Plant Science 143 (1999) 1–8
NADH: nitrate reductase and NAD(P)H: nitrate reductase activities in
mustard seedlings
Altaf Ahmad, M.Z. Abdin *
Centre for Biotechnology, Faculty of Science, Hamdard Uni6ersity, New Delhi
110 062
, India
Received 22 September 1998; received in revised form 20 January 1999; accepted 27 January 1999
Abstract
Mustard (Brassica juncea L. Czern and Coss cv. Pusa Jai Kisan) seedlings were grown for 10 days in nutrient solutions
containing different concentrations of nitrate (10, 20, 30, 40 and 50 mM). Nitrate reductase activity was determined in the
presence of either NADH or NADPH as electron donor. NADH- and NADPH-supported nitrate reductase activity varied
significantly in the seedlings grown at different nitrate concentrations. The NR activity was higher with NADH than NADPH in
the seedlings grown at 10 and 20 mM nitrate levels. With the increase in nitrate concentration in nutrient solution, NADPH-sup-
ported NR activity gradually increased, while NADH-supported NR activity remained nearly constant. Seedlings grown at 30
mM nitrate had almost equal NR activity when either NADH or NADPH was used as electron donor. When NR activity was
assayed in the presence of an equimolar concentration of both NADH and NADPH, it was greater than the one observed either
with NADH or NADPH alone. By the use of affinity chromatography using blue-Sepharose, two nitrate reductases, viz.
NADH:NR (EC 1.6.6.1) and NAD(P)H:NR (EC 1.6.6.2) were partially purified. The trend of activities of purified NADH:NR
and NAD(P)H:NR were almost similar to that observed in the crude extract. The NAD(P)H:NR has a K
m
of 2.5 mM for KNO
3
,
K
m
of 0.05 mM for NADPH and K
m
of 0.20 mM for NADH. The NADH:NR has a K
m
of 0.10 mM for KNO
3
, K
m
of 0.01mM
for NADH. The pH optima for both the NRs was 6.8. © 1999 Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Brassica juncea; NADH:NR; NAD(P)H:NR; Nitrate reduction; Nitrate reductase
1. Introduction
Nitrate reductase (NR) is a key enzyme involved
in the first step of nitrate assimilation in plants
[1–3]. It catalyzes the reduction of nitrate to ni-
trite with pyridine nucleotide in higher plants. The
NR molecule is a complex protein, containing
flavin adenine dinucleotide, cytochrome b 557, and
molybdenum cofactor as prosthetic groups and
exists as a homodimer, each monomer having a
molecular mass of about 100–110 kDa [4]. In
most higher plants, nitrate reduction is catalyzed
by NADH:NR (EC 1.6.6.1), an enzyme which is
specific for NADH as the electron donor and has
a relatively low K
m
for nitrate [1]. The presence of
NADPH-utilizing NRs has been demonstrated in
rice seedlings [5], soybean leaf [6,7], soybean
cotyledons [8,9], maize [10–14], birch [15,16] and
barley [17–20]. In these studies, the NADPH-uti-
lizing NR has been found to be a bispecific en-
zyme, using both NADPH and NADH as electron
donor, but showing highest activity with NADPH
* Corresponding author. Fax.: + 91-11-6988874.
E-mail address
:
root@hamduni.ren.nic.in (M.Z. Abdin)
0168-9452/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168-9452(99)00016-3