- Email: [email protected]
Mechanism of nitrate reductase reversible inactivation by ammonia in Chlamydomonas
Plant Science Letters, 1 (1973) 31--37 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
MECHANISM OF NITRATE REDUCTASE REVERSIBLE INACTIVATION BY AMMONIA IN CHLAMYDOMONAS
M. LOSADA, J. H E R R E R A , J.M. M A L D O N A D O
and A. P A N E Q U E
Department of Biochemistry, Faculty of Sciences, CSIC, Universityof Sevilla(Spain) (ReceivedAugust 10th, 1972)
SUMMARY
Ammonia promotes in vivo the conversion of the active form of nitrate reductase into its inactive form by indirectly causing the reduction of the enzyme. Apparently ammonia acts as an uncoupler of noncyclic photophosphorylation, a process which in turn leads to a rise in the level of reducing power in the cell. The transformation is reversible, and, upon ammonia removal, the enzyme becomes again oxidized and active. Nitrate reductase inactivation by ammonia requires light and does not occur when the noncyclic electron flow is blocked or when the photosynthetically generated reducing power is being simultaneously oxidized. In the dark and in the absence of ammonia, the enzyme appears either active or inactive in response to the degree of aerobicity or anaerobicity of the culture.
INTRODUCTION
We have previously shown that the addition of ammonia to a suspension of Chlorella fusca or Chlamydomonas reinhardi cells growing in the light on nitrate determines the rapid inactivation of the second moiety (i.e., FNH2 -NO3 Rase) of the NADH--nitrate reductase complex, and that the subsequent removal of ammonia brings about an equally rapid reactivation of the inactive enzyme ~-4. Interconversion of the active and inactive forms of nitrate reductase could also be achieved in vitro by reducing and oxidizing the enzyme with its physiological or artificial substrates, using both crude extracts and partially purified preparations 4 -6. Vennesland and her associates have made studies of the nitrate reductase of Chlorella vulgaris and found that the enzyme in the cell-free extracts is largely in an inactive form, which can be extensively activated in the presence of added nitrate and phosphate buffer of low pH; addition of NADH in the absence of nitrate led to a loss df enzyme activity 7,s. More recently, the same group has reported that the activation of the inactive form requires an oxidizing agent and is inhibited 9 by CO. Vennesland and her coworkers have repeatedly called attention to the fact
31
that no trace of activation can be observed with extracts of Chlorella fusca under identical conditions 8,, o Rigano has studied the nitrate reductase of the acidophilic thermophilic unicellular alga Cyanidium caldarium and has shown that in vivo the active enzyme can be reversibly transformed into the inactive species b y ammonia ~ In vitro, the latent nitrate reductase could be reactivated by heating ~' , and by treatments with phosphate, urea and pCMB 12 The present paper describes further studies of our laboratory on the mechanism of the light-induced reversible inactivation of nitrate reductase by ammonia in Chlamydomonas cells. MATERIALS AND METHODS
Chlamydomonas reinhardi was grown in the light in a stream of 5% CO2 in air with 8 raM KNO3, as previously described 4. The experiments were performed with cells previously kept for 4 h in a fresh medium under the same conditions b u t with 4 mM nitrate 4. Harvesting o f the cells, preparation of cell-free extracts and estimation of enzyme activities were also as previously described 4 RESULTS AND DISCUSSION Nitrate can act, more or less directly, in the manner of a Hill reagent, as a terminal electron acceptor of the noncyclic electron-transport chain o f photosynthesis, b o t h in whole cells and in chloroplast systems. The photochemical reduction of nitrate to ammonia proceeds in two steps and is stoichiometrically coupled with the evolution of oxygen and the formation of ATP: nitrate is first reduced to nitrite b y the NADH--NO3 Rase complex, and nitrite is then reduced to ammonia b y ferredoxin--nitrite reductase. Ammonia can thus be considered as the end p r o d u c t of the p a t h w a y catalyzed b y the assimilatory nitrate-reducing system 3 Ammonia has the interesting property of acting in an u n k n o w n w a y as an effective uncoupler of noncyclic p h o t o phosphorylation 3,13. Another uncoupler of noncyclic photophosphorylation is arsenate, which acts as a substitute for phosphate leading to the formation of an unstable arsenyl comp o u n d 13. Both uncouplers have the c o m m o n feature of stimulating electron flow 3,13. The finding that these c o m p o u n d s behave similarly in promoting the reversible inactivation of nitrate reductase in vivo is therefore of great significance. Fig. 1 shows that, upon addition of either ammonia or arsenate to Chlamydomonas cells growing on nitrate, nitrate reductase proper (assayed as FNH2 --NO3 Ease or NADH--NO3 Rase) became rapidly inactivated, and that, upon removal o f the uncoupling agent, the inactive form of the enzyme changed back into its original active form. In contrast with nitrate reductase itself, the first m o i e t y of the NADH--nitrate reductase complex (i.e., NADH-diaphorase) was n o t at all affected b y the same treatments. In all the experi-
32
r-
Cl v
>~-
f * NH3 100'*. . . .
/ .
100
~
÷ As043.
.
. &
A • O
U
60
>.. ~
20.
200
0
;
i
o
TIME (hours)
i
Fig. 1. Reversible inactivation of nitrate reductase by ammonia (left) or arsenate (right) in Chlamydomonas cells. In each case, the first arrow indicates the time when ammonia (16 raM) or arsenate (30 raM) was added to cells growing on nitrate under the conditions described in Methods. The second arrow indicates the time when the cells were harvested and resuspended in the original nitrate medium. Enzyme activities were estimated in the corresponding cell-free extracts at the times indicated. 4-- -- --4, NADH--diaphorase; o -o, N A D H - - N O 3 R a s e ; * ' ,,,, F N H 2 - - N O s R a s e . ments described in this paper, conversion of the inactive form into the active one by oxidation with ferricyanide 3 -6,9 was established. In agreement with previous results 4 -6, Fig. 2 shows that inactivation of the active enzyme in crude extracts depended on its reduction by NADH and was proportional to the concentration of the nucleotide. NADPH was also effective in inducing this transformation. These data together with those presented in Tables I and II seem to support the interpretation that both ammonia and arsenate bring about inactivation of nitrate reductase by raising the level of reducing power in the cell as a result or their uncoupling action. Namely, no inactivation was achieved in uivo in the dark, or when vitamin K (which photooxidizes the reducing power formed ~3,~4 ) or DCMU (which inhibits the noncyclic electron flow ~3 ) were added. The CO2 requirement for inactivation under these conditions remains a puzzling phenomenon, particularly since it could be avoided when the accumulation of reducing power was ackieved by other means (see p. 35). Although the possibility exists t h a t a carbon derivative can potentiate the physiological inactivation rate of the reduced 33
E
O.
100
U
\
\
60"
" W t~ hi
0
_~
\
20-
o
o
hi
0
a()3
0.()6
0.()9 '1 013 [NADH] (mM)
0.6
!
0.9
Fig. 2. Effect of NADH on the inactivation of Chlamydomonas nitrate reductase in cellfree extracts. Enzyme activity was estimated after incubation for 3 h at 0° of an active crude extract from cells growing on nitrate, as described under Methods, with NADH at the concentration indicated.
TABLE I E F F E C T OF LIGHT, MENADIONE, DCMU AND CO2 ON THE IN VIVO INACTIVATION OF CHLAMYDOMONAS N I T R A T E R E D U C T A S E BY AMMONIA Cells growing on nitrate as described under Methods were subjected for 1 h to the treatments indicated. Enzyme activities were then estimated in the corresponding crude extracts. Ammonia, 16 raM; Menadione, 0.1 raM; DCMU, 3-(3',4'-Dichlorophenyl)-l,l-dimethylurea, 10 , M . CO2 was removed from the insufflation gas stream by bubbling through a saturated solution of barium hydroxide. Treatment
Relative specific activity (per cent,) NADH-NADH-FNH2 ~ diaphorase NO 3 R ~ e NO3Rase
Control
100 104 101 94 100 102
plus plus plus plus plus
34
ammonia ammonia, ammonia, ammonia, ammonia,
dark menadione DCMU minus CO2
100 5 96 102 94 105
100 4 92 103 91 102
TABLE II EFFECT OF LIGHT, MENADIONE, DCMU AND CO2 ON THE IN VIVO INACTIVATION OF CHLAMYDOMONAS NITRATE REDUCTASE BY ARSENATE The experimental conditions were the same as in Table I, except that arsenate (30 raM) was used instead of ammonia. Relative specific activity (per cent) Treatment
NADH-diaphorase
NADH-NO 3Rase
FNH2 -NO3Rase
Control
100 106 103 98 100 104
100
100
plus plus plus plus plus
arsenate arsenate, arsenate, arsenate, arsenate,
dark menadione DCMU minus CO2
5
6
85 101 101
80 103 100
98
99
enzyme, there are many other factors o f a complicated nature which may also be involved in the process 1- 12. A very important regulatory effect of ammonia on carbon metabolism in Chlorella p y r e n o i d o s a cells has been f o u n d by Bassham and his associates to occur at the reaction which converts phosphoenolpyruvate to pyruvate 14. Although these authors have sugg~s~d that this effect is caused b y a direct activation of pyruvate kinase b y ammonia, t h e p h e n o m e n o n might also be interpreted as a result of the just described uncoupling action of ammonia on photophosphorylation, which would lead to a drop in the energy charge of the cell and thus stimulate the rate o f the ADPdependent reaction catalyzed b y pyruvate kinase. The experiments shown in Fig. 3 corroborate the assumption that in vivo ~ inactivation of nitrate reductase depends on the reduction of the enzyme as p r o m o t e d b y a variety of circumstances. By interrupting aeration o f the cell culture, the enzyme became fully inactive in a b o u t 15 rain; if, subsequently, air was allowed to bubble through the culture, the enzyme was reoxi " d i ~ , and recovered its original activity in ~ b o u t the same short period. For a comparison, the t i m e course o f nitrate reductaseinactivation b y ammonia followed b y reactivation upon its removal is also shown in the same figure. It can be seen in the experiments o f Table III that light or CO2 are n o t required for inactivation when air is excluded, in contrast t o the experiments above described using ammonia or arsenate. Actl]a!ly, air, CO2, ammonia and light symbolize some of the well-known nutritional o r environmental factors affecting nitrate reductase activity n o t only in algae is, b u t also in f u n ~ 16 and higher plants 1~
35
r" U
100i~
h
0
L,.= 20" "J
e~
o O ~
O """ • --
i
0
30
"'O
• . i
.
.
i
.
.
.
• i
60 90 TIME ( m i n u t e s )
120
Fig. 3. Time course of the reversible inactivation of nitrate reductase in Chlamydomonas cells by switching off aeration of the culture or by adding ammonia to it. When aeration was withheld, a stream of 5% CO2 in argon was bubbled through the culture. Other experimental conditions were the same as in Fig. 1.
TABLE III E F F E C T O F LIGHT, C O 2 A N D A E R A T I O N O N T H E IN V I V O I N A C T I V A T I O N CHLAMYDOMONA8 NITRATE REDUCTASE
OF
Cells growing on nitrate as deseribed under Methods were subjected for 15 rain to the treatments indicated. E n z y m e activitieswere then estimated in the corresponding crude extracts. Air and (or) C O 2 were removed from the inflow gas stream as described in Fig. 3 and Table I. Treatment
Relative specific activity (per cent) NADH-NADH-FNH2 -diaphorase NO 3 Rase NO 3 Rase
Control dark minus minus minus minus minus
100 104 102 98 101 99 101
36
C02 air air,dark air, minus CO2 air, minus CO2, dark
100 99 104 9 9 7 10
100 96 105 7 7 5 7
ACKNOWLEDGEMENTS
This w o r k was aided b y a grant from Philips Research Laboratories (Eindhoven, T h e Netherlands). W e should like to thank Profs. E. Palaci~m and J.M. Ve ga for useful discussion and criticism. REFERENCES 1 M. Losada, A. Paneque, P.J.Aparicio,J.M. Vega, J. C~rdenas and J. Herrera,Biochem. Biophys. Res. Commun., 38 (1970) 1009. 2 M. Losada, The assimilatorynitratereducing system and itsregulationby ammonia in Chlorella,in: 1st InternationalSymposium on Metabolic Interconversionof Enzymes, S. Margherita, Italy,1970, p. 59. 3 M. Losada, La Fotosfntesisdel Nitr6geno N{trico,Real Academia de Ciencias,Madrid, 1972. 4 J. Herrera,A. Paneque, J.M. Maldonado, J.L. Barea and M. Losada, Biochem. Biophys. Res. Commun., 48 (1972) 1002. 5 J.M. Vega, J. Herrera,A.M, Relimpio and P.J.Aparicio,Physiol. Veg.,10 (1972) 637. 6 C.G. Moreno, P.J. Aparicio,E. Palaci~nand M. Losada, FEBSLetters, 26 (1972) 11. 7 B. Vennesland and C. Jetschmann, Biochim. Biophys. Acta, 227 (1971) 554. 8 L.P. Solomonson and B. Vennesland, Biochim. Biophys. Acta, 267 (1972) 544. 9 K. Jetschmann, L.P. Solomonson and B. Vennesland, Biochim. Biophys. Acta, 275 (1972) 276. 10 B. Vennesland and L.P. Solomonson, Plant Physiol., 49 (1972) 1029. 11 C. Rigano, Arch. Mikrobiol., 76 (1971) 265. 12 C. Rigano and U. Violante, Biochem. Biophys. Res. Commun., 47 (1972) 372. 13 M. Losada and D.I. Amon, Selective inhibitors of photosynthesis, in: R.H. Hochster and J.H. Quastel (Eds.), Metabolic Inhibitors, Vol. 2, Acaden~Jc Press, New York and London, 1963, p. 559. 14 J.A. Bassham, Proc. Natl. Acad. Sci. (U.S.), 68 (1971) 2877. 15 A. Thaeker and P.J. Syrett, New Phytologist, 71 (1972) 435. 16 A.G. Morton, J. Expti. Botany, 7 (1956) 97. 17 M.I. Candela, E.G. Fisher and E.J. Hewitt, Plant Physiol., 32 (1957) 280.
37
MECHANISM OF NITRATE REDUCTASE REVERSIBLE INACTIVATION BY AMMONIA IN CHLAMYDOMONAS
M. LOSADA, J. H E R R E R A , J.M. M A L D O N A D O
and A. P A N E Q U E
Department of Biochemistry, Faculty of Sciences, CSIC, Universityof Sevilla(Spain) (ReceivedAugust 10th, 1972)
SUMMARY
Ammonia promotes in vivo the conversion of the active form of nitrate reductase into its inactive form by indirectly causing the reduction of the enzyme. Apparently ammonia acts as an uncoupler of noncyclic photophosphorylation, a process which in turn leads to a rise in the level of reducing power in the cell. The transformation is reversible, and, upon ammonia removal, the enzyme becomes again oxidized and active. Nitrate reductase inactivation by ammonia requires light and does not occur when the noncyclic electron flow is blocked or when the photosynthetically generated reducing power is being simultaneously oxidized. In the dark and in the absence of ammonia, the enzyme appears either active or inactive in response to the degree of aerobicity or anaerobicity of the culture.
INTRODUCTION
We have previously shown that the addition of ammonia to a suspension of Chlorella fusca or Chlamydomonas reinhardi cells growing in the light on nitrate determines the rapid inactivation of the second moiety (i.e., FNH2 -NO3 Rase) of the NADH--nitrate reductase complex, and that the subsequent removal of ammonia brings about an equally rapid reactivation of the inactive enzyme ~-4. Interconversion of the active and inactive forms of nitrate reductase could also be achieved in vitro by reducing and oxidizing the enzyme with its physiological or artificial substrates, using both crude extracts and partially purified preparations 4 -6. Vennesland and her associates have made studies of the nitrate reductase of Chlorella vulgaris and found that the enzyme in the cell-free extracts is largely in an inactive form, which can be extensively activated in the presence of added nitrate and phosphate buffer of low pH; addition of NADH in the absence of nitrate led to a loss df enzyme activity 7,s. More recently, the same group has reported that the activation of the inactive form requires an oxidizing agent and is inhibited 9 by CO. Vennesland and her coworkers have repeatedly called attention to the fact
31
that no trace of activation can be observed with extracts of Chlorella fusca under identical conditions 8,, o Rigano has studied the nitrate reductase of the acidophilic thermophilic unicellular alga Cyanidium caldarium and has shown that in vivo the active enzyme can be reversibly transformed into the inactive species b y ammonia ~ In vitro, the latent nitrate reductase could be reactivated by heating ~' , and by treatments with phosphate, urea and pCMB 12 The present paper describes further studies of our laboratory on the mechanism of the light-induced reversible inactivation of nitrate reductase by ammonia in Chlamydomonas cells. MATERIALS AND METHODS
Chlamydomonas reinhardi was grown in the light in a stream of 5% CO2 in air with 8 raM KNO3, as previously described 4. The experiments were performed with cells previously kept for 4 h in a fresh medium under the same conditions b u t with 4 mM nitrate 4. Harvesting o f the cells, preparation of cell-free extracts and estimation of enzyme activities were also as previously described 4 RESULTS AND DISCUSSION Nitrate can act, more or less directly, in the manner of a Hill reagent, as a terminal electron acceptor of the noncyclic electron-transport chain o f photosynthesis, b o t h in whole cells and in chloroplast systems. The photochemical reduction of nitrate to ammonia proceeds in two steps and is stoichiometrically coupled with the evolution of oxygen and the formation of ATP: nitrate is first reduced to nitrite b y the NADH--NO3 Rase complex, and nitrite is then reduced to ammonia b y ferredoxin--nitrite reductase. Ammonia can thus be considered as the end p r o d u c t of the p a t h w a y catalyzed b y the assimilatory nitrate-reducing system 3 Ammonia has the interesting property of acting in an u n k n o w n w a y as an effective uncoupler of noncyclic p h o t o phosphorylation 3,13. Another uncoupler of noncyclic photophosphorylation is arsenate, which acts as a substitute for phosphate leading to the formation of an unstable arsenyl comp o u n d 13. Both uncouplers have the c o m m o n feature of stimulating electron flow 3,13. The finding that these c o m p o u n d s behave similarly in promoting the reversible inactivation of nitrate reductase in vivo is therefore of great significance. Fig. 1 shows that, upon addition of either ammonia or arsenate to Chlamydomonas cells growing on nitrate, nitrate reductase proper (assayed as FNH2 --NO3 Ease or NADH--NO3 Rase) became rapidly inactivated, and that, upon removal o f the uncoupling agent, the inactive form of the enzyme changed back into its original active form. In contrast with nitrate reductase itself, the first m o i e t y of the NADH--nitrate reductase complex (i.e., NADH-diaphorase) was n o t at all affected b y the same treatments. In all the experi-
32
r-
Cl v
>~-
f * NH3 100'*. . . .
/ .
100
~
÷ As043.
.
. &
A • O
U
60
>.. ~
20.
200
0
;
i
o
TIME (hours)
i
Fig. 1. Reversible inactivation of nitrate reductase by ammonia (left) or arsenate (right) in Chlamydomonas cells. In each case, the first arrow indicates the time when ammonia (16 raM) or arsenate (30 raM) was added to cells growing on nitrate under the conditions described in Methods. The second arrow indicates the time when the cells were harvested and resuspended in the original nitrate medium. Enzyme activities were estimated in the corresponding cell-free extracts at the times indicated. 4-- -- --4, NADH--diaphorase; o -o, N A D H - - N O 3 R a s e ; * ' ,,,, F N H 2 - - N O s R a s e . ments described in this paper, conversion of the inactive form into the active one by oxidation with ferricyanide 3 -6,9 was established. In agreement with previous results 4 -6, Fig. 2 shows that inactivation of the active enzyme in crude extracts depended on its reduction by NADH and was proportional to the concentration of the nucleotide. NADPH was also effective in inducing this transformation. These data together with those presented in Tables I and II seem to support the interpretation that both ammonia and arsenate bring about inactivation of nitrate reductase by raising the level of reducing power in the cell as a result or their uncoupling action. Namely, no inactivation was achieved in uivo in the dark, or when vitamin K (which photooxidizes the reducing power formed ~3,~4 ) or DCMU (which inhibits the noncyclic electron flow ~3 ) were added. The CO2 requirement for inactivation under these conditions remains a puzzling phenomenon, particularly since it could be avoided when the accumulation of reducing power was ackieved by other means (see p. 35). Although the possibility exists t h a t a carbon derivative can potentiate the physiological inactivation rate of the reduced 33
E
O.
100
U
\
\
60"
" W t~ hi
0
_~
\
20-
o
o
hi
0
a()3
0.()6
0.()9 '1 013 [NADH] (mM)
0.6
!
0.9
Fig. 2. Effect of NADH on the inactivation of Chlamydomonas nitrate reductase in cellfree extracts. Enzyme activity was estimated after incubation for 3 h at 0° of an active crude extract from cells growing on nitrate, as described under Methods, with NADH at the concentration indicated.
TABLE I E F F E C T OF LIGHT, MENADIONE, DCMU AND CO2 ON THE IN VIVO INACTIVATION OF CHLAMYDOMONAS N I T R A T E R E D U C T A S E BY AMMONIA Cells growing on nitrate as described under Methods were subjected for 1 h to the treatments indicated. Enzyme activities were then estimated in the corresponding crude extracts. Ammonia, 16 raM; Menadione, 0.1 raM; DCMU, 3-(3',4'-Dichlorophenyl)-l,l-dimethylurea, 10 , M . CO2 was removed from the insufflation gas stream by bubbling through a saturated solution of barium hydroxide. Treatment
Relative specific activity (per cent,) NADH-NADH-FNH2 ~ diaphorase NO 3 R ~ e NO3Rase
Control
100 104 101 94 100 102
plus plus plus plus plus
34
ammonia ammonia, ammonia, ammonia, ammonia,
dark menadione DCMU minus CO2
100 5 96 102 94 105
100 4 92 103 91 102
TABLE II EFFECT OF LIGHT, MENADIONE, DCMU AND CO2 ON THE IN VIVO INACTIVATION OF CHLAMYDOMONAS NITRATE REDUCTASE BY ARSENATE The experimental conditions were the same as in Table I, except that arsenate (30 raM) was used instead of ammonia. Relative specific activity (per cent) Treatment
NADH-diaphorase
NADH-NO 3Rase
FNH2 -NO3Rase
Control
100 106 103 98 100 104
100
100
plus plus plus plus plus
arsenate arsenate, arsenate, arsenate, arsenate,
dark menadione DCMU minus CO2
5
6
85 101 101
80 103 100
98
99
enzyme, there are many other factors o f a complicated nature which may also be involved in the process 1- 12. A very important regulatory effect of ammonia on carbon metabolism in Chlorella p y r e n o i d o s a cells has been f o u n d by Bassham and his associates to occur at the reaction which converts phosphoenolpyruvate to pyruvate 14. Although these authors have sugg~s~d that this effect is caused b y a direct activation of pyruvate kinase b y ammonia, t h e p h e n o m e n o n might also be interpreted as a result of the just described uncoupling action of ammonia on photophosphorylation, which would lead to a drop in the energy charge of the cell and thus stimulate the rate o f the ADPdependent reaction catalyzed b y pyruvate kinase. The experiments shown in Fig. 3 corroborate the assumption that in vivo ~ inactivation of nitrate reductase depends on the reduction of the enzyme as p r o m o t e d b y a variety of circumstances. By interrupting aeration o f the cell culture, the enzyme became fully inactive in a b o u t 15 rain; if, subsequently, air was allowed to bubble through the culture, the enzyme was reoxi " d i ~ , and recovered its original activity in ~ b o u t the same short period. For a comparison, the t i m e course o f nitrate reductaseinactivation b y ammonia followed b y reactivation upon its removal is also shown in the same figure. It can be seen in the experiments o f Table III that light or CO2 are n o t required for inactivation when air is excluded, in contrast t o the experiments above described using ammonia or arsenate. Actl]a!ly, air, CO2, ammonia and light symbolize some of the well-known nutritional o r environmental factors affecting nitrate reductase activity n o t only in algae is, b u t also in f u n ~ 16 and higher plants 1~
35
r" U
100i~
h
0
L,.= 20" "J
e~
o O ~
O """ • --
i
0
30
"'O
• . i
.
.
i
.
.
.
• i
60 90 TIME ( m i n u t e s )
120
Fig. 3. Time course of the reversible inactivation of nitrate reductase in Chlamydomonas cells by switching off aeration of the culture or by adding ammonia to it. When aeration was withheld, a stream of 5% CO2 in argon was bubbled through the culture. Other experimental conditions were the same as in Fig. 1.
TABLE III E F F E C T O F LIGHT, C O 2 A N D A E R A T I O N O N T H E IN V I V O I N A C T I V A T I O N CHLAMYDOMONA8 NITRATE REDUCTASE
OF
Cells growing on nitrate as deseribed under Methods were subjected for 15 rain to the treatments indicated. E n z y m e activitieswere then estimated in the corresponding crude extracts. Air and (or) C O 2 were removed from the inflow gas stream as described in Fig. 3 and Table I. Treatment
Relative specific activity (per cent) NADH-NADH-FNH2 -diaphorase NO 3 Rase NO 3 Rase
Control dark minus minus minus minus minus
100 104 102 98 101 99 101
36
C02 air air,dark air, minus CO2 air, minus CO2, dark
100 99 104 9 9 7 10
100 96 105 7 7 5 7
ACKNOWLEDGEMENTS
This w o r k was aided b y a grant from Philips Research Laboratories (Eindhoven, T h e Netherlands). W e should like to thank Profs. E. Palaci~m and J.M. Ve ga for useful discussion and criticism. REFERENCES 1 M. Losada, A. Paneque, P.J.Aparicio,J.M. Vega, J. C~rdenas and J. Herrera,Biochem. Biophys. Res. Commun., 38 (1970) 1009. 2 M. Losada, The assimilatorynitratereducing system and itsregulationby ammonia in Chlorella,in: 1st InternationalSymposium on Metabolic Interconversionof Enzymes, S. Margherita, Italy,1970, p. 59. 3 M. Losada, La Fotosfntesisdel Nitr6geno N{trico,Real Academia de Ciencias,Madrid, 1972. 4 J. Herrera,A. Paneque, J.M. Maldonado, J.L. Barea and M. Losada, Biochem. Biophys. Res. Commun., 48 (1972) 1002. 5 J.M. Vega, J. Herrera,A.M, Relimpio and P.J.Aparicio,Physiol. Veg.,10 (1972) 637. 6 C.G. Moreno, P.J. Aparicio,E. Palaci~nand M. Losada, FEBSLetters, 26 (1972) 11. 7 B. Vennesland and C. Jetschmann, Biochim. Biophys. Acta, 227 (1971) 554. 8 L.P. Solomonson and B. Vennesland, Biochim. Biophys. Acta, 267 (1972) 544. 9 K. Jetschmann, L.P. Solomonson and B. Vennesland, Biochim. Biophys. Acta, 275 (1972) 276. 10 B. Vennesland and L.P. Solomonson, Plant Physiol., 49 (1972) 1029. 11 C. Rigano, Arch. Mikrobiol., 76 (1971) 265. 12 C. Rigano and U. Violante, Biochem. Biophys. Res. Commun., 47 (1972) 372. 13 M. Losada and D.I. Amon, Selective inhibitors of photosynthesis, in: R.H. Hochster and J.H. Quastel (Eds.), Metabolic Inhibitors, Vol. 2, Acaden~Jc Press, New York and London, 1963, p. 559. 14 J.A. Bassham, Proc. Natl. Acad. Sci. (U.S.), 68 (1971) 2877. 15 A. Thaeker and P.J. Syrett, New Phytologist, 71 (1972) 435. 16 A.G. Morton, J. Expti. Botany, 7 (1956) 97. 17 M.I. Candela, E.G. Fisher and E.J. Hewitt, Plant Physiol., 32 (1957) 280.
37