n c u b a t i o n i n a n u l t ra v i o l e t U V ) c u v e t t e a n d c h e c k t h e r e a c t i o n u s i n g a

s c a n n i n g s p e c t r o p h o t o m e t e r F ig . 2 , l ef t) .

T h e sm a l l -s c a le p r e p a r a t i v e p r o c e d u r e F ig . 2 , r ig h t ) h a s t h e a d v a n t a g e

t h a t i t is o p t im i z e d f o r m i n i m a l f o r m a t i o n o f b y - p r o d u c t s a n d f o r p u r i f i c a t io n

u s in g s t a n d a r d - s i z e d h i g h -p r e s su r e l iq u id c h r o m a t o g r a p h y H P L C ) c o l u m n s

2 5 X 0 .4 6 c m ) . T o a v o i d s e c o n d a r y r e a c t i o n s t h a t c o n v e r t t h e in i t ia l h y d r o -

p e r o x i d e p r o d u c t t o d i h y d r o p e r o x i d e s a n d o x o d i e n e d e r i va t iv e s , l o w c o n -

c e n t r a t io n s o f e n z y m e g e n e r a l ly -


4/10

[281

ALLENE OXIDE SYNTHASE 53

E x t r a c t i o n o f t h e P r o d u c t

U s e o f c o l d s o l u t i o n s , w i t h t h e s a m p l e k e p t o n i c e , h e l p s w i t h t h e

q u a n t i t a t iv e r e c o v e r y o f u n d e g r a d e d f a t t y a c id h y d r o p e r o x i d e . A c i d i f y t h e

s o l u ti o n to p H 3 - 4 a n d e x t r a ct o n c e w i t h 0 .5 v o l u m e o f m e t h y l e n e c h l o r i d e

o r c h l o r o f o r m , e t h y l a c e t a t e , o r d i e t h y l e t h e r ) . C o l l e c t t h e o r g a n i c p h a s e ,

w a s h o n c e o r t w i c e w i t h w a t e r t o r e m o v e t r a c e s o f a c i d) , e v a p o r a t e t o

d r y n e ss , a n d d i ss o lv e t h e h y d r o p e r o x i d e p r o d u c t i n E t O H ~ 5 - 1 0 m g / m l ) .

K e e p o n i c e a n d s t o re a t - 2 0 u n d e r n i t r o g e n o r a r g o n .

P u r i f i c a t i o n o f F a t t y A c i d H y d r o p e r o x i d e

A s t a n d a r d 5 - o r 1 0- /z m s il ic a H P L C c o l u m n 2 5 0 .4 6 c m ) i s s u i t a b l e

f o r th e p u r if i c at io n o f a m o u n t s o f ~ 0 . 5 - 1 m g p e r i n je c ti o n. T h e H P L C

s y s t e m is e s s e n ti a ll y t h e s a m e a s s h o w n u n d e r s t ra i g h t- p h a s e S P ) - H P L C

a n a ly s is o f A O S - d e r i v e d p r o d u c t s F ig . 4 B ) . A s e m i p r e p a r a t i v e c o l u m n

e .g ., A l l t e c h E c o n o s i l 1 0 / x m s ili ca , 2 5 1 c m ) c a n b e u s e d w i t h t h e s a m e

s o l v e n t s a n d w i t h s a m p l e l o a d a n d f l o w r a t e s s c a l e d u p f i v e f o l d .

Q u a n t i ta t io n o f F a t ty A c i d H y d r o p e r o x i d e

T h e m o l a r e x t i n c t i o n c o e f f i c ie n t o f th e h y d r o p e r o x y f a t t y a c id s is

2 3,0 00 . l A s t h e m o l e c u l a r w e i g h t s a r e 3 0 8, 3 1 0, a n d 3 3 6 f o r h y d r o p e r o x y -

C18 . 2 , -C18 . 3 , and -C20 . 4 , r e spec t i ve ly , 100 t xM g ives an abso rbance a t 235

n m o f 2 .3 a b s o r b a n c e u n i ts A U ) a n d c o r r e s p o n d s to a p p r o x i m a t e l y 3 0 / x g /

m l o f p r o d u c t .

D e t e c t i o n o f A l le n e O x i d e S y n t h a s e : U V A s s a y

T h e a s s a y o f a c t i v i t y i n f r a c t i o n s f r o m e n z y m e p u r i f i c a t i o n i s e a s i l y

c a r r i e d o u t u s i n g t h e r e d u c t i o n i n U V a b s o r b a n c e a t 2 3 5 n m a s s o c i a t e d

w i t h t h e f o r m a t i o n a n d a l m o s t i n s t a n t a n e o u s h y d r o l y s is o f th e a l le n e o x i d e

m a i n l y to n o n - U V - a b s o r b i n g o t -k e t o ls F i g . 3 ) . 11

I n c r u d e s a m p l e s t h e U V a s s a y is a ls o e x t r e m e l y u se f u l, a l th o u g h c a u t i o n

m u s t b e a p p l i e d i n i t ia l ly a s t h e r e e x is ts t h e p o s s i b i li ty o f i n t e r f e r e n c e f r o m

o t h e r p a t h w a y s o f d e g r a d a t i o n o f t h e f a t t y a c id h y d r o p e r o x i d e . I n p a r ti c u la r ,

d i s ap p e a r a n c e o f th e c o n j u g a t e d d i e n e c h r o m o p h o r e o f th e h y d r o p e r o x i d e

is a ls o c a t a ly z e d b y t h e h y d r o p e r o x i d e l y a se s t h a t a r e a b u n d a n t i n s o m e

p l a n t t is s u e s . 12 T o d i s t i n g u i s h t h e s e a c t i v it i e s r e q u i r e s H P L C a n a l y s i s o r

lo M. J. G ibian and P. V anden berg,

Anal Biochem

163 , 343 1987).

11 W .-C. Son g and A. R. Bra sh, Science253 , 781 1991).

~2 H. W. G ardn er, Biochim Biophys Acta 1084, 221 1991).


5/10

2 5 4 P L AN T P 4 5 0 s [ 2 8 ]

235 nm

1 AU

full scale

~ n o enzyme

i . . . . 2 o

r i i

1 2

I '

...... i

~ e 0.5 AU

~ g i J fu llscale

1 2

M i n u t e s M i n u te s

Fro . 3. U V as say of allene oxide synthase. Conditions: 13S-hydroperoxylinolenic acid

subs t ra te 20 /z l o f 1 mg/m l E tO H s tock so lu t ion) i s mixed wi th 0 .96-0 .97 ml o f 50 m M

pho sph ate buffer, pI-I 7 , and ab sorban ce a t 235 nm is reco rded for 2 min following the add it ion

of enzyme f ina l reac t ion vo lume, 1 ml) . Lef t ) Over la id record ings 235 nm) f rom sepa ra te

assays wi th no enzym e and a f te r add i t ion o f 10 o r 20 /x l o f an ex t rac t o f 20 mg/m l ace tone

pow der of f taxseed. A ctivity is quantified as the init ia l ra te of decrea se in 235 nm absorb ance.

Righ t) R ecord ings on an expanded sca le show tha t the assay has the sens i tiv i ty to d e tec t

reactio n by 0.1/xl add ed from a 10-fold dilution) of the sam e flaxseed extract . A ssay precis ion

is ev idenced by a lmos t ind is t ingu ishab le dup l ica te a ssays o f the 20 /z l o f ex t rac t .

a n o t h e r p h y s ic o c h e m i c al t e c h n iq u e s u c h as t h e N A D P H c o u p l e d U V a s s a y

d e v e l o p e d b y V i c k J 3

D e t e c t i o n o f A O S P r o d u c t s b y H P L C

T h e s y n t h e s i s o f a l l e n e o x i d e i s c a r r i e d o u t e s s e n t i a l l y as in t h e U V

a s s a y F i g . 3 ) , a l t h o u g h i t i s c o n v e n i e n t t o s c a l e u p c o n s i d e r a b l y , e .g . , r e a c t

1 m g f a t t y a c id h y d r o p e r o x i d e , a d d e d i n a s m a l l v o l u m e o f E t O H 2 0 - 5 0

/z l) , t o i m l o f f l a x s e e d a c e t o n e p o w d e r e x t r a c t . A f t e r 5 m i n a t r o o m

t e m p e r a t u r e , a d j u s t p H t o ~ 3 - 4 , e x t r a c t i n t o m e t h y l e n e c h l o r id e , w a s h t h e

o r g a n i c p h a s e o n e t o tw o t i m e s w i t h w a t e r , a n d e v a p o r a t e t o d r y n e s s .

A n a l y s e s o f 2 - 5 a l i q u o t s o f su c h a n i n c u b a t i o n a r e i l l u s t r a t e d i n F i g . 4 A

[ r e v e r s e d - p h a s e R P ) - H P L C ] a n d F ig . 4 B S P - H P L C ) . F o r m a t i o n o f t h e

d i f f e r e n t k e t o l s f r o m t h e a l l e n e o x i d e is p H d e p e n d e n t J 4

13 B. A . Vick, Lipids 26, 315 1991).

14 A . N. G rechkin , R. A . K uram shin, E. Y. Safonova, S. K. Latyp ov, and A . V. I lyasov,

Biochim. Biophys. Acta 1086, 317 1991).


6/10

[ 2 8 ] A L L E N E O X I D E S Y N TH A SE 2 5 5

RP HPLC

. ~ ~ 235nm

~ _ _ 220 nm

a-ketol I

I I cyc lopentenone

~ - ke to . _ j ~ . ~ _ 2 0 5 n m

I . . . . . . . . ~ . . . . . . I

0 5 1 0 1 5 0

R e t e n t io n t i m e m i n )

SP HPLC

235 nm

. . ~ 220 nm

c ~ k e to lc y c l o p e n t e n o n e

_ _ , ~ J ~ . . 205 nm

5 1 0 1 5

R e t e n t io n t i m e r a in )

F IG . 4 . H P L C a n a ly s is o f a l le n e o x i d e - d e r i v e d p r o d u c t s . L e f t ) R e v e r s e d - p h a s e H P L C

a n a l y si s . C o l u m n : B e c k m a n 5 - /x m O D S U l t r a s p h e r e 2 5 0 . 46 c m ) w i t h B i o - R a d 5 - p, m

O D S g u a r d c a r tr i d g e 5 0 .4 c m ) r u n w i t h a so l v e n t o f m e t h a n o l : w a t e r : g l a c i a l a c e t ic ac id

8 0 : 2 0 : 0 . 0 1 , b y v o l u m e ) a t a f l ow r a t e o f 1 m l / m i n . R i g h t ) S t r a i g h t - p h a s e H P L C a n a ly s i s.

C o l u m n : A l l t e c h 5 - / x m s il ic a c o l u m n 2 5 x 0 .4 6 c m ) r u n w i t h a s o l v e n t o f h e x a n e : i s o p r o p a -

n o l : g l a c ia l a c e t i c a c i d 1 0 0 : 1 .5 : 0 . 1, b y v o l u m e ) a t a f l o w r a t e o f 2 m l / m i n . T h e 2 0 5 - , 2 2 0- ,

a n d 2 3 5 - n m c h a n n e l s a r e s e t a t th e s a m e s e n s it i v it y . T h e a r r o w i n t h e 2 3 5 - n m c h a n n e l s p o i n t s

t o a s m a l l a m o u n t o f u n m e t a b o l i z e d 1 3 S - h y d r o p e r o x y l i n o l en i c a c id s u b s t r a te . T h e y - k e t o l

i s v e r y s tr o n g l y r e t a i n e d o n S P - H P L C ; it e l u t e s at a r e t e n t i o n t i m e o f 36 r a in = 7 2 m l

e l u t i o n v o l u m e ) .

Qu antitation o f ol-Ketol y-Ketol and Cyc lopentenone

T h e p r o m i n e n t o t- ke to l s h o w s o n l y e n d a b s o r p t i o n i n t h e U V a n d c a n n o t

b e q u a n t i f i e d r e l i a b l y b y t h i s m e t h o d ; e i t h e r w e i g h i n g o r , p r e f e r a b l y , m e a -

s u r e m e n t o f s p e c if ic ra d i o a c t i v i t y s t a r ti n g f r o m [ 1 4 C ] h y d r o p er o x y li n o le n i c

a c i d i s r e q u i r e d . N o n e t h e l e s s , a v e r y u s e f u l a p p r o x i m a t i o n o f t h e r e l a t i v e

l e v e ls o f t h e p r o d u c t s o n H P L C i s g i v e n b y t h e s i g n a ls a t 2 0 5 nm ; t h i s

r e f l e c t s f a i r l y w e l l t h e r e l a t i v e a b u n d a n c e s o f t h e h y d r o p e r o x i d e a n d a l l

i t s der ivat ives .

A r e a s o n a b l e e s t i m a t e o f t h e m o l a r e x t i n c t i o n c o e f f ic i e n t s f o r th e t w o

c o n j u g a t e d k e t o n e s ( c y c l o p e n t e n o n e a n d y - k e t o l ) i s 1 0 , 0 0 0 - 1 2 , 0 0 0 , b a s e d

o n m e a s u r e m e n t 3 a n d t h e r e p o r t e d v a l u e s f o r p r o st a g l a n d in a n a l o g s ( p r o s ta -

g l a n d in A 2 a n d 1 5 - o x o p r o s t a g la n d i n s ) . 15 I n S P - H P L C s o l v e n t ( 9 8 h e x -

a n e ) , t h e A m,x a r e o b s e r v e d a t 2 1 7 n m ( c y c l o p e n t e n o n e ) a n d 2 23 n m ( y -

k e t o l ) . I n m o r e p o l a r s o l v e n t s , t h e s e c h r o m o p h o r e s a r e s h i f t e d t o s l i g h t l y

l o n g e r w a v e l e n g t h , e . g . , o n c h a n g i n g t o R P - H P L C s o l v e n t ( M e O H : H 2 0 ,

8 0 : 2 0 , v / v ) , t h e b a t h o c h r o m i c s h i f t s a r e 6 - 7 n m ( c y c l o p e n t e n o n e ) a n d 3 -

4 nm ( - / -keto l ) .

15 j . E . P i k e , F . H . L i n c o l n , a n d W . P . S c h n e i d e r , J Org Chem 34 , 3552 1969) .


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256 PLANT P450s [28]

Isolation a nd Purification of AOS

The purification procedure we developed was optimized by monitoring

enzymatic activity through several open column and fast protein liquid

chromatography (FPLC) methods. The method may be applicable to puri-

fication of allene oxide synthase from other plant sources. In addition to

the flaxseed AOS, we achieved purification of the AOS from maize and

obtained N-terminal and CNBr peptide sequences using essentially the

same chromatographic steps.

Prepa rat ion o f the Ini t ial Tissue Extrac t

An acetone powder preparation is convenient and the AOS activity is

stable on storage at -20 for years. Twenty grams of flaxseeds is homoge-

nized using a Polytron in 200 ml of cold acetone (-10 to -20). After

allowing the solids to settle for about 10 sec, the tan-colored cloudy superna-

tant is decanted and the remaining solids are rehomogenized with fresh

cold acetone. The pooled fine suspension is filtered on a Buchner funnel

under vacuum and washed with 500 ml cold acetone. After removal of

most of the acetone under a strong vacuum for 10-20 sec, the solids are

scraped off the filter paper into a tube normally used for holding desiccants

for gas drying (Aldrich, Cat. No. 37, 123-8). The flaxseed proteins are dried

to a powder by passing through a stream of nitrogen for ~15 min until

there is no longer a smell of acetone in the effluent. About 2 g acetone

powder is recovered and stored at -20

A m m oni um Su l f a t e P r ec i p i t a t i on

The acetone wash removes the microsomal lipids, and aqueous extrac-

tion of the acetone powder (50 mM phosphate buffer, pH 7, stirred with

50 mg/ml powder, for 30 min on ice) leaves the AOS activity in a 100,000-g

supernatant. As shown originally by Z immerman and Vick, 16 AOS activity

with the most enriched specific activity is precipitated by 30 (NH4)2SO4,

although use of 42 saturation is required to recover most of the activity.

We collected the 0-45 NH4)2SO4pellet, n After this initial ammonium

sulfate fract ionation of this extract, the resuspended AOS exists in protein

microaggregates and requires treatment with detergent prior to chromatog-

raphy.

So l ub il iz a t ion o f A O S A c t i v i t y

We tested the solubilizing efficiency of various detergents by measuring

the percentage recovery of AOS activity after passing a 1-ml aliquot through

16 D. C. Zimmerman and B. A. Vick,

Plant Physiol

46, 445 (1970).


8/10


9/10

2 5 8 P L AN T P 4 5 0 S 1 2 8 ]

T A B L E I

PURIFIC TION OF FL XSEED LLE NE OXID E SYNTH SE a

T o t a l p r o t e i n S p e c if ic a c t iv i t y R e c o v e r y P u r i f i c a t io n

P u r i f ic a t io n s t e p ( m g ) ( A U / m g / m i n ) ( ) ( f o ld )

F l a x e x t r a c t 3 3 9 8 3 2 1 0 0

A m m o n i u m s u l f at e 1 30 8 6 4 7 8 2

( 0 - 4 5 )

O c t y l - S e p h a r o s e 5 0 9 8 9 4 6 3 1

( C L - 4 B )

A n i o n e x c h a n g e 4 .2 9 ,9 9 0 3 9 3 15

( M o n o - Q )

C h r o m a t o f o c u s i n g 0 .9 1 5 ,4 8 0 1 3 4 9 1

( M o n o - P )

V a l u e s a r e f r o m a n e x p e r i m e n t s ta r t i n g w i t h 2 0 g a c e t o n e p o w d e r . E n z y m e a c ti v it y w a s

m e a s u r e d u s i n g th e s p e c t r o p h o t o m e t r i c a s s a y (F ig . 3) . P r o t e i n w a s d e t e r m i n e d w i t h t h e

B i o - R a d B r a d f o r d m e t h o d ; c o m p a r e d t o t h e r e s u l ts o f a m i n o a c i d a n al y s is , t h i s c o l o r i m e t -

t i c a s s a y s i g n i fi c a n tl y o v e r e s t i m a t e d t h e p r o t e i n l e v e l s i n t h e f i n a l s t e p s o f p u r i f ic a t i o n .

Absorbance

280 nm

M o n o - Q

o s

ctivity

10 20 30 40 5O

Time min)

M o n o - P

6 0 1 0 2 0 3 0 4 0 5 0

Time min)

F I o . 5. P u r i f ic a t io n o f f la x s e ed A O S o n a n i o n - e x c h a n g e c h r o m a t o g r a p h y a n d c h r o m a t o f o -

c u s i n g . ( L e f t ) M o n o - Q c o l u m n , H R 5 / 5 , ( P h a r m a c i a ) : C o n d i t i o n s a r e d e s c r i b e d i n t h e t e x t .

( R i g h t ) A M o n o - P c o l u m n , H R 5 /2 0 , (P h a r m a c i a ) w a s e q u i li b r a te d w i th 2 5 m M b i s - T r is , p H

6 .7 , 0 .1 E m u l g e n 9 11 , a n d t h e n t h e s a m p l e w a s in j e c t e d i n 7 m l o f t h e s a m e b u f f e r a n d t h e

c o l u m n w a s e lu t e d w i t h 4 0 m l o f P o l y b u f f e r 7 4 ( P h a r m a c i a ) , p H 5 .0 , 0 .1 E m u l g e n 9 1 1 a t 1

m l / m i n . E n z y m e a c ti v it y e l u t e d a s t w o p e a k s a t p l 5 .5 a n d 5 .4 . ( U p p e r t r a c e s ) U V a b s o r b a n c e

a t 2 8 0 n m ; t h e v e r y l a r g e p e a k o n i n j e c t i o n i n A ( i n B , n o t r e c o r d e d ) i s m a i n l y d e t e r g e n t

c o n c e n t r a t e d f r o m t h e p r e v i o u s c h r o m a t o g r a p h i c st e p. ( L o w e r t r ac e s ) A O S a c ti v it y , m e a s u r e d

b y a d e c r e a s e i n 2 3 5 n m a b s o r b a n c e .


10/10

[291 CINNAMIC ACID HYDROXYLASE 25 9

s o l u t i o n c o n t a in i n g 0 .5 M N a C l b u f f e r B ) . T h e A O S a c t i v i ty f r o m f l a x s e e d

e l u t e d a t ~ 2 0 m i n F i g. 5 A ) .

Chromatofocusing FP LC Mono-P Column)

Fina l ly , fo l lowing bu f fe r exchang e us ing a ge l f i lt r a t ion PD -10 co lum n,

P h a r m a c i a ) , t h e e n z y m e w a s p ur if ie d o n a M o n o - P H R 5 /2 0 c h ro m a t o f o c u s -

i n g c o l u m n P h a r m a c i a ) F ig . 5 B a n d T a b l e I ).

T h e t w o i s o z y m e s d i f f e r i n a s i n g l e a m i n o a c i d a t t h e N t e r m i n u s ,

a n d t h e m a j o r i s o zy m e w a s c l o n e d a n d s e q u e n c e d 2 T h e i s o zy m e s ha v e

ind i s t ingu i shab le UV-VIS spec t r a , typ ica l o f a P450 in the h igh - sp in f e r r i c

s t a te , al E a c h c a t a l y z e s a l le n e o x i d e s y n t h e s i s w i t h a t u r n o v e r n u m b e r o f

~>1000 se cq . u

Emulgen Detergent Removal

F o r m i c r o s e q u e n c i n g , r e m o v a l o f E m u l g e n 9 11 is d e s i r a b le . I t c a n n o t

b e r e m o v e d e f f e c t i v e l y b y d i a ly s is o r w i t h C e n t r i c o n s p i n c o n c e n t r a t o r s .

I n j e c ti o n o f t h e s a m p l e o n a M o n o - Q c o l u m n , f o l lo w e d b y e x t e n s iv e w a s h in g

w i t h d e t e r g e n t - f r e e b u f f e r , a n d t h e n e l u t i o n w i t h a s a l t g r a d i e n t i n c l u d i n g

o c t y l g l u c o s i d e 2 0 m M ) is e f f e c ti v e . O c t y l g l u c o s i d e c a n b e d i a l y z e d . T h e

d e n a t u r e d e n z y m e c a n al so b e r e so l v e d f ro m E m u l g e n 91 1 o n R P - H P L C

V y d a c C 4 c o l u m n a n d a w a t e r / a c e t o n i t r i l e g r a d i e n t ) , a l t h o u g h t h i s i s n o t

e f f e c t i v e o n a p r e p a r a t i v e s c al e .

A c k n o w l e d g m e n t

This work was supported by NIH Grant GM-49502.

[ 2 9 ] C i n n a m i c A c i d H y d r o x y l a s e A c t i v i t y

i n P l a n t M i c r o s o m e s

By FRANCIS DURS T IRI~NE BENVENISTE MICH EL SCHALK

and DANII~LE WERCK-REICHH ART

I n t r o d u c t i o n

C y t o c h r o m e P 4 5 0 P 4 5 0 ) e n z y m e s w e r e c h a r a c t e r iz e d i n p l a n t t is s ue s

i n t h e e a r l y 1 9 7 0 s . F o r a l m o s t t w o d e c a d e s t h e s e e n z y m e s a t t r a c t e d l i t t l e

a t t e n t i o n a n d p r o g r e s s w a s s lo w . T h e t w o m a i n r e a s o n s f o r t h is w e r e 1 )

t e c h n i c a l d i f f i c u l t i e s i n d e a l i n g w i t h p o o r l y a b u n d a n t m e m b r a n e p r o t e i n s

Copyright 1996 by Academic Press Inc.

METHODS IN ENZYMOLOGY VOL. 272 All rights of reproduction in any form reserved.

1996-detection, assay, and isolation of allene oxide synthase

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