Planta
Planta 137, 2 3 1 - 2 3 4 (1977)
9 by Springer-Verlag 1977
Studies on the Regulation of Assimilatory Nitrate Reductase in Ankistrodesmus braunii J. Diez, A. Chaparro, J.M. Vega, and A. Relimpio Departamento de Bioquimica, Facultad de Ciencias y CSIC. Universidad de Sevilla, Sevilla, Spain
Abstract. In the green alga Anlcistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH2- or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg 2+, presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH. Key words: Ankistrodesmus - Diaphorase - Enzyme inactivation - Nitrate reductase.
Introduction The assimilatory reduction of nitrate to nitrite in the green alga Ankistrodesmus braunii is catalyzed by NAD(P)H-nitrate reductase (EC 1.6.6.2.), an enzyme complex, M.W. 475,000 (Ahmed and Spiller, 1976) which contains FAD, molybdenum (Zumft et al., 1972) and b-type cytochrome (Ahmed and Spiller, 1976) as prosthetic groups. It has been proposed Abbreviations: A D P - A d e n o s i n e - 5 ' - d i p h o s p h a t e ; A M P = A d e n o -
sine- 5'-monophosphate; A T P - - Adenosine- 5'-triphosphate ; F A D = F l a v i n adenine dinucleotide; F M N H 2 = F l a v i n adenine mononucleotide, reduced form ; G D P = Guanosine-5'-diphosphate; M V H = M e t h y l viologen, reduced form; N A D H - N i c o t i n a m i d e adenine dinucleotide, reduced form; N A D P H = Nicotinamide adenine dinucleotide phosphate, reduced form
(Zumft et al., 1972) that in the transfer of electrons from NAD(P)H to nitrate, catalyzed by this enzyme, two different enzymatic activities participate sequentially: the first is a FAD-dependent NAD(P)H-diaphorase, and the second a Mo-dependent nitrate reductase, which may be assayed with flavin nucleotide or methyl viologen (MV) chemically reduced with dithionite as electron donor. The interconversion of nitrate reductase has been extensively studied in this laboratory, both at cellular and molecular levels, using the green algae Chlorella fusca and Chlamydomonas reinhardii (Losada, 1974). The addition of ammonia to cells growing in the light with nitrate promotes a rapid inactivation of the second moiety of the NADH-nitrate reductase complex (i.e., FNH2-nitrate reductase). Apparently ammonia, acting as an uncoupler of non-cyclic photophosphorylation, produces a rise in the intracellular level of reducing power (Chaparro et al., 1976) and ADP (Losada, 1974), which in turn produces nitrate reductase inactivation. Vennesland and her coworkers have: suggested a metabolic regulation system for Chlorella vulgaris NADH-nitrate reductase based on the reversible inactivation by cyanide of the reduced enzyme (Lorimer et al., 1974). In the present paper we have investigated, at the cellular and molecular levels, the interconversion of nitrate reductase in Ankistrodesmus braunii, in order to find out which model of inactivation mechanism seems to operate in this alga.
Material and Method A. braunii (202-7c from G6ttingen University's culture collection was grown in the light on 8 mmol 1-1 KNO3 as previously described (Kessler et al., 1963) except that silicone was added as an antifoaming agent. Air and 5% COz were bubbled through the media. In the experiments in which aeration was witheld, the media were bubbled with argon during 2 h before inoculation, and the cultures were bubbled with a stream of 5% COz in argon during the treatment.
232 The cells were ground at 4~ with alumina in a mortar, a n d the broken material was extracted with 50 mmoI I -x Tris-HC1, pH 7.5 (8 mI buffer/wet g of ceils). The h o m o g e n a t e was centrifuged at 27,000 • during 15 min, a n d the supernatant used as crude extract. For enzyme purification, the cells were broken in a vibration h o m o g e n a t o r with glass beads, and nitrate reductase was purified from the homogenate by following basically the m e t h o d of Vega et al. (t972) except that the protamine sutfate step was avoided and, after the a m m o n i u m sulfate precipitation treatment, the enzyme preparation was desalted by filtration through a Sephadex G-25 column equilibrated with 50 m m o l 1-1 Tris-HC1 buffer, pH 7.5. NAD(P)H-nitrate reductase activities were measured as follows : the reaction mixture contained in 1 m l ; 0 1 mol 1-1 Tris-HCl, pH 7.5; 10 m m o l I -x K N O 3 ; and 0.3 mmoI I z NAD(P)H. After 1 0 m i n of incubation at 30~ 0.1 ml of 10 mmoI 1-1 potassium ferricyanide was added to remove the excess of N A D ( P ) H , which interferes with nitrite estimation. After one additional min, the nitrite formed was determined colorimetrically according to Snell and Snell (1949). N A D ( P ) H - d i a p h o r a s e activities and F M N H 2 or MVH-nitrate reductase activities were assayed as previously described (Vega et aI., 1972) using m a m m a l i a n eytochrome c as acceptor and 2 m m o l I ~ F M N or 0.15 mmol 1 ~ MV, respectively, as electron carrier. A unit of activity is the a m o u n t of enzyme which catalyzes the formation of 1 pmol of nitrite or the reduction of 1 gmoI of cytochrome c per min. Protein were measured by the method of Lowry et al. (1951).
Results and Discussion
Repression-induction of Nitrate Reductase All the activities of A. braunii NAD(P)H-nitrate reductase complex are nutritionally repressed by ammon i a - t h e end product of the assimilatory nitrate reduction p a t h w a y - or methylamine, an ammonia derivative which acts as an uncoupler without being assimilated (Chaparro et al., 1976), even in the presence of nitrate (Table 1). When either ammonia or methylamine were removed from the culture medium containing nitrate, the nitrate reductase complex was synthetized de novo without a lag period, and after 3 h all the activities of the comptex reached maximum [eveI (not shown). The formation of the nitrate reductase complex depended on the addition of an adequate source of nitrogen, nitrate being the most effective (not shown). In the absence of a nitrogen source, about 60% of enzyme was formed, indicating that nitrate is not essential as an inducer for the synthesis of this enzyme in Ankistrodesmus. These results are in agreement with previous reports for this alga (Zumft et al., 1972 ; Syrett and Hipkin, 1973). It has been proposed that in nitrogen-deficient Ankistrodesmus celts the oxidation of nitrogen compounds leads to the intracellular formation of nitrate and nitrite, which may explain the high level of nitrate reductase found under nitrogen-starved conditions (Oesterheld, 1971).
J. Diez et al. : Regulation of Ankistrodesmus Nitrate Reductase T a b l e L Repression by a m m o n i a or methylamine of the nitrate reductase complex in dnkisrrodesmus braunii. Ceils growing on nitrate, as described under Methods, were collected by centrifugation, washed with a culture medium lacking nitrogen compounds, and resuspended in new media with the indicated nitrogen source: nitrate, 8 mmo] 1-1 ; ammonia, 8 m m o l I L; methylamine, 1 5 m n r o l l - t After 18h treatment, the specific activities of the enzyme complex were determined in the corresponding crude extracts. Specific activities at 100% for N A D H NO3Rase, MVH-NO3Rase, and N A D H - d i a p h o r a s e (using cytochrome c) were in nanomoles of NO2 - formed or cytochrome c reduced per rain per m g protein: 85, 75 and 225, respectively
Nitrogen
Relative specific activities (per cent)
source
NO~NH2 NO~- + N H ~ NO~- + methylamine
NADHNO3Rase ~
MVHNO3Rase
NADHdiaphorase"
100 7 15 4
100 7 16 6
100 i5 21 16
a Similar results were obtained when N A D P H was used in place of N A D H as electron donor
Table 2. Effect of aeration, menadione, and nitrogen source on the in vivo inactivation of Ankistrodesmus braunii nitrate reductase. Cells growing on nitrate, as described under Methods, were collected by centrifugation, washed with a culture medium lacking nitrogen compounds, and resuspended on new media with the indicated nitrogen source: nitrate, 10 mmoI I i ; ammonia, I6 mmoI 1-1; methylamine, 3 0 m m o l l 1. Where indicated, 0 . 3 m m o l 1 - 1 menadione was added. After 90min, specific activities were determined in the extracts of the corresponding culture. Specific activities at 100% for N A D H - N O 3 R a s e , MVH-NO3Rase, and N A D H - d i a p h o r a s e were 64, 58, and 326 m U / m g protein, respectively Treatment
NO~NO~ minus air NO~- + N H ~ NO5 + N H ~ minus air NO~- + N H 2 + menadione NO~- + methylamine NO;- + methylamine + menadione N-Free
Relative specific activities (per cent) NADHNO3Ras&
MVHNO3Rase
NADHdiaphorase"
100 72 19 15
100 78 14 9
100 92 90 74
115 20
l 1t 24
85 I06
101 65
112 54
91 92
" Similar results were obtained when N A D P H was used in place of N A D H as electron donor
In vivo Interconversion of Nitrate Reductase Table 2 shows in vivo inactivation of nitrate reductase after addition of ammonia or methylamine to Ankistrodesmus cells growing in the light with nitrate. It is noteworthy that the diaphorase activity of the
J. Diez et al. : Regulation of Ankistrodesmus Nitrate Reductase NAD(P)H-nitrate reductase complex is not at all affected by the control exerted in vivo by ammonia or methylamine. As can also be seen in the table, the removal of air from the culture media has a certain inactivating effect on the enzyme that increases when ammonia is simultaneously present. N o inactivation was achieved in vivo when menadione, which photooxidizes the reducing power formed (Losada, 1974), was added, together with either ammonia or methylamine. This protection by menadione suggests that the reducing power level may play a role in keeping inactive nitrate reductase in vivo. Since the absence of a nitrogen source brings about a relatively small inactivation of nitrate reductase activity as compared with the ammonia or methylamine effect, the in vivo ammonia-promoted inactivation can not be exclusively explained as a consequence of an inhibition of the uptake of nitrate by the cells. In addition, ammonia prevents nitrate utilization by Ankistrodesmus cells. The concentration of nitrate in the medium analyzed, after removal of the cells, according to the method of Cawse (1967), remains constant while ammonia is present and only decreases when ammonia has been completely assimilated (not shown). Figure 1 shows the in vivo inactivation of nitrate reductase after addition of ammonia to Ankistrodesmus cells growing on nitrate, as well as the reactivation of the inactive enzyme that followed ammonia removal from the medium. This in vivo reactivation of the inactive enzyme required essentially light and was strikingly stimulated by the addition of menadione to the culture medium.
233
/!
+NH~ 1013
\
//
80
60 I/
4o
"
# #
~
20
-N H ~
/.
i/-i i1.
..........
/.
"...............
o
TIME (hours)
Fig. 1. Time course of the reversible inactivation by ammonia of the nitrate reductase complex in Ankistrodesmus braun#. The first arrow indicates the time when ammonia (16 mmol 1 1) was added to cells growingon nitrate (8 mmol l- 1). The second arrow indicates when the cells were harvested, washed and resuspended in new media with nitrate (Smmol1-1) alone (....... ), +0.3mmolI menadione (.... ), +dark ( . . . . . ). Specific activities at 100% for NADH-NO3Rase and FMNH2-NO3Rase were, in mU/mg protein, 115 and 87, respectively. Similar results were obtained when methylamine was used instead of ammonia as inactivating agent
~10C
~8c
,~ 6c
In vitro Interconversion o f Nitrate Reductase
Figure 2 shows that the inactivation of Ankistrodesmus nitrate reductase, using a partially purified preparation, depends on its reduction by N A D ( P ) H , its physiological electron donor. N A D P H is less effective in its inactivating effect on the enzyme than N A D H . Although it is not shown in this figure, the diaphorase activity of the enzyme was essentially unaffected by this treatment. It has been previously shown that A D P cooperates with N A D H in the in vitro inactivation of Chlorella nitrate reductase (Losada, 1974). The results reported in Figure 3 indicate that the A D P concentration which cooperates with N A D H to inactivate Ankistrodesmus nitrate reductase seems to be critical. Under the experimental conditions used, the inactivating effect was maximum at 0.3 mmol 1- ~ of ADP, equimolecular with N A D H . This cooperation seems to be specific, because under similar conditions, AMP,
w 40
,z
~'.ol
doa
o'1 fNAD(P)H] (raM)
o'.a
' 1.0
Fig. 2. Effect of NAD(P)H concentration on the inactivation of Ankistrodesmus braunii nitrate reductase. The enzyme, partially
purified as indicated under Methods, was incubated at 0~ for 100min with NADH (--o--) or NADPH (--9 at the indicated concentrations. The NADH-nitrate reductase activity 'was determined on aliquots of the incubation mixtures. Specific activity at 100% for NADH-NO3Rase was 69 mU/mg protein
ATP, and G D P were much less effective (not shown). N o cooperation was found with N A D P H and ADP, altough N A D P H is also an effective electron donor for this enzyme (Zumft et al., 1972; Ahmed and Spiller, 1976).
234
J. Diez et al. : Regulation of Ankistrodesmus Nitrate Reductase
dinitrophenol (Ahmed and Morris, 1967) to Ankistrodesmus cells inhibits nitrate assimilation. ..~101
Acknowledgments. The authors wish to thank Prof. Losada for helpful advice, criticism, and encouragement. The collaboration of Dr. J.M. Maldonado in the early stages of this work is also appreciated. This work was supported by grants from Philips Research Laboratories (The Netherlands), the Comisaria Asesora de Investigaci6n Cienfifica y T@nica (Spain) and The National Science Foundation (GF-44115) (USA). The skillful technical assistance of Mr. Josh Moreno is gratefully recognized.
>
~ O: ~ -
one
i 25 References 0
//
0.35
0'.2 013
[ADP] (raM)
016 110
210
Fig. 3. Cooperative effect of ADP with NADH on the reversible inactivation of nitrate reductase complex in Ankistrodesmus. The experimental conditions were the same as in Figure 2 when NADH, 0.3mmoll 1, was used, except that MgCIz, 10mmoll 1, and ADP were added as indicated
Nitrate reductase inactivated by any of the treatments cited above can be reactivated by the addition of ferricyanide-an electron acceptor for the diaphorase activity (Zumft et al., 1972)-at a concentration high enough to reoxidize the excess of reducing power. The simplest interpretation of the results reported in this paper is that ammonia or methylamine induce a rise in the reducing power level of Ankistrodesmus cells by a mechanism which may involve their ability to act as uncouplers of non-cyclic photophosphorylation (Losada, 1974). NAD(P)H by itself, or in cooperation with other metabolites (probably ADP), determines the in vivo inactivation of nitrate reductase. This hypothesis is based upon the following facts: (1) When added to A. braunii cells, methylamine produces an inactivation of nitrate reductase similar to that promoted by ammonia. Concomitantly with the in vivo inactivation of the enzyme, methylamine induces an increase in the cellular level of NAD(P)H, as shown by Chaparro et al. (1976) in Chlorella. Furthermore, the interconversion of the enzyme in vitro supports a possible role of ADP in the inactivation in vivo (Fig. 3). (2) The NAD(P)H-dependent in vitro inactivation of nitrate reductase was performed with a partially purified nitrate reductase preparation, in which the presence of cyanide traces that could contribute to the inactivation process, is highly improbable. Finally, it is interesting to note that the addition of other uncouplers, such as carbonylcyanide-m-chlorophenylhydrazone (CCCP) (Ullrich, 1974) or 2,4-
Ahmed, J., Morris, I. : Inhibition of nitrate and nitrite reduction by 2,4-dinitrophenol in Ankistrodesmus. Arch. Mikrobiol. 56, 219-224 (1967) Ahmed, J., Spiller, H.: Purification and some properties of the nitrate reductase fore Ankistrodesmus braunii. Plant & Cell Physiol. 17, 1 10 (1976) Cawse, P.A.: The determination of nitrate in soil solutions by ultraviolet spectrophotometry. Analyst 92, 311-315 (1967) Chaparro, A., Maldonado, J.M., Diez, J., Relimpio, A.M., Losada, M.: Nitrate reductase inactivation and reducing power and energy charge in Chlorella cells. Plant Sci. Lett. 6, 335-342 (1976) Kessler, E., Langner, W., Ludewig, I., Wiechmann, H.: Bildung von Sekundgr-Carotinoiden bei Stickstoffmangel und Hydrogenase-Aktivit/it als taxonomische Merkmale in der Gattung Chlorella. In: Studies on microalgae and photosynthetic bacteria, pp. 7-20. Tokyo: University of Tokyo Press 1963 Lorimer, G.H., Gewitz, H., Voker, V., Solomonson, L.P., Vennesland, B. : The presence of bound cyanide in the naturally inactivated form of nitrate reductase of Chlorella vulgaris. J. Biol. Chem. 249, 6074 6079 (1974) Losada, M.: Interconversion of nitrate and nitrite reductase of the assimilatory type. In: Metabolic interconversion of enzymes. III. Intern. Symp., p. 257, Fischer, E.H., Krebs, E.G., Neurath, H., Stadtman, E.R. eds. Seattle 1973. Berlin-HeideL berg-NewYork: Springer 1974 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 275 (1951) Oesterheld, H.: Das Verhalten yon Nitratreductase, Nitritreductase, Hydrogenase, und anderen Enzymen von Ankistrodesmus braunii bei Stickstoffmangel. Arch. Mikrobiol. 79, 2 5 4 3 (1971) Snell, F.D., Snell, C.T.: Colorimetric method of analysis. New York: D. Van Nostrand 1949 Syrett, P.J., Hipkin, C.R.: The appearance of nitrate reductase activity in nitrogen-starved cells of Ankistrodesmus braunii. Planta 111, 57 64 (1973) Ullrich, W.R.: Die nitrat- und nitritabhS.ngige photosynthetische O2-Entwicklung in N2 bei Ankistrodesmus braunii. Planta 116, 143-152 (1974) Vega, J.M., Herrera, J., Relimpio, A.M., Aparicio, P.J. : NADHnitrate r6ductase de Chlorella: nouvelle contribution fi l'6tude de ses propri6t6s. Physiol. V6g. 10, 637-652 (1972) Zumft, W.G., Spiller, H., Yeboat-Smith, I. : Eisengehalt und Electronendonator-Spezifitfit der Nitratreductase von Ankistrodesmus. Planta 102, 228 236 (1972)
Received 25 May; accepted 10 August 1977
Planta 137, 2 3 1 - 2 3 4 (1977)
9 by Springer-Verlag 1977
Studies on the Regulation of Assimilatory Nitrate Reductase in Ankistrodesmus braunii J. Diez, A. Chaparro, J.M. Vega, and A. Relimpio Departamento de Bioquimica, Facultad de Ciencias y CSIC. Universidad de Sevilla, Sevilla, Spain
Abstract. In the green alga Anlcistrodesmus braunii, all the activities associated with the nitrate reductase complex (i.e., NAD(P)H-nitrate reductase, NAD(P)H-cytochrome c reductase and FMNH2- or MVH-nitrate reductase) are nutritionally repressed by ammonia or methylamine. Besides, ammonia or methylamine promote in vivo the reversible inactivation of nitrate reductase, but not of NAD(P)H-cytochrome c reductase. Subsequent removal of the inactivating agent from the medium causes reactivation of the inactive enzyme. Menadione has a striking stimulation on the in vivo reactivation of the inactive enzyme. The nitrate reductase activities, but not the diaphorase activity, can be inactivated in vitro by preincubating a partially purified enzyme preparation with NADH or NADPH. ADP, in the presence of Mg 2+, presents a cooperative effect with NADH in the in vitro inactivation of nitrate reductase. This effect appears to be maximum at a concentration of ADP equimolecular with that of NADH. Key words: Ankistrodesmus - Diaphorase - Enzyme inactivation - Nitrate reductase.
Introduction The assimilatory reduction of nitrate to nitrite in the green alga Ankistrodesmus braunii is catalyzed by NAD(P)H-nitrate reductase (EC 1.6.6.2.), an enzyme complex, M.W. 475,000 (Ahmed and Spiller, 1976) which contains FAD, molybdenum (Zumft et al., 1972) and b-type cytochrome (Ahmed and Spiller, 1976) as prosthetic groups. It has been proposed Abbreviations: A D P - A d e n o s i n e - 5 ' - d i p h o s p h a t e ; A M P = A d e n o -
sine- 5'-monophosphate; A T P - - Adenosine- 5'-triphosphate ; F A D = F l a v i n adenine dinucleotide; F M N H 2 = F l a v i n adenine mononucleotide, reduced form ; G D P = Guanosine-5'-diphosphate; M V H = M e t h y l viologen, reduced form; N A D H - N i c o t i n a m i d e adenine dinucleotide, reduced form; N A D P H = Nicotinamide adenine dinucleotide phosphate, reduced form
(Zumft et al., 1972) that in the transfer of electrons from NAD(P)H to nitrate, catalyzed by this enzyme, two different enzymatic activities participate sequentially: the first is a FAD-dependent NAD(P)H-diaphorase, and the second a Mo-dependent nitrate reductase, which may be assayed with flavin nucleotide or methyl viologen (MV) chemically reduced with dithionite as electron donor. The interconversion of nitrate reductase has been extensively studied in this laboratory, both at cellular and molecular levels, using the green algae Chlorella fusca and Chlamydomonas reinhardii (Losada, 1974). The addition of ammonia to cells growing in the light with nitrate promotes a rapid inactivation of the second moiety of the NADH-nitrate reductase complex (i.e., FNH2-nitrate reductase). Apparently ammonia, acting as an uncoupler of non-cyclic photophosphorylation, produces a rise in the intracellular level of reducing power (Chaparro et al., 1976) and ADP (Losada, 1974), which in turn produces nitrate reductase inactivation. Vennesland and her coworkers have: suggested a metabolic regulation system for Chlorella vulgaris NADH-nitrate reductase based on the reversible inactivation by cyanide of the reduced enzyme (Lorimer et al., 1974). In the present paper we have investigated, at the cellular and molecular levels, the interconversion of nitrate reductase in Ankistrodesmus braunii, in order to find out which model of inactivation mechanism seems to operate in this alga.
Material and Method A. braunii (202-7c from G6ttingen University's culture collection was grown in the light on 8 mmol 1-1 KNO3 as previously described (Kessler et al., 1963) except that silicone was added as an antifoaming agent. Air and 5% COz were bubbled through the media. In the experiments in which aeration was witheld, the media were bubbled with argon during 2 h before inoculation, and the cultures were bubbled with a stream of 5% COz in argon during the treatment.
232 The cells were ground at 4~ with alumina in a mortar, a n d the broken material was extracted with 50 mmoI I -x Tris-HC1, pH 7.5 (8 mI buffer/wet g of ceils). The h o m o g e n a t e was centrifuged at 27,000 • during 15 min, a n d the supernatant used as crude extract. For enzyme purification, the cells were broken in a vibration h o m o g e n a t o r with glass beads, and nitrate reductase was purified from the homogenate by following basically the m e t h o d of Vega et al. (t972) except that the protamine sutfate step was avoided and, after the a m m o n i u m sulfate precipitation treatment, the enzyme preparation was desalted by filtration through a Sephadex G-25 column equilibrated with 50 m m o l 1-1 Tris-HC1 buffer, pH 7.5. NAD(P)H-nitrate reductase activities were measured as follows : the reaction mixture contained in 1 m l ; 0 1 mol 1-1 Tris-HCl, pH 7.5; 10 m m o l I -x K N O 3 ; and 0.3 mmoI I z NAD(P)H. After 1 0 m i n of incubation at 30~ 0.1 ml of 10 mmoI 1-1 potassium ferricyanide was added to remove the excess of N A D ( P ) H , which interferes with nitrite estimation. After one additional min, the nitrite formed was determined colorimetrically according to Snell and Snell (1949). N A D ( P ) H - d i a p h o r a s e activities and F M N H 2 or MVH-nitrate reductase activities were assayed as previously described (Vega et aI., 1972) using m a m m a l i a n eytochrome c as acceptor and 2 m m o l I ~ F M N or 0.15 mmol 1 ~ MV, respectively, as electron carrier. A unit of activity is the a m o u n t of enzyme which catalyzes the formation of 1 pmol of nitrite or the reduction of 1 gmoI of cytochrome c per min. Protein were measured by the method of Lowry et al. (1951).
Results and Discussion
Repression-induction of Nitrate Reductase All the activities of A. braunii NAD(P)H-nitrate reductase complex are nutritionally repressed by ammon i a - t h e end product of the assimilatory nitrate reduction p a t h w a y - or methylamine, an ammonia derivative which acts as an uncoupler without being assimilated (Chaparro et al., 1976), even in the presence of nitrate (Table 1). When either ammonia or methylamine were removed from the culture medium containing nitrate, the nitrate reductase complex was synthetized de novo without a lag period, and after 3 h all the activities of the comptex reached maximum [eveI (not shown). The formation of the nitrate reductase complex depended on the addition of an adequate source of nitrogen, nitrate being the most effective (not shown). In the absence of a nitrogen source, about 60% of enzyme was formed, indicating that nitrate is not essential as an inducer for the synthesis of this enzyme in Ankistrodesmus. These results are in agreement with previous reports for this alga (Zumft et al., 1972 ; Syrett and Hipkin, 1973). It has been proposed that in nitrogen-deficient Ankistrodesmus celts the oxidation of nitrogen compounds leads to the intracellular formation of nitrate and nitrite, which may explain the high level of nitrate reductase found under nitrogen-starved conditions (Oesterheld, 1971).
J. Diez et al. : Regulation of Ankistrodesmus Nitrate Reductase T a b l e L Repression by a m m o n i a or methylamine of the nitrate reductase complex in dnkisrrodesmus braunii. Ceils growing on nitrate, as described under Methods, were collected by centrifugation, washed with a culture medium lacking nitrogen compounds, and resuspended in new media with the indicated nitrogen source: nitrate, 8 mmo] 1-1 ; ammonia, 8 m m o l I L; methylamine, 1 5 m n r o l l - t After 18h treatment, the specific activities of the enzyme complex were determined in the corresponding crude extracts. Specific activities at 100% for N A D H NO3Rase, MVH-NO3Rase, and N A D H - d i a p h o r a s e (using cytochrome c) were in nanomoles of NO2 - formed or cytochrome c reduced per rain per m g protein: 85, 75 and 225, respectively
Nitrogen
Relative specific activities (per cent)
source
NO~NH2 NO~- + N H ~ NO~- + methylamine
NADHNO3Rase ~
MVHNO3Rase
NADHdiaphorase"
100 7 15 4
100 7 16 6
100 i5 21 16
a Similar results were obtained when N A D P H was used in place of N A D H as electron donor
Table 2. Effect of aeration, menadione, and nitrogen source on the in vivo inactivation of Ankistrodesmus braunii nitrate reductase. Cells growing on nitrate, as described under Methods, were collected by centrifugation, washed with a culture medium lacking nitrogen compounds, and resuspended on new media with the indicated nitrogen source: nitrate, 10 mmoI I i ; ammonia, I6 mmoI 1-1; methylamine, 3 0 m m o l l 1. Where indicated, 0 . 3 m m o l 1 - 1 menadione was added. After 90min, specific activities were determined in the extracts of the corresponding culture. Specific activities at 100% for N A D H - N O 3 R a s e , MVH-NO3Rase, and N A D H - d i a p h o r a s e were 64, 58, and 326 m U / m g protein, respectively Treatment
NO~NO~ minus air NO~- + N H ~ NO5 + N H ~ minus air NO~- + N H 2 + menadione NO~- + methylamine NO;- + methylamine + menadione N-Free
Relative specific activities (per cent) NADHNO3Ras&
MVHNO3Rase
NADHdiaphorase"
100 72 19 15
100 78 14 9
100 92 90 74
115 20
l 1t 24
85 I06
101 65
112 54
91 92
" Similar results were obtained when N A D P H was used in place of N A D H as electron donor
In vivo Interconversion of Nitrate Reductase Table 2 shows in vivo inactivation of nitrate reductase after addition of ammonia or methylamine to Ankistrodesmus cells growing in the light with nitrate. It is noteworthy that the diaphorase activity of the
J. Diez et al. : Regulation of Ankistrodesmus Nitrate Reductase NAD(P)H-nitrate reductase complex is not at all affected by the control exerted in vivo by ammonia or methylamine. As can also be seen in the table, the removal of air from the culture media has a certain inactivating effect on the enzyme that increases when ammonia is simultaneously present. N o inactivation was achieved in vivo when menadione, which photooxidizes the reducing power formed (Losada, 1974), was added, together with either ammonia or methylamine. This protection by menadione suggests that the reducing power level may play a role in keeping inactive nitrate reductase in vivo. Since the absence of a nitrogen source brings about a relatively small inactivation of nitrate reductase activity as compared with the ammonia or methylamine effect, the in vivo ammonia-promoted inactivation can not be exclusively explained as a consequence of an inhibition of the uptake of nitrate by the cells. In addition, ammonia prevents nitrate utilization by Ankistrodesmus cells. The concentration of nitrate in the medium analyzed, after removal of the cells, according to the method of Cawse (1967), remains constant while ammonia is present and only decreases when ammonia has been completely assimilated (not shown). Figure 1 shows the in vivo inactivation of nitrate reductase after addition of ammonia to Ankistrodesmus cells growing on nitrate, as well as the reactivation of the inactive enzyme that followed ammonia removal from the medium. This in vivo reactivation of the inactive enzyme required essentially light and was strikingly stimulated by the addition of menadione to the culture medium.
233
/!
+NH~ 1013
\
//
80
60 I/
4o
"
# #
~
20
-N H ~
/.
i/-i i1.
..........
/.
"...............
o
TIME (hours)
Fig. 1. Time course of the reversible inactivation by ammonia of the nitrate reductase complex in Ankistrodesmus braun#. The first arrow indicates the time when ammonia (16 mmol 1 1) was added to cells growingon nitrate (8 mmol l- 1). The second arrow indicates when the cells were harvested, washed and resuspended in new media with nitrate (Smmol1-1) alone (....... ), +0.3mmolI menadione (.... ), +dark ( . . . . . ). Specific activities at 100% for NADH-NO3Rase and FMNH2-NO3Rase were, in mU/mg protein, 115 and 87, respectively. Similar results were obtained when methylamine was used instead of ammonia as inactivating agent
~10C
~8c
,~ 6c
In vitro Interconversion o f Nitrate Reductase
Figure 2 shows that the inactivation of Ankistrodesmus nitrate reductase, using a partially purified preparation, depends on its reduction by N A D ( P ) H , its physiological electron donor. N A D P H is less effective in its inactivating effect on the enzyme than N A D H . Although it is not shown in this figure, the diaphorase activity of the enzyme was essentially unaffected by this treatment. It has been previously shown that A D P cooperates with N A D H in the in vitro inactivation of Chlorella nitrate reductase (Losada, 1974). The results reported in Figure 3 indicate that the A D P concentration which cooperates with N A D H to inactivate Ankistrodesmus nitrate reductase seems to be critical. Under the experimental conditions used, the inactivating effect was maximum at 0.3 mmol 1- ~ of ADP, equimolecular with N A D H . This cooperation seems to be specific, because under similar conditions, AMP,
w 40
,z
~'.ol
doa
o'1 fNAD(P)H] (raM)
o'.a
' 1.0
Fig. 2. Effect of NAD(P)H concentration on the inactivation of Ankistrodesmus braunii nitrate reductase. The enzyme, partially
purified as indicated under Methods, was incubated at 0~ for 100min with NADH (--o--) or NADPH (--9 at the indicated concentrations. The NADH-nitrate reductase activity 'was determined on aliquots of the incubation mixtures. Specific activity at 100% for NADH-NO3Rase was 69 mU/mg protein
ATP, and G D P were much less effective (not shown). N o cooperation was found with N A D P H and ADP, altough N A D P H is also an effective electron donor for this enzyme (Zumft et al., 1972; Ahmed and Spiller, 1976).
234
J. Diez et al. : Regulation of Ankistrodesmus Nitrate Reductase
dinitrophenol (Ahmed and Morris, 1967) to Ankistrodesmus cells inhibits nitrate assimilation. ..~101
Acknowledgments. The authors wish to thank Prof. Losada for helpful advice, criticism, and encouragement. The collaboration of Dr. J.M. Maldonado in the early stages of this work is also appreciated. This work was supported by grants from Philips Research Laboratories (The Netherlands), the Comisaria Asesora de Investigaci6n Cienfifica y T@nica (Spain) and The National Science Foundation (GF-44115) (USA). The skillful technical assistance of Mr. Josh Moreno is gratefully recognized.
>
~ O: ~ -
one
i 25 References 0
//
0.35
0'.2 013
[ADP] (raM)
016 110
210
Fig. 3. Cooperative effect of ADP with NADH on the reversible inactivation of nitrate reductase complex in Ankistrodesmus. The experimental conditions were the same as in Figure 2 when NADH, 0.3mmoll 1, was used, except that MgCIz, 10mmoll 1, and ADP were added as indicated
Nitrate reductase inactivated by any of the treatments cited above can be reactivated by the addition of ferricyanide-an electron acceptor for the diaphorase activity (Zumft et al., 1972)-at a concentration high enough to reoxidize the excess of reducing power. The simplest interpretation of the results reported in this paper is that ammonia or methylamine induce a rise in the reducing power level of Ankistrodesmus cells by a mechanism which may involve their ability to act as uncouplers of non-cyclic photophosphorylation (Losada, 1974). NAD(P)H by itself, or in cooperation with other metabolites (probably ADP), determines the in vivo inactivation of nitrate reductase. This hypothesis is based upon the following facts: (1) When added to A. braunii cells, methylamine produces an inactivation of nitrate reductase similar to that promoted by ammonia. Concomitantly with the in vivo inactivation of the enzyme, methylamine induces an increase in the cellular level of NAD(P)H, as shown by Chaparro et al. (1976) in Chlorella. Furthermore, the interconversion of the enzyme in vitro supports a possible role of ADP in the inactivation in vivo (Fig. 3). (2) The NAD(P)H-dependent in vitro inactivation of nitrate reductase was performed with a partially purified nitrate reductase preparation, in which the presence of cyanide traces that could contribute to the inactivation process, is highly improbable. Finally, it is interesting to note that the addition of other uncouplers, such as carbonylcyanide-m-chlorophenylhydrazone (CCCP) (Ullrich, 1974) or 2,4-
Ahmed, J., Morris, I. : Inhibition of nitrate and nitrite reduction by 2,4-dinitrophenol in Ankistrodesmus. Arch. Mikrobiol. 56, 219-224 (1967) Ahmed, J., Spiller, H.: Purification and some properties of the nitrate reductase fore Ankistrodesmus braunii. Plant & Cell Physiol. 17, 1 10 (1976) Cawse, P.A.: The determination of nitrate in soil solutions by ultraviolet spectrophotometry. Analyst 92, 311-315 (1967) Chaparro, A., Maldonado, J.M., Diez, J., Relimpio, A.M., Losada, M.: Nitrate reductase inactivation and reducing power and energy charge in Chlorella cells. Plant Sci. Lett. 6, 335-342 (1976) Kessler, E., Langner, W., Ludewig, I., Wiechmann, H.: Bildung von Sekundgr-Carotinoiden bei Stickstoffmangel und Hydrogenase-Aktivit/it als taxonomische Merkmale in der Gattung Chlorella. In: Studies on microalgae and photosynthetic bacteria, pp. 7-20. Tokyo: University of Tokyo Press 1963 Lorimer, G.H., Gewitz, H., Voker, V., Solomonson, L.P., Vennesland, B. : The presence of bound cyanide in the naturally inactivated form of nitrate reductase of Chlorella vulgaris. J. Biol. Chem. 249, 6074 6079 (1974) Losada, M.: Interconversion of nitrate and nitrite reductase of the assimilatory type. In: Metabolic interconversion of enzymes. III. Intern. Symp., p. 257, Fischer, E.H., Krebs, E.G., Neurath, H., Stadtman, E.R. eds. Seattle 1973. Berlin-HeideL berg-NewYork: Springer 1974 Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265 275 (1951) Oesterheld, H.: Das Verhalten yon Nitratreductase, Nitritreductase, Hydrogenase, und anderen Enzymen von Ankistrodesmus braunii bei Stickstoffmangel. Arch. Mikrobiol. 79, 2 5 4 3 (1971) Snell, F.D., Snell, C.T.: Colorimetric method of analysis. New York: D. Van Nostrand 1949 Syrett, P.J., Hipkin, C.R.: The appearance of nitrate reductase activity in nitrogen-starved cells of Ankistrodesmus braunii. Planta 111, 57 64 (1973) Ullrich, W.R.: Die nitrat- und nitritabhS.ngige photosynthetische O2-Entwicklung in N2 bei Ankistrodesmus braunii. Planta 116, 143-152 (1974) Vega, J.M., Herrera, J., Relimpio, A.M., Aparicio, P.J. : NADHnitrate r6ductase de Chlorella: nouvelle contribution fi l'6tude de ses propri6t6s. Physiol. V6g. 10, 637-652 (1972) Zumft, W.G., Spiller, H., Yeboat-Smith, I. : Eisengehalt und Electronendonator-Spezifitfit der Nitratreductase von Ankistrodesmus. Planta 102, 228 236 (1972)
Received 25 May; accepted 10 August 1977
