Cytosolic V-1-ATPase complex in pea (Pistum sativum L.) root cells was characterized by immunoblotting, immuno-electron microscopy and measurement of ATP hydrolysis activity. Using antibodies against the subunits A and B of V-type H+-ATPase (V-ATPase) from mung bean as primary antibodies, both immuno-blotting and immuno-electron microscopy showed that subunit A and subunit B existed in the cytosol. Measurement of ATPase activity further showed that cytosolic proteins had NO3--sensitive ATP hydrolysis activity. All these suggested that V-1-ATPase complex occurred in the cytosol. This is the direct proof that V-1-ATPase occurs in the cytosol of the plant cells for the first time.
To study the function and adaptive mechanism of tonoplast H + ATPase under salt stress, pea ( Pisum sativum L.) seedlings were treated with different concentrations of salt (100-250 mmol/L NaCl) and with 100 mmol/L NaCl for different days (1-3 d). The ATP hydrolytic activity and the proton transport activity and the changes of the amount of tonoplast H + ATPase (subunit A) were measured. ATP hydrolytic activity of H + ATPase prepared from plants treated with 250 mmol/L NaCl was reduced by about 25% compared to that of control plants, but that of stressed plants treated with 100 mmol/L and 200 mmol/L NaCl was unchanged. The activity from plants treated with 100 mmol/L NaCl for up to 3 d was lower than that of control plants by 20%. But the proton transport activity was increased under the same salt stresses as above. These results showed that the changes of the hydrolytic activity and the proton transport activity were not in proportion and salt stress may cause the change of the coupling ratio of H + transport activity to ATP hydrolysis. The protein amount kept unchanged and reduced a little only when pea was treated with 100 mmol/L NaCl for 3 d. These results indicated that salinity stimulated the increase of the pump efficiency of the V_ATPase from pea roots, which was due to the change of the coupling ratio, but not due to the increase of ATP hydrolysis and the amount of V_ATPase.
