- Email: [email protected]
The inhibition of nitrate assimilation by ammonium in chlorella
566
rata
BIOCHIMICA ET IJIOPItY$1CA-ACTA
xzx53 THE
INHIBITION OF NITRATE ASSIMILATION BY AMMONIUM IN CHLORELLA P. J. SYRETT AND I. MORRIS
llotany Department. University College, London (Great Britain) fReceived Augunt lottt, 19(>2)
.~UgIMARY
Ctdorella vulgaris growing with a m m o n i u m n i t r a t e as ni t rogen source p r e f e r e n t i a l l y assimilates a m m o n i u m . N i t r a t e assimilation ceases c o m p l e t e l y w hen a m m o n i u m is a d d e d a n d r e c o m m e n c e s as soon as a m m o n i u m has disappeared. A m m o n i u m does n o t inhibit n i t r a t e r e d u c t i o n b y cells unable to assimilate a m m o n i u m b e e a u ~ t h e y lack c a r b o n source. T h u s t h e inhibition is n o t due t o a m m o n i u m j~er n'e b u t is c o : m e c t e d with its a.~;imilation. T h e inhibition is not t h o u g h t t o result f r o m c o m p e t i t i o n for r e d u c e d p y r id in e nucleotide becat~se n i t r a t e r e d u c t a s e of Chlorella is specific for D P N while glutamic d e h y d r o g e n a s e is specific for T P N . N i t r i t e a d d i t i o n also inhibits n i t r a t e assimilation c o m p l e t e l y b u t a m m o n i u m onl y p a r t i a l l y inhibits n i t r i t e assimilation. INTRODUCTION I t has often been obs e r ved t h a t algae grogSng w i t h a m m o n i u m n i t r a t e as n i t r o g e n source a~simi|ate a m m o n i u m ions p r e f e r e n t i a l l y , ~.itrate not being utilized until a m m o n i u m has air,. s t d i s a p p e a r e d t-~. Several fungi b e h a v e similarly 4. I t is clear from studies with tSN-labelled substrate*,S t h a t this b e h a v i o u r does n o t result f r o m an equilibrium reaction involving n i t r a t e a n d a m m o n i u m which m a i n t a i n s t h e level of n i t r a t e c o n s t a n t until a m m o n i u m has disappeared. R a t h e r m u s t it result f r o m an inhibition o f one stage in n i t r a t e r e duc t ion. MORTON AND MACMILLAN¢ obsel-Ved t h a t while a m m o n i u m c o m p l e t e l y i nhi bi t ed t he assimilation of n i t r a t e by the fungus ScoptdarieObsis bvevicaulis, it h a d little effect on t h e a.~similation o f nitrite. T h e y t h e r e f o r e suggested t h a t a m m o n i u m a c t e d b y inhibiting t he r e d u c t i o n of n i t r a t e to nitrite. In f u r t h e r studies with fungi, MORTON* concluded t h a t t h e inhibition b y a m m o n i u m did n o t occur unless it was assimilated. I n this p a p e r some effects o f a m m o n i u m on n i t r a t e assimilation b y t he alga Chlorella vulgar'is are described a n d it is show n t h a t a n a m o n i u m inhibits n i t r a t e a-~similation as it does in t h e fungi s t udi ed b y MORION AND MACMIL1.A,~#. Moreover, t h e ev id en ce s u g g ~ t s t h a t the inhibitor is a p,'oduet o f a m m o n i u m a~irnJlation which inhibits t h e r educt i on o f n i t r a t e t o nitrite. I n a n o t h e r p a p e r 7 a f u r t h e r effect of a m m o n i u m will b~ di.~ussed, n a m e l y it_s repressive effect on t h e synt hesi s of th~ e n z y m e n i t r a t e rc~luctase. B i ~ l ~ m . Biopkys. A ~ .
67 { t963} 566-575
I N H I B I T I O N OF NITRATE AGSIMILATION IN CIILORELLA
~6 7
METHOD~
Growth of organism P u r e cultures of Chorella t,ulgaris (Pearsall's strain) wtwe gro~vn in a m e d i u m containing, per litre glass-distilled water, 0.2 g a n h y d r o u s MgSO~, 7.76g I-[H~P04. 2.32 g K I H P O , , x ml ~4 t r a c e - e l e m e n t solution ~ a n d xo mg Fe as F e - E D T A (see ref. 9). 28o mg nitrogen per litre was supplied a~ either {NH4)~SO4, KNO= or NH4NO~. T h e p H o f t h e m e d i u m , af t e r autoclaving, was 6.r. Cultures were n o r m a l l y gr ow n for 4 d a y s in wash-t×~ttles at z5 ° with a light i n t e n s i t y o f 600 ft-candles at t h e surface o f the cultures. T he cultures were v i g , r o u s l y a e r a t e d with air containing 0. 5 o~ (v/v) e~arbon dioxide xu. When the cells were growing e x p o n e n t i a l l y the g e n e r a t i o n time was 8 h. C a r b o h y d r a t e - s t a r v e d ceils were pr epar e d by aerating cultures fi,r 18 h in darknes~ b ef o r e harvesting. N i t r o g e n - s t a r v e d cultures were p r e p a r e d b y remo~'ing the cells b y c e n t r i f u g a t i o n at 400 × g, wa_~hing and resusvending t h e m in fnll g r o w t h m e d i u m w i t h o u t a nitr~gen source. T h e c ul tures were t h e n illuminated and a e r a t e d with air c o n t a i n i n g o . 5 % (v/v) c a r b o n dioxide for t6 h before being har,:ested for e x p e r i m e n t a t i o n to. B ef o r e the e x p e r i m e n t s , the celLs were h a t e r , ted by cent ri fugat i on :tt 4oo x g, washed and s u s p e n d e d in nitrogen-free g r o w t h medium. T h e cell d e n s i t y was adj u s t e d turbidometric~ally t o 3- 8 mgl'ml d e p e n d i n g on the e x p e r i m e n t .
Gas exchange This was m e a s u r e d by c o n v e n t i o n a l ~,Varburg m a n o m e t r y at 25 °. For light exp e r i m e n t s , th e flasks were i i l u m i n a t e d with tluor~-~cent tubes giving a light inteltsity in t h e fla~l~s o f a b o u t 800 ft-candles.
Ammonium-N This was d e t e r m i n e d colorimetrically b y a Ne.~ler m e t h o d tt after separat i on f r o m th e cells a n d m e d i u m b y t h e m e t h o d o f CO,WAy ~z.
N i tral~ N This was d e t e r m i n e d in samples of t he m e d i u m a f t e r r e m o v a l of the cells b y c e n t r i f u g a t i o n . A phenol-disaflphonie acid colorimetric m e t h o d was used 13. T he light a b s o r p t i o n o f the eolourcd c o m p o u n d was read at 44o m # a n d c o m p a r e d with t h a t o f freshly p r e p a r e d s t a nda r ds .
Nitrite-N Th is was d e t e r m i n e d colorimetrically with Griess-I!osway reagents an on samples o f the m e d i u m a f t e r r em ova l of the cells. T h e colour was read a t 53 ° m/~, 20 rain after t h e a d d i t i o n o f the reagents.
PrL'pas'ation of ¢ell-frt~ extracts Cells were harve_~ted, w a s h e d a n d r e suspended in ice-cold o.z M Tris buffer ( p H 7.4)- T h e cell d e n s i t y was 6--re mg/ml. T h e cell suspension was st ored at -- x4 ° for 2 . 4 - 2 8 il- Af ter thawing, t h e cell suspension was passed t h r o u g h a F r e n c h p r e s s ~ to B~&Z~.
Bio~/Jys. A~:/~. 67 (xg63) 56b-575
5(~8
P.J.
S Y R E T T , I. M O R R I S
s m a s h t h e cells. D u r i n g this process t h e t e m p e r a t u r e rose to z7-zo ~ b u t t h e s u s p e n sion l e a v i n g t h e press w a s i m m e d i a t e l y i m m e r s e d in ice. T h e s u s p e n s i o n w a s cent r i f u g e d at x3 tx~, × g for zo rain a t z ~. T h e clear, green, s u p e r n a t a n t w a s d e c a n t e d , s t o r e d a t - - : 4 ° a n d u s e d for m e a s u r e m e n t s o f e n z y m e a c t i v i t y .
Assay of ,fftrate reductase activity N i t r a t e r e d u c t a ~ a c t i v i t y wa.g m e a s u r e d b y e s t i m a t i n g t h e n i t r i t e f o r m e d a f t e r i n c u b a t i o n o~-the cell-free e x t r a c t w i t h K N O ~ a n d D P N H . Full details are g i v e n in a later p a p e r L
Measurement of pyridine m~clcotid~ oxidation by flz~orimrtrv l ~ t h n i t r a t e reducta_~e a n d g i u t a m i c d e h y d r o g e n a s e a c t i v i t i ~ o f cell-free e x t r a c t s were d e t e r m i n e d b y following t h e o x i d a t i o n o f r e d u c e d p y H d i n e n u c h ; o t i d e fluorimetrieallv. T h e r e a g e n t s w e r e c o n t a i n e d in a b o u t x ml in a q u a r t z c u v e t t e in a ' l . ~ c a r t e ' f l u o r i m e t e r . T h e p r i m a r y filter w a s L F / 2 a n d t h e s e c o n d a t T L F / 3 / z . D e t a i l s f~f t h e a s s a y m i x t u r e s are g i v e n in t h e l e g e n d t o Fig. 6.
7"]:e protein conteJ,t T h e protc:itt ¢:~ntent o f cell-free e x t r a c t s w a s d e t e r m b , e d b y the meth¢~l, o f L o w a v et al. xs u.~ing b o v i n e - a l b u m i n s t a n d a r d s . RES[~LTS
I. ?'he preferential assimilatio~ of ammonium Fig. x s h o w s tile d i s a p p e a r a n c e o f a m m o n i u m n i t r o g e n a n d n i t r a t e n i t r o g e n f r o m (:ulture_~ o f C. vldgaris g r o w i n g w i t h a m m o n i u m n i t r a t e ,L~ n i t r o g e n source. 150
-
o%
,oo \
\,
~o~
, o
1 ~ ) o f.c.) 0~. <3
o F i g . ~. T h e a . s s i m i l a t i o n o f a m m o n i u m
1 Time ( h }
and
nitrate
Ioo
&.
~
~oo
b y e u l t u r e ~ o f C. vtd.qaria g r o w i n g
at 3oo or
~mo ft-candles light inten~/ty wi~h aiumonium nitrate as nitrogen .source. Note tlmt nitrate i:, not a-,~qimilated until almost ~.1 the ammonium has disappeared. Temperature 25 ~. Cultures acrata, d with 0 . 5 % (v/v) COI in air. 13Wehim. Bio~hys. Acta, 6 7 ( x ~ )
566-575
INHIBITIO.~ OF NITRztTE A.~%IM~LA'rlo.x" IN CHLORELLA
5[JC)
C u l t u r e ~ w e r e g r o w n a t t w o d i f f e r e n t light inten~i~ie~ G r o w t h is f,3_ster a t 6 o o i'tcan~Hes t h a n at 3on ft-candle.~ a n d a m m o , 6 u m nitrotzetx d i s a p p e a r s m n r e r a p i d l y . I n n e i t h e r culturt~ i~ ... :"~,,~-N * hue.; *,,,,e,, • n i t r a t e "'*:'"" ,,,,d,.ed u n t i l ,t . . . . . . . .,............ . . . . ~L~:dmflated. ~. T h e i n h i b i i i o n o[ n i t r a t e a s s i ~ g i l a t i o ~ by the z, d d i t z , n ~¢{ a m , t o n i t : m - N
Fig. 2 a s h o w s t h e r e s u l t o f a d d i n g a s m a l l q u a n t i t y o f a m m ( m i u m - N t~ a .~usp e n s i o n wifich is a s s i m i l a t i n g nitrate, in d.',rkness w i t h g l u c o s e a_s c;trb~n +our~.,:. N i t r a t e a~ssimilation t'~'a~es as s o o n as a r n m - n i u m - N is a d d e d . The: amm~niun~-.N" is r a p i d l y a.%sirnilated. A~ s~um as it h a s :~t! g~me. n i t - a t e a~
.
Nr,4~to 2
o
"g7
-a-~...'2
\.,,
a
,.
F i g . 2. T h e e f f e c t o f 8 . m r . l ~ n i u m additior~ o n n i t r a t e a ~ i m i l a t t , m b y [a) n i t r a t e - g r o w n cell~ in d a r k in p r e s e n c e o f t %~ {w/v) g l u c o s e , lb} nitratt~-gro~vn ccli~ iu l i g h t i n p r e s e n c e o f 3 % ~ . : v ) caa'tmn d i o x i d e i n air, (c) n i t r o g ( ~ n - s t a r v c t l cell~ lit d a r k tn pr~.~;ence o f t % (w/'~-] glu~-o.~. ~----O, n i t a ' a t e - N in c u l t u r e t whicix doe~ n o t retzeive a m m o n i u m ; A - - - ~ k . n i t r a t e - N i n c u l t u r e 2 w h i c h r e c e i v e s a m m o n i u m a t t i m e i t t d i e a t e d ; ~IV--~1F. a m m o n a u m - N irt c u l t u r e z, Cell den.~ity. 5.o m g d r y w t , l m l . L i g h t intera~ity. Boo f t - c a n d l e s . T ~ e f i g u r e s repr~.-~ent / s t n i t r a t t ; o r a m m o n i u m - . ~ p e r mi f o r ~..x|=ts, a a n t i b, anti kst~ N t>¢r 0. 5 lt:tl fi)r E x p t . c. T e m p e r a t u r e , zS ~.
U R H ^ S tt r e p o r t e d t h a t n i t r o g e n - s t a r v e d cells o f CkloreUa a n d Scetiedestlaus assimilated ammonium and nitrate simultaneously. However, under the conditions o f o u r e x p e r i m e n t s , a m m o n i u m i n h i b i t s n i t r a t e a:~.~imilatio'h b y n i t r o g e n - s t a v t ' e d cells j u s t as it d o e s t h a t o f n o r m a l cells (Fig. 2c) 3. T ~ e effect o f n i t r i t e o n n i t r a t e a s s i m i l a t i v ~ ,
Fig. 3 a s h o w s t h e effect o f t h e a d d i t i o n o f n i t r i t e ~n n i t r a t e ~ s i ~ i l a t i o n . It c a n b e s e e n t h a t its effect is s i m i l a r t o t h a t o f a m m o n i u m a d d i t i o n , i.e. it pr~duc~'s a c o m p l e t e i n h i b i t i o n o f n i t r a t e ,xssimilation w h i c h p e r s i s t s u n t i l all t h e n i t r i t e h a s been assimilated. 4- T h e effext o f a m , v a n i u m
on nitrite assimilatio;,
T h e a d d i t i o n o f a m m o n i u m - N also i n h i b i t s n i t r i t e a s s i m i l a t i o n b u t t h e i n h i b i t i o n is n o t c o m p l e t e (Fig. 3b). I n t h r e e experiment.~ o f this t y p e t h e p e r c e n t a g e i n h i b i t i o n l:lio,'him. Biophy,~. ,'[cta, 67 (tq£)3) 56G-575
~7 0
P. J . SYRETT, I. MORRIS
o f n ' t r i t e assirnil;ttion w a s 75, 84 a n d 85 ~/o g i v i n g a m e a n v a l u e o f a b o u t 8o ~/o inhibition.
5. T!te dependence of inhibition u~on the assimilation of ammonium I n t h e experi~;~ents d e s c r i b e d a b o v e , t h e i n h i b i t i o n o f n i t r a t e a s s i m i l a t i o n b y a m m o n i u m c o u l d be d u e e i t h e r t o t h e p r e s e n c e o f a i n m o a i u m or to t h e a m m o n i u m as~hnilation w h i c h ~-'cur~ i m m e d i a t e l y a f t e r a m m o n i u m is a d d e d . T h e results described in this s e c t i o n s~mggest t h a t t h e ; n h i b i t i o n is n o t d u e t¢+ t h e p r e s e n c e o f a m m o n i u m per se b u t t o a m m o n i u m a~similation. 60
•
4~L
h~
,.¢ ~
A
J.
o
"+..m~.-- , ~
I 4t~eoy to2
";
~o " , b o " ~so " ~bo " "(';me (,'n~)
D.
t.+
Fig. 3- A. T h e e f f e c t o f n i t r i t e o n n i t r a t e a ~ s i m i ] a t i o n bst n i t , r a t e - g r o w n eel]g+ T h e c u l t u r ~
~n-
tained b 5 mg dry wt. cells per m: and a % Iw/v) glucose, i ~ 4 1 , nitrate-N in culture x which received no nitrite; & - - A , nitxate-N in culture • which reccive~i ]o/ag nib-ite-N per mi at time indicated; V ' - - ~ . nittl.te-N in culture z. B. The effect of ammonium on nttrtte a.q~simi~tion by nitrate-grown cells. The cultures contained 3 mg dry wt. cells per ml and t % [w/v) glucose. Culture z received zz ttg xmmonium-N per ml at the time indicated. E x p e r i m e n t s ~,'ith cell-free e x t r a c t s containi,~g n i t r a t e r e d u c t a s e (section 6) sh~>w t h a t a m m n n i u m s u l p h a t e in a final c o n c e n t r a t i o n o f 3" xo - t M h a s no effect a t all o n t h e a c t i v i t y o f n i t r a t e r e d u c t a x e . T h i s a m m o n i u m c o n c e n t r a t i o n L~ t h i r t y t i m e s t h a t w h i c h i n h i b i t s n i t r a t e a s s i m i l a t i o n b y i n t a c t cells a n d t h e a b s e n c e o f i n h i b i t i o n wi~h cell-free e x t r a c t s * s u g g e s t s t h a t a m m o n i u m a l o n e does n o t i n h i b i t n i t r a t e reduction. This s u g g e s t i o n was s u p p o r t e d b y e x p e r i m e n t s c a r r i e d o u t w i t h i n t a c t cells w h i c h h a d been p r e v i o u s l y :~tarved o f c a r b o h y d r a t e . BOSGEag t¢ founci t h a t c a r b o h y d r a t e - s t a r v e d cells of S c e n e d e s m u s w o u l d r e d u c e n i t r a t e t o a m m o n i u m q u a n t i t a tiwely in light in ttle a b s e n c e o f c a r b o n dioxide. S u c h cells p r e s u m a b l y l a c k c a r b o n r e s e r v e s w i t h w h i c h t o m a k e c a r b o n s k e l e t o n s for a m m o n i u m a s s i m i l a t i o n . Fig. 4A(i) s h o w s t h a t in light, in t h e a b s e n c e o f c a r b o n d i o x i d e , c a x b o h y d r a t e starw~d ceils o f C. vulgaris r e d u c e n i t r a t e t o a m m o n i u m w h i c h a c c u m u l a t e s . T h e a d d i t i o n o f o . o o 5 M a m m o n i u m h a s n o effect o n this r e d u c t i o m H o w e v e r , w h e n e i t h e r c a r b o n d i o x i d e o r g l u c o s e is p r e s e n t t o p r o v i d e a c a r b o n s o u r c e (Fig. 4All a n d iii) n o a m m o n i u m a c c u m u l a t e s since it is a s s i m i l a t e d a n d , u n d e r t h e s e c o n d i t i o n s , t h e a d d i t i o n o f a m m o n i u m i n h i b i t s n i t r a t e d i s a p p e a r a n c e . I n fact, w h e n t h e c a r b o n so,arce is 5 % (v/v) c a r b o n d i o x i d e in t h e g a s p h a s e , a , , , , o n i u ~ addition i-hibits [ ? i o c h l m . B ~ l ~ h y s . . 4 ¢ 1 , ~ + 57 l t 9 6 3 )
566-575
INHIBITION
O F NITRATE: A S S I M I L A T I O N IN C H L O R E L L A
57z
n i t r a t e r e d u c t i o n c o m p l e t e l y (Fig. 4A]ii). T h e effect ,,f t h e p r e s e n c e o f g l u c o s e i~ o f i n t e r e s t . W h e n g l u c , ~ i~ a d d e d , in t h e a b s e n c e o f c a r b o n d i o x i d e , i.e. t h e W a r b u r g fla~ks i n w h ~ h th'- e x p e r i m e n t s a r e c a r r i e d o u t c o n t a i n c a u s t i c p o t a s h , t h e i n h i b i t i o n o f c i t r a t e r e d u c t i o n b y a m m o n i u m is p r e s e n t b u t i n c o m p l e t e (Figs. 4Aii a n d 4Bii). I f c a u s t i c p o t a s h is o m i t t e d f r o m t h e W a r b u r g flasks tb~.- i n h i b i t i o n in t h e p r e s e n c e o f g l u c o s e is m u c h m o r ~ p r o n o u n c e d (Fig. 4Bill). Thi~ m a y Ix: a r e f l e c t i o n ~,f t h e f a c t t h a t a m m o n i u m a s s i m i l a t i o n t o a m i n o a c i d s is a c c o m p a n i e d b y cot~:dderable c a r b o n d i o x i d e fixation~e; it m i g h t , h o w e v e r , i n d i c a t e a mor~" d i r e c t r e l a t i o n s h i p b e t w e e n a m m o n i u m , c a r b e m d i o x i d ~ a n d t h e ,.'nhibit¢,r ~.:f n i t r a t e r e d u c t i o n . Fig. 4 B also
f°l._,l
,Ira
"
!]I[[ i,[1 I, rfit~rte
F~g• 4- The effect of ammonium on nitrate reduc~on and as~imil&tion by ca.cbohydra.tv-atarved cel/~ in light. The plus and minus sign~ below the c o l , m n denote the presence or absence of added a m m o n i u m - N (7° ~ / m l ) . The e.xperiments were carried out ia Waxburg flasklg, eaclt containing 2.o ml cell suspension {3 mg dry .yr. cells/ml)~ The iMtia.I concentration of nitrlte-N was 7o Fg/ml. The flask contenta were analysed 2 h after the ~tddJtion ot nitrate land/or ammonium)+ L i g h t iuten.~ity, 8oo t~-candle,. Temperature. 25 °. --COn indicates tkat the ~,Varburg flasks contained caustic potaoh in the centre wells. )n experiment 4 B Iiii). this was omitted; +CO~ indicates manometer~ flushed ~ t h 5% COn in a.ir. When glucc~se was present, its final concentration was ! % (w/v). Solid column° n2trate-N (lisappeating from su.~l~--nsion in 2 h ; c~oss-hatched co|u,nn. ammonium-N appearing in suspension in 2 b; open column in Iowex figure, arnmonium-N disgppem-ing from suspension in ~ h in absence c~f nitrate. The percentage inhibition of nitrate rcxlucUon by presence of ummonium is shown. s h o w s t h e a m o u n t o f a m m o n i u m a s s i m i l a t i o n caJTied o u t b y t h e cells i n t h e a b s e n c e o f n i t r a t e . I t is g r e a t e s t w h e n 5~/o CO 2 is t h e c a r b o n .source (i) a n d l e a s t w i t h g l u c o s e i n t h e a b s e n c e o f c a r b o n d i o x i d e (iii). I t is c l e a r t h a t t h e i n h i b i t o r y effect o f a m m o n i u m o n n i t r a t e r e d u c t i o n is mo:~t p r o n o u n c e d u n d e r t h o s e c o n d i t i o n s w h i c h m o s t favour ammonium assimilation. T h e e x p e r i m e n t s i l l u s t r a t e d in Fig. 4 w e r e c a r r i e d o u t i n W a x b t t r g m a n o m e t e r s , t h e flask c o n t e n t s being a n ~ y s e d at the e n d of the e x p e r i m e n t . T h e g a s e x c h a n g e w h i c h o c c u r s d u r i n g a n e x p e r i r a e n t o f t h i s tyF~, L~ ~itown in Fig. 5- I n light, n i t r a t e r e d u c t i o n is a c c o m p a n i e d b y a c o n s i d e r a b l e e v o l u t i o n o f o x y g e n t ° , ~. T h u s g a s e x c h a n g e is a s e n s i t i v e i n d i c a t o r o f t h e r a t e o f n i t r a t e r e d u c t i o n . I t c a n b e s e e n f r o m
572
p,
J.
SYREI~,
1. M O R R I S
Fig. 5 a, t h a t , in t h e a b s e n c e o f g l u c o s e a n d c a r b o n d i o x i d e , t h e a d d i t i o n o f N H ~ + h a s no effect on g a s exehar~ge in t h e p r e s e n c e o f n i t r a t e . T h i s r e s u l t is in s h a r p c o n t r a s t to t h a t s h o w n in Fig. 5e w h e r e t h e a d d i t i o n o f N H4 ÷ to ceils ~ s i m i l a t i n g n i t r a t e in t h e p r e s e n c e o f 5 % ( v / v ) c a r b o n d i o x i d e , l o w e r s t h e r a t e o f g a s o u t p u t t o the l e v e l o f t h a t o f cells w i t h N H~* a l o n e ; t h i s c o r r e s p o n d s t o t h e a l m o s t c o m p l e t e i n h i b i t i o n of n i t r a t e r e d u c t i o n s h o w n in Figs. 4 A (iii) a n d 4 B (i). I n t h e p r e s e n c e o f g l u c o s e and absence of carbon dioxide, the gas-exchange measurements indicate an incomp l e t e i n h i b i t i o n o f n i t r a t e r e d u c t i o n b y N H ~ ~ (Fig. 5b) corre.~ponding to t h a t s h o w n in Figs. 4A (it) a n d ~ B (it). -CO~
i
<> i
'
o
~1
@l,~cor~
-
-, Ga,a¢o~
-" ~
lb) p ,
-COI
[
l / / -
=co o
, oo
*COz •
I~)#o
-Otu.¢ol,~ / I
/
ji.i ~o o
loo
~o
T I m t (ral, n}
1"i,,'*. 5. t-.as exchange accompanying nitrate mim'.latiorl by ca.rbohydrate-starvcd cells in light. Lighl intensity, ,~oo ft-tu~lltll~.~. Temperature, 25 °. Nitrate-gro~r~ cells star,.c,l of earlmhydrste t o t i 6 h. Cell .~us!ril~iort contained 3.o l~g d r y ~ ' t . c e l l s p e r m L (a} a n d (b), t l a ~ k s contained 2.0 m l
cell ~u~pe.~ion, Caustic potash in cent.re well, in (b} t ~/o (wlv) glcccmc w ~
addo;l. 8 4 pig N O i - N
or ~)61zg N t l i ' - N added at times indic~t~xl. (c), Flasks corttaiu-.d i.o ml cell suspension. Gas phase. 5~h (v/v) CO= in air. lo 4 pg NO=-N or 57lag NI4i*-N added at times it=die, ted+ T h e s e e x p e r i m e n t s , t h e n , s u g g e s t t h a t it is a m m o n i u m a k x i m i l a t i o n r a t h e r t h a n a m m o n i u m i t s e l f w h i c h i n h i b i t s n i t r a t e a s s i m i l a t i o n . MCmTON e c a m e to a s i m i l a r c o n c l u s i o n for diflb.rent r e a s o n s .
7"he. pyri$ine nucleotide specifcity of nitrate reductas¢ and glutamic dehydrogenase in C. v,dgaris 6.
] h e p r e s e n c e of b o t h n i t r a t e reducta.~e e n d g l u t a m i c d e h y d r o g e l , a s e w a s dem o n s t r a t e d in ceil-free e x t r a c t s o f cell~ g r o w n w i t h K N O s ,~s n i t r o g e n s o u r c e (Fig. 6a). N i t r a t e reducta.~e requirext I ) P N H as e l e c t r o n d o n o r a n d d o e s n o t utilize T P N H . T h e r e v e r s e is t r u e for g l u t a m i c d e h y d r o g e n a s e (Fig. 6b).
7. Nitrate assimilation by cells grown u ~ h N H I N O , as nilrogen sot~rc¢ Fig. 7 shov,.~ t h e t i m e c o u r s e o f n i t r a t e a . ~ i m i l a t i o n o f ceils ~ r o w n w i t h N H i N O s as n i t r o g e n s o u r c e in c o n t r a s t t o t h o ~ e g r o w n ~ q t h K N O = . W h e r e a s t h e celts g r o w n on K N O = a s s i m i l a t e n i t r a t e i m m e d i a t e l y a f t e r it is a d d e d , t h e cells g r o w n on N H i N O , s h o w a l o n g I ~ r l o d o f 3 h b e f o r e n i t r a t e a s s i m i l a t i o n s t a r t s . T h i s l o n g ~-.riod is c o n siderab!:,- ~ h o r t e n e d i f t h e cells ~re a e r a t e d for 3 h in a N - f r e e m e d i u m b e f o r e n i t r a t e i~. a d d e d . T h a t t h e d i f f e r e n c e ]n b e h a v k m r o f t h e s e c u l t u r e ~ is n o t d u e t o d i f f e r e n c e s Biochim. Btophys..4~la, 6 7 ( x 9 6 ] } 36¢~-375
INHIBITION
.
12 "~j 11
573
O F N I T R A T E ASSI511LATION IN C H L O R E L L A
v"---r
~
v--v
"-"--v-.-v~ v -v
~I¢
J
z
l
L
&
-1
0
~
a
(~
~.
~0
J[/ L
a.9
0
t .--4~_~L~
~'
4
--1
I
d-
8
TO
~2
~
t;rr~ ( ~ ; n ) F i g . 6. T h e p y r i d i n , : i ~ u c l e o t i d e ~pe~:ificity o f ( a } n i t r ~ t e r e , l u c t ~ e a n d (b) g l u t a m J e d ~ . h y d r n g e n a - ~ e ; t h e o x i d a t i o n o f r e d u c e d p T r ~ d i n e : m c l e o t i d e w a s ::lea~t~re~i fluo::taaeLri~zaily~ T h e ..-~s~ty m i x t u r e s c o n t o r t e d o . ~ m l c e i l - f r e e e x t r a c t c o n t z i n i n g o. t5 m g p r o t e i n , o . z hal o.a .~i Tri.~ b u f f e r !pt[ 7-4] ; o . t m l a 0 o z M ~ a p p r n x . ) D P N H o r T P N H . A str-arx~ o f ox~'~,'w-rree, n i t r o g e n w a s b u b b l e d t l : r o u g h t h e m i x t u r e b e t w e e n r e a d i n g s ; t h i s r e d u c e d t h e ..tx(log(rncu~ t ~ x i d a t i o n o f r e d u c e d p y r i d i n e n u e . l e o t t d e . I n (a) 2 o # t m o l v s KI-IO~ w e r e a d d e d a t t h e t i m e indicatt:cl ; i n {b) -~o l t m o l e ~ ~ - o x o ~ h a t a r a t e w e r e i n c l u d e d i n t h e r e a c t i o n mixttlrt~ zttzd 2 0 / ~ m o l e ~ ( _ ~ l l l ) ~ t O ~ a d d c t l ~ t t h e t i m e inctic~tted. Temperature, 3 t ~. I n f l u o r i m e t e r u n i t s approx. 40 ml~mole~ PNH.
F c.
103
=' go * ' N
v "e
~o-
0
50
1QD
150
200
250
T ; r n e O'~in)
F i g . 7" N i t r a t e a s s i ~ i ; a t i o n b y ¢~tls g r o w n witl-, a m m o n i u m rut,at,." a s N - s o u r c e . C u l t u r e z c 0 n tain_*~d 13 m l c e l l s u s p e n s i o n g-rowan i n p o t a s s i u m n i t r a t e ; t h e celia a s s i m i l a t e r ~ i t r a t e ~ks s o o n a.~ i t is a d d e d . C u l t u r c m z a n d 3 e a c h c o n t a i n 8 m l c e l l .~u.qr,,m~i<,n g r o w n i n a ~ m m n n i u m n i t r a t e b u t culture 3 was shaken for 3 h in -zitxogen-free medium helot c mtratc was added. Note that culture 2 o n l y as.Mmil~tt-~ t l ~ a d d e d nitr-.~te a ~ e r ~, l e g o f ~ b o u t 3 b : i n c u l t u r e 3 t h e l a g h ~ I x ' c a c o u $1derably reduee,t. All ftmsks cents/ned Chlorell~ suspension, 7 mg dry wt./ml, with glucose added t o g i v e a f i n a l c o n e e n t r a ~ z o n o f g °/6 ( w ] v ) . T -~'- f l a s k s w e r e s h a k e n i n d a r k n e s s a t z5 °. A l l r e c e i v e d x o o / 4 w o l e s K N O ~ ~ t z e r o t i m e . S a m p l e s o f e a c h tyt>c o f cell w e r e p a s s e d t h r o u g h a F r e n c ~ p r e s s and nitrate reductase activity, assayed. The enzyme levels arc shown below. Note that the n i t r a t e r e d u e t a . s e l e v e l o f t h e a n ~ m o n i u r n n i t r a t e - g r o w n c e l l s is u n c h a n g e d b y 3 h a e r a t i o n i n nitrogen- fr~ medium. Nava~t vd'_~e'_e -..-'ioityo, ~U-lv~
.~,a,~
l,~r
KNOm-grown cells , " T H ~ N O s - g T o v : n cell~ N'HeNOi-gaow-n ceils after 3 h in N-free medium Bioek~m.
loms~l ~ mare m K Ov,~'~s
z5,6 4.0 4.8 /~io~kys. Aaa.
67 t,z g ~ 3 ) 5 6 ~ - - 5 7 5
574
e . . [ . SYRET']', !. MORRIS
in t h e a m o u n t o f n i t r a t e r e d u c t a s e p r e s e n t is s h o w n b y t h e d a t a i n c l u d e d in Fig. 7It is clear t h a t t h e cells g r o w n o n N H a N O s posses n i t r a t e r e d u c t a s e ( a l t h o u g h less t h a n t h a t in t h e K N O s cells) a n d t h a t t h e i r n i t r a t e r e d u c t a s c c o n t e n t does n o t a l t e r a p p : e c i a b l y d u r i n g t h e 3-h p e r i o d o f n i t r o g e n s t a r v a t i o n . N e v e r t h e l e s s t h e p~riod o f n i t r o g e n s t a r v a t i o n c o n s i d e r a b l y increases t h e i r a b i l i t y to a_~similate n i t r a t e . DISCUSSION N i t r a t e a.~imilation b y C. vtdgaris is c o m p l e t e l y i n h i b i t e d as scxm a.~ a sm al l q u a n t i t y o f a m m o n i u m is a d d e d a n d t h e i n h i b i t i o n is r e l i e v e d as soon as all t h e a m m o n i u m has b een a s s i m i l a t e d . Since a m m o n i u m o n l y paxtially i n h i b i t s t h e a s s i m i l a t i o n o f n i t r i t e a n d since n o a p p r e c i a b l e q u a n t i t y o f n i t r i t e e v e r a c c u m u l a t e s in o u r c u l t u r e s , a m m o n i u m m u s t i n h i b i t t h e first s t e p o f n i t r a t e assi m i l at i on, n a m e l y t h e r e d u c t i o n i~f n i t r a t e t o n itr i t e . T h e a d d i t i o n o f n i t r i t e also i n h i b i t s n i t r a t e a~sirnilation; t h i s effect m a y well be a r e s ul t o f t h e c o n v e r s i o n o f n i t r i t e t o a m m o n i u m so t h a t b o t h act in a c o m m o n mariner. I n m a n y r e s p e c t s o u r re~ults r e s e m b l e t h o . ~ o f MORTON A.WD MAt:MII.LA.~4 w i t h fungi. "I hey. t oo, f o u n d t h a t a m m o n i u m c o m p l e t e l y i n h i b i t e d n i t r a t e aggimilation a n d p a r t i a l l y i n h i b i t e d n i t r i t e assimilation, b u t in t h e i r w o r k , n i t r i t e a p p a r e n t l y h a d little effect on t h e r e d u c t i o n o f n i t r a t e . A m m o n i u m a ppe a xs to i n h i b i t n i t r a t e a s s i m i l a t i o n o n l y w h e n it is itself assim i l a t e d . Thu.~ a m m o n i u m has no effect on n i t r a t e ~educLion b y c a r b o h y d r a t e s t a r v e d cells unless a c a r b o n solxr¢~ is a d d e d w h i c h enable~ t h e cells t o a s s i m i l a t e a m m o n i u n L MORTOXs, too, s u g g e s t e d t h a t a x n m o n i u m m u x t be a s s i m i l a t e d b e f o r e it can inhibit n i t r ~ t c r e d u c t i o n . T h e r e ar c at least t w o w a y s in w h i c h a m m o n i u m a s s i m i l a t i o n m i g h t i n h i b i t n i t r a t e r e d u c t i o n . F i r s t l y , t h e t w o pr oc e s s e s m i g h t c o m p e t e for t h e a v a i l a b l e s u p p l y o f r e d u c e d p y r i d i n e n u c l e o t i d e in t h e cells. S e c o n d l y , a m m o n i u m m i g h t be assim i l a t e d t o an o r g a n i c n i t r o g e n o u s c o m p o u n d w h i c h i n h i b i t s n i t r a t e r e d u c t i o n in s o m e way . T h e ~ c o n d o f these, p o ~ i b i l i t i e s a p p e a r s t h e m o r e likely. I t is t r u e t h a t t h e p r i m a r y s tep s in b o t h a m m o n i u m a n d n i t r a t e a s s i m i l a t i o n r e q u i r e r e d u c e d p y r i d m c n u c l e o t i d e , in t h e firsL for t h e r e d u c t i v e a m i n a t i o n o f a - o x o g l u t a r a t e t o g l u t a m i c acid nard, in t h e second, for t h e r e d u c t i o n o f n i t r a t e t o ni t ri t e. B u t in C. v~dg~,is, t h e p y r i d i n e n u c l e o t i d e specificity of t h e e n z y m e s c a t a l y s i n g t h e s e t w o r e a c t i o n s a p p e a r s t o differ. Ghitaanic d e h y d r o g e n a t e r e q u i r e s T P N H while n i t r a t e r e d u c t a s ~ r e q u i r e s DP,N'H. Thu:~ a d i r e c t c o m p e t i t i o n a p p e a r s u n l i k e l y a l t h o u g h it c a n n o t be c o m p l e t e l y r a l e d out. M or eover , t h e e x p e r i m e n t w i t h cells g r o w n on a m m o n i u m n i t r a t e suggests t h a t s o m e t h i n g i n h i b i t i n g n i t r a t e a s s i m i l a t i o n a c c u m u t a t e s m ceils w hi ch axe a s s imilatin g a m m o n i u m . T h e s e cells, a t t h o u g h Possessing n i t r a t e r e d u c t a s e , do n o t begin t o r e d u c e n i t r a t e until t h e y h a v e b e e n a e r a t e d for 3 h in a n a m m o n i u m free m e d i u m . It looks as i f t h e i n h i b i t o r is r e m o v e d d u r i n g t hi s period. P r e s u m a b l y , in t h e e x p e r i m e n t s in w h i c h r a t h e r a small q u a n t i t y o f a m m o n i u m w a s a d d e d , t h e q u a n t i t y o f i n h i b i t o r f o r m e d w o u l d be. small a n d w o u l d d i s a p p e a r soon a f t e r all t h e a m m o n i u m h a d been as s i m i l a t e d. I f such an i n h i b i t o r is f o r m e d d u r i n g a m m o n i u m a s s i m i l a t i o n it m i g h t i n h i b i t n i t r a t e r e d u c t i o n in one o f t w o ways. F i r s t l y it m i g h t , i~ s o m e w a y , p r e v e n t t h e e n t r y o f n i t r a t e i n t o t h e cell o r i n t e r f e r e w i t h its access t o t h e e n z y m e , n i t r a t e red u c t a s e . S e c o n d l y it m i g h t act b y i n h i b i t i n g n i t r a t e r e d u c t a s e itself. W e p r e f e r t!:e Bio¢i~ivn. Biopl, y~. Acta, 67 (If763) 566-575
;.NI-ilBI'I+ION O F N I ' I R A T E ASS[MII+ATION I N C H L O R E L L A
5~r3
s e c o n d o f t h e s e po.~sihilitie5 a n d h a v e .~>mo e v i d e n c e to .~upport itL W e h a v e failed t o find e v i d e n c e o f a n y effect o f ananmniurv, a s s i m i l a t i o n o n .*.lie e n t r y o f n i t r a t e #nto t h e cells b u t since the q u a n t i t y of n i t r a t e in Ch!orella is a l w a y s e x t r e m e l y low t h e r e s u l t s are i n c o n c l u s i v e W e h a v e a l ~ ,+xarnlned t h e effect ~f a v a r i e t y o f n i t r o g e n o u s c o m p o u n d s on t h e n i t r a t e reddcta_se a c t i v i t y ~f cell-free e x t r a c t s ; tile r e s u l t s a r e p r e s e n t e d in a l a t e r p a p e r L AC K N ()'~,'l,-[. 1- 1)1 ; |- .~i l," N'.T"
W e s h o u l d like t(, t h a n k the D e p a r t m e n t a g r a n t to o n e o f us {I.M.).
of Scientifit: a n d I n d u s t r i a l Rtrsearch for
REFERENCES t C . A. l . u D w i , ~ . A m + J+ / / o t a n y . ~5 ( t 9 3 8 ) 448. s l ~ + Pan-arT ^ N D J . Fore(,, . . | m J+ 130tdl+ly, 27 [ t o . t o ) /35+ * J . F'. S C I ' I U L E R . V+ I~1+ I)ILI.I-.K ~'+Xt~+ | l J . KEILSLF-..~, I>I+¢P'~t l*i''+"~lOl., Z8 [ [ : ~ $ 3 ) g~J~+ 4 A . G. M o l t ' t o N ^NI21 A+ ~ [ A C M I I . L . t . N . J . E x p P I 13or.. 5 (1+~5-1) .'3-'C. C. D t ~ t , w t c x ~ , J+ B i o l . C k e m . , t & ) { 1 9 S t ) I 0 7 " A. G. M o m T o m , J+ E , , p t l . B o t . 7 ( ~ 9 5 ~) 97+ = I+ M o R z t s ^ s I > P . J+ %vm~.'rr. i n p r e p a r a t i , ~ n . ~1 D- I. AllNON, ,,is'a+ J+ B o l a n y . z 5 (193~) 322. t L . JAcoz*~lo.~. Plff~'! .z'h.vsi,,l. 26 {I¢.}31 ) .$11. t." V. J- S'¢RImTZ, . , I n n . B o t a n y f'l.ondo~,J, I 7 (|0.53) r *l O . [?OLIN AND ttV+ I)ENiS+ J . o+ol. (.;/lelB.. 26 I t g t c , ) 4 7 3 . I I E . J . CO:i%;'AY, Aficro.dztJuMo~* .~nalys+, . ~ l d v:,':t.~'.c!.-:,, L o n d o n , 1947.
N o s t r a n d , t 9 4 9 . p+ 785-8o7+ t¢ H+ ~kV+ MILNER, .~. S+ L^V,°R~NCP+ ,'~,'wt, C. S. FK~.~CIt. Nc.i.,!r+ce. [ t l {195o) 0 3 3 . '+ ('). H . ~ClXVRy, +NT.J . I~O51"~BROUGH+ .~+ L+ F,~I+.R AND IL J R.~+~U,XL, J+ I++oL C + e m . . ' 9 3 (,<~fl) z+5+ LI (). [."RHAN, _[altrb. wi$$. Bolan+, 75 l. l o ~ z ) 1. tT I.. H. J. Bo~'c,r.as, Mcgedel+ Landbouu,hoge+ch,',ot D'aeening,+. 5 6 ( i 9 5 u ) I. z. F+ J . S r n E T T , P h y s i o L Plantavto~L 9 ',t95¢,1 z65 lb ( ) , "I.~PARI~IURG A-hID ~+ ~ ' I P G K I . E | N . l'fiochem. Z., i t o tt+-~o3 6q+~ t~ j . M Y ~ t a s , i n J . F m x . ~ c g ,x.~t> W . E . L o o ~ t s , Ph,+t,++ynthe++.+ +~ l~lant+, l o w + S -t~t¢ C,+H,+ge i-'re.,:s, A m e s , I o w a , zg.++j, p. 3 4 9
BsocAi.,. ~iophys+ . 4 a a , 67 ( 1 ~ 3 )
56¢~-575
rata
BIOCHIMICA ET IJIOPItY$1CA-ACTA
xzx53 THE
INHIBITION OF NITRATE ASSIMILATION BY AMMONIUM IN CHLORELLA P. J. SYRETT AND I. MORRIS
llotany Department. University College, London (Great Britain) fReceived Augunt lottt, 19(>2)
.~UgIMARY
Ctdorella vulgaris growing with a m m o n i u m n i t r a t e as ni t rogen source p r e f e r e n t i a l l y assimilates a m m o n i u m . N i t r a t e assimilation ceases c o m p l e t e l y w hen a m m o n i u m is a d d e d a n d r e c o m m e n c e s as soon as a m m o n i u m has disappeared. A m m o n i u m does n o t inhibit n i t r a t e r e d u c t i o n b y cells unable to assimilate a m m o n i u m b e e a u ~ t h e y lack c a r b o n source. T h u s t h e inhibition is n o t due t o a m m o n i u m j~er n'e b u t is c o : m e c t e d with its a.~;imilation. T h e inhibition is not t h o u g h t t o result f r o m c o m p e t i t i o n for r e d u c e d p y r id in e nucleotide becat~se n i t r a t e r e d u c t a s e of Chlorella is specific for D P N while glutamic d e h y d r o g e n a s e is specific for T P N . N i t r i t e a d d i t i o n also inhibits n i t r a t e assimilation c o m p l e t e l y b u t a m m o n i u m onl y p a r t i a l l y inhibits n i t r i t e assimilation. INTRODUCTION I t has often been obs e r ved t h a t algae grogSng w i t h a m m o n i u m n i t r a t e as n i t r o g e n source a~simi|ate a m m o n i u m ions p r e f e r e n t i a l l y , ~.itrate not being utilized until a m m o n i u m has air,. s t d i s a p p e a r e d t-~. Several fungi b e h a v e similarly 4. I t is clear from studies with tSN-labelled substrate*,S t h a t this b e h a v i o u r does n o t result f r o m an equilibrium reaction involving n i t r a t e a n d a m m o n i u m which m a i n t a i n s t h e level of n i t r a t e c o n s t a n t until a m m o n i u m has disappeared. R a t h e r m u s t it result f r o m an inhibition o f one stage in n i t r a t e r e duc t ion. MORTON AND MACMILLAN¢ obsel-Ved t h a t while a m m o n i u m c o m p l e t e l y i nhi bi t ed t he assimilation of n i t r a t e by the fungus ScoptdarieObsis bvevicaulis, it h a d little effect on t h e a.~similation o f nitrite. T h e y t h e r e f o r e suggested t h a t a m m o n i u m a c t e d b y inhibiting t he r e d u c t i o n of n i t r a t e to nitrite. In f u r t h e r studies with fungi, MORTON* concluded t h a t t h e inhibition b y a m m o n i u m did n o t occur unless it was assimilated. I n this p a p e r some effects o f a m m o n i u m on n i t r a t e assimilation b y t he alga Chlorella vulgar'is are described a n d it is show n t h a t a n a m o n i u m inhibits n i t r a t e a-~similation as it does in t h e fungi s t udi ed b y MORION AND MACMIL1.A,~#. Moreover, t h e ev id en ce s u g g ~ t s t h a t the inhibitor is a p,'oduet o f a m m o n i u m a~irnJlation which inhibits t h e r educt i on o f n i t r a t e t o nitrite. I n a n o t h e r p a p e r 7 a f u r t h e r effect of a m m o n i u m will b~ di.~ussed, n a m e l y it_s repressive effect on t h e synt hesi s of th~ e n z y m e n i t r a t e rc~luctase. B i ~ l ~ m . Biopkys. A ~ .
67 { t963} 566-575
I N H I B I T I O N OF NITRATE AGSIMILATION IN CIILORELLA
~6 7
METHOD~
Growth of organism P u r e cultures of Chorella t,ulgaris (Pearsall's strain) wtwe gro~vn in a m e d i u m containing, per litre glass-distilled water, 0.2 g a n h y d r o u s MgSO~, 7.76g I-[H~P04. 2.32 g K I H P O , , x ml ~4 t r a c e - e l e m e n t solution ~ a n d xo mg Fe as F e - E D T A (see ref. 9). 28o mg nitrogen per litre was supplied a~ either {NH4)~SO4, KNO= or NH4NO~. T h e p H o f t h e m e d i u m , af t e r autoclaving, was 6.r. Cultures were n o r m a l l y gr ow n for 4 d a y s in wash-t×~ttles at z5 ° with a light i n t e n s i t y o f 600 ft-candles at t h e surface o f the cultures. T he cultures were v i g , r o u s l y a e r a t e d with air containing 0. 5 o~ (v/v) e~arbon dioxide xu. When the cells were growing e x p o n e n t i a l l y the g e n e r a t i o n time was 8 h. C a r b o h y d r a t e - s t a r v e d ceils were pr epar e d by aerating cultures fi,r 18 h in darknes~ b ef o r e harvesting. N i t r o g e n - s t a r v e d cultures were p r e p a r e d b y remo~'ing the cells b y c e n t r i f u g a t i o n at 400 × g, wa_~hing and resusvending t h e m in fnll g r o w t h m e d i u m w i t h o u t a nitr~gen source. T h e c ul tures were t h e n illuminated and a e r a t e d with air c o n t a i n i n g o . 5 % (v/v) c a r b o n dioxide for t6 h before being har,:ested for e x p e r i m e n t a t i o n to. B ef o r e the e x p e r i m e n t s , the celLs were h a t e r , ted by cent ri fugat i on :tt 4oo x g, washed and s u s p e n d e d in nitrogen-free g r o w t h medium. T h e cell d e n s i t y was adj u s t e d turbidometric~ally t o 3- 8 mgl'ml d e p e n d i n g on the e x p e r i m e n t .
Gas exchange This was m e a s u r e d by c o n v e n t i o n a l ~,Varburg m a n o m e t r y at 25 °. For light exp e r i m e n t s , th e flasks were i i l u m i n a t e d with tluor~-~cent tubes giving a light inteltsity in t h e fla~l~s o f a b o u t 800 ft-candles.
Ammonium-N This was d e t e r m i n e d colorimetrically b y a Ne.~ler m e t h o d tt after separat i on f r o m th e cells a n d m e d i u m b y t h e m e t h o d o f CO,WAy ~z.
N i tral~ N This was d e t e r m i n e d in samples of t he m e d i u m a f t e r r e m o v a l of the cells b y c e n t r i f u g a t i o n . A phenol-disaflphonie acid colorimetric m e t h o d was used 13. T he light a b s o r p t i o n o f the eolourcd c o m p o u n d was read at 44o m # a n d c o m p a r e d with t h a t o f freshly p r e p a r e d s t a nda r ds .
Nitrite-N Th is was d e t e r m i n e d colorimetrically with Griess-I!osway reagents an on samples o f the m e d i u m a f t e r r em ova l of the cells. T h e colour was read a t 53 ° m/~, 20 rain after t h e a d d i t i o n o f the reagents.
PrL'pas'ation of ¢ell-frt~ extracts Cells were harve_~ted, w a s h e d a n d r e suspended in ice-cold o.z M Tris buffer ( p H 7.4)- T h e cell d e n s i t y was 6--re mg/ml. T h e cell suspension was st ored at -- x4 ° for 2 . 4 - 2 8 il- Af ter thawing, t h e cell suspension was passed t h r o u g h a F r e n c h p r e s s ~ to B~&Z~.
Bio~/Jys. A~:/~. 67 (xg63) 56b-575
5(~8
P.J.
S Y R E T T , I. M O R R I S
s m a s h t h e cells. D u r i n g this process t h e t e m p e r a t u r e rose to z7-zo ~ b u t t h e s u s p e n sion l e a v i n g t h e press w a s i m m e d i a t e l y i m m e r s e d in ice. T h e s u s p e n s i o n w a s cent r i f u g e d at x3 tx~, × g for zo rain a t z ~. T h e clear, green, s u p e r n a t a n t w a s d e c a n t e d , s t o r e d a t - - : 4 ° a n d u s e d for m e a s u r e m e n t s o f e n z y m e a c t i v i t y .
Assay of ,fftrate reductase activity N i t r a t e r e d u c t a ~ a c t i v i t y wa.g m e a s u r e d b y e s t i m a t i n g t h e n i t r i t e f o r m e d a f t e r i n c u b a t i o n o~-the cell-free e x t r a c t w i t h K N O ~ a n d D P N H . Full details are g i v e n in a later p a p e r L
Measurement of pyridine m~clcotid~ oxidation by flz~orimrtrv l ~ t h n i t r a t e reducta_~e a n d g i u t a m i c d e h y d r o g e n a s e a c t i v i t i ~ o f cell-free e x t r a c t s were d e t e r m i n e d b y following t h e o x i d a t i o n o f r e d u c e d p y H d i n e n u c h ; o t i d e fluorimetrieallv. T h e r e a g e n t s w e r e c o n t a i n e d in a b o u t x ml in a q u a r t z c u v e t t e in a ' l . ~ c a r t e ' f l u o r i m e t e r . T h e p r i m a r y filter w a s L F / 2 a n d t h e s e c o n d a t T L F / 3 / z . D e t a i l s f~f t h e a s s a y m i x t u r e s are g i v e n in t h e l e g e n d t o Fig. 6.
7"]:e protein conteJ,t T h e protc:itt ¢:~ntent o f cell-free e x t r a c t s w a s d e t e r m b , e d b y the meth¢~l, o f L o w a v et al. xs u.~ing b o v i n e - a l b u m i n s t a n d a r d s . RES[~LTS
I. ?'he preferential assimilatio~ of ammonium Fig. x s h o w s tile d i s a p p e a r a n c e o f a m m o n i u m n i t r o g e n a n d n i t r a t e n i t r o g e n f r o m (:ulture_~ o f C. vldgaris g r o w i n g w i t h a m m o n i u m n i t r a t e ,L~ n i t r o g e n source. 150
-
o%
,oo \
\,
~o~
, o
1 ~ ) o f.c.) 0~. <3
o F i g . ~. T h e a . s s i m i l a t i o n o f a m m o n i u m
1 Time ( h }
and
nitrate
Ioo
&.
~
~oo
b y e u l t u r e ~ o f C. vtd.qaria g r o w i n g
at 3oo or
~mo ft-candles light inten~/ty wi~h aiumonium nitrate as nitrogen .source. Note tlmt nitrate i:, not a-,~qimilated until almost ~.1 the ammonium has disappeared. Temperature 25 ~. Cultures acrata, d with 0 . 5 % (v/v) COI in air. 13Wehim. Bio~hys. Acta, 6 7 ( x ~ )
566-575
INHIBITIO.~ OF NITRztTE A.~%IM~LA'rlo.x" IN CHLORELLA
5[JC)
C u l t u r e ~ w e r e g r o w n a t t w o d i f f e r e n t light inten~i~ie~ G r o w t h is f,3_ster a t 6 o o i'tcan~Hes t h a n at 3on ft-candle.~ a n d a m m o , 6 u m nitrotzetx d i s a p p e a r s m n r e r a p i d l y . I n n e i t h e r culturt~ i~ ... :"~,,~-N * hue.; *,,,,e,, • n i t r a t e "'*:'"" ,,,,d,.ed u n t i l ,t . . . . . . . .,............ . . . . ~L~:dmflated. ~. T h e i n h i b i i i o n o[ n i t r a t e a s s i ~ g i l a t i o ~ by the z, d d i t z , n ~¢{ a m , t o n i t : m - N
Fig. 2 a s h o w s t h e r e s u l t o f a d d i n g a s m a l l q u a n t i t y o f a m m ( m i u m - N t~ a .~usp e n s i o n wifich is a s s i m i l a t i n g nitrate, in d.',rkness w i t h g l u c o s e a_s c;trb~n +our~.,:. N i t r a t e a~ssimilation t'~'a~es as s o o n as a r n m - n i u m - N is a d d e d . The: amm~niun~-.N" is r a p i d l y a.%sirnilated. A~ s~um as it h a s :~t! g~me. n i t - a t e a~
.
Nr,4~to 2
o
"g7
-a-~...'2
\.,,
a
,.
F i g . 2. T h e e f f e c t o f 8 . m r . l ~ n i u m additior~ o n n i t r a t e a ~ i m i l a t t , m b y [a) n i t r a t e - g r o w n cell~ in d a r k in p r e s e n c e o f t %~ {w/v) g l u c o s e , lb} nitratt~-gro~vn ccli~ iu l i g h t i n p r e s e n c e o f 3 % ~ . : v ) caa'tmn d i o x i d e i n air, (c) n i t r o g ( ~ n - s t a r v c t l cell~ lit d a r k tn pr~.~;ence o f t % (w/'~-] glu~-o.~. ~----O, n i t a ' a t e - N in c u l t u r e t whicix doe~ n o t retzeive a m m o n i u m ; A - - - ~ k . n i t r a t e - N i n c u l t u r e 2 w h i c h r e c e i v e s a m m o n i u m a t t i m e i t t d i e a t e d ; ~IV--~1F. a m m o n a u m - N irt c u l t u r e z, Cell den.~ity. 5.o m g d r y w t , l m l . L i g h t intera~ity. Boo f t - c a n d l e s . T ~ e f i g u r e s repr~.-~ent / s t n i t r a t t ; o r a m m o n i u m - . ~ p e r mi f o r ~..x|=ts, a a n t i b, anti kst~ N t>¢r 0. 5 lt:tl fi)r E x p t . c. T e m p e r a t u r e , zS ~.
U R H ^ S tt r e p o r t e d t h a t n i t r o g e n - s t a r v e d cells o f CkloreUa a n d Scetiedestlaus assimilated ammonium and nitrate simultaneously. However, under the conditions o f o u r e x p e r i m e n t s , a m m o n i u m i n h i b i t s n i t r a t e a:~.~imilatio'h b y n i t r o g e n - s t a v t ' e d cells j u s t as it d o e s t h a t o f n o r m a l cells (Fig. 2c) 3. T ~ e effect o f n i t r i t e o n n i t r a t e a s s i m i l a t i v ~ ,
Fig. 3 a s h o w s t h e effect o f t h e a d d i t i o n o f n i t r i t e ~n n i t r a t e ~ s i ~ i l a t i o n . It c a n b e s e e n t h a t its effect is s i m i l a r t o t h a t o f a m m o n i u m a d d i t i o n , i.e. it pr~duc~'s a c o m p l e t e i n h i b i t i o n o f n i t r a t e ,xssimilation w h i c h p e r s i s t s u n t i l all t h e n i t r i t e h a s been assimilated. 4- T h e effext o f a m , v a n i u m
on nitrite assimilatio;,
T h e a d d i t i o n o f a m m o n i u m - N also i n h i b i t s n i t r i t e a s s i m i l a t i o n b u t t h e i n h i b i t i o n is n o t c o m p l e t e (Fig. 3b). I n t h r e e experiment.~ o f this t y p e t h e p e r c e n t a g e i n h i b i t i o n l:lio,'him. Biophy,~. ,'[cta, 67 (tq£)3) 56G-575
~7 0
P. J . SYRETT, I. MORRIS
o f n ' t r i t e assirnil;ttion w a s 75, 84 a n d 85 ~/o g i v i n g a m e a n v a l u e o f a b o u t 8o ~/o inhibition.
5. T!te dependence of inhibition u~on the assimilation of ammonium I n t h e experi~;~ents d e s c r i b e d a b o v e , t h e i n h i b i t i o n o f n i t r a t e a s s i m i l a t i o n b y a m m o n i u m c o u l d be d u e e i t h e r t o t h e p r e s e n c e o f a i n m o a i u m or to t h e a m m o n i u m as~hnilation w h i c h ~-'cur~ i m m e d i a t e l y a f t e r a m m o n i u m is a d d e d . T h e results described in this s e c t i o n s~mggest t h a t t h e ; n h i b i t i o n is n o t d u e t¢+ t h e p r e s e n c e o f a m m o n i u m per se b u t t o a m m o n i u m a~similation. 60
•
4~L
h~
,.¢ ~
A
J.
o
"+..m~.-- , ~
I 4t~eoy to2
";
~o " , b o " ~so " ~bo " "(';me (,'n~)
D.
t.+
Fig. 3- A. T h e e f f e c t o f n i t r i t e o n n i t r a t e a ~ s i m i ] a t i o n bst n i t , r a t e - g r o w n eel]g+ T h e c u l t u r ~
~n-
tained b 5 mg dry wt. cells per m: and a % Iw/v) glucose, i ~ 4 1 , nitrate-N in culture x which received no nitrite; & - - A , nitxate-N in culture • which reccive~i ]o/ag nib-ite-N per mi at time indicated; V ' - - ~ . nittl.te-N in culture z. B. The effect of ammonium on nttrtte a.q~simi~tion by nitrate-grown cells. The cultures contained 3 mg dry wt. cells per ml and t % [w/v) glucose. Culture z received zz ttg xmmonium-N per ml at the time indicated. E x p e r i m e n t s ~,'ith cell-free e x t r a c t s containi,~g n i t r a t e r e d u c t a s e (section 6) sh~>w t h a t a m m n n i u m s u l p h a t e in a final c o n c e n t r a t i o n o f 3" xo - t M h a s no effect a t all o n t h e a c t i v i t y o f n i t r a t e r e d u c t a x e . T h i s a m m o n i u m c o n c e n t r a t i o n L~ t h i r t y t i m e s t h a t w h i c h i n h i b i t s n i t r a t e a s s i m i l a t i o n b y i n t a c t cells a n d t h e a b s e n c e o f i n h i b i t i o n wi~h cell-free e x t r a c t s * s u g g e s t s t h a t a m m o n i u m a l o n e does n o t i n h i b i t n i t r a t e reduction. This s u g g e s t i o n was s u p p o r t e d b y e x p e r i m e n t s c a r r i e d o u t w i t h i n t a c t cells w h i c h h a d been p r e v i o u s l y :~tarved o f c a r b o h y d r a t e . BOSGEag t¢ founci t h a t c a r b o h y d r a t e - s t a r v e d cells of S c e n e d e s m u s w o u l d r e d u c e n i t r a t e t o a m m o n i u m q u a n t i t a tiwely in light in ttle a b s e n c e o f c a r b o n dioxide. S u c h cells p r e s u m a b l y l a c k c a r b o n r e s e r v e s w i t h w h i c h t o m a k e c a r b o n s k e l e t o n s for a m m o n i u m a s s i m i l a t i o n . Fig. 4A(i) s h o w s t h a t in light, in t h e a b s e n c e o f c a r b o n d i o x i d e , c a x b o h y d r a t e starw~d ceils o f C. vulgaris r e d u c e n i t r a t e t o a m m o n i u m w h i c h a c c u m u l a t e s . T h e a d d i t i o n o f o . o o 5 M a m m o n i u m h a s n o effect o n this r e d u c t i o m H o w e v e r , w h e n e i t h e r c a r b o n d i o x i d e o r g l u c o s e is p r e s e n t t o p r o v i d e a c a r b o n s o u r c e (Fig. 4All a n d iii) n o a m m o n i u m a c c u m u l a t e s since it is a s s i m i l a t e d a n d , u n d e r t h e s e c o n d i t i o n s , t h e a d d i t i o n o f a m m o n i u m i n h i b i t s n i t r a t e d i s a p p e a r a n c e . I n fact, w h e n t h e c a r b o n so,arce is 5 % (v/v) c a r b o n d i o x i d e in t h e g a s p h a s e , a , , , , o n i u ~ addition i-hibits [ ? i o c h l m . B ~ l ~ h y s . . 4 ¢ 1 , ~ + 57 l t 9 6 3 )
566-575
INHIBITION
O F NITRATE: A S S I M I L A T I O N IN C H L O R E L L A
57z
n i t r a t e r e d u c t i o n c o m p l e t e l y (Fig. 4A]ii). T h e effect ,,f t h e p r e s e n c e o f g l u c o s e i~ o f i n t e r e s t . W h e n g l u c , ~ i~ a d d e d , in t h e a b s e n c e o f c a r b o n d i o x i d e , i.e. t h e W a r b u r g fla~ks i n w h ~ h th'- e x p e r i m e n t s a r e c a r r i e d o u t c o n t a i n c a u s t i c p o t a s h , t h e i n h i b i t i o n o f c i t r a t e r e d u c t i o n b y a m m o n i u m is p r e s e n t b u t i n c o m p l e t e (Figs. 4Aii a n d 4Bii). I f c a u s t i c p o t a s h is o m i t t e d f r o m t h e W a r b u r g flasks tb~.- i n h i b i t i o n in t h e p r e s e n c e o f g l u c o s e is m u c h m o r ~ p r o n o u n c e d (Fig. 4Bill). Thi~ m a y Ix: a r e f l e c t i o n ~,f t h e f a c t t h a t a m m o n i u m a s s i m i l a t i o n t o a m i n o a c i d s is a c c o m p a n i e d b y cot~:dderable c a r b o n d i o x i d e fixation~e; it m i g h t , h o w e v e r , i n d i c a t e a mor~" d i r e c t r e l a t i o n s h i p b e t w e e n a m m o n i u m , c a r b e m d i o x i d ~ a n d t h e ,.'nhibit¢,r ~.:f n i t r a t e r e d u c t i o n . Fig. 4 B also
f°l._,l
,Ira
"
!]I[[ i,[1 I, rfit~rte
F~g• 4- The effect of ammonium on nitrate reduc~on and as~imil&tion by ca.cbohydra.tv-atarved cel/~ in light. The plus and minus sign~ below the c o l , m n denote the presence or absence of added a m m o n i u m - N (7° ~ / m l ) . The e.xperiments were carried out ia Waxburg flasklg, eaclt containing 2.o ml cell suspension {3 mg dry .yr. cells/ml)~ The iMtia.I concentration of nitrlte-N was 7o Fg/ml. The flask contenta were analysed 2 h after the ~tddJtion ot nitrate land/or ammonium)+ L i g h t iuten.~ity, 8oo t~-candle,. Temperature. 25 °. --COn indicates tkat the ~,Varburg flasks contained caustic potaoh in the centre wells. )n experiment 4 B Iiii). this was omitted; +CO~ indicates manometer~ flushed ~ t h 5% COn in a.ir. When glucc~se was present, its final concentration was ! % (w/v). Solid column° n2trate-N (lisappeating from su.~l~--nsion in 2 h ; c~oss-hatched co|u,nn. ammonium-N appearing in suspension in 2 b; open column in Iowex figure, arnmonium-N disgppem-ing from suspension in ~ h in absence c~f nitrate. The percentage inhibition of nitrate rcxlucUon by presence of ummonium is shown. s h o w s t h e a m o u n t o f a m m o n i u m a s s i m i l a t i o n caJTied o u t b y t h e cells i n t h e a b s e n c e o f n i t r a t e . I t is g r e a t e s t w h e n 5~/o CO 2 is t h e c a r b o n .source (i) a n d l e a s t w i t h g l u c o s e i n t h e a b s e n c e o f c a r b o n d i o x i d e (iii). I t is c l e a r t h a t t h e i n h i b i t o r y effect o f a m m o n i u m o n n i t r a t e r e d u c t i o n is mo:~t p r o n o u n c e d u n d e r t h o s e c o n d i t i o n s w h i c h m o s t favour ammonium assimilation. T h e e x p e r i m e n t s i l l u s t r a t e d in Fig. 4 w e r e c a r r i e d o u t i n W a x b t t r g m a n o m e t e r s , t h e flask c o n t e n t s being a n ~ y s e d at the e n d of the e x p e r i m e n t . T h e g a s e x c h a n g e w h i c h o c c u r s d u r i n g a n e x p e r i r a e n t o f t h i s tyF~, L~ ~itown in Fig. 5- I n light, n i t r a t e r e d u c t i o n is a c c o m p a n i e d b y a c o n s i d e r a b l e e v o l u t i o n o f o x y g e n t ° , ~. T h u s g a s e x c h a n g e is a s e n s i t i v e i n d i c a t o r o f t h e r a t e o f n i t r a t e r e d u c t i o n . I t c a n b e s e e n f r o m
572
p,
J.
SYREI~,
1. M O R R I S
Fig. 5 a, t h a t , in t h e a b s e n c e o f g l u c o s e a n d c a r b o n d i o x i d e , t h e a d d i t i o n o f N H ~ + h a s no effect on g a s exehar~ge in t h e p r e s e n c e o f n i t r a t e . T h i s r e s u l t is in s h a r p c o n t r a s t to t h a t s h o w n in Fig. 5e w h e r e t h e a d d i t i o n o f N H4 ÷ to ceils ~ s i m i l a t i n g n i t r a t e in t h e p r e s e n c e o f 5 % ( v / v ) c a r b o n d i o x i d e , l o w e r s t h e r a t e o f g a s o u t p u t t o the l e v e l o f t h a t o f cells w i t h N H~* a l o n e ; t h i s c o r r e s p o n d s t o t h e a l m o s t c o m p l e t e i n h i b i t i o n of n i t r a t e r e d u c t i o n s h o w n in Figs. 4 A (iii) a n d 4 B (i). I n t h e p r e s e n c e o f g l u c o s e and absence of carbon dioxide, the gas-exchange measurements indicate an incomp l e t e i n h i b i t i o n o f n i t r a t e r e d u c t i o n b y N H ~ ~ (Fig. 5b) corre.~ponding to t h a t s h o w n in Figs. 4A (it) a n d ~ B (it). -CO~
i
<> i
'
o
~1
@l,~cor~
-
-, Ga,a¢o~
-" ~
lb) p ,
-COI
[
l / / -
=co o
, oo
*COz •
I~)#o
-Otu.¢ol,~ / I
/
ji.i ~o o
loo
~o
T I m t (ral, n}
1"i,,'*. 5. t-.as exchange accompanying nitrate mim'.latiorl by ca.rbohydrate-starvcd cells in light. Lighl intensity, ,~oo ft-tu~lltll~.~. Temperature, 25 °. Nitrate-gro~r~ cells star,.c,l of earlmhydrste t o t i 6 h. Cell .~us!ril~iort contained 3.o l~g d r y ~ ' t . c e l l s p e r m L (a} a n d (b), t l a ~ k s contained 2.0 m l
cell ~u~pe.~ion, Caustic potash in cent.re well, in (b} t ~/o (wlv) glcccmc w ~
addo;l. 8 4 pig N O i - N
or ~)61zg N t l i ' - N added at times indic~t~xl. (c), Flasks corttaiu-.d i.o ml cell suspension. Gas phase. 5~h (v/v) CO= in air. lo 4 pg NO=-N or 57lag NI4i*-N added at times it=die, ted+ T h e s e e x p e r i m e n t s , t h e n , s u g g e s t t h a t it is a m m o n i u m a k x i m i l a t i o n r a t h e r t h a n a m m o n i u m i t s e l f w h i c h i n h i b i t s n i t r a t e a s s i m i l a t i o n . MCmTON e c a m e to a s i m i l a r c o n c l u s i o n for diflb.rent r e a s o n s .
7"he. pyri$ine nucleotide specifcity of nitrate reductas¢ and glutamic dehydrogenase in C. v,dgaris 6.
] h e p r e s e n c e of b o t h n i t r a t e reducta.~e e n d g l u t a m i c d e h y d r o g e l , a s e w a s dem o n s t r a t e d in ceil-free e x t r a c t s o f cell~ g r o w n w i t h K N O s ,~s n i t r o g e n s o u r c e (Fig. 6a). N i t r a t e reducta.~e requirext I ) P N H as e l e c t r o n d o n o r a n d d o e s n o t utilize T P N H . T h e r e v e r s e is t r u e for g l u t a m i c d e h y d r o g e n a s e (Fig. 6b).
7. Nitrate assimilation by cells grown u ~ h N H I N O , as nilrogen sot~rc¢ Fig. 7 shov,.~ t h e t i m e c o u r s e o f n i t r a t e a . ~ i m i l a t i o n o f ceils ~ r o w n w i t h N H i N O s as n i t r o g e n s o u r c e in c o n t r a s t t o t h o ~ e g r o w n ~ q t h K N O = . W h e r e a s t h e celts g r o w n on K N O = a s s i m i l a t e n i t r a t e i m m e d i a t e l y a f t e r it is a d d e d , t h e cells g r o w n on N H i N O , s h o w a l o n g I ~ r l o d o f 3 h b e f o r e n i t r a t e a s s i m i l a t i o n s t a r t s . T h i s l o n g ~-.riod is c o n siderab!:,- ~ h o r t e n e d i f t h e cells ~re a e r a t e d for 3 h in a N - f r e e m e d i u m b e f o r e n i t r a t e i~. a d d e d . T h a t t h e d i f f e r e n c e ]n b e h a v k m r o f t h e s e c u l t u r e ~ is n o t d u e t o d i f f e r e n c e s Biochim. Btophys..4~la, 6 7 ( x 9 6 ] } 36¢~-375
INHIBITION
.
12 "~j 11
573
O F N I T R A T E ASSI511LATION IN C H L O R E L L A
v"---r
~
v--v
"-"--v-.-v~ v -v
~I¢
J
z
l
L
&
-1
0
~
a
(~
~.
~0
J[/ L
a.9
0
t .--4~_~L~
~'
4
--1
I
d-
8
TO
~2
~
t;rr~ ( ~ ; n ) F i g . 6. T h e p y r i d i n , : i ~ u c l e o t i d e ~pe~:ificity o f ( a } n i t r ~ t e r e , l u c t ~ e a n d (b) g l u t a m J e d ~ . h y d r n g e n a - ~ e ; t h e o x i d a t i o n o f r e d u c e d p T r ~ d i n e : m c l e o t i d e w a s ::lea~t~re~i fluo::taaeLri~zaily~ T h e ..-~s~ty m i x t u r e s c o n t o r t e d o . ~ m l c e i l - f r e e e x t r a c t c o n t z i n i n g o. t5 m g p r o t e i n , o . z hal o.a .~i Tri.~ b u f f e r !pt[ 7-4] ; o . t m l a 0 o z M ~ a p p r n x . ) D P N H o r T P N H . A str-arx~ o f ox~'~,'w-rree, n i t r o g e n w a s b u b b l e d t l : r o u g h t h e m i x t u r e b e t w e e n r e a d i n g s ; t h i s r e d u c e d t h e ..tx(log(rncu~ t ~ x i d a t i o n o f r e d u c e d p y r i d i n e n u e . l e o t t d e . I n (a) 2 o # t m o l v s KI-IO~ w e r e a d d e d a t t h e t i m e indicatt:cl ; i n {b) -~o l t m o l e ~ ~ - o x o ~ h a t a r a t e w e r e i n c l u d e d i n t h e r e a c t i o n mixttlrt~ zttzd 2 0 / ~ m o l e ~ ( _ ~ l l l ) ~ t O ~ a d d c t l ~ t t h e t i m e inctic~tted. Temperature, 3 t ~. I n f l u o r i m e t e r u n i t s approx. 40 ml~mole~ PNH.
F c.
103
=' go * ' N
v "e
~o-
0
50
1QD
150
200
250
T ; r n e O'~in)
F i g . 7" N i t r a t e a s s i ~ i ; a t i o n b y ¢~tls g r o w n witl-, a m m o n i u m rut,at,." a s N - s o u r c e . C u l t u r e z c 0 n tain_*~d 13 m l c e l l s u s p e n s i o n g-rowan i n p o t a s s i u m n i t r a t e ; t h e celia a s s i m i l a t e r ~ i t r a t e ~ks s o o n a.~ i t is a d d e d . C u l t u r c m z a n d 3 e a c h c o n t a i n 8 m l c e l l .~u.qr,,m~i<,n g r o w n i n a ~ m m n n i u m n i t r a t e b u t culture 3 was shaken for 3 h in -zitxogen-free medium helot c mtratc was added. Note that culture 2 o n l y as.Mmil~tt-~ t l ~ a d d e d nitr-.~te a ~ e r ~, l e g o f ~ b o u t 3 b : i n c u l t u r e 3 t h e l a g h ~ I x ' c a c o u $1derably reduee,t. All ftmsks cents/ned Chlorell~ suspension, 7 mg dry wt./ml, with glucose added t o g i v e a f i n a l c o n e e n t r a ~ z o n o f g °/6 ( w ] v ) . T -~'- f l a s k s w e r e s h a k e n i n d a r k n e s s a t z5 °. A l l r e c e i v e d x o o / 4 w o l e s K N O ~ ~ t z e r o t i m e . S a m p l e s o f e a c h tyt>c o f cell w e r e p a s s e d t h r o u g h a F r e n c ~ p r e s s and nitrate reductase activity, assayed. The enzyme levels arc shown below. Note that the n i t r a t e r e d u e t a . s e l e v e l o f t h e a n ~ m o n i u r n n i t r a t e - g r o w n c e l l s is u n c h a n g e d b y 3 h a e r a t i o n i n nitrogen- fr~ medium. Nava~t vd'_~e'_e -..-'ioityo, ~U-lv~
.~,a,~
l,~r
KNOm-grown cells , " T H ~ N O s - g T o v : n cell~ N'HeNOi-gaow-n ceils after 3 h in N-free medium Bioek~m.
loms~l ~ mare m K Ov,~'~s
z5,6 4.0 4.8 /~io~kys. Aaa.
67 t,z g ~ 3 ) 5 6 ~ - - 5 7 5
574
e . . [ . SYRET']', !. MORRIS
in t h e a m o u n t o f n i t r a t e r e d u c t a s e p r e s e n t is s h o w n b y t h e d a t a i n c l u d e d in Fig. 7It is clear t h a t t h e cells g r o w n o n N H a N O s posses n i t r a t e r e d u c t a s e ( a l t h o u g h less t h a n t h a t in t h e K N O s cells) a n d t h a t t h e i r n i t r a t e r e d u c t a s c c o n t e n t does n o t a l t e r a p p : e c i a b l y d u r i n g t h e 3-h p e r i o d o f n i t r o g e n s t a r v a t i o n . N e v e r t h e l e s s t h e p~riod o f n i t r o g e n s t a r v a t i o n c o n s i d e r a b l y increases t h e i r a b i l i t y to a_~similate n i t r a t e . DISCUSSION N i t r a t e a.~imilation b y C. vtdgaris is c o m p l e t e l y i n h i b i t e d as scxm a.~ a sm al l q u a n t i t y o f a m m o n i u m is a d d e d a n d t h e i n h i b i t i o n is r e l i e v e d as soon as all t h e a m m o n i u m has b een a s s i m i l a t e d . Since a m m o n i u m o n l y paxtially i n h i b i t s t h e a s s i m i l a t i o n o f n i t r i t e a n d since n o a p p r e c i a b l e q u a n t i t y o f n i t r i t e e v e r a c c u m u l a t e s in o u r c u l t u r e s , a m m o n i u m m u s t i n h i b i t t h e first s t e p o f n i t r a t e assi m i l at i on, n a m e l y t h e r e d u c t i o n i~f n i t r a t e t o n itr i t e . T h e a d d i t i o n o f n i t r i t e also i n h i b i t s n i t r a t e a~sirnilation; t h i s effect m a y well be a r e s ul t o f t h e c o n v e r s i o n o f n i t r i t e t o a m m o n i u m so t h a t b o t h act in a c o m m o n mariner. I n m a n y r e s p e c t s o u r re~ults r e s e m b l e t h o . ~ o f MORTON A.WD MAt:MII.LA.~4 w i t h fungi. "I hey. t oo, f o u n d t h a t a m m o n i u m c o m p l e t e l y i n h i b i t e d n i t r a t e aggimilation a n d p a r t i a l l y i n h i b i t e d n i t r i t e assimilation, b u t in t h e i r w o r k , n i t r i t e a p p a r e n t l y h a d little effect on t h e r e d u c t i o n o f n i t r a t e . A m m o n i u m a ppe a xs to i n h i b i t n i t r a t e a s s i m i l a t i o n o n l y w h e n it is itself assim i l a t e d . Thu.~ a m m o n i u m has no effect on n i t r a t e ~educLion b y c a r b o h y d r a t e s t a r v e d cells unless a c a r b o n solxr¢~ is a d d e d w h i c h enable~ t h e cells t o a s s i m i l a t e a m m o n i u n L MORTOXs, too, s u g g e s t e d t h a t a x n m o n i u m m u x t be a s s i m i l a t e d b e f o r e it can inhibit n i t r ~ t c r e d u c t i o n . T h e r e ar c at least t w o w a y s in w h i c h a m m o n i u m a s s i m i l a t i o n m i g h t i n h i b i t n i t r a t e r e d u c t i o n . F i r s t l y , t h e t w o pr oc e s s e s m i g h t c o m p e t e for t h e a v a i l a b l e s u p p l y o f r e d u c e d p y r i d i n e n u c l e o t i d e in t h e cells. S e c o n d l y , a m m o n i u m m i g h t be assim i l a t e d t o an o r g a n i c n i t r o g e n o u s c o m p o u n d w h i c h i n h i b i t s n i t r a t e r e d u c t i o n in s o m e way . T h e ~ c o n d o f these, p o ~ i b i l i t i e s a p p e a r s t h e m o r e likely. I t is t r u e t h a t t h e p r i m a r y s tep s in b o t h a m m o n i u m a n d n i t r a t e a s s i m i l a t i o n r e q u i r e r e d u c e d p y r i d m c n u c l e o t i d e , in t h e firsL for t h e r e d u c t i v e a m i n a t i o n o f a - o x o g l u t a r a t e t o g l u t a m i c acid nard, in t h e second, for t h e r e d u c t i o n o f n i t r a t e t o ni t ri t e. B u t in C. v~dg~,is, t h e p y r i d i n e n u c l e o t i d e specificity of t h e e n z y m e s c a t a l y s i n g t h e s e t w o r e a c t i o n s a p p e a r s t o differ. Ghitaanic d e h y d r o g e n a t e r e q u i r e s T P N H while n i t r a t e r e d u c t a s ~ r e q u i r e s DP,N'H. Thu:~ a d i r e c t c o m p e t i t i o n a p p e a r s u n l i k e l y a l t h o u g h it c a n n o t be c o m p l e t e l y r a l e d out. M or eover , t h e e x p e r i m e n t w i t h cells g r o w n on a m m o n i u m n i t r a t e suggests t h a t s o m e t h i n g i n h i b i t i n g n i t r a t e a s s i m i l a t i o n a c c u m u t a t e s m ceils w hi ch axe a s s imilatin g a m m o n i u m . T h e s e cells, a t t h o u g h Possessing n i t r a t e r e d u c t a s e , do n o t begin t o r e d u c e n i t r a t e until t h e y h a v e b e e n a e r a t e d for 3 h in a n a m m o n i u m free m e d i u m . It looks as i f t h e i n h i b i t o r is r e m o v e d d u r i n g t hi s period. P r e s u m a b l y , in t h e e x p e r i m e n t s in w h i c h r a t h e r a small q u a n t i t y o f a m m o n i u m w a s a d d e d , t h e q u a n t i t y o f i n h i b i t o r f o r m e d w o u l d be. small a n d w o u l d d i s a p p e a r soon a f t e r all t h e a m m o n i u m h a d been as s i m i l a t e d. I f such an i n h i b i t o r is f o r m e d d u r i n g a m m o n i u m a s s i m i l a t i o n it m i g h t i n h i b i t n i t r a t e r e d u c t i o n in one o f t w o ways. F i r s t l y it m i g h t , i~ s o m e w a y , p r e v e n t t h e e n t r y o f n i t r a t e i n t o t h e cell o r i n t e r f e r e w i t h its access t o t h e e n z y m e , n i t r a t e red u c t a s e . S e c o n d l y it m i g h t act b y i n h i b i t i n g n i t r a t e r e d u c t a s e itself. W e p r e f e r t!:e Bio¢i~ivn. Biopl, y~. Acta, 67 (If763) 566-575
;.NI-ilBI'I+ION O F N I ' I R A T E ASS[MII+ATION I N C H L O R E L L A
5~r3
s e c o n d o f t h e s e po.~sihilitie5 a n d h a v e .~>mo e v i d e n c e to .~upport itL W e h a v e failed t o find e v i d e n c e o f a n y effect o f ananmniurv, a s s i m i l a t i o n o n .*.lie e n t r y o f n i t r a t e #nto t h e cells b u t since the q u a n t i t y of n i t r a t e in Ch!orella is a l w a y s e x t r e m e l y low t h e r e s u l t s are i n c o n c l u s i v e W e h a v e a l ~ ,+xarnlned t h e effect ~f a v a r i e t y o f n i t r o g e n o u s c o m p o u n d s on t h e n i t r a t e reddcta_se a c t i v i t y ~f cell-free e x t r a c t s ; tile r e s u l t s a r e p r e s e n t e d in a l a t e r p a p e r L AC K N ()'~,'l,-[. 1- 1)1 ; |- .~i l," N'.T"
W e s h o u l d like t(, t h a n k the D e p a r t m e n t a g r a n t to o n e o f us {I.M.).
of Scientifit: a n d I n d u s t r i a l Rtrsearch for
REFERENCES t C . A. l . u D w i , ~ . A m + J+ / / o t a n y . ~5 ( t 9 3 8 ) 448. s l ~ + Pan-arT ^ N D J . Fore(,, . . | m J+ 130tdl+ly, 27 [ t o . t o ) /35+ * J . F'. S C I ' I U L E R . V+ I~1+ I)ILI.I-.K ~'+Xt~+ | l J . KEILSLF-..~, I>I+¢P'~t l*i''+"~lOl., Z8 [ [ : ~ $ 3 ) g~J~+ 4 A . G. M o l t ' t o N ^NI21 A+ ~ [ A C M I I . L . t . N . J . E x p P I 13or.. 5 (1+~5-1) .'3-'C. C. D t ~ t , w t c x ~ , J+ B i o l . C k e m . , t & ) { 1 9 S t ) I 0 7 " A. G. M o m T o m , J+ E , , p t l . B o t . 7 ( ~ 9 5 ~) 97+ = I+ M o R z t s ^ s I > P . J+ %vm~.'rr. i n p r e p a r a t i , ~ n . ~1 D- I. AllNON, ,,is'a+ J+ B o l a n y . z 5 (193~) 322. t L . JAcoz*~lo.~. Plff~'! .z'h.vsi,,l. 26 {I¢.}31 ) .$11. t." V. J- S'¢RImTZ, . , I n n . B o t a n y f'l.ondo~,J, I 7 (|0.53) r *l O . [?OLIN AND ttV+ I)ENiS+ J . o+ol. (.;/lelB.. 26 I t g t c , ) 4 7 3 . I I E . J . CO:i%;'AY, Aficro.dztJuMo~* .~nalys+, . ~ l d v:,':t.~'.c!.-:,, L o n d o n , 1947.
N o s t r a n d , t 9 4 9 . p+ 785-8o7+ t¢ H+ ~kV+ MILNER, .~. S+ L^V,°R~NCP+ ,'~,'wt, C. S. FK~.~CIt. Nc.i.,!r+ce. [ t l {195o) 0 3 3 . '+ ('). H . ~ClXVRy, +NT.J . I~O51"~BROUGH+ .~+ L+ F,~I+.R AND IL J R.~+~U,XL, J+ I++oL C + e m . . ' 9 3 (,<~fl) z+5+ LI (). [."RHAN, _[altrb. wi$$. Bolan+, 75 l. l o ~ z ) 1. tT I.. H. J. Bo~'c,r.as, Mcgedel+ Landbouu,hoge+ch,',ot D'aeening,+. 5 6 ( i 9 5 u ) I. z. F+ J . S r n E T T , P h y s i o L Plantavto~L 9 ',t95¢,1 z65 lb ( ) , "I.~PARI~IURG A-hID ~+ ~ ' I P G K I . E | N . l'fiochem. Z., i t o tt+-~o3 6q+~ t~ j . M Y ~ t a s , i n J . F m x . ~ c g ,x.~t> W . E . L o o ~ t s , Ph,+t,++ynthe++.+ +~ l~lant+, l o w + S -t~t¢ C,+H,+ge i-'re.,:s, A m e s , I o w a , zg.++j, p. 3 4 9
BsocAi.,. ~iophys+ . 4 a a , 67 ( 1 ~ 3 )
56¢~-575