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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

in a



containing 3


3 mM Mg SO4, 50 mM KCl, 0.02 % Triton X-100, 2


ATPase Activity

mM sodium molybdate and 1 mM Na Azide. The

(In mM)

(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


0.42 +-0.10

of reagent (ammonium heptamolybdate 0.5% in


0.46 +-0.08

H2SO4 1N and ascorbic acid 2% in H2SO4 1N).


050 +- 0.15

Driouich et al.

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Relationship between ATPase activity and phosphoenolyruvate carboxylase (PEPC) capacity of durum wh  
Relationship between ATPase activity and phosphoenolyruvate carboxylase (PEPC) capacity of durum wh  

The effect of NaCl on the membrane ATPase activity was studied at the root level in durum wheat (Triticum durum Desf. Var...