ment inactived by heating for 10 min at 56°C. Plasma was stored at − 20°C until use.
Antibody specifity was determined by Western blotting of crude extracts subjected to SDS-
PAGE. After electrophoresis, proteins were trans- ferred to nitrocellulose blots according to the
procedure of Towbin et al. [23]. Protein blots were reacted with anti-GOGAT antibodies and visual-
ized
with peroxidase-conjugated
anti-rabbit antibodies.
The immunoprecipitation
curve of
nodule NADH-GOGAT activity was performed using
aliquots of crude extract 100 ml incubated with increasing amounts of anti-GOGAT from 5 to 60
ml, PBS 50 mM potassium phosphate at pH 7.2 containing 150 mM NaCl up to 0.4 ml and 100 ml
of Protein A Sepharose 10 wv 100 ml at 4°C for 12 h. The samples were centrifuged at 2500 × g
for 10 min and NADH-GOGAT activity was as- sayed in the supernatant. Pre-immune serum was
incubated under identical conditions as a control.
2
.
8
. Protein extraction Nodule, root and leaf protein for Western blots
and enzyme assays were ground in a mortar and pestle with an extraction medium containing 100
mM maleic acid-KOH, pH 6.8, 100 mM sucrose, 2
vv 2-mercaptoethanol,
and 15
vv ethylene glycol. The homogenate was centrifuged
at 3500 × g at 2°C for 8 min and afterwards at 30 000 × g for 20 min. The bacteroid protein was
extracted as described by Delgado et al. [24].
2
.
9
. Protein determination The soluble proteins were determined by Brad-
ford [25] assay, using bovine serum albumin Merck, fraction V as the standard.
3. Results
The NADH-GOGAT of V. faba cv. Alborea nodules was purified 74-fold after the final gel
filtration chromatography step to a specific activ- ity of about 3 mmol min
− 1
mg protein
− 1
with a final yield of 32 Table 1. Ammonium sulfate
precipitation was used for concentrating the crude preparation of enzyme, while also removing
leghemoglobin and other proteins, resulting in a 2.6-fold increase in specific activity. Ion exchange
chromatography gave the highest purification, with a further 21-fold increase in specific activity.
The peak of maximal activity was eluted from the column at KCl concentration of about 0.24 M
Fig. 1A. Higher purification 385-fold could be obtained by including affinity chormatography on
Blue Sepharose, instead of gel filtration, but about 85 of remaining activity was lost during this step
and moreover the binding of the protein to the column was erratic data not shown.
The proteins at different stages during the purification procedures were run on SDS-poly-
acrylamide gels Fig. 2A. The NADH-GOGAT preparations passed through gel filtration chro-
matography showed a major protein band after SDS-PAGE, indicating that the major component
of the fractions was NADH-GOGAT.
Only one peak of NADH-GOGAT activity was clearly distinguishable in the eluate from Q-Sep-
harose column, eluted at 0.36 M KCl Fig. 1B. Native molecular weight of V. faba nodule
NADH-GOGAT was estimated by both non-de- naturing PAGE and gel filtration chromatography
on Sephacryl S-300. Native PAGE stained for activity revealed a single band of 236 9 3 kDa
Fig. 2B. The estimation by gel filtration was a little lower, 222 9 5 kDa, and the subunit molecu-
Table 1 Purification of faba bean root nodule NADH-GOGAT from 50 g of nodules
a
Total protein Total activity
Specific activity mmol min
− 1
mg Purification factor
Purification Recovery
step -fold
protein
− 1
mg mmol min
− 1
100 0.036
25.0 695
1.0 Crude extract
249 NH
4 2
SO
4
23.0 0.092
92 2.6
15.7 7.87
DEAE-Sep- 63
55 2.00
hacel 32
74 2.66
7.9 Sephacryl S-
2.97 300
a
Data are representative of six separate purifications, all purifications were completed within 48 h.
Fig. 1. Elution profile of V. faba nodule NADH-GOGAT activity from a DEAE-Sepharose column A or a Q-Sep-
harose columnB. Fig. 2. A. SDS gel electrophoresis pattern of partially purified
NADH-GOGAT from V. faba nodules. Lane 1, purified by DEAE-sephacel column 25 mg protein; lane 2, purified by
Sephacryl S-300 column 9.3 mg protein; lane 3, molecular weight markers; lane 4, crude extract 23 mg protein; lane 5,
concentrated by ammonium sulfate precipitation 30 – 60 saturation 25 mg protein. B. Non-denaturing linear gradient
4.5 – 10 PAGE of the NADH-GOGAT partially purified 100 mg proteinlane and stained by activity. The position,
and size in molecular weight of protein markers are indicated. The native molecular weight 236 kDa was determined by
regression analysis y = − 1.482x + 6.047; r = 0.999 against native-PAGE separated protein markers.
corresponded in weight 195 kDa to purified GOGAT. Western blot also revealed a comparable
195 kDa polypeptide in root extracts Fig. 4B, lane 1 but not in those of leaves or bacteroids
Fig. 4B, lanes 3 and 4.
Antibodies to
faba bean
nodule NADH-
GOGAT recognize a 195 kDa polypeptide from nodules of five inbred lines VF51, VF61, VF52,
lar weight, estimated by SDS-PAGE, was 195 9 2 kDa.
Fig. 3 shows the NADH-GOGAT activity im- munoinhibition curve. The addition of the anti-
bodies raised against purified V. faba nodule NADH-GOGAT to the crude extract of this plant
resulted in the appearance of a precipitate and a decrease in the NADH-GOGAT activity of the
supernatant obtained after centrifugation. The val- ues shown in Fig. 3 correspond to the remaining
NADH-GOGAT activity of the supernatants; with the addition of 20 ml of antiserum 60 of the in
vitro NADH-GOGAT activity was lost, and, with 40 ml of antiserum, all the NADH-GOGAT activ-
ity was lost. Electrophoresis SDS-PAGE of the immunoprecipitate, followed by Western blotting
using anti-GOGAT serum, showed increasing quantities of a 195 kDa polypeptide precipitated
from the nodule extract Fig. 3A.
Polyclonal antibodies were produced against V. faba nodule NADH-GOGAT as shown by the
unique reactive band seen on the Western blots of SDS-PAGE of purified preparations Fig. 4A or
crude nodule proteins Fig. 4B, lane 2, which
Fig. 3. Immunoprecipitation curve of V. faba nodule NADH- GOGAT activity using antisera. A. Western blot of immune
complexes precipitates. B. Curve of NADH-GOGAT activity remaining in the supernatant, using non-immune serum as
control.
Fig. 4. A. Western blot of SDS-PAGE of partially purified V. faba nodule NADH-GOGAT. Lane 1, 5 mg protein; lane 2, 9 mg protein. B. Western blot of SDS-PAGE of soluble proteins 25 mg from root lane 1, effective nodules lane 2, bacteroids lane
3, and leaves lane 4. C. Western blot of SDS-PAGE of nodule soluble proteins of different genotypes of V. faba. Lanes: 1 Alborea; 2 VF51; 3 VF61; 4 VF52; 5 VF60 and VF184 0.083, 0.009, 0.075, 0.023, 0.065 and 0.069 mmol NADH
ox
min
− 1
mg
− 1
protein, respectively. D. Western blot of SDS-PAGE of nodule soluble proteins of various legume species. Lanes: 1 V. faba; 2 V. sati6a; 3 M. sati6a; 4 G. max and 5 P. sati6um 0.065, 0, 0.123, 0.070 and 0.096 mmol NADH
ox
min
− 1
mg
− 1
protein, respectively. For each lane, 35 mg protein.
VF60, and VF184 of V. faba Fig. 4C and for a number of legumes Fig. 4D. The lack of a cross-
reactive band in V. sati6a reflects the lack of activity in the crude extract from nodules Fig. 4.
4. Discussion
