CÓDIGO EXAMEN MACROSCÓPICO EXAMEN MICROSCÓPICO

-173-

5. ^ In tr o d u c tio n

A lo t o f e n z y m e s are in v o lv e d in a m m o n ia a ssim ila to ry p ath w a y s a n d c o n s id e r a b le w o rk h a s b e e n d o n e o n th e s u b c e llu la r lo c alizatio n s o f th e se e n z y m e s . S in c e is o la te d b a c te ro id s e x c re te N H 4 + (B erg ersen an d T u r n e r 1 9 6 7 ), R o b e rtso n e t a /.(1 9 7 5 a ) su g g e s te d th a t N H 4 + is e x c re ted to th e h o s t c e ll c y to p la sm a n d is in c o rp o rated in to g lu ta m in e an d g lu tam ate v ia th e c o m b in e d activ ity o f G S an d G O G A T . S e v e ra l re p o rts h av e sh o w n th a t th e m a jo r ity o f G S is lo c a te d in th e c y to so l w h ile N A D H -G O G A T ex ists m a in ly in th e p la stid s (A w o n a ik e e t al. 1 9 81, B o la n d e t al. 1982, S h elp an d A tk in s 1 9 8 4 , S h e lp e t a l. 1 9 8 3 ). T h u s a l th o u g h N H 4 + is f ir s tly in c o rp o ra te d in to g lu ta m in e b y th e fu n c tio n o f G S in th e c y to so l o f p la n t c e lls o f r o o t n o d u le s , th e sec o n d r e a c tio n in th e " G lu ta m a te S y n th a s e C y c le " (g lu ta m in e + 2 - o x o g lu ta r a te v ia th e a c tiv ity o f G O G A T to fo rm g lu ta m a te ) m ig h t b e c a rr ie d o u t in th e p la stid s . A m o d e l f o r th e p ath w a y a n d s u b c e llu la r lo c a liz a tio n o f a m id e s y n th e s is a n d u r e id e b io g e n e sis in n o d u le s o f u r e i d e - p r o d u c in g le g u m e s h a s b e e n p ro p o s e d by S c h u b e rt (1 9 8 6 ). I n th is m o d e l, th e a u th o r p ro p o s e d th a t th e f ir s t re a c tio n in v o lv e d in " G lu ta m a te S y n th a s e C y cle" o cc u rre d in th e c y to p la s m o f th e n o d u le and g lu ta m in e p ro d u c e d w a s th e n tra n s p o rte d in to th e p la s tid s an d p la s tid - lo c a te d N A D H - G O G A T c a taly z ed th e tra n sa m id a tio n o f glu tam in e a m id o - n itr o g e n to th e a - a m i n o p o s itio n o f 2 - o x o g lu ta r a te to fo rm tw o m o le s o f

-1 74 -

g lu tam a te. O n e m o le o f g lu ta m a te p ro d u c e d w a s tra n sp o rted b a c k to the c y to p la sm f o r th e p ro v is io n o f su b stra te o f G S . T h e o th e r g lu ta m a te w as u s e d to s y n th e s iz e o th e r n itr o g e n - c o n ta in in g c o m p o u n d s. S in c e tw o N A D H - G O G A T is o e n z y m e s h a v e b ee n id e n tifie d and s e p a ra te d in our e x p e rim e n ts , i t is im p o r ta n t to d e te rm in e th e lo c a tio n o f th e s e tw o is& enzym es a n d h e n c e th e lo c a liz a tio n o f th e seco n d r e a c tio n in th e "G lutam ate S y n th a se C ycle".

In th is C h a p te r, s u c ro s e g ra d ie n t c e n trifu g a tio n fo llo w in g the m e th o d d e s c r ib e d b y B o la n d e t al. (1 9 8 2 ) h a s b e e n u s e d to sep arate o rg an elles an d to stu d y th e lo c a tio n o f N A D H -G O G A T . A ra p id isolation o f p la stid s d e s c r ib e d b y B o la n d a n d S c h u b e rt (1 9 8 2 ) w as a d o p te d to d e te rm in e th e s u b c e llu la r lo c a lis a tio n o f th e tw o N A D H - G O G A T isoenzym es.

5 .2 .Results

1. S ep aratio n o f o rg a n e lle s from ro o t nodules on sucrose gradients and determ in atio n o f the su b c ellu la r lo calizatio n of N A D H -G O G A T activity

F re s h ly p ic k e d n o d u le s w e re v ery g en tle p re s s e d a n d b ro k e n in a O.SM su c ro s e c o n ta in in g b u f f e r in o rd e r to is o la te a n d m a in ta in the in te g rity o f th e o rg a n e lle s . T h e e x tra c te d c r u d e o rg a n e lle s o lu tio n was

-1 75 -

lo a d e d o n c o n tin u o u s s u c r o s e g r a d ie n ts a s d e s c rib e d in M e th o d s . In a d d itio n to th e p la n t c y to so l p o rtio n w ith th e ch a ra cte ristic le g h aem o g lo b in , th re e d is tin c t b a n d s w e re v is ib le a f te r c e n trifu g a tio n . T h e s e p a ra tio n o f o r g a n e l le s w a s in d ic a te d b y th e m a r k e r e n z y m e s , f u m a r a s e f o r m i t o c h o n d r i a , t r i o s e p h o s p h a t e i s o m e r a s e o r p h o s p h o g ly c e r a te d e h y d ro g e n a se f o r p la stid s a n d (3 -h y d ro x y b u ty ra te d e h y d ro g e n a s e f o r b a c te ro id s . T h e se o rg a n e lle s w e re fo u n d a t d e n sitie s o f 1 .1 7 , 1.21 an d 1 .2 3 g /m l resp ectiv ely . F ig 5 .1 sh o w s th e d is trib u tio n o f m a rk e r en z y m es f o r th e th re e o rg an elles a n d N A D H - G O G A T o f P .v u lg a ris r o o t n o d u le s fo llo w in g su cro se d en sity g r a d ie n t c e n trifu g a tio n . It ca n b e seen fro m th e fig u re th a t a lo t o f ac tiv ity o f trio se p h o sp h a te iso m e rase (ab o u t 6 0 % ) fro m p la stid s w as p re se n t in th e c y to s o l f ra c tio n s , th is is h ig h e r th a n th e valu e o b ta in e d b y A w o n a ik e (1 9 8 1 ), s u g g e s tin g m u c h m ore b reak a g e o f p la stid s in th is e x p e rim en t. F ractio n s f r o m th e s u c ro s e grad ien ts w ere a s s a y e d fo r a c tiv ity o f N A D H - G O G A T . T h e m a jo r ity o f N A D H -G O G A T a c tiv ity (6 5 % ) w a s fo u n d in th e c y to s o l f r a c tio n s . G O G A T a c tiv ity d e c re a s e d g ra d u a lly as th e su c ro s e d e n s ity in c re a s e d u n til th e ac tiv ity o f N A D H - G O G A T c o u ld n o t b e d e te c te d in fra c tio n s o f a b o u t l . l l g / m l o f su cro se. G O G A T a c tiv ity w a s n o t d e te c ta b le in th e fra c tio n s a s s o c ia te d w ith m ito c h o n d ria fractio n s b e tw e e n 1.15 an d 1 .1 8 g /m l o f su cro se. T h e re w as a d e f in ite ac tiv ity p ea k a s s o c ia te d w ith p la s tid s (20% ) in fra c tio n s b etw e en 1 .1 9 a n d 1.23 g / m l o f s u c ro s e an d 8% o f to t a l ac tiv ity w as o b s e rv e d in f ra c tio n s b etw e en 1.22 a n d 1 .2 5 g /m l o f s u c ro s e , w hich w as an o v e rla p o f p la stid s a n d b ac tero id s. p - h y d r o x y b u ty r a t e d e h y d ro g e n a se a c tiv ity w as

-176-

o n ly d e te c ta b le a f t e r u ltr a s o n ic s o n ic a tio n o f th e s e la tte r fra c tio n s . H o w e v e r, N A D H -G O G A T activ ity w a s n o t in c re a s e d in these fractio n afte r so n ica tio n ; this su g g e ste d th a t N A D H - G O G A T in th e s e fractio n s w as fro m th e p la s tid s . Y e t, i t w as d if f ic u lt to te ll w h ic h is o e n z y m e o f N A D H - G O G A T w as p la stid lo c ated b ec au se th e lo w r e c o v e ry o f N A D H -G O G A T in th e p la stid fra c tio n s m a d e it im p o ss ib le to ru n th is a c tiv ity on th e H P L C co lu m n to determ in e w h ich iso en zy m e w as p resen t.

-177-

F i g .3 .1 . Separation of organelle« from root nodule« by «ucroie denlity gradient centrifugation

The distribution o f mariecr enzymes for mitochondria, plastids, and bacteroids and NADH-GOGAT of P.vuIgMris root nodules. --- triosephosphate isomerase (for plastids, x 0.2), x— x fumarase (for mitochondria), ♦— ♦ P- hydroxybutyrate dehydrogenase (for bacteroids) A— A NADH-GOGAT activity.

D e n s it y (g .m l- 1 )

-178-

2. Isolation o f p lastid from root nodules and determ ination o f the plastid location o f the two NADH-GOGAT isoenzym es.

A m e th o d f o r ra p id p rep ara tio n o f p la stid s re p o rte d b y B o la n d a n d S c h u b e rt (1 9 8 3 b ) w a s ad o p te d to c o n firm th e p la s tid lo c a tio n o f N A D H -G O G A T a n d to d e te rm in e w h ich is o e n z y m e w a s p re s e n t in th is fra c tio n . E x tr e m e c a re w a s ta k e n to a v o id p la s tid s b r e a k a g e d u rin g ex tractio n as d escrib ed in M eth o d s. N o d u le ex tract w a s sp u n d o w n a t 1,000 rp m fo r 4 m in to g et rid o f th e d eb ris an d b ac tero id s. T h e su sp en sio n fro m th e to p o f th e c e n trifu g e tu b e w as lo a d ed o n to th e d is c o n tin u o u s su cro se g radients. T h e p la stid s w e re sep arated b y c e n trifu g in g f o r 3 0 m in a t 1 45,000