Plant Physiol. (1976) 58, 57-59
Specificity for Nicotinamide Adenine Dinucleotide and
Nicotinamide Adenine Dinucleotide Phosphate of Nitrate
Reductase from the Salt-tolerant Alga Dunaliella parvaI
Received for publication January 7. 1976 and in revised form March 15. 1976
YAIR M. HEIMER
Atomic Energy Commission, Nuclear Research Centre-Negev, Beer-Sheva, Israel
ABSTRACT
x 40 cm) was equilibrated with 50 mm K-phosphate (pH 7.5).
containing 1 mM L-cysteine and eluted with a linear gradient of 0
to 0.25 M K2S04 in the equilibrating buffer. Sepharose 4B in a
column (1 .5 x 40 cm) was equilibrated and eluted with the same
buffer as that used for the equilibration of the DEAE-cellulose.
Nitrate reductase activity was assayed as previously described
(14). Activity was determined by measuring either the amount
of nitrite formed or the nitrate-dependent oxidation of the reduced pyridine nucleotides. The incubation mixture for the determination of Pi released from NADPH was free of added Pi
and contained in 1 ml: 60 ,umol tris-HCI (pH 7.5), 10 ,umol
KNO3; 6 ,umol NADPH or NADH, and 2 to 5 mg of enzyme
protein with specific activity of 20 to 40 nmol NO3- reduced/
m mg protein. Incubation period was 30 min. Inorganic phosmin
phate was determined by the method of Fiske and SubbaRow
(5). The incubation mixture for the coupling of nitrate reduction
to glycerol oxidation contained in 1 ml: 50 ,umol tris-HCI (pH
9), 10 ,umol KNO3, 50 nmol NADH or NADPH, 3.3 mmol
glycerol, and 2 to 3 mg crude extract with specific activity of 8 to
12 nmol NO3- reduced/min- mg protein. Protein was determined by the method of Lowry et al. ( 11).
The experiments were repeated at least three times with
similar results.
Nitrate reductase of the salt-tolerant alga Dunaliella parva could
utilize NADPH as well as NADH as an electron donor. The two
pyndine nudeotide-dependent activities could not be separated by
either ion exchange chromatography on DEAE-cellulose or gel filtration
on Sepharose 4B. The NADPH-dependent activity was not inhibited by
phosphatase inhibitors. NADPH was not hydrolyzed to NADH and
inorganic phosphate in the course of nitrate reduction. Reduction of
nitrate in vitro could be coupled to a NADPH-regenerating system of
glycerol and NADP-dependent glycerol dehydrogenase. It is concluded
that the nitrate reductase of D. parva will function with NADPH as well
as NADH. This is a unique characteristic not common to most algae.
Nitrate reductase of most algae and higher plants is specific
for, or has a preferential requirement for. NADH as electron
donor (1. 6. 10). The ability of the enzyme from several higher
plants to use NADPH as well as NADH (2, 4) was recently
shown ( 13) to be an artifact caused by the presence in the extract
of a phosphatase-like activity which converted NADPH to
NADH and Pi. In an earlier communication (8). it was shown
that the nitrate reductase of the salt-tolerant alga Dunaliella
parva could utilize NADPH as well as NADH as an electron
donor, and that the NADPH-dependent activity was insensitive
to phosphatase inhibitors. Since the ability to utilize both pyridine nucleotides for nitrate reduction represented an uncommon
characteristic among algae, a more detailed study was undertaken to determine the true electron donor specificity of the
RESULTS AND DISCUSSION
Nitrate reductase from D. parva can utilize NADH or
NADPH as electron donors for nitrate reduction (8) with apparent Km values of 10 gM and 20 /LM, respectively (Fig. 1). These
Km values may point to a similar affinity of the enzyme for the
two pyridine nucleotides. However, the ability to utilize
NADPH as electron donor could be an artifact caused by the
presence of a phosphatase in the crude extract which converted
NADPH to NADH and Pi. as was recently shown for several
higher plants (13). The following experiments were designed to
determine whether this was the case for the nitrate reductase of
D. parva as well.
As seen from Figure 2, niether ion exchange chromatography
on DEAE-cellulose nor gel filtration on Sepharose 4B could
eliminate or at least inhibit the NADPH-dependent activity.
This result did not rule out the possibility that the phosphataselike activity was tightly associated with the nitrate reductase. The
effect of phosphatase inhibitors on the NADPH-dependent activity of a partially purified preparation was tested. There was no
preferential inhibition of the NADPH-dependent activity by
either inorganic phosphate or fluoride as compared with the
NADH-dependent activity. Such an inhibition could be expected
if a phosphatase were associated with the ability to use NADPH
(13). Furthermore, NADPH was not hydrolyzed to NADH and
Pi in the course of nitrate reduction. After a 30-min incubation
period in the presence of NADPH, 4050 nmol NO2- and 480
enzyme.
MATERIALS AND METHODS
Cells of D. parva were grown as previously described (7) on a
synthetic medium (12) containing 2 M NaCl. Nitrate reductase
was extracted from cells at midlogarithmic phase as previously
described (8). The crude extract was either dialyzed overnight
against 0.1 M K-phosphate buffer (pH 7.5), containing 1 mM Lcysteine and then used as the enzyme source, or was further
fractionated with ammonium sulfate. The protein fraction,
which precipitated at 50% saturation of ammonium sulfate, was
used as enzyme source after dialysis against 0.1 M K-phosphate
buffer (pH 7.5), containing 1 mM L-cysteine.
Ion exchange chromatography on DEAE-cellulose was carried out according to Wells and Hageman (13). The column (1.5
l A short account of this work was published in Plant Physiol. 56: 50,
August 1975.
57
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1976 American Society of Plant Biologists. All rights reserved.
Plant
HEIMER
58
Physiol. Vol. 58, 1976
nmol Pi were detected in the incubation mixture. The amount of
Pi released from NADPH to the incubation mixture was about
10% of the amount of nitrite formed, which was well below the
100% expected if hydrolysis had taken place.
The coupling of nitrate reduction to a NADPH-regenerating
system would provide the best evidence for the specificity of the
nitrate reductase. We were able to couple nitrate reduction to a
NADPH-regenerating system made of glycerol and the enzyme
NADP-glycerol dehydrogenase (dihydroxyacetone reductase) of
D. parva described by Ben-Amotz and Avron (3) (Fig. 3). It can
be clearly seen that in the presence of a catalytic amount of
NADPH but not NADH, glycerol could provide a source of
electrons for nitrate reduction.
The data presented above indicate that the ability to utilize
NADPH as electron donor by nitrate reductase of D. parva was
not an artifact caused by a phosphatase-like activity in the
extract. Thus, the enzyme will utilize either NADPH or NADH
as an electron donor. A similar conclusion was drawn earlier by
1/ [NAD(P)H] (PuM)
LeClaire
and Grant (9), who used partially purified nitrate
of
of
the
rate
oxida1.
Lineweaver-Burk
FIG.
plot
nitrate-dependent
of Dunaliella tertiolecta assuming it was free of a
reductase
tion of NADH (0) and NADPH (0). as a function of their concentration in the assay mixture. Oxidation was assayed as the decrease of possible contaminating phosphatase. However, in some cases,
such an assumption has been shown to be incorrect (13).
absorbancy at 340 nm.
200
-
160
-
120
A
-
80
E
_
40
-
E
O
B
z
80 _
0
Ec
80
f
l.
-60
E
140
0
20
le
NUMBER
FRACTION
FIG. 2. Gel filtration on Sepharose 4B (A) and ion exchange chromatography on DEAE-cellulose (B) of nitrate reductase. NADH (A) and
NADPH (A) nitrate reductase activities are expressed as nmol N02 formed/ml- 10 min. Broken line indicates the concentration of K2SO4. Fraction
volume was 1 .1 ml.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1976 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 58, 1976
NITRATE REDUCTASE OF DUNALIELLA PARVA
59
most algae is specific only for NADH (10). It remains to be seen
whether this ability is related to the adaptation of this alga to
extreme environmental conditions.
2.0
LITERATURE CITED
1.6
E
41
1.2
E
0
z
0.8
v
0
t
E
0.4
+
l
60
80
~ ~~~~~~~~~~~~l
0
0
20
40
1. BEEVERS. L. AND R. H. HAGEMAN. 1969. Nitrate reduction in higher plants. Annu. Rev.
Plant Physiol. 20: 495-522.
2. BEEVERS. L.. D. FLESHER. AND R. H. HAGEMAN. 1964. Studies on pyridine nucleotide
specificity of nitrate reductase in higher plants and its relationship to sulfhvdryl level.
Biochim. Biophys. Acta 89: 453-464.
3. BEN-AMOTZ, A. AND M. AVRON. 1974. Isolation, characterization and partial purification
of a reduced nicotinamide adenine dinucleotide phosphate dependent dihydroxyacetone
reductase from the halophilic alga Dunaliella parva. Plant Physiol. 53: 628-631.
4. EvANS, H. J. AND A. NASON. 1953. Pyridine nucleotide nitrate reductase from extracts of
higher plants. Plant Physiol. 28: 233-244.
5. FIsKE. C. H. AND Y. SuBsARow. 1925. The colorimetric determination of phosphorous. J.
Biol. Chem. 66: 375-400.
6. HAGEMAN. R. H. AND D. P. HUCKLESBY. 1971. Nitrate reductase from higher plants.
Methods Enzymol. 24: 491-503.
7. HEIMER. Y. M. 1973. The effect of sodium chloride, potassium chloride and glycerol on the
nitrate reductase of a salt tolerant and two non-tolerant plants. Planta 113: 279-281.
8. HEIMER, Y. M. 1975. Nitrate reductase of Dunaliella parva, electron donor specificity and
heat activation. Arch. Mikrobiol. 103: 18 1-183.
9. LECLAIRE. J. A. AND B. R. GRANT. 1972. Nitrate reductase from Dunaliella tertiolecta,
purification and properties. Plant Cell Physiol. 13: 899-907.
10. LOSADA, M. 1974. Interconversion of nitrate and nitrite reductase of the assimilatory type.
In: Metabolic Interconversion of Enzymes. Springer-Verlag. Heidelberg. p. 257.
11. LoWRY, 0. H.. N. J. ROSEBROUGH. A. J. FARR, AND R. J. RANDALL. 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275.
12. McLAcHLAN, J. 1960. The culture of Dunaliella iertiolecta Butcher-a euryhaline organism.
Can. J. Microbiol. 6: 367-379.
13. WELLS. G. N. AND R. H. HAGEMAN. 1974. Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves. Plant Physiol. 54: 136-141.
14. WRAY. J. L. and P. FILNER. 1970. Structural and functional relationships of enzyme
activities induced by nitrate in barley. Biochem. J. 119: 715-725.
100
120
TIME (min.)
FIG. 3. Coupling of nitrate reduction to glycerol oxidation. Amount
of nitrite in the reaction mixture was determined at the indicated time
after proper dilution. NADH (- -A- -), NADH + glycerol (- -A- -),
NADPH (-A-), NADH + glycerol (-A-). Arrow indicates the addition of 10 nmol NADPH to reaction mixture containing NADH.
The ability to use both reduced pyridine nucleotides for nitrate
reduction is a unique characteristic since nitrate reductase from
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1976 American Society of Plant Biologists. All rights reserved.