Our work provides additional evidence that regulation of the phosphorylation status of the PM H ATPase is critical for the response of Arabidopsis to environmental stimuli. Since A. halophytica is a halotolerant and alkaliphilic cyanobacterium, it is of interest to investigate the changes of ATPase activity in response to changes of NaCl concentration and pH of the growth medium. Increasing NaCl concentration in the growth medium up to 2 M led to a progressive increase of ATPase activity in the membrane vesicles . Higher ATPase activity was observed in cells grown at high pH than at low pH at all four concentrations of NaCl tested. When the cells were grown at different pH values, ATPase activity in the membrane vesicles was slightly increased upon increasing the pH from 5.0 to 7.6 . A marked increase of enzyme activity was evident at pH higher than 7.6. Cells grown at 2 M NaCl showed higher enzyme activity than those grown at 0.5 M NaCl at all four pH values tested. Purification of ATPase ATPase is a membrane protein, hence it is necessary to find optimum conditions to solubilize the ATPase from the membranes.
Treatment of membrane vesicles with 7 mM sodium cholate for 30 min resulted in the Proteasome Inhibitor highest yield of ATPase . The two step purification, PEG 6000 precipitation followed by Superose 6 gel filtration, resulted in a 17.5 fold purified enzyme with 6.5% yield . It is noteworthy that more than 85% ATPase activity was recovered in the solubilized membrane protein suggesting that nearly all ATPase proteins were solubilized in their native forms. The enzyme was stable up to one month at 20 C. SDS PAGE analysis revealed some polypeptide bands which could tentatively be identified as ATPase subunits of an F type ATPase, namely a , b , g , a , b ? and c based on the comparison with the typical mobilities and band pattern of an F type ATPase subunits from both Ilyobacter tartaricus and thermoalkaliphilic Bacillus sp. strain TA2.A1 . The subunit c was further characterized by cutting the band on SDS PAGE and digesting with trypsin before analysis by liquid chromatography mass spectrometry . The spectra were recorded on an ESI Q TOF mass spectrometer .
The amino acid sequences of the peptides were obtained using Mascot program . One resultant tryptic peptide ISSGAEGIAR with the molecular mass of 959.5036, by searching the National Center for Biotechnology Information database, was highly identical to mTOR inhibitor the sequence of F type ATPase subunit c of Synechococcus sp. WH 8102 , Synechococcus sp. CC9902 , Synechococcus sp. PCC 7002 and Gloeobacter violaceus with % identity of 90, 90, 80 and 60%, respectively. Catalytic properties of the purified ATPase The activity of ATPase as influenced by various cations was tested and the results are shown in Figure 3A. Increasing Na concentration up to 10 mM caused a progressive increase in enzyme activity. K , Li , and Ca2 had no stimulatory effect on enzyme activity.