ELABORACIÓN DEL MANJAR DE LECHE CON LICOR

2 .7 .1 P r o t e in e s s a y

P r o t e i n was a ssa yed by t h e d y e —b in d in g method o f B ra d fo rd (1 9 7 6 > . P r o t e in s o l u t i o n c o n t a in in g 20 p g t o lOO pg o f p r o t e in in a volume o f lOO p i was p i p e t t e d i n t o chrom ic acid -w ash ed t e s t tu b e s . To t h i s was added S ml o f p r o t e in a s s a y d ye r e a g e n t c o n c e n t r a t e ( B i o r a d ) f w hich was d ilu t e d a c c o r d in g t o t h e m a n u fa c tu re rs * i n s t r u c t i o n s . The m ix tu re was v o r te x e d and a llo w e d t o s ta n d f o r 30 m in u te s . The a b sorb a n ce a t 39S nm was measured u s in g a Shimadzu s p e c tr o p h o to m e te r and a b s o lu te p r o t e i n c o n c e n t r a t io n s in th e unknowns d eterm in e d from a s ta n d a rd c u rv e p re p a re d w it h gamma g l o b u l i n s (B ig m a ). 2 .7 .2 C h lo r o p h y ll assay

C h lo r o p h y ll was e x t r a c t e d from membranes p re p a re d as d e s c r ib e d in S e c t io n 2 .2 . To t h e membrane p e l l e t was added 3 ml o f 90X <v / v ) a c e to n e and t h e e x t r a c t i o n was a llo w e d t o p ro c e e d f o r 30 m inutes in th e d a rk . The d e b r is was rem oved by c e n t r i f u g a t i o n a t to p sp eed in a M8E M inor bench to p

c e n t r i f u g e f o r 5 m in u tes and t h e s u p ern a ta n t removed f o r a s s a y . The a s s a y used was t h a t o f Arnon (1 9 4 9 ). The

ab s o rb a n ce o f t h e s u p e rn a ta n t was d eterm in ed a t 663 nm and 645 nm in q u a r tz c u v e t t e s a g a in s t a blan k c o n t a in in g 90%

(v/v> a c e to n e . The c o n c e n t r a t io n o f c h lo r o p h y ll in th e e x t r a c t was d eterm in e d from t h e f o l l o w i n g e q u a tio n i

(A<.4b x 2 0 .2 ) + ( A u s x 8 .0 2 ) ” C c h lo ro p h yl 13 in pg ml-1 2 . 7 . 3 RUBISCO a s s a y

The RUBISCO a c t i v i t y in pea e x t r a c t s and p u r i f i e d RUBISCO p r e p a r a t io n s was d e te rm in e d u s in g t h e method o f L o rim e r e t a l . (1977) as f o l l o w s . Between SO p i and 100 p i o f t h e enzyme e x t r a c t was in c u b a te d in an E ppen dorf tu b e a t 30*C f o r S m in u tes w ith 30 p i o f NaHCOs (167 mM), ISO

p i o f a s s a y b u f f e r (c o n t a in in g 100 mh T r is - H C l, 16 mM M gCla. 6HaO, pH 8 .2 ) and 5 pCi o f NaH^COs

(56 mCi mmol- 1 ) . The r e a c t i o n was s t a r t e d by th e

a d d it io n o f SO p i o f r i b u l o s e —1 ,3 -b i» p h o s p h a t e (4 mM). In c u b a tio n c o n tin u e d a t 30*C f o r up t o 10 m in u tes and was te r m in a te d b y th e a d d it io n o f 10O p i o f 12 M fo r m ic a c id . An a l i q u o t (200 p i ) o f th e in c u b a tio n m ix tu re was e v a p o ra te d t o d ry n e s s in a g l a s s s c i n t i l l a t i o n v i a l and t h e r a d i o l a b e l l e d p h o s p h o g ly c e r ic a c id in w a te r (1 m l). Beckman EP s c i n t i l l a t i o n f l u i d (lO m l) was added and t h e v i a l cou n ted in a LKB M in lb e ta 1212 s c i n t i l l a t i o n c o u n te r. E f f i c i e n c y o f c o u n tin g was 90%.

2 .7 .4 S u e r o » » d a n a it y g r a d i a n t c e n t r i f u g a t i on

S tro m a l a x t r a c t * p rep a ra d fr o m lig h t - g r o w n p a a * and t o t a l s o l u b l a a x t r a c t s from d a rk -g ro w n p la n t s wara a n a ly s a d on s u c ro s a s ta p g r a d ia n t s (5% t o SOX (w /v) s u c r o s a ). Tha g r a d ia n t s w ara composad o f 4 x 3 ml s ta p s <5X, 20X, 35X and 50X (w /v) s u c r o s a ) and th a s u c ro s a s o l u t i o n s w a r* c a r a f u l l y la y a r e d o n * on to p o f th a o th a r u s in g a f l o w in d u c a r (W atson M arlow L t d . ) Tha s u c ro s a was d i s s o l v a d in th a f o l l o w i n g b u f fa r t

50 mM T r is -H C l

7 mM 2—m a rca p to a th a n o l 1 mM PM8F

pH - 7 .6

I f r a q u ir a d , ATP (lO mM) and MgClz (1 0 mM) wara in c lu d a d in th a b u f f a r . Tha sam pl* (500 u l ) was la y a r a d on t o p o f t h * 5X (w /v) s u c r o s a s o lu t io n and th a g r a d ia n t s c a n t r lf u g a d a t 8 8,000 x g f o r 17 hou rs a t 4*C in a Backman 8W40T1 r o t o r . G ra d ia n ts w a rs f r a c t io n a t a d u s in g a p i p a t t * and f r a c t i o n s (500 |il > w ara s to r a d a t -2 0*C u n t i l r a q u lr a d . 2 .7 .5 A u to ra d io g ra p h y D r ia d g a l s c o n t a in in g L-C SBS 3 -m * th lo n in a -

la b a lla d p o ly p a p t id a s and n l t r o c a l l u l o s a f i l t a r s w it h bound

i o d in a t Bd p r o t e i n s from "W e s te r n " b l o t t i n g weir* exp o sed t o F u ji X -r a y -film a t room te m p e r a tu r e . Whan n i t r o c e l l u l o s e f i l t e r e wara a u t o r a d iograph ad I l f o r d i n t e n e i f y i n g s c r e e n s wara uaad.

F ilm m i s d e v e lo p e d in Kodak LX-24 and -fix ed in Kodak FX-40 Mhich Mara b oth p re p a r e d a c c o r d in g t o th e

m a n u fa c tu re rs ' i n s t r u c t i o n s . The -film Mas than Maahed in ta p M ater and d r i e d .

2 . 7 . 6 P h o to g ra p h y

A u t o r a d iogra p h a and a t a in a d p o ly a c r y la m id e g a la Mare Mare p la c e d on an illu m in a t e d l i g h t box and p h o tograp h ed on P an atom ic X -film (K o d a k ). F ilm Maa d e v e lo p e d in A c u to l

(P a te r a o n P h o to g r a p h ic L t d . ) -for IO m inutea and -fix ed f o r IO m inutea in Kodak K o d a fix (b o th a o lu t lo n a M ere p re p a re d

a c c o r d in g t o t h e m a n u fa c tu re ra ' i n a t r u c t i o n a ) . The n e g a t iv e a M ere Maahed w it h M ater and d r i e d . P r i n t e M ere p re p a re d on Kodak Kodabrome p aper Mhich Mae d e v e lo p e d in I l f o r d C o n tra a t FF and f i x e d in K o d a fix .

2 . B CHEMICALS

A l l c h e m i c a l a w e r e o f t h e h i g h e a t g r a d e c o m m e r c i a l l y

a v a i l a b l e . S u p p l i e r a o f a p e c l f i c r e a g e n t e M e r e aa f o l l o M i i

Ameraham I n t e r n a t io n a l p i c , Amereham, Buck!ngham ahlret L—CSB8J—m e th io n in e (lOOO Cl m m o l-* ), NaH“ *C03

(36 mCi mmol-1) , N a1 M I <13.5 mCi p g ~ » ) . B.D .H . C h em ica ls L t d . , P o o l « , Dormati

A c r y la m id « , ammonium p «r m u lp h a t«, «odiu m d o d «c y l s u lp h a t « . Eaatman Kodak, R o c h «m t«r , N «m Y o rk , USAi

N ,N ' - m « t h y l « n » b im a c r y la m id «, N ,N , N, N ' —t « t r a m « t h y l « n « d ia m in « .

Ph arm acia (GB) L t d . , Londoni

8 «p h a c r y l 8300 s u p e r f in e , DEAE S « p h a c « l , cyan og«n b ro m id e— a c t i v a t e d S c p h a r o u , lo w m o la c u la r w e ig h t p r o t e in k i t -for d e n a tu r in g p o ly a c r y la m id e g e l a , PD—10 d e m a ltin g colum ns. Sigma Chem ical Co. L t d . , P o o le , Dormati

A d e n o s in e t r ip h o s p h a t e (A T P ), r i b u l o s e - 1 ,3 —b i »p h o s p h a te (RuBP) , phenyl m eth yl s u lp h o n y l 'f l u o r i d e (PM8F) , Coom assie b r i l l i a n t b lu e R, a g a r o s e ( t y p e 1 lo w EEO), b a r b i t a l b u f f e r , gamma g l o b u lin s ( b o v i n e ) , b o v in e serum album in (B 8 A ),

B ta p h y lo c o c c u s au reu s P r o t e in A , c h lo ra m in e T , L—m e th io n in e , L - c y s t e i n e .

3.1 CHARACTERI SATION OF THE RUBISCO LSU-BINDINO PROTEIN

3 . 1 . 1 R t d i o U b t l l i n g o f c h l o r o p l a s t p r o t w i n »

Chi o r o p l amts posm as* t h s l r own ONA (R ls 8< P ls u t , 1962) and a l l th s snzym ss n s c s s s a r y f o r p r o t a in s y n th a s is (r a v ie w a d by B o u lt a r a t a l . . 1 9 7 2 ). I s o la t a d i n t a c t

c h l o r o p l a s t s w i l l in c o r p o r a t a r a d i o l a b a lla d a m in o -a c id s in t o p r o t a in s i f in cu b a ta d in t h a l i g h t ( B l a i r It E l l i s , 1973| B o tto m le y a t a l . . 1974| F is h & J a g a n d o r f, 1 9 8 2 ). I n t a c t i s o l a t a d paa c h l o r o p l a s t s hava baan shown t o c a r r y out

i n i t i a t i o n , a lo n g a t io n and t a r m ln a t lo n o f p o ly p a p t id a c h a in s ( H i g h f i a l d Sc E l l i s , 1976) and h ava baan shown t o ba parm aabla t o most a m in o -a c id s (N ob al Sc Wang, 1 97 0). In such s u b c a llu la r systam s th a an a rgy f o r p r o t a in s y n t h a s is i s s u p p lia d by

p h o t o s y n t h a t ic p h o s p h o r y la t io n . T h a r a fo r a th a c h lo r o p la s t s