performed at 4°C as soon as possible to minimize the
The volume of the reaction media is brought to 500 µl
process of proteolysis.
by the Tris-Mes buffer solution of 50 mM, pH 6.5. The blue coloration is developed during 40 min at
Measurement of PEPC capacity
ambient temperature. The tubes are reset to 0°C to
The PEPC capacity is measured in vitro in the crude
stabilize the coloration and optical density is
extract. It represents the activity of the enzyme in
measured by spectrophotometer at 820 nm.
optimum conditions of substrates and cofactors (Queiroz and Morel, 1974). We follow the oxidation
of NADH for at least 10 min at 30°C in buffer solution
The evolution of the membrane ATPase activity of
of ris-bicine pH 8. The reading is performed by
roots in durum wheat variety "Karim" is represented
spectrophotometer (Beckman DU 640) at 340 nm
on the figure 1. It shows that the salt treatment of seedling's roots for 4 days led to ATPase activity
Determination of malate content
stimulation and an acidification of culture media
The malate is assayed in the "crude extract" according
which could be explained by the release of H+
to the method of Hohorst (1970) in the presence of
malate deshydrgenase enzyme. The measurement is carried out in a buffer solution of hydrazine- glycine (400 - 500 mM) pH 9, in 2.5 mm of NAD and 40 µl of the "crude extract".
A first reading of the optical
density (OD1) is made with a spectrophotometer at 340 nm. The second reading (DO2) is obtained after 10 minutes of adding 5 UE of trade MDH (Sigma). Measurement of the ATPase activity The roots are washed in cold distilled water, dried
Fig 1. Variation in the membrane ATPase activity of
and then put in plasmolyse for 40 minutes in a
wheat roots under salt treatment for a period of 4
homogenization buffer solution of Tris-HCl 100
mM pH 7.5 containing 5 mM EDTA, 10 mm betamercaptoethanol, 0.5 M sucrose and 40 µM PMSF.
Transfer of seedlings under normal conditions for 48
Then they are crushed cold and the homogenate is
hours resulted in a return of ATPase activity to
filtered through layers gauze. The filtrate obtained
standard level (Table1). The pH of the culture media
underwent a series of centrifugation until the
has not changed. These results show that the
obtaining of "enzyme extract" (Driouich , 2006).
membrane ATPase activity is reversible.
All extraction steps were also performed at 4°C. The membrane ATPase activity is measured according to
Table 1. Evolution of the ATPase activity after
the method of Chen et al. (1956) on "extract enzyme"
transfer of seedlings in normal conditions during 48h
3 mM Mg SO4, 50 mM KCl, 0.02 % Triton X-100, 2
mM sodium molybdate and 1 mM Na Azide. The
(In µmol. -3.min-1.µg-1 1 protein)
reaction was held at 38°C for 15 min; it is initiated by
0.40 +- 0.12
adding the enzyme extract and stopped at 0°C by 2 ml
of reagent (ammonium heptamolybdate 0.5% in
H2SO4 1N and ascorbic acid 2% in H2SO4 1N).
050 +- 0.15
Driouich et al.
Published on Feb 17, 2014
Published on Feb 17, 2014
The effect of NaCl on the membrane ATPase activity was studied at the root level in durum wheat (Triticum durum Desf. Var...