Evaluación y comparación de los resultados obtenidos

B 4 m l C . 0.6m l D . 22.4m l W a ter 1 2 .85m l S ta c k in g gel A . 1.7 m l B . 0.7m l C . O .lm l E . 1.25ml W ater 6.2 5 m l 2 ).T h e electro d e b u f f e r P e r liter SD S l.Og T ris base 6 .0 g G ly cin e 2 8 .8g T h e g el w a s ru n a t 3 0 m A co n sta n t c u rre n t 3 ).S am p le b u ffer

-6 2 - 1 M T ris-C l p H 6 .8 1m l 10% ( W / V ) SDS 2 m l 20% ( V /V ) g ly c ero l 2m l 4 % B PB lOOpl h2o 5 m l B efo re ru n n in g th e g el, ta k e 2 0 0 p i o f ab o v e m ix tu re a n d a d d 4 p.1 o f p - M E as s am p le b u f f e r . T h e sa m p le s w e re m ix e d w ith th e e q u a l v o lu m e o f sam p le bu ffer. O n c e in sam p le b u ffe r, all sam p les w ere h e a te d to 100* C fo r 3 m in . p r io r to lo a d in g .

4 ). A u to rad io g rap h y and s ta in in g gels

T h e ele c tro p h o re se d g els, o r p o rtio n o f th e m , w ere e ith e r le ft u n f ix e d an d im m e d ia te ly p r e p a r e d f o r W e ste rn b lo ttin g o f p r o te in s o n to n itro c e llu lo s e (d e sc rib e d e ls e w h e re ) o r fix e d in 5 0 % m e th a n o l w ith 3 - 5 tim e s ch a n g in g f o r silv e r s ta in in g o r g els w ere stain ed w ith C o o m a ss ie b lu e s o lu tio n co n tain in g m e th an o l a n d ac etic a c id d ire ctly . G e ls co n ta in in g 3 5 $ _ m e th io n in e la b e lle d p ro te in s w e re f ix e d in 4 5 % ( V / V ) m e th a n o l, 1 0 % ( V / V ) acetic ac id f o r 3 0 m in a n d th e n w e re flu o ro g rap h e d w ith A m p lify f o r 3 0 m in ., a fte r th a t g els w ere d rie d d o w n f o r au to rad io g rap h y .

M o le c u la r w e ig h t m a rk e rs w ere m y o s in ( M r 2 0 0 ,0 0 0 ) , P - g a la c to s id a s e (M r 1 1 6 ,2 5 0 ), p h o s p h o ry la s e B (M r 9 2 ,5 0 0 ), b o v in e s e ru m

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alb u m in (M r 66 ,2 0 0 ), o v alb u m in (M r 4 5 ,0 0 0 ).

8. P rotein electroelution procedure ( L a e m m li, 1 9 7 0 )

1) .A c r y la m id e g el w as s ta in e d v e ry b r ie f ly w ith C o o m a ss ie b lu e ( 5 - 1 0 m in .), th e n rin sed in d e s ta in u n til th e p r o te in b a n d s b e c a m e v is ib le ( 5 - 1 0 m in .). T h e stain in g an d d e sta in in g tim e s w ere k ep t as sh o rt as p o s sib le f o r m a x im u m yields.

2 ) . T h e f la t- b e d tan k w as filled w ith b u f f e r (E lectro p h o re sis b u f f e r w ith 2 m M D l l ) .

3 ) . T h e b a n d o f in te re s t w as c u t o f f a n d p u t in to a d ia ly sis b a g w ith th e b u f f e r f r o m th e ta n k , so th a t th e am o u n t o f th e b u ffe r ju s t co v e re d th e gel slice. T h e tw o en d s w ere clip p ed w ith m e d iclip s and ca re w as ta k en to av o id tra p p in g a n y a ir b u b b le s. T h e g el slic e w as p o sitio n e d o n o n e sid e o f th e d ia ly sis b a g .

4 ) . T h e d ia ly s is b ag w as p la ced o n to th e p la tfo rm o f th e e le c tro p h o re sis tan k . T h e le v el o f th e b u ffe r w as ad ju sted so th a t it ju s t co v e re d th e b ag .

5 ) . E lectro p h o re sis w as ca rrie d o u t f o r ab o u t 20h. at 2 0 m A co n sta n t cu rre n t th en th e cu rre n t w as r e v e rs e d fo r ap p ro x 3 0 seconds.

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6 ) . T h e g e l slice w as rem o v ed fro m th e bag and d isca rd ed . T h e so lu tio n w as ta k e n to a m icro fu g e tu b e and ce n trifu g ed b riefly to p ellet a n y sm all pieces. T h e so lu tio n w as p u t b ac k in to th e dialy sis b a g a n d th e p ro te in so lu tio n w a s d ia ly s e d ag a in s t a t least 5 ch a n g es o f d istille d w a te r ( 4 - 5 liters ea c h ) o v e r 2 - 3 d ay s at 0 * - 4 * C .

7 ) . T h e d ialy sed p ro te in solution w as fro zen an d ly o p h ilised .

9. Enzym e assays

1) A ssay o f N A D H -G O G A T A ctivity A . S p e c tro p h o to m e tric assay.

The ac tiv ity o f N A D H -d e p e n d e n t G O G A T w as assay ed at 18* to 2 2 * C b y m e a s u rin g th e o x id a tio n o f N A D H a t 3 4 0 n m . T h e reactio n m ix tu r e ro u tin e ly c o n ta in e d 25 m M H E P E S b u f f e r p H 7 .5 , 1 m M 2 - o x o g lu t a r a t e , 2 .5 m M L - g l u ta m in e , 0 .1 6 m M N A D H , 1% (5- m e rcap to e th an o l an d 1 0 0 -2 0 0 p l en z y m e solution in a total v olum e o f 1.2ml. T h e re a c tio n w as in itia te d by the ad d itio n o f th e e n z y m e an d th e ch a n g e in A 3 4 0 ( E 3 4 0 “ 6 .2 2 x 10 ® cm m o l“ * ) w as m o n ito re d o v e r a p erio d o f a t le a st 2 m in and w as fo u n d to b e lin e a r ov er th e tim e . A n a ssa y w ith o u t 2 - o x o g lu ta r a te a n d L - g lu ta m in e w as u sed as c o n tr o l. T h e a c tiv itie s are e x p re s s e d in fim ol N A D H o x idized m in “ *. In th e d eterm in atio n o f th e Km v a lu e s o f N A D H -G O G A T fo r th e v ario u s su b stra te s, the co n c en tratio n o f

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th e s u b stra te s w ere v aried in th e reac tio n m ix tu re . F o r p H d eterm in atio n s th e p H o f th e H E P E S b u ffe r w a s v aried . P a rtia lly p u rified e x tracts p rep ared as in th e p u rific a tio n p ro c e d u re s b u t la ck in g th e B lu e S ep h aro se an d fin a l g el filtra tio n step s w ere u se d in th e se d e term in atio n s.

B . R ad io ch em ical assay .

N A D H -G O G A T ac tiv ity w as m e a su re d b y the p ro d u ctio n o f R e ­ la b e lle d g lu ta m a te fro m ^ C - g l u t a m i n e e s s e n tia lly as d e s c r ib e d b y W a llsg ro v e e f a7. (1 9 8 2 ) w ith so m e m o d ifica tio n s. R C - g lu ta m in e , o b ta in ed fro m A m e rs h a m In te r n a tio n a l, w as p u rifie d o n a co lu m n o f D o w e x - 1 - ch lo rid e a n d w as u se d at a sp e c ific activ ity o f 1 0 9 p .C i/m m o l in th e reactio n m ix tu r e . T h e s e p a r a t io n o f g lu ta m a te f r o m g lu ta m in e b y p a p e r electro p h o re sis w as a c h iev ed a t ab o u t 9 0 V fo r a b o u t 4h.

2 ) . A ssay o f F d - G O G A T A ctiv ity

F d - G O G A T a c tiv ity w as m e a s u re d a c co rd in g to th e m e th o d d e s c rib e d b y W a llsg ro v e e r a /. (1 9 8 2 ) b u t w ith th e m o d ific a tio n s as m e n tio n e d above.

3 ) . A ssay o f N itro g e n ase A c tiv ity

N itro g e n a s e ac tiv ity o f n o d u la ted r o o t sy stem s w a s m e asu red b y ac ety le n e re d u c tio n as d escrib ed p rev io u sly (D a rt e t a/., 1972).

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u n iv e rsa l b o ttle c o v e re d w ith a S u b aseal th ro u g h w h ic h 100 JXl o f acety len e w a s in je c te d to g iv e a n a c e ty le n e c o n c e n tr a tio n o f a b o u t 3 .3 % . A fte r sh ak in g , th e b o ttle w a s in cu b ated in th e light. W ith in lh , 1 0 0 p l o f m ix tu re o f th e g ases in th e co n ta in e rs w as ta k en e v e ry 5 m in b y B e c to n D ick in so n p la stip a k sy rin g e a n d th e n in je c te d in to th e g as c h ro m a to g ra p h and C 2H 4 w a s q u a n tifie d b y a fla m e io n isa tio n d etecto r. T h e c o lu m e p a c k in g sy stem u se d w as 8 0 - 1 0 0 m e s h Poropak R in a lm x 0 .0 0 3 m d ia m eter g la ss co lu m n a t 100 C , w ith a n itro g e n c a rr ie r g as flo w ra te o f 2 0 m l /m i n , u sin g a h y d r o g e n /a ir fla m e io n isatio n d etecto r.

A fte r as s a y , th e nodules w e re p ick ed o f f th e ro o t a n d w eig h e d .The n itro g e n a s e ac tiv ity is ex p ressed a s p m o ls e th y le n e p ro d u c e d p e r m in per g ram fresh w eig h t n o d u le .

10. P rotein determ ination

P ro te in , in c e ll- f r e e e x tr a c ts , w as d e te rm in e d u s in g th e B io R ad P ro te in A ss a y D y e - B in d in g re a g e n t, and b o v in e y -g lo b u lin as stan d a rd . P ro te in s ta n d a rd c o n ta in in g 4 m g / m l y - g lo b u l in w as p r e p a r e d a n d a stan d a rd c u rv e w as p erfo rm e d f o r e a c h assay . 1 0 - 1 0 0 p g o f stan d a rd s and lOOpl o f a p p ro p ria te ly dilu ted sa m p le s w ere p u t in to m ic ro fu g e tu b e s and m a d e to 2 0 0 p l w ith a ssay b u ffe r. 1.0m l d ilu te d 5 - f o ld d y e re a g e n t w as ad d e d to ea c h tu b e . A fte r m ix in g , A 5 9 5 w as m e a s u re d v ersu s reag e n t blank. A 5 9 5 v e rsu s c o n c e n tra tio n o f stan d a rd s w as p lo tte d . U n k n o w n s w ere read fro m th e stan d a rd c u rv e (Fig 2.4.).

A

59

5

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11. M arker proteins assay

F o r d e te rm in a tio n o f N A D H -G O G A T m o le c u la r w e ig h t, th e H PLC g e l filtra tio n co lu m n (T S K G 4 0 0 0 S W 7 .5 x 6 0 0 m m ) w as c a lib ra te d using a n u m b e r o f g lo b u la r p ro te in s . T h e co lu m n w a s p re e q u ilb ra te d w ith H PL C