100 NITRATE REDUCTION
Dr. Sarvajeet Singh
Asst. Professor, Botany, Rajkiy Mahila Mahavidhyala, Shahganj, Jaunpur
Abstract - Nitrate reductase is the first enzyme of reduction path way nitrate to ammonia.
It is substrate inducible enzyme and occur in bacteria, fungi, algae including blue-green and higher plants (Kessler, 1964). Nitrate reduction in algae occur in 2 steps, first the reduction of nitrate to nitrite and than nitrite to ammonia (Vennesland and Guessen, 1979;
Guerroro et at. ‘1981).
In the present study an attempt made to see the effect of different factor on nitrate reductase. Nostoc showed and enhanced nitrate reduction in light, temperature reduce the activity, transcriptions and translation inhibit the activity of Nitrate reductase.
1 INTRODUCTION
Nitrate red uctase is the key enzyme in nitrogen metabolism and catalyse the reduction of Nitrate to nitrite.
This enzyme has been studied in some blue-green algae (Hattori and Mayeres 1967, Manzane et al. 1976 Ortege et al. 1977, Rao 1978, Gupta and Talpassayi 1904 Parmeswaran 1985 Jacok 1979) made a detailed comprative study of this enzyme in several blue green algae.
In present investigation an attempt has been made to study the effect of light darkness and temperature of the algae on nitrate reduction.
The reduction of Nitrate by the algae was studied under the following factor
1. Effect of light and darkness 2 Effect of temperature
2 MATERIAL METHODS
2.1 Estimation of Nitrate Reductase Activity
In vivo nitrate reductase activity was estimated by the method of Camm and Stein (1974). The activity was based on total nitrate formed in a definitive volume of culture suspension. For the assay of enzyme a known amount of algal suspension was centrifuged, washed with glass distilled water and incubated in basal medium containing 10 mM KNO3
(pH 7.5). Samples were taken at suitable intervals and the nitrite formed was determined by the diazocoupling method of Lowe and Evaans (1964).
Reaction mixture consisted of the following:
Control Test
Sample Nil 1mI
Potassium nitrate 1 ml Nil KNO3 (0.02 M)
Sulfanilamide (1 gm in 100 ml of (1:4 HCI) 2 ml 2 ml
(N-1)-naphthyl ethylene Diamine dihydrochloride
(0.2%) 2 ml 2 ml First sulfanilamide was added and solution was mixed well and after an interval of 15 mm a-(N-1)-naphthyl- ethylenediamine dihydrochloride was added. After 15 mm the absorbance of pink colour was estimated at 540 nm.
Enzyme activity was expressed as mg N02- formed mg1 chl a using the standard curve of nitrate.
Nitrate grown cultures were incubated in 40% argon or 60% °2’ in fluorescent or blue light. For argon and O2
treatment, we used culture tubes of 7 ml capacity containing I ml of culture suspension.
Monochromatic blue (450 nm) light w. & tainted by passing incandescent light (from 150 Watt Phillips reflector lamp) through coloured glass filter (Carolina Biological Supply Co., Burlington: U.S.A.). The half-band-width of blue filter was ca. 22 nm. To avoid overheating, the cultures were incubated in a water-jacked box with the desired light quality. Equal energy level (1 W/m-2) of fluorescent and blue light was adjusted by changing the distance between the light source and the sample.
3 RESULTS
3.1 Effect of Light and Temperature In this experiment the nitrate reduction by the alga Nostoc and Anabaena was studied in light and darkness.
After every 30 mm -5 ml sample was taken and tested for nitrate by the addition of 2 ml suiphanilamide and 2 ml
101 N-Cl - naphthyl) - ethylene diamine
hydrochloride. The absorbency of the sample was taken at 540 nm after the development of the pink colour. Fresh medium was added after every sample taken to maintain constant volume. At the end of each cay, the column was washed 45 times with double distilled water and Kept in fresh CHU-10 to be used again.
A similar column covered with other paper was used for nitrate reduction in darkness.
This experiment was conducted under normal laboratory condition with light (2500 lux). The medium used was CHU-10 and it was passed over the algal bygravity.
All results were calculated after deduction of the lilution factor. The results are expressed as µM m1 medium h-1.
Table 1 Effect of light and darkness on reduction of nitrate by Nostoc
cells Time interval
(days) Nitrate formed µM m1 medium h-1 Light Dark 1
2 8 9 17 18 19 20 22 23 24
14 15.4 76.3 61.6 19.5 21.0 32.1 13.4 40.3 29.9 5.04
6.5 4.3 5.5 5.5 1.36
1.5 9.1 4.2 4.6 3.6 1.24 Figure – Effect of light and darkness on reduction of nitrate by Nostoc cells
Table 2 Effect of light darkness on reduction of Nitrate by Anabaena cells
Time interval
(days)
Nitrate formed µM m1 medium h-1 Light Dark 1
2 3 6 8 10
1.61 5.86 3.08 3.04 2.03 1.85
0.00 0.86 1.05 0.76 1.06 0.98
Figure – Effect of light and darkness on reduction of nitrate by Anabaena cells Nostoc
The results are presented in table and Figure. In Ught high nitrate reduction was observed in comparison to darkness maximum reduction in light was observed on the 8th, 9th and 22nd days.
In dark the reduction of nitrate was low all days and decreased to 24 µm m-l 1 medium h-1 on the 25th day.
3.2 Anabaena
Similar behaviour was also found with Anabaena in light high nitrate reduction was observed on 2nd, 3rd and 6th day (5.88, 3.08 and 3.04 p.m m11 medium1) respectively. The nitrate reduction by the alga showed a linear declined with only 1.00 µm m-l 1 medium h-1 being reduced on 10th day in presence of light.
In dark the activity was low on all days being nill on 1st day. The nitrate reduced was only 0.98 µm m-l 1 medium h-1on the 10th day.
(b) Effect of temperature
The aim of this experiment was to study the effe of temperature on the reduction of nitrate at different temperature.
Table 3 Effect of temperature on reduction of nitrate on Nostoc Inculation
Temperature (°C)
Time Interval
(mm)
Nitrate formed µm mg-1 chla 30
45 50
30 60 30 60 30 60
2.78 2.96 5.57 6.00 6.44 6.79
Chi Conc: 0.8 µm m-l, light intensity: 8500 lux medium used: CHU-10
The alga Nostoc was used in the present study. Alga kept over night in a nitrate containing medium (CHU-10) to induce the nitrate reductase activity. They were washed 4-5 times with double distilled water before starting the experiment.
The samples were taken of the required temperature (30, 45 and 50°C) with the light (8500 lux) for 30 minutes and 60 minutes. The medium after incubation was separated and tested for nitrate by the addition of suiphanilmide and N- (1-naphthyl) - ethelene diamine hydrochloride.
To test for nitrate reduction in the alga 1 ml of K2CO3 was added to solublised the alga.
The solublised alga were mixed with the above samples and reagents for nitrate test were added after the colour was developed the sample was centrifuged and absorbance taken at 540 nm. The
102 equal no. of alga were taken for
chlorophyll estimation. The results are expressed as im mg-1 chia
The results are presented in table and figure maximum nitrate reductase was observed at 50°C at 30 and 60 mm incubation (6.44 and 6.7 µm mg-1 chia) respectively.
Comparatively, more nitrate was reduced at 60 mm incubation at 30, 45 and 50°C (2.96 and 6.79 tm mm1 chia respectively).
From the above two experiments, we can conclude that, the nitrate formed by the enzyme activity of the algae could be detected in the medium.
Light stimulation of the enzyme nitrate reductase was characteristics of both Nostoc and Anabaena.
In dark, with Nostoc and Anabaena less enzyme activity was observed as there was non-availability of photosynthetic reducing power and energy to device the reaction.
The enzyme activity higher at 50°C than at 30°C and 45°C,
3.3 Effect of different factors on nitrate Reductase activity on Anabaena In the present investigation we have attempted to study the regulation of nitrate reductase in Anabaena. Here in we report some kinetics of nitrate production by Anabaena with emphasis on aspect of the regulation of NR activity. We have also studied the’ repression - depression mechanism of NR in Anabaena. An attempt has also been made to see the effect of argon and blue light on NR activity.
NR activity was determined as described in materials and methods.
Figure shows the kinetics of induction of NR activity following transfer of N2- grown cells into No3 supplemented medium.
No2’ began to appear this there after and then increased linearly.
Addition of the protein synthesis inhibitor chloramphenicol at 0 hr resulted in complete inhibition of NR activity.
However, if chloramphenicol was added at 12 hrs, the NR activity was not completely inhibited. Similar response was observed for rifamycin and rifampicin, NR activity being strongly inhibited by rifamycin and rifampicin.
Rifamycin (1.0 mg) produced a 92% inhibition of NR while 1.0 mg
rifampicin caused only about 45%
inhibition.
Table 4 Effect of various Inhibitors on nitrate reductase activity on Anabaena
Compound Concentratio n mg/ml
% inhibitio
n of NR activity
% inhibitio
n of growth Chloramphenic
ol 5
10 20 5
30 90 100
25
60 25 100
55
Kanamycin 10
20 1
65 100
45
90 100
80 Rifampicin 2.5
5 1
85 100
90
90 100
90 Rifamycin 2.5
5
55 100
100 100 The NR activity of the contract without added inhibitors was taken as 100%.
Like chioramphenicol, Kanamy Gin also inhibited the NR. 10.0 mg of chloramphenicol caused a 50% inhibition:
The same conc. of Kanamycin caused about 65% inhibition.
The effect of monochromatic light on nitrate production is shown in figure.
The NR activity was higher in fluorescent light than in blue light. In dark there was no induction of NR activity.
Figure shows the effect of argon 40% and 60% (02) on NR activity in fluorescent light and blue light.
Incubation of culture in 40%
argon stimulated the activity both in fluorescent and blue light. By comparison, if the culture was incubated in 60% O2 the activity was inhibited both in fluorescent and blue light.
Further more incubation in argon - oxygen mixture (40% 60%) showed a significant effect on enzyme activity as it was formed that the °2 inhibition of NR activity was restored by argon.
Figure - shows the repression and derepression of NR activity. Addition of ammonium at other inhibited the activity significantly. The activation of the enzyme after 12 hrs was prevented by the addition of NH4 Cl (1 m) at point B also but not significantly. The inhibitory action of NH4+
indicate NR repression when NH4+ + NO3
supplemented medium, no induction of NR occured but the addition of 5 mm MSO derepressed the NR activity even in NH4+ + NO3- medium.
103 Table - shows the activity of NR on
the effects of various amino acid.
Glutamine, glutamate, histidine, phenyl alanine and tryptophan repressed the NR activity. Arginine, aspartic acid and proline were without any significant effect.
As is evident from Figure, tyrosine affects induction of NR. Tysosine (0.5 m) resulted in a 1.5 to 2.0 fold higher NR activity. The stimulation was lesser in 1 mm as compared to 0.5 m.
Table 5 Effect of various samino acids (0.5 mm) on Nractivity
Amino acid Effects on NR activity Arginine
Aspoarticacid Glutamine a- Keto glutahic acid
Histidinne L-Phenylalannine
L-Tryptophan L-Tyrosine
Proline Serine
NSE NSE Inhibits Inhibits Inhibits Inhibits Inhibits Stimulates
NSE Inhibits NSE- No signification effects.
Fig. 1 Effect of Chloramphenicol on induction of NR activity. (O), Control (Δ) 10 µg/ml chloramphenicol added at point A (□) 10 µmg/ml chloramphenicol
added at point B.
Fig. 2 Effect of tyrosine on induction of nitrate reductase activity. (O) Control,
(▲) 0.5 mM tyrosine, (Δ) 1 mM tyrosine.
4 DISCUSSION
Nitrate reduction (assimilation) like other aspect of algal metabolism has received increased attention and several reviews have been published (Fogg, 1953, 1959;
Fogg and Wife 1954, Kessler, 1959 1976;
Zumft, 1976; Hewitt and. Cutting 1979.
Beevers and Hogeman 1980, Guerrero et al. 1981, Ullrich, 1983).
Nitrate reductase is the first enzyme of reduction path way nitrate to ammonia. It is substrate inducible enzyme and occur in bacteria, fungi, algae including blue-green and higher plants (Kessler, 1964). Nitrate reduction in algae occur in 2 steps, first the reduction of nitrate to nitrite and than nitrite to ammonia (Vennesland and Guessen, 1979; Guerroro et at. ‘1981).
In the present study an attempt made to see the effect of different factor on nitrate reductase. Nostoc showed and enhanced nitrate reduction in light (Table and Figure) as had been demonstro.ted in many other higher plant and algae (Kessler 1957, Hattori 1962; Hattori and Meyers, 1966) Moriss and Ahmad 1963, Vijay raghavan et at 1979) Parmeswarn, 1985). Similar result was also obtained with Anabae, (Table and Figure). In dark, there was very little reduction because the photosynthetic activities were less and
104 hence, the production of energy and
reducing power for nitrate reduction was less.
Studied on nitrate reduction by Nostoc showed that at 50°C that at 50°C maximum nitrate was reduced (Table and Figure) at all temperature studies move nitrate was reduced at 60mm, results were obtained with enzyme acivity being at 50°C in comparison to 30° and 45°C.
Our results shown in figure 3, 4 indicate that inhibitions of transcription and translation cause inhibition of nitrate induced increase in NR. Chloramphenicol and Kanamycin inhibit the NR activity indicating that NR may be synthesized on 70s Nbosomes. Rifamycin and rifampicin (inhibitors of RNA Polymerase) Completely inhibited NR activity.
Figure - 5 indicates the possible role of mono chromatic light in the regulation of NR activity Fluorescent light appears to be needed for the full induction of NR activity in vivo. The possible effects of light might be involved in the movement of nitrate to the site of enzyme synthesis similar results were obtained for induction of NR activity in Anabaena variabilis (Avissar, 1985) and in Eukary.
The antagonistic effect of NH4+ on NR know in many organism.
Figure 6 suggest that NH4+ acts as essor of NR in Anbaena.
Atranscriptional control of NR by ammonium has been thought operate in Anabaena dolium and Nostoc strain 6719.
(Herreret et al. 2001) and Anabaena guadruplicatum (Stevens and Van Baalen et al 1974 Ohmori et al., (1977) have questioned the role of NH4 as a repressor in Anabaena cyllndrica.
Figure 7 shows that MSO causes depression of NR activity. This suggest some direct or indirect involvement of CS in the regulation of NR Regarding the mechanism of ammonium promoted in activation of NR, it has been suggested that in enzyme complex, there is a labile moiety bearing an inhibitor site for an organic nitrogen compound resulting from NH4 metabolism.
(Vennesland and Guerrero, 1979).
We have shown here that blue light inhibits NR activity. A physiological role for the quality of light in modulating NR activity has been suggested, the flavin
prosthetic group being the light absorbing.
Pigment (Aparicio et al., 1976) Tischner and Hutterrnann (1978) concluded that there is no denovo synthesis of NR, but rather a light mediated activation of enzyme in Chlorella sps.
Increase of NR under anaerobic incubation (argon) andits loss under higher level of O2 shows anaerobic affinity of NR enzyme.
The NR activity of argon incubated culture was about 20-25%, higher.
Our results also indicate that the NR activity in Anabaena is apparent ly inhibited by °2 and that this inhibition is relived by argon. Although we have no adequate explanation for this stimulation of NR by argon, conceivably the higher NR in argon alone is some how related to H2 recycling. Nitrogen fixing cyanobactria are known to produce H2 at higher rates when incubated in argon.
It has long been known that and product inhibit both the turn over and activity of NR in prokarryotes. The NR activity in Anabaena is sensitive to inhibition by a few amino acids.
The repressive effect of Keto glutarte and glutamine point to some feedback inhibition.
Tyrosine stimulated NR activity and shortened the induction period.
Glutamine has been suggested as the putative repressor of NR in the fungus Neurospoara crass (Prem Kumar et al).
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